This study demonstrated the presence and development of structures utilized in the adhesion and feeding of Haematoloechus longiplexus in its infection of Rana catesbeiana lungs.  The structures considered included the oral and ventral suckers as well as tegumental spines.  It was found that this particular species of trematode has an aspinose tegument.  This finding is particularly important, for there is a great deal of controversy surrounding this characteristic in the literature.  It was also observed that the ventral sucker of H. longiplexus is not nearly as prominent as the oral sucker.  This suggests that the majority of feeding occurs at the anterior end of the fluke through the oral sucker.  The combination of a weak ventral sucker and an aspinose tegument suggest that this species is not greatly reliant upon adhesion mechanisms to remain in its host.  This is due to the fact that H. longiplexus infect the alveoli of the bullfrog host’s lungs and remain in close contact with the epithelium, thus adhesion mechanisms are not required.

     Haematoloechus has been described as one of the most commonly encountered genera of frog trematodes, as infection has been shown to be quite wide spread (Kennedy, 1981).  Haematoloechus longiplexus is a digenetic trematode known to infect the lungs of Rana catesbeiana (Shields, 1987).  Infection occurs when the bullfrogs ingest infected damselfly nymphs and adults.  The ingested metacercariae migrate from the frog’s stomach to its lungs via the glottis (Shields, 1987).  The parasites then burrow into the lung parenchyma where they will commence life as a blood feeder in the alveoli (Shields, 1987).  The trematode’s lifecycle continues as the fully embryonated eggs are carried out of the lungs, via the ciliary action of the lungs, into the glottis where they are swallowed and eventually voided in the feces (Krull, 1933).  These eggs then hatch upon being swallowed by a snail host (host unknown for Haematoloechus longiplexus) (Krull, 1933).  Cercariae are then released from the snail into the water where they are taken up by naiads (nymphs) of damselflies wherein they become metacercariae (Krull, 1933).  The lifecycle is complete when the bullfrogs ingest these infected nymphs or the metamorphosed adults (Krull, 1933). (Figure 0 shows the lifecycle of Haematoloechus)
 

There is a great deal of controversy in regards to the spiny characteristic of the tegument of this particular species.  There exists literature, which claim that H. longiplexis is aspinose and spinose in nature (Leon-Regagnon, et al., 1999).  It has also been stated that these spines may be lost during the development of the trematode or as a result of fixation (Leon-Regagnon, et al., 1999).  It has thus been determined that the presence of spines (or lack thereof) is not a useful means for differentiating between the various species of this genus (Leon-Regagnon, et al., 1999).
    This study attempts to determine whether or not Haematoloechus longiplexus does in fact possess a spiny tegument by looking at the surface of the trematode with a scanning electron microscope; this should no doubt allow for the detection of any presence of spines.  As well, a comparison will be made between flukes as they undergo maturity taking into particular consideration the development of the oral and ventral suckers.

    The specimens utilized in this study were supplied by Dr. Tim Goater who had collected the trematodes from the lungs of bullfrogs from Robin’s Pond in Nanaimo, British Columbia.  These specimens had been cleaned and then stored in an AFA (Alcohol-Formalin-Acetic acid) and ethanol solution.  The specimens were then dehydrated in 100% ethanol prior to undergoing critical point drying.  All specimens were mounted on specimen stubs with colloidal silver paste, sputter coated and viewed under the SEM.

