Using combined morphological, allometric and molecular approaches to identify species of the genus Raillietiella (Pentastomida)

Abstract

Taxonomic studies of parasites can be severely compromised if the host species affects parasite morphology; an uncritical analysis might recognize multiple taxa simply because of phenotypically plastic responses of parasite morphology to host physiology. Pentastomids of the genus Raillietiella are endoparasitic crustaceans primarily infecting the respiratory system of carnivorous reptiles, but also recorded from bufonid anurans. The delineation of pentastomids at the generic level is clear, but the taxonomic status of many species is not. We collected raillietiellids from lungs of the invasive cane toad (Rhinella marina), the invasive Asian house gecko (Hemidactylus frenatus), and a native tree frog (Litoria caerulea) in tropical Australia, and employed a combination of genetic analyses, and traditional and novel morphological methods to clarify their identity. Conventional analyses of parasite morphology (which focus on raw values of morphological traits) revealed two discrete clusters in terms of pentastome hook size, implying two different species of pentastomes: one from toads and a tree frog (Raillietiella indica) and another from lizards (Raillietiella frenatus). However, these clusters disappeared in allometric analyses that took pentastome body size into account, suggesting that only a single pentastome taxon may be involved. Our molecular data revealed no genetic differences between parasites in toads versus lizards, confirming that there was only one species: R. frenatus. This pentastome (previously known only from lizards) clearly is also capable of maturing in anurans. Our analyses show that the morphological features used in pentastomid taxonomy change as the parasite transitions through developmental stages in the definitive host. To facilitate valid descriptions of new species of pentastomes, future taxonomic work should include both morphological measurements (incorporating quantitative measures of body size and hook bluntness) and molecular data.

Figure 1. Molting Raillietiella frenatus that has retained the hooks of two previous instars.

(a) Anterior view of molting R. frenatus from Hemidactylus frenatus, scale bar is 0.1 mm. (b) Close-up of posterior hooks from the same individual showing retained posterior hooks from two previous instars. The oldest hook is marked O, the intermediate aged hook is marked I, the newest hook is marked N. Photograph is compiled from five focus-stacked images of the same field of view, scale bar is 20 µm.

Figure 2. Photographs of Raillietiella frenatus showing key features.

(a) Female Raillietiella frenatus (bearing morphological resemblance to Raillietiella indica) from Rhinella marina showing entire specimen. Note sharp anterior hooks and intermediately sharp posterior hooks. Photograph is complied from five images stitched longitudinally together. (b) Anterior view of male R. frenatus from Hemidactylus frenatus, note buccal cadre (BuCa), sharp anterior hooks (AH), relatively blunt posterior hooks (PH) and copulatory spicules (CS). (c) Anterior view of female R. frenatus from H. frenatus, note blunt posterior hooks. Scale bars are all 0.1 mm.

Figure 3. Area of posterior hook tips of Raillietiella frenatus graphed against pentastome body length.

Small areas indicate relatively sharp hooks, large areas indicate relatively blunt hooks (see example pictures inset with sharp, intermediate and blunt hook tips).

Figure 4. Copulatory spicules of Raillietiella frenatus and Raillietiella orientalis.

(a) Copulatory spicule of male Raillietiella frenatus from Rhinella marina. Photograph is compiled from three focus-stacked images of the same field of view, scale bar is 0.05 mm. (b) Copulatory spicule of male R. frenatus from Hemidactylus frenatus. Photograph is compiled from two focus-stacked images of the same field of view, scale bar is 0.05 mm. (c) Paired copulatory spicules of male Raillietiella orientalis from R. marina. Note flared ornamented bases, scale bar is 0.1 mm.

Figure 5. Barb length (AB) versus overall length (BC) of posterior hooks of Raillietiella frenatus from Rhinella marina, Hemidactylus frenatus, and Litoria caerulea.

(a) Raw hook measurements uncorrected for pentastome body size, note two visible clusters. (b) Hook measurements corrected for pentastome body size (by calculating residual scores from a linear regression of hook measurements against pentastome body size), note that clusters now disappear.

Figure 6. Barb length (AB) versus overall length (BC) of posterior hooks of Raillietiella frenatus from the current study compared to the literature.

Kelehear et al. (2011, current study) measured R. frenatus from Rhinella marina and Hemidactylus frenatus; Ali et al. (1981) measured R. frenatus from H. frenatus; and Barton and Riley (2004) measured “R. indica” from R. marina. All measurements corrected for pentastome body size by calculating residual scores from a linear regression of hook measurements against pentastome body size.

Figure 7. Protocol for measuring pentastome hooks.

Photograph of right posterior hook of Raillietiella frenatus demonstrating standard procedure for measuring barb length (AB) and overall length (BC), and a new procedure for measuring bluntness of hooks.