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Parasitic life and environment of monogenean: geometric morphometric study of haptoral anchors in seven Diplorchis species (Monogenea: Polystomatidae)

BMC Zoology volume 10, Article number: 5 (2025) Cite this article

Abstract

Background

The development of larger monogeneans and their survival on more active hosts is thought to have led to the emergence of haptoral suckers and, in some instances, anchors, enabling a more stable anchorage. Because of their strict host specificity, the morphological variation of anchors in genus Diplorchis (Monogenea: Polystomatidae) may be determined to a large degree by adaptation to the host species, its habitat and ecological environment to ensure stable attachment.

Methods

In this study, we estimated the interspecific and intraspecific differences of haptoral anchors and other morphological characteristics in six recorded species of Diplorchis and one unidentified species parasitizing Sylvirana maosonensis (Bourret, 1937) in China using geometric morphometrics.

Results

Geomorphometric analyses revealed significant differences in the shape and size of the anchors among the seven species, indicating that the morphological differences in anchors can be used as a basis for species identification within the genus Diplorchis. In addition, we found that the same Diplorchis species collected from different localities not only differed significantly in anchor form, but also in body size and haptor size, as well as haptoral sucker size. This may reflect the effect of different habitat environments on biological/behavioral activities of the same host, thus further affecting the stable attachment of flatworms within species. Interestingly, in two species collected from the same localities, we found no significant differences in anchor or sucker size, while body size and haptor size all differed significantly. Meanwhile, the significant differences in anchor shape may suggest that the attachment mechanism of the different Diplorchis species is related to the variation in anchor shape.

Conclusions

From the perspective of morphological adaptation to the environment, the study not only indicated that the morphological variation of Diplorchis anchors can be used as an auxiliary tool to distinguish species, but also found that the morphological differences in the anchors were influenced by factors such as host species, habitat and ecological environment. This may provide a basis for a better understanding of host-parasite interaction.

Graphical abstract

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Background

Monogenean flatworms are parasites attached to the host’s flexible and uneven surfaces, such as infecting the gills, body surfaces of fish or the oral cavity, urinary bladder, and/or conjunctival sacs of amphibians and freshwater turtles [1,2,3]. The morphological structure of the attachment organ in monogenean parasites, especially the haptor, is thought to play a crucial role in parasite specialization and adaptation to host species [4,5,6,7]. Different from monogeneans parasitizing fishes, the flatworms mainly parasitize the urinary bladder of anuran hosts. Their haptor not only have sclerotized attachment apparatuses such as anchors, but also haptoral suckers. The general hypothesis is that the development of larger monogeneans and their survival on more active hosts led to the emergence of haptoral suckers and in some instances one or two pairs of larger hooks (anchors), providing a more stable anchorage [8].

In addition, the morphological variation of sclerotized attachment apparatuses within the haptor have also attracted the attention of many scholars. In 2002, Šimková et al. [9] calculated that the linear distances of the attachment organ between nine Dactylogyrus species, and the results revealed that the morphology of the haptor reflects the adaptation of the parasite to the host considerably, and even to specific sites within their hosts. Furthermore, other studies indicate that haptoral morphology is driven by a combination of both adaptive selection and phylogenetic constraints [10, 11]. Moreover, Cruz-Laufer et al. [12] further indicated that the morphological variation of the attachment organ is influenced by host and environmental parameters. This indicates that the sclerotized attachment apparatuses of the haptor, especially the anchors, the morphological variation of which may be determined by adaptation to the host species, its habitat and ecological environment. Unfortunately, there is limited research on the morphological variation of haptoral anchors of monogeneans parasitizing anuran hosts.

Species of Diplorchis Ozaki, 1931 (Monogenea: Polystomatidae Gamble, 1896) infect the urinary bladder of anurans [13, 14], and have been recorded in seven species, all in China (six) and Japan (one) [15,16,17,18,19,20]. Because of the strict host specificity (restricted to one single host), the taxonomic status of the host is often used as part of the basis for species identification of their parasites [21, 22]. However, the reliability of host specificity as the basis for species identification has also been questioned. For example, under experimental conditions, the oncomiracidium of some polystomatid flatworms can infect both natural hosts and substitute hosts [23]. Subsequently, polystomatid cross-infection has been recorded in South Africa and Europe [24, 25]. In morphological studies, body size, intestines, haptors, suckers, hamuli, gonads and genital spines can generally be used for species identification in Diplorchis [24, 26]. However, the life cycle of the parasite, the degree of maturity and the treatment of the specimen may lead to morphological variation, which makes accurate species identification difficult [15, 27]. The wide application of molecular phylogenetic methods has facilitated the study of classification, systematics and evolutionary relationships of monogeneans [27,28,29,30,31,32]. Nonetheless, regarding specimens in our existing collections, the longest storage time may be close to 40 years (collected in 1984), and many of them have not been collected in time for the molecular data. In addition, the collection of molecular data is affected by the preservation, fixation methods, and the difficulty of acquiring further specimen. Therefore, the lack of molecular data will not only increase the difficulty of the identification of species with similar morphological characteristics, but also reduce the potential use of collected specimens.

