Presence of Ribeiroia ondatrae in the developing anuran limb disrupts retinoic acid levels
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- Szuroczki, D., Vesprini, N.D., Jones, T.R.B. et al. Parasitol Res (2012) 110: 49. doi:10.1007/s00436-011-2451-z
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The widespread reports of malformed frogs have sparked interest worldwide to try and determine the causes of such malformations. Ribeiroia ondatrae is a digenetic trematode, which has been implicated as one such cause, as this parasite encysts within the developing tadpole hind limb bud and inguinal region causing dramatic limb malformations. Currently, the mechanisms involved in parasite-induced limb deformities remain unclear. We sought to investigate whether the level of retinoic acid (RA), a morphogenetic factor known to play a critical role in limb bud formation, is altered by the presence of R. ondatrae within the infected tadpole. Alteration of RA levels within the limb bud caused by the presence of the parasite may be achieved in three ways. First, metacercariae are actively secreting RA; second, cercariae, upon entering the limb/inguinal region, may release a large amount of RA; finally, the metacercariae may induce either an increase in the synthesis or a decrease in the degradation of the host’s endogenous retinoic acid levels. Here, we show through high performance liquid chromatography and mass spectrometry that limb bud tissue of Lithobates sylvaticus, which has been parasitised, contains 70% more RA compared to the unparasitised control. Furthermore, parasites that have encysted within the limb buds appear to contain substantially less RA (56%) than the free swimming cercariae (defined as the infectious stage of the parasite). Taken together, these data illustrate for the first time that encystment of R. ondatrae leads to an increase in RA levels in the tadpole limb bud and may offer insight into the mechanisms involved in parasite-induced limb deformities.
Global loss of amphibian biodiversity has been linked to many factors, such as pollution, habitat loss, climate change, invasive species and emerging infectious diseases (e.g. chytrid, ranavirus and parasitism; Collins and Storfer 2003). One particular parasite species, Ribeiroia ondatrae, a digenetic trematode that infects multiple hosts (for a complete review of the life cycle, see Szuroczki and Richardson 2009), has gained worldwide attention as a possible ecological driver behind amphibian declines. This is in part due to the severe and grotesque limb/body malformations caused by infection (Blaustein and Johnson 2003; Johnson et al. 1999). For example, Johnson et al. (2002) reported a significant association between severe limb malformations and Ribeiroia infection in nine species of natural amphibian populations distributed across the Western USA. Within infected anurans, the majority of metacercariae (i.e. cercarial bodies, which have encysted and are maturing) were found embedded in the basal and tail resorption area. Encystment of the parasite within the basal tissue of hind limbs caused multiple malformations including skin webbings, extra digits, missing limbs and limb duplications (Johnson et al. 2002). It is important to note that the types of malformations, which result from successful encystment of parasites in limb tissue, are highly dependent on tadpole developmental stage (Schotthoefer et al. 2003).
Currently, there are two prevailing hypotheses as to how R. ondatrae induces limb malformations in amphibians. First, metacercariae, which encyst within the tissues of the developing limb buds, may cause mechanical disruption of the spatial organisation of cells leading to abnormal cellular outgrowth through a process called intercalation [a process whereby neighbouring cells produce daughter cells mitotically to fill in missing positional values (Johnson et al. 2004; Stopper et al. 2002)]. This has been shown in the laboratory by inserting resin beads into the developing limb bud, which caused supernumerary limb structures similar to parasite-induced limb abnormalities observed in nature (Sessions and Ruth 1990).
The second hypothesis is that the parasite may itself actively produce and release growth or morphogenetic factors that alter the molecular signals directing limb development (Gardiner et al. 2003; Johnson et al. 1999, 2004). This hypothesis was initially proposed by Johnson et al. (1999); however, no attempt to date has been made to determine if R. ondatrae may be producing growth factors to disrupt normal limb patterning. One morphogenetic factor that has been suggested to play a role in parasite-induced limb malformations is the vitamin A metabolite, retinoic acid (Johnson et al. 1999; Johnson and Sutherland 2003; Maden 1996).
