Phylogenetic evidence of a possible Trichuris globulosa species complex in Arabian camels from Kuwait

Abstract

During a 1-year study, Trichuris adults were obtained after necropsy of Arabian camels (Camelus dromedarius) from a slaughterhouse in Kuwait. Morphological and molecular identification was performed to confirm the identity of the Trichuris specimens obtained from C. dromedarius. Fifteen male Trichuris specimens were selected, and molecular identification was performed using mitochondrial cytochrome c oxidase subunit I, 12S ribosomal RNA, 16S ribosomal RNA genes and the nuclear internal transcribed spacer 2 (ITS2) region. Through phylogenetic analysis, 2 distinct groups were obtained using the mitochondrial genes, where group 1 showed a close relationship to Trichuris globulosa while group 2 showed a close relationship to Trichuris ovis, providing molecular evidence of a possible T. globulosa species complex. Additionally, the nuclear ITS2 region did not provide enough resolution to distinguish between the 2 groups of Trichuris specimens. Observation of morphological characters revealed variations in the shape of the male spicule sheath, where specimens present either a globular posteriorly truncated swelling or the absence of posteriorly truncated swelling. Moreover, the variations in male spicule sheath does not corroborate with the results of molecular data, suggesting the limited use of this character for identification of T. globulosa. In conclusion, molecular analysis suggests a possible species complex in T. globulosa, with the mitochondrial genetic markers successfully differentiating between the 2 groups. The limited use of the male spicule sheath as a diagnostic character for identification of T. globulosa is suggested.

Introduction

The genus Trichuris Roederer, 1761 includes numerous species that parasitize humans and animals, and have a cosmopolitan distribution (Doležalová et al., 2015). Species identification has generally been based on morphological characters and morphometrical measurements, including host species as a guide (García-Sánchez et al., 2019). Molecular phylogenetics are also providing evidence of species complexes within Trichuris, where it is highly possible that Trichuris can harbour cryptic species due to their wide geographic distribution and capability to parasitize variety of host species (Callejón et al., 2012; Ravasi et al., 2012; Robles et al., 2014; Rivero et al., 2021). In ruminants, more than 23 species of Trichuris have been described, including Trichuris globulosa (Linstow, 1901), Trichuris ovis (Abildgaard, 1795), Trichuris skrjabini Baskakov, 1924 and Trichuris discolor (Linstow, 1906) (Knight, 1974; Cutillas et al., 1995). In camels specifically, 9 species of Trichuris have been found. They are Trichuris barbetonensis Ortlepp, 1937, T. globulosa, Trichuris infundibulus (Linstow, 1906), Trichuris lani (Artjuch, 1948), T. skrjabini, Trichuris tenuis Chandler, 1930, Trichuris vulpis (Froelich, 1798), Trichuris raoi Alwar and Achutan, 1960 and Trichuris cameli (Chandler, 1930; Knight, 1971; Sazmand and Joachim, 2017). Aside from parasitizing camels, some species have also been found in other hosts, for example, T. globulosa have also been found in sheep and goats, while T. skrjabini have been reported in other large and small ruminants (sheep, deer, goats, elk) (Knight, 1971). Similarly, T. ovis and T. discolor, which are parasites of sheep and cattle respectively, were also found in many hosts globally (Chandler, 1930; Knight, 1971; Cutillas et al., 1995; Oliveros et al., 2000; Wang et al., 2012).