Different from linear measure-based morphometrics, the advantage of geometric morphometrics is that the shape analysis relies on homologous landmarks, and the coordinate data are independent. In this way, the size and shape of the sample can be discussed separately [33,34,35]. In addition, the technique also provides visualization tools [36], such as shape deformation grids, that facilitates the interpretation of shape changes. As a consequence, studying the overall shape of sclerotized haptoral structures with geometric morphometrics is more prone to detect differences among groups rather than linear measure-based morphometrics [37]. In recent years, many studies have tried to quantify intraspecific and interspecific variation based on geometric morphometrics of haptoral anchors, and to explore the relative influence of host species, habitat and evolutionary history in shaping species’ form [11, 38,39,40,41,42]. However, the focus of research conducted to date has been on monogeneans parasitizing fishes, and there are few studies on the geometric morphometrics of anchors of monogeneans parasitizing anuran hosts.

In this study, we collected and collated six recorded species of the genus Diplorchis in China, including Diplorchis grahami Fan et al., 2007 [15], D. hangzhouensis Zhang & Long, 1987 [16], D. latouchii Zhang & Long, 1987 [16], D. lividae Song et al., 2008 [17], D. nigromaculatus Lee, 1936 [18], D. shilinensis Fan et al., 2005 [19]) and one unidentified species (Diplorchis sp.)Footnote 1 parasitizing Sylvirana maosonensis (Bourret, 1937). Meanwhile, we analyzed the morphological variation of anchors using geometric morphometrics. The purpose of this study was to (a) evaluate the role of geometric morphometrics of haptoral anchors in distinguishing interspecific differences and for species identification; (b) evaluate the relationship between the morphological variation of anchors and stable attachment; (c) explore the relationship between anchor shape and factors such as host and habitat. In addition, we also tried to interpret the significance of the morphological variation of anchor as the attachment organ for the adaptation of Diplorchis species to endoparasitic phases.

Methods

Parasite samples, hosts and locality

For this study, we sampled seven species of the genus Diplorchis held at the School of Life Sciences, Yunnan Normal University, and the Laboratory of Fish Parasitology, School of Life Sciences, South China Normal University. The information of specimens, host species, locality are shown in Table 1 (complete information can be found in Additional file 1: Table S1). Finally, 82 specimens were used in the study, after excluding any anchors showing apparent deformation, tears or ruptures.

Table 1 Parasite species, host species, locality (geographical coordinates) and the sample size of Diplorchis spp.
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Geomorphometric analyses

All specimens were photographed using an Olympus BX53 light microscope and cellSens ver.2.2 imaging software (Olympus Corporation, Tokyo, Japan). We used only specimens, for which the whole body was mounted on a slide, and only marked the right anchor of the flatworm to avoid data duplication. Images were input into tpsUtil32, and output as a TPS file [43], then the landmarks (LMs) were collected using tpsDig232 [44]. Six homologous LMs were used as fixed points, including the tip of the hamuli, the upper and lower bases of the hook tip, the base of the prominent crest, the tip of the guard and tip of the handle. In addition, five semi-landmarks were placed from the most prominent point of the guard to the lowest point of the shaft, and nine semi-landmarks were placed from the base of prominent crest to the tip of the handle. The start and end positions for the semi-landmarks were placed in close proximity to fixed landmarks three and four as reference points (Fig. 1). Because some slides had been preserved for a long time and the fact that the layer where the anchor is obstructed by the distribution of eggs or other tissues, the edge between roots of anchor that was not easy to identify accurately. Therefore, in this study, we did not set the landmark in that area. After placing all semi-landmarks, the “resample curves by length” option in tpsDig232 was used to place them at equidistant points. All landmarks were placed by one person (TJ). The full landmark data file for all species is shown in Additional file 2: Dataset S1.