From an ecological stand point, it would be beneficial for R. ondatrae to actively induce malformations within its second intermediate host (the tadpole), as it may facilitate faster transmission into the definitive host (typically a semi-aquatic bird). This is not a novel strategy, as many parasite species can alter their intermediate hosts in order to enhance trophic transmission (as reviewed by Moore 2002). The presence of abnormalities in adult frogs, for example, has been hypothesised to increase predation by the definitive bird host (Johnson et al. 2004).
Retinoic acid (RA) has been well studied for its role in genetic regulation during development (Maden 1999, 2007; Mark et al. 2009). Specifically, its role in the patterning of tissue during limb formation has been well documented (e.g. Elinson et al. 2008; Lee et al. 2004). RA exists as two isoforms, namely, all-trans RA (atRA) or 9-cis RA, which regulate gene expression by binding to retinoic acid receptors (RAR) and retinoid X receptors (RXR), respectively (Maden 2007). Both RARs and RXRs are nuclear receptors that bind to DNA at specific sites to influence gene expression at the transcriptional level. Alterations in the retinoid signaling pathway have been shown to disrupt normal limb development and patterning (Lewandoskia and Mackem 2009). For example, as reviewed in Maden (1999) and Maden and Hind (2003), exposure of amphibian tail and limb tissue to excess retinoids has been shown to result in the development of multiple hind limbs. Furthermore, RA has been shown to inhibit intact limb buds and to form two common abnormalities: proximal-distal duplications and mirror-image duplications along the anterior–posterior axis (e.g. Bryant and Gardiner 1992; Maden 1983; Scadding and Maden 1986b).
Here, we determine, using high-pressure liquid chromatography (HPLC) and mass spectrometry (MS) whether the presence of R. ondatrae metacercariae changes the levels of retinoic acid within the developing anuran limb. Additionally, we determine whether free swimming cercariae and encysted metacercariae contain different levels of RA.
Materials and methods
Collection of animals and husbandry
Six Lithobates sylvaticus egg masses were collected in May 2009 from Bat Lake (45°35′ N, 78° 31′ W) at the Wildlife Research Station, Algonquin Park, ON, Canada (45°35′ N, 78° 30′ W). Individual L. sylvaticus egg masses were placed into either 1.5-L glass bowls or, for smaller egg masses, 300 mL glass bowls, which were all placed into two growth chambers and cooled to 5°C prior to hatching (cooling egg masses is required as L. sylvaticus egg masses have very low hatching success at warmer temperatures). Hatchlings were housed in 38 L (61 × 40.6 × 22.2 cm) Rubbermaid® tubs filled with a combination of filtered pond water and animal-ready water (carbon-filtered and aged tap water with pH adjusted to 7.0). As tadpoles developed and grew larger, they were housed in smaller tubs with no more than 20 tadpoles per tub. All tadpoles were maintained on ground Spirulina Algae Discs (Wardley®, Secaucus, NJ, USA).
Throughout May to August 2009, Planorbella trivolvis snails were collected by handpicking snails from vegetation or using net sweeps along the pond floor, from the Glenridge Naturalization Site in the Niagara region, Ontario, Canada (43°7′ N, 79°14′ W). Snails were housed in their own individual 150 mL Styrofoam™ cups filled with approximately 75 mL of animal-ready water and fed lettuce ad libitum. The water in each individual cup containing a snail was screened daily for the presence of R. ondatrae cercariae by placing small aliquots in a Petri dish under a dissecting microscope. R. ondatrae cercariae were identified by the presence of the characteristic esophageal diverticula, distinct swimming, and size (for a more detailed description on how to identify cercariae refer to Szuroczki and Richardson 2009). While it is a possibility that snails could have been harbouring other trematode species while simultaneously shedding R. ondatrae, we are confident that tadpoles were only exposed to cercariae of R. ondatrae for the following reasons. First, a subset of cercariae from each shedding snail was obtained and properly identified daily (as mentioned above) to confirm the presence of R. ondatrae cercariae. In addition, all of the water from each snail shedding R. ondatrae cercariae were collected each day and examined under the microscope; all of the cercariae that were present and which were subsequently hand pipetted into the experimental cups consistently matched the approximate size of R. ondatrae cercariae and exhibited the same distinct “wobble-like” swimming pattern. While the cercariae of another species, namely, Echinostoma triolvis, also exhibit a similar swimming pattern and can look similar to R. ondatrae cercariae under a dissecting microscope, E. trivolvis cercariae are noticeably smaller than R. ondatrae cercariae (Szuroczki and Richardson 2009).