Based on male morphology, the spicule sheath, spicule lengths and spines on the spicule sheath are used as the main criteria for differentiating T. globulosa and T. ovis. Trichuris globulosa was described as having the spicule sheath with a globular posteriorly truncated swelling, with spines longer on the swelling than the rest of the spicule sheath, while in T. ovis, 2 morphological types of spicule sheath were observed: one was having a spherical bulge in the distal part of the spicule sheath (Yevstafieva et al., 2018) and the other with a globular posteriorly truncate sheath (Sarwar, 1945; Cutillas et al., 1995). Although spicule lengths have been cited as a useful criterion by Chandler (1930), overlapping lengths were previously reported from specimens isolated from various hosts, limiting their discriminatory power (Chandler, 1930; Callejón et al., 2015a). In Trichuris females, the structure of the vulva is mainly used for species differentiation. Baylis (1932) found consistent differences between the vulva structure of T. globulosa and T. ovis (Baylis, 1932; Yevstafieva et al., 2018). However, other studies further revealed that the vulva structure cannot be used as a distinguishing morphological character for species differentiation of T. globulosa from T. ovis (Callejón et al., 2015a). Thus, the vagueness of morphological characters and overlap of morphometrical measurements render species identification challenging. Additionally, the existence of T. globulosa and T. ovis in similar hosts can further complicate morphological identification due to host-induced variation and phenotypic plasticity.

With the challenges in morphological identification, molecular genetic markers have aided in Trichuris species identification and have been used in the investigation of genetic variability among different hosts. These include the mitochondrial cytochrome c oxidase subunit I (COI), cytochrome b (cytB), 16S ribosomal RNA (rRNA), complete mitochondrial genomes and nuclear internal transcribed spacer 1 and 2 regions (ITS1 and ITS2) (Callejón et al., 2012, 2015b; Hawash et al., 2015; Rivero et al., 2020; Rivero et al., 2022). Among T. globulosa and T. ovis isolated from sheep and goats, Oliveros et al. (2000) revealed that these 2 species are synonymous using the ITS2 region. However, through a morpho-biometrical and molecular study using mitochondrial COI and cytB markers, it was concluded that T. globulosa constitute a different genetic lineage to T. ovis (Callejón et al., 2015b). Additionally, Salaba et al. (2013) revealed that specimens initially morphologically identified as T. globulosa were later molecularly identified as T. discolor using the nuclear ITS regions (Salaba et al., 2013).

Here, to increase the clarity of the species status of T. globulosa, 3 mitochondrial genetic markers (COI, 12S and 16S rRNA) and the nuclear ITS2 region were used for the molecular identification of Trichuris species from Camelus dromedarius (Arabian camel) in Kuwait. Through molecular analyses, we provide phylogenetic evidence to suggest a probable Trichuris species complex with the specimens obtained from C. dromedarius and suggest that the morphology of the male spicule sheath is not a reliable character for species discrimination.

Materials and methods

Morphological measurements and identification

Fifteen Trichuris males obtained after necropsy of 3 C. dromedarius hosts from a slaughterhouse in Kuwait were used as representative specimens for morphological and molecular identification. Trichuris males were selected as representatives for this study as the morphological characters are useful for species identification. In the Department of Helminthology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand, individual specimens were washed thoroughly in sterile distilled water, and morphological measurements of the body length and body width were obtained using a stereomicroscope (Olympus SZ51). The posterior and anterior portions of each specimen were separated, and the posterior portion was subsequently mounted on a microscope slide with lactophenol. Under an inverted compound microscope (ZEISS Primovert), morphological measurements and characters of the posterior portion (spicule length and width, spicule sheath shape, spicule sheath length and width) were performed following Callejón et al. (2015a). The middle portion of the specimen that was not mounted on a microscope slide with lactophenol was maintained in 70% ethanol and at −20°C for preservation prior to molecular identification.

Molecular identification and phylogenetic analyses

Genomic DNA extraction and polymerase chain reaction (PCR) amplification

Each specimen was individually washed in sterile water and transferred into a 1.7 mL microcentrifuge tube. Total genomic DNA (gDNA) was extracted using the DNeasy^® Blood & Tissue kit (Qiagen, Hilden, Germany) following the manufacturer's recommendations.