Fig. 1
figure 1

Photograph of seven species of Diplorchis (A) and position of the landmarks placed on each hamuli (numbered sequentially) (B). Fixed landmarks are marked in red, and semi-landmarks are marked in blue

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The landmark file (20 coordinates for each specimen) was imported into MorphoJ v. 1.08 [45]. Generalized Procrustes analysis (GPA) was employed to obtain matrices of shape coordinates of anchors. To visualize the variation at the taxonomic and geographical levels, we applied a principal component analysis (PCA) to the coordinates of GPA of the anchors based on the covariation matrix. Additionally, canonical variate analyses (CVA) were performed to maximize the differentiation in anchor shape between groups. The significance of differences between groups was calculated using a permutation test with 10,000 iterations (α = 0.05). The main axes of the PCA and CVA were visualized using wireframe graphs.

Discriminant function analysis (DFA) was used to compare the differences in the shape of haptoral anchor of different species, and the accuracy rate of discrimination was calculated. Furthermore, the Mahalanobis distances were calculated using CVA to investigate the degree of dissimilarity and geographic variations in the shape of anchors of the genus Diplorchis between the study groups [46]. Meanwhile, a neighbor-joining (NJ) tree was constructed with 1000 bootstrap replicates to illustrate the patterns of variation among the species using PAST v. 4.17 [47].

Log-transformed centroid size (log CS) was used as a measure of anchor size, and the distribution of anchor size was carried out and graphs plotted using PAST v. 4.17 [48]. Most notably, the Kruskal-Wallis test was used to compare the anchor size of different species, and the Mann-Whitney U test was used for pairwise comparison.

Influence of size on anchor shape

To investigate the effect of size on shape variation (i.e., interspecific allometry) of the anchors in the seven species of Diplorchis, linear regressions were performed using principal components scores (PC scores) and Procrustes shape coordinates with log CS, and the statistics and drawing were conducted using PAST v. 4.17. PC scores and Procrustes coordinates were exported from MorphoJ v. 1.08. The percentage of size variation in total shape variation was derived from the pooled regression within subgroups of species using MorphoJ v. 1.08. Permutation tests were performed with 10,000 rounds.

Anchor size and other morphological characteristics

To assess the relationship between anchor size and the stable attachment of the flatworm, the relevant morphological characteristics, including body length, body width (maximum body width), haptor length, haptor width (maximum haptor width), haptor-to-body-length ratio and haptoral sucker diameter (the mean value of the diameter of six suckers) on the calibrated photographs of specimens using tpsDig232. Maintaining a high level of accuracy, each specimen was measured six times repeatedly, and the mean value was used in the subsequent analysis. Pearson correlation analysis and plotting were performed in PAST v. 4.17.

Results

Shape of haptoral anchors

The PCA showed that a large proportion of the variation was contained in relatively few dimensions, with the first three PCs accounting for 81.71% of the total shape variance in haptoral anchors (Table 2). Eigenvalues and variance explained by each PC are given in Additional file 1: Table S2.

Table 2 Eigenvalues and variance explained by the first three principal component
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Among them, the first PC (PC1) explained 49.39% of the total shape variance, and the main shape changes corresponded with the anchor having a shorter outer root and a longer inner root, plus a longer point and a thicker shaft (Fig. 2A). The second PC (PC2), accounting for 22.40% of total variance, showed the shape changes on the positive side were related to the anchor having a shorter point, a relatively thinner base with a longer inner and outer root, and a larger curved shaft (Fig. 2A). The third PC (PC3) accounted for 9.92% of the variance, and the shape changes along this axis corresponded with the anchor having a shorter point, a thinner shaft and a relatively thicker base with a slightly longer inner and an outer root extending outward (Fig. 2B). Although PCA could not fully visualize differences of all of them, some species, such as D. shilinensis and Diplorchis sp., could be clearly separated.

Fig. 2
figure 2

Geomorphometric analyses of the difference among the shapes of anchors from seven species of Diplorchis. A, B Scatter plot showing the variation in shape along PC1 and PC2, also PC1 and PC3. Wireframe graphs illustrating the shape changes from overall mean next to each PC, with starting shapes (consensus) in grey and target shapes (changes + 0.1) in black. C, D Scatter plot showing the variation in shape along CV1 and CV2, also CV1 and CV3. Wireframe graphs illustrating the shape changes from the overall mean based on CVA are next to each PC, with starting shapes (consensus) in grey and target shapes (changes + 10.0) in black