Ten cercariae, which were shed anywhere from 3 to 5 h prior to the use in experiments, were pipetted and placed into individual 150 mL Styrofoam™ cups containing approximately 30 mL of animal-ready water. Ten cercariae were chosen, as it has been previously shown that as little as eight R. ondatrae cercariae can cause 90% mortality in L. pipiens tadpoles; Schotthoefer et al. 2003. L. sylvaticus tadpoles between Gosner stages 28 and 31 (Gosner 1960) were randomly chosen from the communal housing container and placed individually into cups containing the cercariae and left for 24 h to allow sufficient time for the cercariae to form cyst walls (or to become metacercariae). After the 24-h period, the tadpoles were euthanised by an overdose of a chemical anesthetic (MS-222; Sigma). In addition, control tadpoles, which never encountered any parasites, were randomly chosen each day, placed into Styrofoam™ cups containing 30 mL of parasite-free, animal-ready water and kept in the cups overnight to control for any housing effects. After 24 h, the control tadpoles were subsequently euthanised by an overdose of a chemical anesthetic (MS-222; Sigma). We decided to use limb tissue from separate control individuals rather than non-encysted limb tissue from individuals exposed to parasites for two reasons. First, we wanted to ensure that we had enough limb tissue that was not infected to serve as a control. Second, if R. ondatrae is not found encysted in the limb bud directly, metacercariae are typically found in the inguinal region, which we speculate could also disrupt the RA levels within the limb bud.
Collection of tissue
Once euthanised, all tadpoles that had been exposed to parasites as well as control tadpoles were first separated according to developmental stage (Gosner stages 28–31) and were pinned out (ventral side up) in a dissection dish and placed under a dissecting microscope. Fine forceps and spring scissors were used to position the animal and to remove the entire limb bud by making an incision perpendicular to the anterior–posterior axis at the base of the limb bud. Each cut was made flush along the surface of the body such that no limb bud tissue was left on the body while also ensuring that the collected limb bud was free of any inguinal tissue. For the parasitised limb tissue, only the limb buds containing metacercariae were collected (as sometimes one limb remained free of metacercariae) and placed into microcentrifuge tubes (Eppendorf™). Typically, limb buds contained anywhere between one and five metacercariae, regardless of developmental stage. To obtain control tissue, limb buds from control tadpoles were removed in the same manner as the “parasitised” limb tissue and placed in separate microcentrifuge tubes.
Collection of cercariae consisted of pipetting free swimming individuals from pond water samples obtained from the cups individually housing P. trivolvis snails. Metacercariae were collected by gently removing the encysted capsules from the inguinal region and limb buds of parasitised animals ranging between Gosner stages 28 and 31. Once removed, all tissue was immediately stored at −80°C until further use.
Retinoid extraction procedure
Retinoid extraction was performed as described previously (Dmetrichuk et al. 2008). Briefly, tissue was thawed and homogenised in 500 μl of ice cold homogenisation buffer (phosphate buffered saline, pH 7.3 containing 0.5% ascorbic acid and 0.5% EDTA). A 50-μl aliquot was taken from each homogenate for protein concentration determination (bicinchoninic acid assay kit, Thermo Inc.). The remaining homogenate was then briefly vortexed with 500 μl of ice cold extraction solvent (8:1 ethyl acetate/methyl acetate, 0.5% butylated hydroxytoluene). Samples were vigorously shaken for 30 min on ice then centrifuged for 5 min at 14,000×g, after which the upper organic phase was collected. Another 500 μl of extraction solvent was added, and this process was repeated twice, pooling the combined organic supernatants. Once pooled, the supernatant was dried under a light stream of nitrogen gas. Once dried, samples were resuspended in 50 μl of HPLC grade acetonitrile.
High-pressure liquid chromatography
Techniques used were performed as described previously (Dmetrichuk et al. 2008). Briefly, chromatographic separation was performed using a Waters Spherisorb S5ODS2 C-18 Column. Injection volumes were 5–10 μl. Elution of samples used an initial mobile phase consisting of 70% acetonitrile and 30% Milli-Q water containing 0.1% formic acid. This phase was changed via a linear gradient to 100% acetonitrile over 30 min with a flow rate of 0.5 ml/min. Retention times were compared with atRA and 9-cis RA standards dissolved in acetonitrile prepared from HPLC grade reagents (Sigma). Detection was set to 350 nm.