PCR amplification was performed for 4 genetic markers – the mitochondrial COI, 12S rRNA, 16S rRNA genes and the nuclear ITS2 region. A T100™ thermocycler (Bio-Rad, California, USA) was used for amplification with a final PCR volume of 30 μL. Each reaction contained 15 μL of 2× i-Taq™ mastermix (iNtRON Biotechnology, Gyeonggi, South Korea), 5–10 μm of each primer and 2 μL of template DNA. The thermocycling profiles and primers used follow the protocol by Callejón et al. (2015a) for the COI gene and ITS2 region, while the protocols for the mitochondrial 12S and 16S rRNA genes follow Chan et al. (2020). Amplicons were visualized on 1% agarose gel stained with SYBR™ Safe (Life Technologies, California, USA). Successful amplicons were purified with the Geneaid PCR Purification kit (Geneaid Biotech Ltd, Taipei, Taiwan) using the manufacturer's recommendations. The purified DNA samples were sent for Sanger sequencing by Macrogen (Seoul, South Korea) with the same primers used for PCR amplification.

Phylogenetic analyses

Electropherograms obtained after Sanger sequencing were checked using Bioedit 7.0, and the sequences were aligned using ClustalX 2.1 with reference Trichuris and Trichinella (used as outgroup) sequences obtained from the NCBI database (Hall, 1999; Thompson et al., 2002). The reference sequences used are presented in Supplementary Table 1. The aligned sequences were checked using Bioedit 7.0 and used to construct neighbour-joining (NJ) and maximum-likelihood (ML) phylogenetic trees in MEGA X (Kumar et al., 2018). NJ phylogenetic tree construction used the pairwise nucleotide distance (p-distance) model while the ML phylogenetic tree used a best-fit nucleotide substitution model with 1000 bootstrap iterations for tree support. FigTree 1.3.1 was used for phylogenetic tree aesthetics (Rambuat, 2009). The sequences obtained in this study with their NCBI accession numbers, and their corresponding specimen and host identity numbers are listed in Table 1.

Table 1. Trichuris specimens from Camelus dromedarius used for analysis

Genetic distance calculation was performed in MEGA X, where the p-distance values for each genetic marker were calculated using the aligned sequences (Kumar et al., 2018). The values were converted into percentage distance based on sequence dissimilarity.

Statistical analysis of morphological measurements

Statistical analysis of the morphological measurements was performed to determine the suitability of the morphological characters for identification between the 2 molecular groups (based on the results after molecular identification, 2 molecular groups were identified). The 7 morphological characters used were – posterior body length, posterior body width, spicule length, spicule width, spicule width at the proximal, spicule sheath length and the maximum spicule sheath width. Comparisons between groups for each morphological character were visualized with a boxplot. The suitability of the characters was then examined by the statistical significance in the means between the 2 groups using either the Mann–Whitney or the independent t test (depending on whether the data were normally distributed). A P value <0.05 indicates that the means were significantly different. All statistical analysis and boxplot visualization were performed in R Studio version 1.2.5033 (RStudio Team, 2021).

Results

Morphology of Trichuris male spicule sheath

The specimens obtained from Arabian camels were morphologically identified as Trichuris, with morphological variations observed in the male spicule sheath. The 2 different types of spicule sheaths are – the first type presents the globular posteriorly truncated swelling (Fig. 1a and b), while the second type does not present a globular posteriorly truncated swelling (Fig. 1c and d). Specimens with both types of spicule sheath were present in host T5, whereas hosts T3 and T4 had only specimens with the spicule that did not present a globular posteriorly truncated swelling.

Figure 1. Male spicule sheath morphology of Trichuris species from camels. In (a and b), a globular posteriorly truncated swelling with spines on the swelling (indicated by the black arrow) longer than the rest of the spicule sheath is shown. In (c and d), the spicule sheath does not have a globular posteriorly truncated swelling.