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Subsequently, CVA was performed on the anchor dataset of all seven species, and 84.57% of the shape variation was explained by CV1, CV2 and CV3 (55.83%, 17.11% and 11.63%, respectively). Compared to PC1, CV1 can distinguish most of the species, such as Diplorchis sp., D. latouchii, D. hangzhouensis and D. shilinensis were along the negative side of axis, while D. grahami and D. nigromaculatus were along the positive side. In addition to the similar changes shown with PC1, CV1 corresponded with the anchor having a more prominent guard and a less-curved shaft (Fig. 2C). Compared to PC2, the shape changes of the anchor shown in CV2 were more pronounced along this axis, and Diplorchis sp. and D. latouchii were separated from D. hangzhouensis, D. lividae and D. shilinensis. As with PC2, CV2 corresponded with a difference in the anchor’s shorter point. Additionally, the different changes of shape were shown, e.g., that the outer root was much longer and the inner was shorter (Fig. 2C). As for PC3, CV3 could further separate D. lividae and D. shilinensis, but the shape changes were quite different from that of PC3. In comparison, CV3 corresponded with the different anchor shapes, such as a longer point, a longer inner root, a shorter outer root, and a larger curved and thicker shaft (Fig. 2D).

“Species” was used as the classification criterion to conduct a permutation test against the null hypothesis of no differences among group means. The results showed that the shapes of the anchor in the seven species of Diplorchis were significantly different from each other (Goodall’s F: F = 9.6698, P < 0.0001; Pillai’s trace: 4.2125, P < 0.0001). The matrix resulting from Mahalanobis and Procrustes distances can be found in Additional file 1: Table S3.

Furthermore, DFA was used to explore whether Diplorchis sp. can be distinguished from other species by geometric morphometrics of the haptoral anchor. On the basis of the pairwise comparison results, the accuracy rate of discrimination with D. latouchii was 90% from cross-validation, and the rest were 100% (Table 3). The complete comparison data are shown in Additional file 1: Table S4.

Table 3 Correct classification rates between Diplorchis sp. and other Diplorchis spp.
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In the NJ tree (Fig. 3A) based on Mahalanobis distances (Additional file 1: Table S5), all clusters were well supported (≥77%). According to the clustering tree, D. grahami and D. lividae, whose hosts were both in the genus Odorrana, were not clustered together. However, Diplorchis sp. and D. latouchii, whose hosts were both in the genus Sylvirana, seemed to be closer based on the shape of the haptoral anchor.

Fig. 3
figure 3

Projection of Neighbour-Joining tree (NJ) generated from the morphometric Mahalanobis distance matrix (A) and distribution of log centroid sizes (log CS) of anchors (B). The bootstrap values are indicated above the nodes

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Next, using the collection localities as the classifier variable, the NJ tree (Fig. 4A) was constructed using Mahalanobis distances (Table S5) of the seven Diplorchis spp. The results showed that the Diplorchis spp. collected in Hangzhou were effectively distinguished from those collected in Dayao and Guangzhou, with a bootstrap value of 100%. The difference can also be seen in the shape variation of the anchor of the seven Diplorchis spp. (Fig. 4B). However, it is worth noting that, through DFA, the shapes of the anchor in two Diplorchis spp. (i.e., D. hangzhouensis and D. latouchii) collected in Hangzhou and Dayao were significantly different (Procrustes distance: 0.073, P = 0.003; Mahalanobis distance: 8.779, t2 = 409.225, P = 0.093), and the accuracy rate of discrimination was more than 58% (discriminant function: 100%, cross-validation: 58.62%). Importantly, the shapes of anchor in D. hangzhouensis collected in Hangzhou and Dayao were also significantly different (Procrustes distance: 0.111, P < 0.001; Mahalanobis distance: 8.560, t2 = 244.254, P=P < 0.001), and the accuracy rate of discrimination was more than 93% (discriminant function: 100%, cross-validation: 93.33%). For the two species of D. hangzhouensis and D. latouchii collected from the same localities (i.e., Dayao, Guangxi), the shapes of the anchor also showed significant differences (Procrustes distance: 0.093, P < 0.001; Mahalanobis distance: 10.531, t2 = 604.970, P < 0.001), and through DFA, the accuracy rate of discrimination from cross-validation was 81.82%.

Fig. 4
figure 4

Projection of Neighbour-Joining tree (NJ) generated from the morphometric Mahalanobis distance matrix about locations (A) and scatter plot showing the variation of location in shape along CV1 and CV2 (B). The bootstrap values are indicated above the nodes. Abbreviations correspond to the locality (Table 1), GLQ, Gulinqing; HZ, Hangzhou; DYS, Dayao; GZ, Guangzhou; WZS, Wuzhi; SL, Shilin; TX, Taixing; LF, Lufeng

