A total of 34 years work in the small area where R. capensis occurs, leads to several conclusions: (i) this fluke species appears to be specific for M. varius, as no brachylaimid has ever been found in any of the other vertebrates inhabiting the same area; (ii) the slug Ariostralis nebulosa is the first intermediate host of this fluke species, as branched, cercariogenous sporocysts resembling those typical of brachylaimids [14, 16, 24, 25] were frequently found but only in this slug among the five terrestrial mollusc species inhabiting the area; this means that this fluke is highly specific at first intermediate host level; (iii) Ariopelta capensis, the only other slug species present in the same area, appears to be the only second intermediate host species, as brachylaimid metacercariae resembling immature juveniles found in the shrews were only found in this mollusc. Although no intracellular early mother sporocyst stage within hepatopancreatic cells  was observed nor any sporocystogenous first generation sporocysts [19–22], the existence of such stages cannot be a priori denied.
The sequencing results verify that the sporocysts, cercariae, metacercariae and adult stages belong to the same species and confirm this life cycle. The mere three mutations found in the metacercariae infecting an Ariopeltis capensis slug individual may be considered as intraspecific genetic variability within the local area, according to the well known evolutionary rates of the mtDNA cox 1 gene in invertebrates and its usefulness for inter- and intrapopulation diversity studies in general . This agrees with the three-host life cycle pattern of Brachylaimidae. The observation that the shrew M. varius preys on both species of slugs supports this view.
However, results of the several years of field work indicate that prevalence and intensity data do not appropriately fit the above statement:
a) in brachylaimid species in general, the prevalences of the first intermediate snail host by cercariogenous sporocysts are always pronouncedly low [14, 16, 17], whereas in the case of R. capensis the prevalence is pronouncedly higher, mainly in autumn-spring;
b)in brachylaimids, prevalences and intensities of the second intermediate snail host by metacercariae are usually very high, even up to 100%, and high infection burdens of up to more than 100 metacercariae occur in the main second snail host [14, 16, 17]; on the contrary, in R. capensis, the prevalence is strikingly low; this may be explained by the second intermediate slug host not being readily available in winter and thus allowing second intermediate slug infections only very rarely;
c) the high prevalences and intensities of R. capensis in the shrews do not correspond with the very low prevalences (and sometimes apparently also intensities) of metacercariae in slugs, nor with the second intermediate slug population densities in the area;
d) the adult stage does not show an egg production capacity higher than that of other Brachylaima species (similar body size, similar size of the gonads, similar uterus extent); the fluke prevalence in the shrews (72.4%) is, however, higher than those usually found for intestinal Brachylaima species in their definitive host species: for instance, 56.7-64.3% in one host species and 21.4-60.0% in another host species for B. ruminae, or 38.5-66.7% for B. mascomai; moreover, M. varius shrew densities are not as high as to argue that a higher egg dissemination ability could be the cause for higher infection rates of the first intermediate slug species.
From the point of view of the r/K selection concept, ecological studies on the compared fitness of the life cycle strategies of Brachylaimidae in general show that these trematodes follow a compensation pattern within the different transmission phases . According to such a rule, the surprising prevalence and intensity data at the levels of first and second intermediate hosts, markedly opposite to what is found in other brachylaimids, can be understood, although the route of transmission may still remain an open question.
The question immediately arises about the possibility of direct definitive host infection by ingestion of mature cercariae still inside sporocysts carried by the first intermediate mollusc host. Although definitive host infection by mature cercariae resulting in the development of mature adults, without being exposed to metacercariae, could not be proven, observations in three brachylaimid species suggest that this could be possible. In the species Z. spearei, juvenile specimens presenting a cercarial tail were found in the urinary bladder of the toad Bufo marinus and it was concluded that this may be indicative of the possibility that the cercaria in the first intermediate host could be directly infective to the final host. This theory is further supported by the statement that in the smallest of these individuals the genital system was only represented by a single primordium . In Brachylaima ruminae, such a cercarial tail was also found in several juvenile specimens from the intestine of garden doormice Eliomys quercinus (Valero and Mas-Coma, unpublished data). In D. frontalis, a cercarial tail was found in a juvenile fluke  as well as in three gravid fluke specimens (Valero and Mas-Coma, unpublished data) from the nasal sinuses of the same garden dormouse species. However, a cercarial tail such as this appears to be sometimes retained for a period after having penetrated the second intermediate snail host , so that it cannot be ruled out that tailed adults may also derive from tailed metacercariae infecting a second intermediate snail host. The numerous tailed metacercariae found in one Ariopelta capensis indicate that this phenomenon may also take place in R. capensis.