Analysis was performed using a Bruker Model HCT Ultra high capacity trap and liquid chromatography (LC/MS) system using electrospray in negative ion mode. This was connected online to an Agilent 1100 LC system with the conditions described above. As described previously (Dmetrichuk et al. 2008), the mass spectrometer was set up to detect 255 m/z, which is a fragment observed in MS2 when the parent ion (299 m/z) (corresponding to either atRA or 9-cis RA) is fragmented according to the following scheme: molecular anion (299)→(255) + CO2. This specific fragmentation behaviour has been previously documented in our earlier work (unrelated to current study) and has been shown to correspond specifically to both isomers of RA.
To determine the approximate concentration of RA in samples, a calibration curve was created using three 9-cis and atRA standards ranging from 50 to 1,000 pg/μl. Using QuantAnalysis, the area of the 255 m/z peak was determined, and the corresponding sample concentration was determined. Our lower detection limit was estimated to be approximately 50 pg/μl.
All methods presented were approved by the Brock University Research Committee on Animal Care Use (AUPP 09-01-01).
We next sought to determine whether the retinoid concentration in the tissue from each separate developmental stage would be detectable, in an attempt to determine whether parasitised limb tissue varied in RA levels depending on developmental stage. We performed an initial HPLC analysis using tissue from animals at Gosner developmental stage 29 (approximately 28 and 36 parasitised and unparasitised limbs, respectively). While a detectable peak was apparent at 12.4 min, corresponding to the elution of atRA, no peak was observed for the 9-cis isomer (data not shown). Furthermore, there was an abundance of background peaks, making the precise determination of RA retention difficult. Lastly, while continued MS analysis revealed the 255.1 m/z fragmented molecule, numerous other molecules were detected, suggesting that a number of other molecules generating ion intensity at m/z 299.1 were present in the samples. This suggested that reliable detection of retinoids using lower numbers of tissue samples (generated at each developmental stage) was not possible. As such, in an attempt to maximise retinoid concentration and acquire an accurate detection signal, tissue from all developmental stages (Gosner stages 28, 30 and 31) were pooled together for the parasitised tissue and then separately pooled for unparasitised (control) tissue. Due to the low concentration of endogenous retinoic acid, which is also typically observed in many other systems (Dmetrichuk et al. 2008; Maden et al. 1998), each HPLC sample required approximately one hundred individuals (118 parasitised and 103 unparasitised limb buds) to generate a reliable detection signal. As such, the parasite vs. unparasitised tissue samples were homogenised separately and run in duplicate, as is standard practice with HPLC/MS analysis.
In this study, we were able to demonstrate that the presence of R. ondatrae metacercariae, encysted within the developing anuran limb bud, leads to a 70% increase in limb bud atRA relative to tissue devoid of metacercariae. This is an important finding, as infection by R. ondatrae has been shown to cause limb malformations in anurans (e.g. Johnson et al. 1999; Johnson et al. 2002; Johnson and Sutherland 2003; Schotthoefer et al. 2003). The exact mechanism of how the parasite induces these malformations remains unclear, though the mechanical perturbation hypothesis has been the most widely accepted to date. The results of the current study cannot rule out the possibility of mechanical perturbation. However, since we have shown that atRA is present in limb bud tissue at 1.7 times the normal concentration when metacercariae are present, this suggests that parasite-induced malformations may involve RA in some capacity. In fact, it would not be surprising to discover that both mechanical perturbation and a disruption of the RA pathway are occurring in concert to produce the wide array of malformation phenotypes observed in nature.