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Size of haptoral anchors

The Kruskal-Wallis test showed a significant difference in the anchor size of the seven species of Diplorchis (H = 62.61, df = 6, P < 0.0001). Subsequently, the Mann-Whitney U test showed via the pairwise comparisons that there were significant differences (Additional file 1: Table S6) in most anchor sizes, except for D. grahami-D. lividae (U = 9, Z = 1.308, P = 0.191), D. hangzhouensis-D. latouchii (U = 106, Z = 0.497, P = 0.619), D. hangzhouensis-D. shilinensis (U = 16, Z = 0.957, P = 0.339), D. latouchii-Diplorchis sp. (U = 24.5, Z = 1.404, P = 0.160), D. latouchii-D. shilinensis (U = 5, Z = 1.958, P = 0.050), and D. lividae-D. shilinensis (U = 1, Z = 1.789, P = 0.074). Only D. nigromaculatus, with or without Holm-Bonferroni correction, had a significantly smaller anchor size than the others (Additional file 1: Table S6).

In addition, the size of haptoral anchors in the seven Diplorchis spp. was reordered according to the means of log CS (Fig. 3B), which seemed to correspond to the NJ tree constructed using the seven species of Diplorchis (Fig. 3). In this figure, the log CS values of D. hangzhouensis and D. latouchii were dispersed to both ends, which might be related to the fact that these two species were collected from multiple localities. Analyzing the size of Diplorchis haptoral anchors from different localities at the intraspecific level using the Mann-Whitney U test showed that in D. hangzhouensis collected from Hangzhou and Dayao (excluding Guangzhou, n = 2), the haptoral anchor sizes were significantly different (U = 4.5, Z = 2.454, P = 0.014). Without considering the interspecific differences, the haptoral anchor sizes of the two species (i.e., D. hangzhouensis and D. latouchii), which were collected from Hangzhou (n = 7) and Dayao (n = 22), were significantly different based on the different localities (U = 25.5, Z = 2.601, P = 0.009). More interestingly, when the same collection locality (i.e., Dayao) was further analyzed, there was no significant difference (U = 57, Z = 0.165, P = 0.869) in anchor size between the two species.

Influence of size on anchor shape

The multivariate regression of PCs on log CS (Fig. 5) showed that log CS was significantly correlated with PC1 (r = − 0.547, P < 0.0001) (Fig. 5A) and PC3 (r = 0.330, P = 0.003) (Fig. 5B), indicating that allometry had a significant effect on the overall variation of shape of haptoral anchor in the seven Diplorchis spp. Pooled regression with subgroups of species was conducted on the datasets of PC1 and PC3 with log CS, which provided evidence for an allometric relationship between shape and size in haptoral anchor (PC1: r = −0.182, P < 0.0001 and PC3: r = 0.055, P = 0.004), accounting for 34.20% and 10.05% of the total shape variation of anchor, respectively. The multivariate regression of Procrustes coordinates on log CS also showed that there was an allometric relationship between shape and size in haptoral anchor (overall statistics: R2 = 0.1732, MSE = 0.0001; MANOVA: Wilks’ λ = 0.046, F = 21.35, P < 0.0001).

Fig. 5
figure 5

Results from multivariate regression of PC1 (A) and PC3 (B) against log centroid size (log CS)

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Anchor size and other morphological characteristics

The measurement values of morphological characteristics can be found in Additional file 1: Table S7. On the basis of the correlation analysis (Fig. 6A), the anchor size (log CS) in the seven species was significantly positively correlated with body length (r = 0.74, P < 0.001), body width (r = 0.66, P < 0.001), haptor length (r = 0.78, P < 0.001), haptor width (r = 0.78, P < 0.001) and haptoral sucker diameter (r = 0.89, P < 0.001), but not with haptor-to-body-length ratio (r = 0.07, P = 0.533). In addition, for the anchor size of D. latouchii and D. hangzhouensis, which were obtained from multiple collection localities, there was a similar result (Fig. 6B, C), including a significant positive relationship with body length (r = 0.72, P = 0.001; r = 0.61, P = 0.020), haptor length (r = 0.82, P < 0.001; r = 0.64, P = 0.013), haptor width (r = 0.70, P = 0.002; r = 0.58, P = 0.031) and haptoral sucker diameter (r = 0.84, P < 0.001; r = 0.64, P = 0.014). However, anchor size was unrelated to haptor-to-body-length ratio (D. hangzhouensis: r = 0.15, P = 0.576; D. latouchii: r = 0.36, P = 0.210) and body width (D. hangzhouensis: r = 0.43, P = 0.085; D. latouchii: r = 0.38, P = 0.184). Additionally, the correlation plot showed that the diameter of the major attachment structure (i.e., haptoral sucker) of Diplorchis was unrelated to the haptor-to-body-length ratio (r = 0.13, P = 0.262; D. hangzhouensis: r = 0.12, P = 0.659; D. latouchii: r = 0.33, P = 0.254), regardless of the parasite species or the collection locality.