Moreover, the presence in mature cercariae of R. capensis of a genital complex already divided, with visible terminal male duct ending in a small ventral genital pore, and a genital anlagen already forming the three gonads, should be highlighted. Such a precociousness is not typical of Brachylaimidae. In species of this family, only a rounded, postacetabular, undifferentiated genital primordium appears in the mature cercariae [14, 16, 17]. This suggests that mature cercariae may develop sufficiently inside the sporocyst as to become infective for the definitive host (i.e., do not need further genital maturation in a second intermediate host). Additionally, the very small increase in fluke size during their transmission through the three hosts (length of mature cercariae inside the sporocyst, metacercariae in slug kidney and smallest adult in the shrew of 330–436 μm, 528–694 μm, and 930 μm, respectively), suggests that a develpment transit in a second intermediate host may not be necessary. In other terms, a larger size development of metacercariae in the second intermediate slug host could become an impediment for a subsequent successful intraorganic migration within the shrew.
The capacity of self-infection of the first intermediate mollusc host individual with metacercariae shed by the sporocysts harboured by the same molluscan individual also implies the reduction of the life cycle from a three-host pattern to a two-host pattern. Such a capacity is apparently related to an adaptation strategy of the characteristics of both intermediate mollusc host and habitat environment . Such self-infection seems to be prevented by a kind of premunition in species such as B. ruminae, which is consequently an obligatory three-host brachylaimid . In the species D. frontalis inhabiting dry habitats, 90.6-100% of the snails bearing sporocysts also harbour metacercariae in the pericardium while only 19.9-40.0% of the snails, of the same species, lacking sporocysts carry metacercariae . Similarly, in a P. pericardicum inhabiting wet habitats, the relative figures are 50.0% and 39.2% .
In another brachylaimid, Serpentinotrema laruei (= Postharmostomum laruei; = P. helicis) [19–21, 55–57], the discovery of a precociously developed metacercaria within a sporocyst  also indicates the secondary possibility for a two-host life cycle. In Brachylaimidae, however, the genus Parabrachylaima presents a two-host life cycle, as the adult stage fully develops to maturity and egg laying within a terrestrial snail . Progenesis within a former second intermediate snail host has been the interpretation used to justify its inclusion into the subfamily Brachylaiminae . All in all, Brachylaimidae appear to be a versatile group where the elimination of one or another of the typical hosts in the life cycle might be possible. It thus seems as although a triheteroxenous life cycle is indicated for the new South African fluke species, the possibility of the second intermediate host being eliminated in current transmission cannot be ruled out.
A reduction from a three-host life cycle to a two-host life cycle has been described in species in other families included within Brachylaimoidea, namely Hasstilesiidae from mammals, and Leucochloridiidae and Leucochloridiomorphidae from birds. In all of them, metacercariae develop in branched sporocysts infecting snails (terrestrial snails in hasstilesiids and leucochloridiids, and aquatic snails in leucochloridiomorphids) [13, 15, 59–61]. Leucochloridiidae and Leucochloridiomorphidae seem to be morphologically and biologically distant from the South African fluke, but hasstilesiid species of the genus Strzeleckia from the intestine of marsupials appear to be not as distant morphologically . Another genus, Michajlovia, comprises parasites which infect the intestine of passerine birds and whose life cycle is still unknown. Michajlovia adults present a ventral genital atrium just posterior to the gonads or in the region of the posterior testis. Michajlovia is included in Brachylaimoidea as incertae sedis although close to Leucochloridiidae and Leucochloridiomorphidae [31, 63], and also shows several similarities with the South African fluke. Except for the extent of vitellaria, the morphological similarities of Michajlovia with the panopistine genus Dasyurotrema, whose type species D. mascomai infects the alimentary tract and associated organs of marsupials , are evident.
Fluke transmission, monotesticular forms and their origin
The not uncommon monotesticular forms of R. capensis adults merit an additional analysis to elucidate fluke transmision. In these curious adults, the single postovarian testis is relatively much larger and elongated than either the anterior or posterior testes of the bitesticular specimens .