Exactly how the parasite induces an increase in RA concentrations within the limb bud remains unclear and will require further investigation. However, we propose three potential mechanisms as to how RA concentrations increase within the limb bud once parasitised by R. ondatrae. One mechanism is that the metacercariae actively secrete RA, perhaps to aid in penetration of the host tissue, which in turn perturbs the normal RA concentration gradients within the limb bud of the intermediate host. It would be advantagous for R. ondatrae to actively manipulate its intermediate host in such a way as to enhance trophic transmission, as a frog with limb malformations may succumb to predation by an avian predator quicker than a healthy counterpart. This is not a novel strategy, as a wide variety of other parasites have been shown to secrete different hormones and even neurotransmitters that affect their host behaviour in order to enhance trophic success (as reviewed by Thomas et al. 2005). For example, the parasitic hairworm Spinochordodes tellini, which requires water to complete its life cycle, actively secretes proteins that change its host’s response to water. Grasshoppers that are infected with this parasite are more likely to jump into water than non-infected individuals (Biron et al. 2005).
Metabolically producing and actively secreting RA for the sole purpose of facilitating quicker trophic transmission presumably requires a significant energetic investment on the part of the parasite, at least compared to a mechanism such as mechanical perturbation. Deformaties caused by mechanical perturbation are simply a result of colonisation, which occur with no additional host manipulation. Thus, for such a mechanism (actively secreting RA) to have evolved, the energetic cost required to actively produce and secrete RA would have to be offset substantially with significantly quicker transmission success. In addition, metacercariae of numerous parasite species have been thought of as an inactive dormant stage (e.g. Fried 1997; Huffman and Fried 1990; Johnson et al. 2004; Olsen 1974), and as such, it would not be expected that they remain metabolically active in order to produce and secrete RA. However, our examination of live E. trivolvis (another digenetic trematode that infects tadpoles) and R. ondatrae metacercariae encysted within host tissue when viewed under a dissecting microscope has revealed substantial movement of the internal organs within the cyst (unpublished observations). This suggests that the metacercarial stage may not be truly inactive or in a resting state. A logical step forward would be to determine whether R. ondatrae metacercariae have the capacity to actively synthesise and secrete RA. More specifically, the detection of retinol dehydrogenase and retinaldehyde dehydrogenase activities, which are the enzymes required to produce retinoic acid from dietary retinol (see review by Maden 2007) may provide support that metacercariae are metabolically active during encystment.
A second proposed mechanism for how parasitised host RA levels are increased is that the cercariae upon entering the limb/inguinal region may release a large amount of RA, or upon encystment, RA may simply be slowly diffusing across the cyst membrane (as RA is a lypophilic molecule) into the surrounding limb tissue, thereby increasing the RA levels within the limb bud. One of the most surprising results obtained within this study was that the free-swimming cercariae contained 56% more RA than the metacercariae. This suggests that either cercariae introduce RA upon entering the host tissue or, perhaps, the cercariae require a certain concentration of RA for their own development into the metacercariae. Taken together, the process of encystment is complex and is still not fully understood (Fried 1997), and the involvement of any biochemical molecule such as RA is unclear.
A third equally plausible mechanism is that upon encystment, the metacercariae induce either an increase in the synthesis or a decrease in the degradation of the host’s endogenous retinoic acid levels. That is, the increase seen in parasitised limb tissue may be due to a host response to the parasite. This may help to explain the wide array of malformations observed which range from bony triangles to limb reductions (Degitz et al. 2000; Gardiner et al. 2003).
While the mechanism of how R. ondatrae disrupts the RA gradient within the limb remains highly unclear, the results of the current study suggest that when tadpoles become infected with R. ondatrae, the amount of RA within the limb tissue becomes altered in some way. Considering that no other trematode species that we know of shows the same overwhelming and consistent preference for encystment within the developing limb/inguinal region as R. ondatrae, it seems likely that R. ondatrae could be causing limb malformations by altering the endogenous RA levels within the limb. To our knowledge, only two other studies have attempted to link other species of trematodes to limb malformations. Sessions and Ruth (1990) associated Manodistomum syntomentera with limb deformities, as these parasites were present in malformed frogs. However, subsequent studies have failed to show any association between this parasite species and limb malformations. In fact, Dalzell and Sutherland (1998) showed that this parasite was more prevalent in normal frogs than deformed frogs and concluded that this parasite is not likely the causative agent of deformities. Another study to this end has shown that a monostome-type cercaria from Sri Lanka tends to encyst within the tail region of tadpoles and causes limb malformations in dose-dependant manner (Rajakaruna et al. 2008). The authors, however, were unable to identify the trematode species, which makes it difficult to validate their results and conclusions.