Fig. 6
figure 6

Correlation matrix plots are between anchor size (log CS) and other morphological characteristics. A The correlation matrix plot about 7 species of Diplorchis. B The correlation matrix plot of D. hangzhouensis. C The correlation matrix plot of D. latouchii. D The correlation matrix plot of D. hangzhouensis collected from Hangzhou. E The correlation matrix plot of D. hangzhouensis collected from Dayao. F The correlation matrix plot of D. latouchii was collected from Dayao. Abbreviations BL body length, BW body width, HL haptor length, HW haptor width, HL/BL haptor to body length ratio, HSD haptoral sucker diameter. The “Ellipses” function shows the correlation coefficients r as ellipses with major axis of unity, and minor axis d according to Schilling. Correlation values are given in the middle of the ellipses. The grey boxes show a significant correlation (P < 0.05) between morphological characteristics. “Red” represents negative correlation, while “blue” represents positive correlation. Then the darker the color, the greater the correlation

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For D. hangzhouensis (Fig. 6D, E), which were collected from two of the main collection localities (i.e., Hangzhou and Dayao), the anchor size was significantly positively correlated with haptor length (Hangzhou: r = 0.94, P = 0.018, Dayao: r = 0.63, P = 0.049) at both localities. But with haptoral sucker diameter, there was a significant positive correlation (r = 0.94, P = 0.016) from Hangzhou only, and from Dayao showed a positive correlation trend, yet it was not significant (r = 0.51, P = 0.129). Next, we compared the morphological characteristics of D. hangzhouensis from two collection localities by Mann-Whitney U pairwise comparison, and the results showed that the differences were significant, including body length (U = 3, Z = 2.633, P = 0.008), haptor length (U = 2, Z = 2.756, P = 0.006), haptor width (U = 5, Z = 2.388, P = 0.017) and haptoral sucker diameter (U = 0, Z = 3.001, P = 0.003), but there were no significant differences in body width (U = 18, Z = 0.796, P = 0.426) or haptor-to-body-length ratio (U = 23, Z = 0.184, P = 0.854).

Comparing the correlation plots (Fig. 6E, F) between D. hangzhouensis and D. latouchii collected from Dayao, the anchor size of both species was significantly positively correlated with haptoral length (D. hangzhouensis: r = 0.63, P = 0.049; D. latouchii: r = 0.72, P = 0.008). Moreover, comparing the main morphological characteristics of the two species by Mann-Whitney U pairwise comparisons showed that there were no significant differences in body width (U = 36, Z = 1.550, P = 0.121), haptor length (U = 34, Z = 1.681, P = 0.093), haptor-to-body-length ratio (U = 44, Z = 1.022, P = 0.307) or haptoral sucker diameter (U = 31, Z = 1.879, P = 0.060). Conversely, there were significant differences in body length (U = 20, Z = 2.605, P = 0.009) and haptor width (U = 22, Z = 2.473, P = 0.013).

Discussion

Species identification and distinguishing interspecific differences

Monogeneans infecting anurans rely on the haptor to provide a firm attachment to the flexible bladder wall of their hosts. This is not only to ensure stability of the anterior end for feeding [49], but also to respond the sudden contraction of the bladder epithelium and reduce the immediate risk of being swept away from the attachment site during host urination [8, 50]. Although suction by the muscular suckers enables powerful adhesion, previous studies have illustrated that haptoral suckers are vulnerable to detachment on highly contractile surfaces [50, 51]. Nevertheless, in these circumstances, the point of the anchor (i.e., hamuli) can remain embedded in host tissue. That is to say, even if all other points of contact are detached with the strength of the bladder contraction, the anchor is sufficient to maintain attachment until the suckers regain their grip on the now-altered surface area of bladder tissue. As in the study of chelonian polystomatids, Tinsley and Tinsley (2016) demonstrated that monogeneans found in the urinary bladder rely more on the hamuli to provide a firm attachment to the bladder wall [50]. Additionally, species of Diplorchis parasitizing anuran hosts are strict specialists [15]. Therefore, the morphological variation of the anchor in the different species should be related to their specific hosts. In other words, the morphological variation of anchors should be used as a criterion for distinguishing different species of Diplorchis, as supported by the results of this study—i.e., the seven species of Diplorchis were distinguishable based on the shape and size of the anchor. Diplorchis sp. was also distinguishable from the other six species of Diplorchis identified by geometric morphometrics of the anchor, with the accuracy of over 90%.