The phenomenon of neoteny, as previously defined , seemingly applies to these individuals seeing that the testis matures in the adult without the larval genital anlagen having separated into two independent testes beforehand. This fits the concept of cercariae having the capacity to infect the definitive host, as the genital primordium in brachylaimid cercariae does not differentiate into the three separate gonads prior to the development of the mature metacercaria within the second intermediate snail host [14, 16, 17]. Monotesticular forms of R. capensis thus also support a direct transmission from first intermediate snail host to definitive host, as such forms may derive from intrasporocystic cercariae already infective but with division of the genital primordium still not complete.
Monotesticular specimens have also been found, but rarely, in other brachylaimid species. Interestingly, modern brachylaimid species such as Brachylaima ruminae, it is always the posterior testis that is absent in the monotesticular forms. Contrarily, in presumably archaic brachylaimids such as Ityogonimus ocreatus from moles (Talpidae insectivores) and Dollfusinus frontalis from hedgehogs (Erinaceidae insectivores) and dormice (Glirimorph rodents), it is always the anterior testis which is absent. Noteworthy is that certain adult specimens of D. frontalis show a rare triangular arragement of the gonads in which the ovary appears anterior to both testes (Valero and Mas-Coma, unpublished data), thus remimiscent of the gonad arrangement typical of Zeylanurotrema.
A strong argument to support a direct cercarial origin for monotesticular specimens is found in the monorchid species Parabrachylaima euglandensis. In this peculiar brachylaimine, the progenetic adult stage fully develops to maturity and egg laying in a terrestrial snail, and shows only one postovarian, sacculate testis with a pair of anterior projections from which the two respective vasa efferentia arise . Additional to its peculiar life cycle, the following features of Parabrachylaima, however, rules out a close relationship with the South African fluke: (i) both suckers close together in the anterior part of the body; (ii) caeca unequal, with right caecum terminating in middle third of body; (iii) very long excretory vesicle reaching anteriorly to level of acetabulum; and (iv) cirrus pouch present and including a poorly developed cirrus.
If a definitive-host-infection capacity is accepted for intrasporocystic mature cercariae of R. capensis, massive infections of the shrews by adult flukes are easily explained (one or a very few sporocyst-carrying slugs ingested would be sufficient), but on the contrary a question mark is posed by the shrews infected by only a few adult flukes. Such low infections could be the consequence of (i) shrews swallowing sporocysts including only a few mature cercariae at that moment (i.e., all other intrasporocystic cercariae present were still immature at that moment, not able to migrate inside the shrew’s body and hence eliminated digestively after ingestion), (ii) most cercariae may get lost in the definitive host infection, most probably expelled by the shrew with their faeces; thus, the massive sporocyst infection of the first intermediate slug host and the very long sporocyst branches filled by mature cercariae could be interpreted as a fluke strategy to mitigate the great losses; (iii) a crowding effect not allowing the penetration into the kidney and ureters by all migrating cercariae because of lack of microhabitat space, (iv) massive infections becoming too pathogenic for shrews which would thus be quickly eliminated from natural populations, and of course also (v) ingestion of slugs only carrying a few metacercariae. In addition to all this, an influence of innate and cellular immunity on establishment of infection may also play a role in such individual cases.
Mitochondrial DNA cox 1 gene sequence variability and transmission modalities
It is evident that the number of slugs and shrews, from which respective larval stages and fluke adults have been molecularly analysed, is insufficient to conclude significant results. Unfortunately conservation restriction laws will never allow the collection of a sufficient host number, mainly of Ariopelta capensis, to obtain sufficient amount of metacercariae from different slug individuals, to perform the necessary study, given the pronouncedly low metacercarial prevalences in this slug species.
The characteristics of the three mutations found in the metacercariae are surprising. In mtDNA coding genes, when intraspecific mutations appear they are mostly silent and only a small percentage give rise to amino acid changes . In R. capensis, among three mutations only one is silent. Whether such a rare genetic characteristic may be interpreted as two different biological strains of R. capensis coexisting in the study area may always remain a question mark. But there is the temptation to suggest that there coexists a majority of haplotype R.cap-cox 1a strain following a two-host life cycle modality with a less frequent R.cap-cox 1b strain following a three-host life cycle modality. Such a parallel circulation of the two biological strains could be perhaps related to climatic seasonality, due to Ariopelta capensis not being readily available in winter.