	Figure 1. Adult trematode at low magnification. The oral and ventral suckers of the trematode are indicated. 28X magnification
	Figure 2. Oral sucker of an adult trematode. The dimensions of the oral sucker are indicated.  The “junk” present inside the sucker is host lung tissue. 300X magnification
	Figure 3. Ventral sucker of an adult trematode. The dimensions of the ventral sucker are indicated. 475X magnification
	Figure 4. Full body view of an adult trematode. Again the location of the oral and ventral suckers are indicated.  Host lung tissue is evident on the entire surface of the trematode. 30X magnification
	Figure 5. Adult trematode’s oral sucker. The dimensions of the sucker are indicated. 325X magnification
	Figure 6. Adult trematode’s ventral sucker. The dimensions of the sucker are indicated. 325X magnification
	Figure 7. Full body view of an immature adult trematode. Again the oral and ventral suckers are labeled. 28X magnification
	Figure 8. Oral sucker of an immature adult trematode. The size of the sucker has been indicated. 500X magnification
	Figure 9. Host lung epithelial tissue. A close-up of the host epithelial tissue that was present on the surface of all specimens studied.  This particular specimen’s (immature adult trematode) ventral sucker was obstructed by the tissue and was therefore not visible. 300X magnification
	Figure 10. Full body view of an immature trematode. The oral and ventral suckers have again been labeled. 42X magnification
	Figure 11. Oral sucker of an immature trematode. The size of the sucker has been indicated. 800X magnification
	Figure 12. Ventral sucker of an immature trematode. The size of the sucker has been indicated. 700X magnification
	Figure 13. The oral sucker of another immature trematode has been shown. The dimensions are again provided. 850X magnification
	Figure 14. The ventral sucker of another immature trematode has been shown. The dimensions of the sucker are again provided. 1600X magnification
	Figure 15. The tegument surface of an adult trematode. This demonstrates the aspinose nature of the trematode. 2000X magnification
	Figure 16. The tegumental surface of an immature trematode. This too demonstrates the aspinose nature of the trematode in an earlier stage of its lifecycle. 1600X magnification

 

     It was obvious from this study that the ventral sucker of this particular species is not as prominent as the oral sucker.  This signifies that the acetabulum (ventral sucker) is not as important in the adhesion and feeding of this fluke.  It has previously been stated that the ventral sucker of Haematoloechus is smaller than the oral sucker (Kennedy, 1981).  It was found that in all specimens studied the ventral sucker was indeed smaller, but the degree to which the suckers differed in size varied between the specimens.  It seemed as though there was not a significant difference in the overall size of the suckers, while their prominence and degree of invagination differed greatly.  This is especially obvious when viewing Figures 4, 5, and 6 of the adult trematode (although it was observed for all five specimens).  Figures 5 and 6 show the size of the oral and ventral suckers respectively and it can be seen that they differ very slightly in dimensions (273.6µm and 262.4µm respectively).  Figure 4 however, divulges the fact that the oral sucker is exceedingly more prominent, as the ventral sucker is hardly visible.  With the increased prominence came an increase in the musculature of the oral suckers. This would allow for more efficient blood feeding.  H. longiplexus is known to feed via continuous blood flow as a result of suction provided by a plug of tissue drawn into the oral sucker and the pulsatory action of the pharynx (Shields, 1987).  Thus it is known that the oral sucker is important in feeding.  This “plug” of host tissue is quite obvious in the micrographs of the oral suckers, but especially in Figure 8. The fact that there are no spines or adhesive structures surrounding the oral sucker is representative of the degree of suction which must be produced here.  It was found that the suckers increased in size as the flukes matured, as would be expected.
    A great deal of host epithelial tissue from the lumen of the lung was still present on the specimens after cleaning and fixation had taken place.  This shows just how strong the adhesive properties of the fluke’s surface really are.
    In regards to the controversy over the presence of spines on the tegument it can be concluded that in fact Haematoloechus longiplexus is an aspinose species.  Figures 15 and 16 demonstrated a lack of spine presence throughout the lifecycle of the fluke.  The lack of spines indicates that H. longiplexus must not require an additional means of adhesion in order to remain in the host’s lung tissue.  There must therefore be something else that is responsible for the strong adhesion of the trematode to its host’s lung tissue.  Further studies should be carried out to determine what, if anything, is in fact responsible for the adhesive properties of Haematoloechus longiplexus.

 I would like to thank Dr. Tim Goater for supplying the specimens and reference materials used in this study.  I would also like to thank Doug Bray and Alan Box for all of their help with the preparation of the specimens as well as their extensive knowledge of the scanning electron microscope.

Kennedy, M.J. 1981. A revision of species of the genus Haematoloechus Looss, 1899 (Trematoda: Haematoloechidae) from Canada and the United States. Can. J. Zool, 59:1836-1846.
Krull, W.H. 1932. Studies on the life history of Pneumobites longiplexus. Zoolo. Anz, 99:231-239.
Leon-Regagnon, V.; D.R. Brooks and G. Perez-Ponce de Leon. 1999. Diferentiation of Mexican species of Haematoloechus Looss, 1899 (Dignea: Plagiorchiformes): Molecular and morphological evidence. J. Parasitol, 85(5):935-946.
Shields, J.D. 1987. Pathology and Mortality of the lung fluke Haematoloechus longiplexus (Trematoda) in Rana catesbeiana. J. Parasitol, 73(5):1005-1013.