It is worth noting that the multivariate regression results regarding the effect of size on anchor shape in the seven species of Diplorchis provide evidence for allometric relationships between shape and size. This also suggests that it might be necessary to further consider the effect of evolutionary allometry when we evaluate the phylogenetic signal in anchor shape and size of Diplorchis, which has been mentioned in the study of the evolutionary morphology of haptoral anchors in monogeneans parasitizing fishes [11, 38].

Morphological variation of anchors and stable attachment

The thin, soft urinary bladder lining is effectively sucked into the sucker cups of Diplorchis parasites of anurans, providing a very firm and secure attachment. When there is a risk that the parasite will be displaced from its host, the suckers will double over and attach to other suckers or to the body of the parasite [8]. However, when the host urinates, the bladder will suddenly contract from a highly expanded surface. As the surface area changes, and converts the flat bladder epithelium into irregular folds, this will change the relative positions of the suckers, resulting in detachment [50]. At the same time, the detachment risk will also be increased by massive expulsion of the urine around the flatworm. Therefore, to prevent being swept away, the anchors and suckers need to be coordinated to ensure stable attachment [51].

On the basis of the above considerations, we speculate that, same as Tinsley and Tinsley (2016) [50], the mechanical stresses acting to detach the parasite, including sudden changes in bladder surface and liquid pressures, should be proportional to the body size of the flatworm. Additionally, the strength of attachment (i.e., the size of the attachment organ) should also have a positive relationship with parasite size [50]. The results of this study support this, i.e., the anchor size of the seven species of Diplorchis was significantly positively correlated with the body length, maximum body width, haptor length, haptor width and haptoral sucker diameter. In other words, for these species, those with larger anchors generally have significantly larger suckers, and their body size and haptor size are also larger. Yet it is worth noting that neither anchor size or haptor size are correlated with the haptor-to-body-length ratio, which may indicate that the growth of the body and haptor should also have a certain standard and range while having strong anchoring ability, and exceeding this range may affect stable attachment by the parasite. Certainly, the same results are also reflected in the two species of Diplorchis (D. hangzhouensis and D. latouchii) obtained from multiple collection localities.

Moreover, there was no significant difference in the size of the attachment organs (i.e., anchor and sucker), but there were significant differences in the shape of anchor, body size and haptor size when comparing the results of D. hangzhouensis and D. latouchii collected from Dayao. This may suggest that the stable attachment of different species of flatworms parasitizing the urinary bladder of their specific hosts is related to the variation of anchor shape. Meanwhile, the influence of convergence cannot be entirely excluded regarding the similarities in size of the attachment organ, and this needs further study.

Relationships between anchor form, host, habitat and environment

Different from monogeneans parasitizing fish, the post-larva of Diplorchis will leave the gill cavity when their anuran hosts metamorphose, migrate through the digestive tract to the cloaca, and eventually enter the urinary bladder of the host to settle in the adult form (including fully developed anchors and equal-sized three pairs of suckers) as an endoparasite [20, 52]. The internal environment is, in general, more predictable than the external environment, because all conspecific hosts are similar in general body plan and function, with organs performing the same function or secreting the same chemicals [53]. Therefore, it is generally believed that phylogenetically more closely related host species may have more similar parasites [54]. This assumption is consistent with the partial results of the clustering tree, that is, Diplorchis sp. and D. latouchii which parasitize hosts in the genus Sylvirana are clustered together in this study. However, the hosts of D. grahami and D. lividae in this study belong to genus Odorrana, but they are not clustered in the same branch. This suggests that even though D. grahami and D. lividae are strictly host specific, the influence of their evolutionary history on the anchor shape of the parasite may only be a part of the reason. The morphological similarity in anchors may be an determined more by adaptation to the environment (convergent evolution) than a common evolutionary history (phylogenetic constraints) [11, 38].