Molecular identification of Trichuris species from camels

Based on the molecular phylogenies of the Trichuris specimens obtained from camels, the specimens were grouped into 2 distinct clusters using the mitochondrial COI, 12S rRNA and 16S rRNA genes. The phylogenies across the 3 mitochondrial genetic markers were congruent, revealing 2 distinct genotypes with strong bootstrap support. Figs 2–4 present the phylogenies inferred from the mitochondrial genetic markers. In the first cluster (group 1), the specimens were phylogenetically placed together with T. globulosa, while in the second cluster (group 2), the specimens were closely placed with T. ovis. Additionally, 3 Trichuris specimens isolated from the same host (T5) were found in groups 1 and 2. Of the 9 specimens isolated from T5, 5 were phylogenetically placed in group 1 while 4 were placed in group 2. Conversely, no distinct groups were obtained based on the nuclear ITS2 phylogeny (Fig. 5). All 15 specimens were clustered together with the reference T. globulosa sequences, with T. ovis in a separate clade.

Figure 2. Phylogeny of Trichuris spp. based on the mitochondrial COI gene. The phylogenetic tree was inferred using the ML and NJ algorithms in MEGA X. The numbers at the nodes indicate bootstrap support obtained through 1000 replications (ML/NJ). Trichuris specimens in this study are indicated in blue (group 1) and red (group 2) colours. The final alignment used for phylogenetic tree construction was 390 bp in length.

Figure 3. Phylogeny of Trichuris spp. based on the mitochondrial 12S rRNA gene. The phylogenetic tree was inferred using the ML and NJ algorithms in MEGA X. The numbers at the nodes indicate bootstrap support obtained through 1000 replications (ML/NJ). Trichuris specimens in this study are indicated in blue (group 1) and red (group 2) colours. The final alignment used for phylogenetic tree construction was 420 bp in length.

Figure 4. Phylogeny of Trichuris spp. based on the mitochondrial 16S rRNA gene. The phylogenetic tree was inferred using the ML and NJ algorithms in MEGA X. The numbers at the nodes indicate bootstrap support obtained through 1000 replications (ML/NJ). Trichuris specimens in this study are indicated in blue (group 1) and red (group 2) colours. The final alignment used for phylogenetic tree construction was 190 bp in length.

Figure 5. Phylogeny of Trichuris spp. based on the nuclear ITS2 region. The phylogenetic tree was inferred using the ML and NJ algorithms in MEGA X. The numbers at the nodes indicate bootstrap support obtained through 1000 replications (ML/NJ). Trichuris specimens in this study are indicated in blue (group 1) and red (group 2) colours. The final alignment used for phylogenetic tree construction was 320 bp in length.

Compared among the other Trichuris species, the nuclear and mitochondrial phylogenies obtained showed that T. discolor was a sister group to T. globulosa and T. ovis, supporting a clade of Trichuris species belonging to ruminants.

Genetic variability among T. globulosa

As shown in Table 2, the genetic distance obtained from the ITS2 region ranged from 0 to 0.49% within our Trichuris specimens and the reference T. globulosa sequences, while the genetic distance between T. globulosa and T. ovis ranged from 3.47 to 3.96%. For the mitochondrial genetic markers, genetic distances between groups 1 and 2 ranged from 6.32 to 7.42% with the COI gene. The genetic distances for the 12S and 16S rRNA genes were smaller, ranging from 3.03 to 3.86% and 1.35 to 2.70%, respectively. Within group genetic distances were also smaller than between group genetic distances for each of the mitochondrial genetic markers.

Table 2. Genetic distance (% difference) comparison between genetic markers for Trichuris globulosa and Trichuris ovis

The average genetic distances are indicated in parentheses, and NA indicates not applicable.

Comparison of morphological characters between groups 1 and 2

Based on the molecular results from the Trichuris specimens obtained in this study, 2 molecular groups were present. Table 3 presents the morphological characteristics and measurements of the Trichuris specimens used in this study, allocated based on their molecular groupings. Of the 7 morphological characters that had continuous data, statistical analysis revealed that there was significant difference (P = 0.038) between the means of the spicule sheath length, with group 1 averaging a length of 0.656 mm and group 2 with 0.364 mm (Fig. 6). No statistical significance was observed with the other 6 morphological characters.

Table 3. Morphological measurements of Trichuris from each group used in this study

Measurements are given in mm. The mean values are shown, while the minimum and maximum values are in parentheses.

An asterisk (*) indicate statistical significance (P < 0.05) between the means of groups 1 and 2.