Intraorganic migration, adult microhabitat and egg shedding
The occurrence of R. capensis in the urinary system of a mammal host is a unique phenomenon for the family Brachylaimidae. Within Brachylaimoidea, only the species of Zeylanurotrema parasitising amphibians and reptiles show a similar microhabitat: Z. lyriocephali in the urinary bladder of the agamid lizard Lyriocephalus scutatus in Sri Lanka  and Z. spearei in the urinary bladder of the cane toad Bufo marinus. However, there are fundamental differences between the urinary system of mammals and that of amphibians and reptiles. In mammals, the urinary system is isolated from the alimentary tract, so that a renal helminth following an oral way of infection unavoidably has to traverse tissues, then find the kidney and actively penetrate it. On the contrary, in lizards the urinary system opens into the cloaca forming a direct connection along which the parasite can migrate between the two systems. Similarly, in amphibians the urinary bladder is practically merely an evagination of the alimentary tract. Furthermore, the distinct anatomy of Zeylanurotrema having a very anterior, postbifurcal acetabulum, pretesticular, lobed ovary, opposite testes and terminal genital pore rules out any close relationships with R. capensis.
Within Brachylaimoidea, another additional exception presenting a similar microhabitat, although inside a definitive snail host, is the “progenetic” adult stage of Parabrachylaima euglandensis, which also develops in the lumen of the kidney sac .
That an active, tissue-traversing, intraorganic migration within the shrew takes place during its infection by R. capensis, is the only way to understand the observation of both (i) a young individual attached to the outer surface of the shrews kidney, and (ii) other young individuals in some cases seen just under the surface of the kidney which may be interpreted as immature flukes just penetrated and on their way to the deeper areas of the kidney and the ureters.
Concerning the intraorganic route followed by the infective stage to the kidney, the knowledge about such a life cycle phase in other renal helminths suggest that a probable intermediate migratory step through the liver could be envisaged. This is the case in the nematode Dioctophyme renale, a renal parasite of carnivores , as well as, interestingly, that of another trematode from European insectivores (shrews and moles) but also archaic glirimorph rodents (dormice), Nephrotrema truncatum[67, 68]. In both cases, the infective stage crosses the intestinal wall and migrates through the general body cavity to penetrate the right hepatic lobes which cover the right kidney before penetrating the latter. Such a liver phase appears to be important from the trophic point of view . Contrary to N. truncatum where the adult stage only infects the right kidney, in R. capensis the adult stages infect both kidneys and ureters without any apparent lateral preference . This poses the question whether R. capensis migrates throught the choledoc duct up to the biliary ducts to finally reach both kidneys after crossing the distal parenchyma of the liver lobes. Such hypothesis is supported by the hepatic duct microhabitat of species of another close brachylaimid genus, namely Scaphiostomum, although a more simple migration only including intestinal wall crossing and direct migration through the general body cavity to enter the two kidneys of the shrew by penetrating their surface can a priori not be denied.
Should one or another intraorganic migration route be followed in the definitive host, the infecting stage would evidently need the capacity to traverse host tissues. This is a feature worth emphasizing, because there are no other species of Brachylaimidae known to have acquired such a capacity to reach their final microhabitat. In Brachylaimidae, the adult stage of the great majority of species develops in the digestive tract (mainly intestine, rarely oesophagous and stomach). Only very few species present other microhabitats (Scaphiostomum in ducts of the liver and pancreas; Dollfusinus in nasal and frontal sinuses), but these are all directly connected to the digestive tract [14, 16]. As already mentioned, the same applies to Zeylanurotrema, where both the reptilian and amphibian urinary bladders are part of the alimentary canal. In Brachylaimidae in general, it thus is the cercaria and not the metacercaria that presents a high tropism and migration capacity, a fact reflected by the existence of two postacetabular lateral aggregations of large penetration glands in cercariae. Nothing of this kind appears in the metacercariae [14, 16, 17]. This also supports a direct definitive-host-infection capacity by intrasporocystic cercariae and, thus, definitive host infection by predation of the first intermediate slug host in the case of R. capensis.
Owing to the adult stage microhabitats of kidneys and ureters, eggs are shed by the shrews by urinating. A priori this poses the problem of understanding how the first intermediate slug host may become infected by ingesting the fluke eggs dispersed throughout the external milieu. In other brachylaimids, eggs are in all cases shed with faeces and the fluke transmission is assured because of snails being attracted by the deposited stool materials, with many terrestrial gastropods showing coprophagous trends. Unfortunately, to our knowledge, nothing is known about potential snail attraction by urine. However, the shrew M. varius always urinate during the process of defaecation, therefore concomitantly depositing eggs with faecal material . This fact has been corroborated by the finding of eggs of R. capensis on the shrew’s droppings.