According to Todd (2007), endoparasitic helminths of amphibians require an aquatic environment for the development and transmission of their infective stages [55]. The urinary bladder of amphibians provides a high-humidity environment for monogeneans of the genus Diplorchis [52]. However, anurans frequently and intermittently travel between water and land. When entering aquatic environments, they tend to urinate more frequently and the salinity of their urine declines [8], so that the haptor of the parasite should readily detach and then reattach [51]. Generally, parasite evolution is considered in relation to adaptations to contrasting micro-environmental conditions within the body of the host [50], but considered in functional terms, the morphometric differences correlate with the micro-conditions at the infection sites [50]. For D. hangzhouensis collected from multiple localities, mainly from Hangzhou (Zhejiang Province) and Dayao Mountain (Jinxiu County, Guangxi Province), there are significant differences in the shape and size of the anchor, the size of the body and the haptor, or the size of the haptoral sucker. These differences may reflect the different micro-conditions of host ‘s urinary bladder. In addition, for the endoparasite, the state of the urinary bladder of anuran may also be affected by the host’s lifestyle and the habitat environment [56, 57]. We speculate that, for the same host species, the ecological characteristics of the habitat may have an impact on the micro-conditions within the urinary bladder. For example, in this study, Dayao Mountain has significant characteristics of mountain climate of subtropical regions. That is, warm in winter (not less than 8℃) and cool in summer (at most 28.5℃), the lowest sunshine levels in China (the sunshine percentage is only 29%), more rainy days (190d), and high relative humidity (about 83%) [58]. At the same time, Hangzhou is in the subtropical monsoon zone, with distinct seasons and abundant sunshine. Differences in climatic conditions may affect the life activities of the hosts, and further lead to the morphometric differences of D. hangzhouensis which parasitizing the urinary bladder of host.

Conclusions

As an important structure reflecting the adaptation of Diplorchis to parasitic life in the host, the haptor, especially the suckers and anchor, play a role in stable anchoring and grip function [49]. In this study, the relationship between the morphological variation of anchors of Diplorchis and the parasitic life was analyzed from the perspective of morphological adaptation to the environment. The geomorphometric analyses in this study indicated that the morphological variation of Diplorchis anchors can be used as an auxiliary tool to distinguish species. At the same time, we found that the morphological differences in the anchors were influenced by factors such as host species, habitat and ecological environment. We believe that such a prototypical framework of functional adaptation based on the shape and size of the haptoral anchor can lay a theoretical foundation for further exploration of the relative weight of convergence and phylogeny in shaping the morphology of Diplorchis. Additionally, at present, proposals of preserving frog diversity in China pose challenges to specimen collection, which will affect further study. Nevertheless, it is undeniable that the potential value of our specimens in existing collections will not be fully demonstrated, if more new methods and techniques cannot be introduced. Therefore, the results of this study are likely to open up avenues for the future study of parasitism in frogs.

Data availability

Data is provided within the manuscript or supplementary information files.

Notes

  1. A report about Diplorchis sp. as a new species (Diplorchis yunnanensis sp. nov.) is currently in writing; therefore, we describe it here only as an unidentified species.

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Acknowledgements

We are grateful to Professor Jiaying Liu (School of Life Science, East China Normal University, Shanghai 2000241, China) and Professor Xuejuan Ding (School of Life Sciences, South China Normal University, Guangzhou 510631, Guangdong, China) who provided material of Chinese recorded species. Then we thank Clio Reid, PhD, from Liwen Bianji (Edanz) (http://www.liwenbianji.cn) for editing the English text of a draft of this manuscript.

Funding

This research was supported by the National Natural Science Foundation of China (Nos 32060115, 31260507, 31560589); Scientific and Research Fund Project of Yunnan Provincial Education Department (2025).

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Authors and Affiliations

  1. School of Life Sciences, Yunnan Normal University, Kunming, Yunnan, 650092, China

    Ting Jia, Fei-Yan Meng, Wei-Jiang Xu & Li-Xian Fan

  2. Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming, 650500, China

    Fei-Yan Meng, Wei-Jiang Xu & Li-Xian Fan

  3. Key Lab of Yunnan Province for Biomass Energy and Environmental Biotechnology, Kunming, 650500, China

    Fei-Yan Meng, Wei-Jiang Xu & Li-Xian Fan

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Contributions

TJ conceived the study, designed the analyses and wrote the first draft of the manuscript. FYM was involved in the acquisition of data. WJX performed the biometrical analysis. LXF revised the manuscript and supervised the project.

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Correspondence to Li-Xian Fan.

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Additional file 1: Table S1 Sample collection information. Table S2 Eigenvalues and variance explained by each principal component. Table S3 Mahalanobis distances matrix and Procrustes distances matrix obtained from anchor dataset among 7 Diplorchis spp. Table S4 Correct classification rates between groups. Table S5 Mahalanobis distances matrix and Procrustes distances matrix obtained from anchor dataset among different location. Table S6 Results of Mann–Whitney U-test for comparisons of the log CS among the studied groups. Table S7 The measurement value about the morphological characteristic

Additional file 2: Dataset S1 The full landmark data file for seven species of Diplorchis

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Jia, T., Meng, FY., Xu, WJ. et al. Parasitic life and environment of monogenean: geometric morphometric study of haptoral anchors in seven Diplorchis species (Monogenea: Polystomatidae). BMC Zool 10, 5 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s40850-025-00226-2

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