It could also be argued that other trematode species encysting elsewhere in the body may also release RA which could might potentially alter RA levels within the limbs. However, based on other studies, it is far more likely that perturbation of the RA signaling pathway, leading to limb malformations, would require local alterations in RA, specific to the limb bud (Maden 1996; 1999; 2007). For example, the generation of the RA gradient that is essential for proper limb formation is created locally in the limb bud tissue. We feel it is unlikely that encystment of a parasite in, for example the tail, would lead to an RA increase in the limb bud tissue. Precise RA signaling is critical for development of various structures, such as the forelimbs, digits, brain and retina, and as such, is tightly regulated in local domains (Maden 1996; 1999; 2007). However, as we did not test any other trematode species in this study, a full investigation of our hypothesis and interpretation of our results will need to be investigated in the future, and we cannot completely rule out the possibility that changes in RA signaling can be induced by encystment of other trematode species in other regions of the body.
It is also not clear at this time whether the amount of RA potentially released by R. ondatrae would be sufficient to induce limb malformations. However, it is well known that patterning in the limb involves the establishment of a number of local chemical gradients and perturbation of these minute chemical gradients can disrupt normal limb patterning (Maden 1996, 1999, 2007). Extremely small alterations to retinoid signalling in the limb bud also have the capacity to disrupt limb development and induce limb malformations. For example, it has been shown that 33% of tadpoles exposed to a minute concentration (2.78 μg/L) of a synthetic RA analogue, TTNPB, develop limb malformations (Ndayibagira et al., unpublished work). However, further studies will be required to validate our initial findings and will need to thoroughly examine whether such changes in RA levels in the limb bud are sufficient to induce the level and type of limb malformations observed with R. ondatrae.
Finally, it is important to shed some light on the role of retinoids in parasite-induced malformations, which have been previously overlooked. It has been argued that the range of malformation phenotypes observed in nature far outweigh the different phenotypes produced by retinoids in laboratory studies. Furthermore, the most common RA-induced duplications observed in laboratory studies are proximal-axis duplications, which are not found in nature (Meteyer et al. 2000; Sessions et al. 1999). These previous findings were some of the reasons why RA involvement was dismissed and mechanical perturbation by the parasite has been the prevailing explanation. However, we should point out that the majority of RA-induced malformations obtained in laboratory studies are observed following exposure of regenerating anuran limbs to retinoids and not developing limbs (Scadding and Maden 1986a, b). One cannot assume that developing limbs will respond in the same manner as regenerating limbs. For example, Scadding and Maden (1986a) demonstrated that in developing limbs, retinoids produced a completely different suite of abnormalities when compared to regenerating limbs, namely, deletions of skeletal elements, reduction in limbs and even total inhibition of limb development.
Furthermore, many other factors such as developmental stage and species need to be considered (Degitz et al. 2000). For example, Schotthoefer et al. (2003) showed that infection of tadpoles by R. ondatrae during different developmental stages resulted in differential responses. Namely, tadpoles that became infected in the pre-limb bud stage experienced high mortality rates ranging between 47.5% and 97.5%, whereas those individuals that became infected in the limb-bud stage displayed a high limb malformation rate and 40 different limb malformation phenotypes were observed.
In summary, this study provides preliminary support for a potential role of RA in limb bud malformations in the developing tadpole. In addition, our findings highlight the importance of the need to further investigate other trematode species and their capacity to release compounds such as RA as well as examine alternate hypotheses beyond mechanical disruption to understand how R. ondatrae causes limb deformities. Whether R. ondatrae actively secretes RA into the limb tissue still remains unclear; however, we can clearly say that the mechanisms behind R. ondatrae induced malformations are far more complicated than previously hypothesised.
We would especially like to thank Alysen Lougheed for all of her help in the seemingly never ending snail hunt and preparation of samples and Amanda Lepp for her assistance in the earlier experimental design phase. We would also like thank Dr. Janet Koprivnikar for all of her valuable input on earlier drafts of the manuscript. This research was funded by the Department of Biological Sciences, Brock University and the Natural Sciences and Engineering Research Council (DG# 238373 to G.E.S and DG# 2802-07 to R.L.C).