Figure 6. Boxplot of the 7 morphological characters of the Trichuris specimens. The morphological characters are (a) posterior body length, (b) posterior body width, (c) spicule length, (d) spicule width, (e) spicule width at proximal, (f) spicule sheath length and (g) spicule sheath width. An asterisk (*) indicates statistical significance (P < 0.05) between the means of groups 1 and 2. The black solid dots indicate outliers. The solid line of the boxplot indicates the mean value obtained from the morphological measurements.

Additionally, both types of male spicule sheath were found in each molecular group. The morphological variations of the male spicule sheath did not corroborate with the molecular phylogenies, thus limiting the use of the male spicule sheath as a diagnostic character for species identification of T. globulosa.

Discussion

Our findings demonstrated the use of the mitochondrial COI, 12S rRNA, 16S rRNA genes and the nuclear ITS2 region for molecular identification of Trichuris from C. dromedarius. Two distinct clusters of Trichuris were obtained from the mitochondrial phylogenies (group 1 closer to T. globulosa while group 2 closer to T. ovis), providing molecular evidence of a possible T. globulosa species complex. Also, 2 forms of male spicule sheath that did not corroborate with molecular data were observed, suggesting the limited use of the male spicule sheath as a morphological character for species identification.

Molecular evidence of possible T. globulosa species complex

With the T. globulosa specimens obtained in this study, molecular phylogenies suggest the possibility of a T. globulosa species complex. Firstly, the phylogenies obtained from the mitochondrial genes revealed 2 distinct clusters within T. globulosa. Secondly, no distinction between groups 1 and 2 was observed in the nuclear ITS2 phylogeny.

The species status of T. globulosa has been questioned by some authors, with mitochondrial genetic markers revealing that T. globulosa and T. ovis are different species, while morphology, isoenzymatic and the nuclear ITS2 region support the synonymy of both species (Cutillas et al., 1995; Oliveros et al., 2000). Based on the nuclear ITS2 region, Oliveros et al. (2000) demonstrated that no sequence variation was present between T. ovis and T. globulosa isolated from sheep and goats (Oliveros et al., 2000). Contrarily, using the mitochondrial cytB and COI genes, Callejón et al. (2015a) showed that T. globulosa isolated from C. dromedarius in Iran and T. ovis isolated from Ovis aries in South Africa were 2 distinct species (Callejón et al., 2015a). Moreover, the overlap in morphological characters such as spicule lengths and size between the 2 species render accurate species identification challenging (Knight, 1974; Callejón et al., 2015a).

Here, with the 2 distinct groups present based on the mitochondrial genetic markers, the T. globulosa specimens obtained from this study could present a possible species complex. Specimens obtained from the same host were also found in groups 1 and 2. Moreover, specimens from each group could not be differentiated based on their morphological characters. Species complexes are no stranger among helminths, and molecular evidence has also facilitated the discovery of closely related species that are morphologically similar but genetically different (Callejón et al., 2013; Xie et al., 2018). Among Trichuris, Rivero et al. (2021) revealed the presence of 2 different genotypes corresponding to different lineages within Trichuris trichiura obtained from humans and non-human primates using the mitochondrial COI and cytB genetic markers, suggesting the existence of a T. trichiura species complex (Rivero et al., 2021). The mitochondrial genetic markers are known to contain high sequence variation for cryptic species delimitation, with the ability to differentiate closely related members within species complexes (Thaenkham et al., 2022). Moreover, they have proven to be useful for Trichuris species differentiation in instances where specimens were unable to be morphologically identified to the species level (Callejón et al., 2016; Di Filippo et al., 2020). Contrarily, although the nuclear ITS2 region can be used for species differentiation, the genetic marker is relatively conserved among members of species complex and closely related species partly due to concerted evolution in play (Thaenkham et al., 2022). Likewise, low levels of sequence variation were observed for our specimens using the ITS2 region, with no distinction between groups 1 and 2, supporting the possibility that the 2 groups are genetically closely related. Also, genetic differences obtained using the full-length ITS2 region between T. globulosa and T. ovis ranged from 3.47 to 3.96%, disagreeing with previous studies suggesting that T. globulosa and T. ovis are synonymous.

Through congruence of the mitochondrial phylogenies supporting distinction between the 2 groups of T. globulosa, insufficient ITS2 sequence variation to differentiate between groups, along with the complicated species status between T. globulosa and T. ovis based on previous studies, a species complex is thus plausible.

Limited use of morphological characters for T. globulosa species identification

From the T. globulosa specimens used in this study, 2 morphological variations of the male spicule sheath were observed, and they did not correspond to the groups based on the mitochondrial phylogenies (groups 1 and 2). The shape and spines of the male spicule sheath has been used as a criterion distinguishing T. globulosa from other Trichuris, particularly with the closely related T. ovis (Sarwar, 1945; Cutillas et al., 1995). Previous studies have reported that T. globulosa isolated from camels, sheep and goats present a male spicule sheath with a globular posteriorly truncated swelling with spines on the swelling longer than the rest of the spicule sheath (Sarwar, 1945; Cutillas et al., 1995; Callejón et al., 2015a; Yevstafieva et al., 2018). Cutillas et al. (1995) also added that while T. ovis does not have a posterior swelling, some may also present a globular posteriorly swelling. However, the spines on the globular posterior swelling are shorter than or equal to the rest of the spicule sheath, rendering T. ovis being morphologically different from T. globulosa.

Species identification of Trichuris using morphological characters is often challenging for taxonomists due to similar and overlapping characters for both males and females. Distinct Trichuris genotypes were found from morphologically similar Trichuris species isolated from rodents (Robles et al., 2014). For T. globulosa, aside from spicule sheath, spicule lengths have also been used as another criterion. However, overlapping lengths have been observed between T. globulosa and T. ovis (Callejón et al., 2015a). Additionally, assumptions of species identity are often based on the types of hosts that they are found in, but evidence has revealed their capacity to infect wide varieties of hosts for some species. As both T. globulosa and T. ovis have been found in similar hosts such as camels, sheep and goats, host-induced phenotypic plasticity can be present, thus limiting the use of morphological characters. Here, with the morphological variations of the male spicule sheath observed among T. globulosa, along with their capability to infect various hosts, identification of T. globulosa requires reliance on molecular genetic markers for accurate species identification.

Limitations

Firstly, only male Trichuris specimens were selected for morphological and molecular analysis in this study. The inclusion of Trichuris females in the analysis could provide more information regarding the genetic variation among the Trichuris specimens obtained. Secondly, similar to any molecular study, the accuracy of species identification is subjected to the accuracy of reference sequences in the database. The sequences included for comparison were thus limited due to the small number of sequences and molecular studies that have been performed on T. globulosa. Thirdly, as compared to the phylogeny obtained using the ITS region by Betson et al. (2015) and Cavallero et al. (2015), Trichuris suis and T. trichiura, although being morphologically similar and closely related, they each formed their own subclade within the monophyletic clade containing both species (Betson et al., 2015; Cavallero et al., 2015). Contrarily in our ITS2 phylogeny, no genetic difference was observed between the T. globulosa groups 1 and 2 specimens. Thus, a nuclear genetic marker from another loci or the whole genome can be utilized to further investigate the phylogenetic relationships among T. globulosa. Lastly, as the Trichuris specimens were obtained from Arabian camels from the slaughterhouse, sequence variation of Trichuris between localities could not be compared.

Conclusion

Mitochondrial phylogenies revealed 2 groups of T. globulosa, while the nuclear ITS2 region did not have sufficient sequence variation to discriminate between the 2 T. globulosa groups. Molecular evidence thus suggests the possibility of a T. globulosa species complex, with potential implications on the administration of the appropriate anthelmintic treatment for ruminants. Additionally, 2 morphological variations of the male spicule sheath were revealed, suggesting the limited use of the male spicule sheath as a diagnostic character for species identification of T. globulosa.