Canadian Journal of Zoology

Host resistance and tolerance to parasitism: development- dependent fitness consequences in common hourglass tree (: cruciger) tadpoles exposed to two larval trematodes

Journal: Canadian Journal of Zoology

Manuscript ID cjz-2018-0126.R3

Manuscript Type: Article

Date Submitted by the 06-Jan-2019 Author:

Complete List of Authors: Pathirana, Nuwandi; University of Peradeniya, Faculty of Science, Zoology Meegaskumbura,Draft Madhava; Guangxi University, College of Forestry Rajakaruna, Rupika; University of Peradeniya, Faculty of Science

Is your manuscript invited for consideration in a Special Not applicable (regular submission) Issue?:

cercariae, malformations, resistance, Tolerance, survival, Polypedates Keyword: cruciger, common hourglass treefrog

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Host resistance and tolerance to parasitism: development-dependent fitness

consequences in common hourglass tree frog (Rhacophoridae: )

tadpoles exposed to two larval trematodes

N.U.K. Pathirana1, 2, 3, M. Meegaskumbura 4, 5 and R.S. Rajakaruna1, 2

1Department of Zoology, University of Peradeniya, Peradeniya, Sri Lanka,

[email protected]

2Postgraduate Institute of Science, University of Peradeniya, Peradeniya, Sri Lanka

3 Freshwater Fish Group & Fish Health Unit, Centre for Sustainable Aquatic Ecosystems,

School of Veterinary & Life Sciences,Draft Murdoch University, Australia

4Guangxi Key Laboratory of Forest Ecology and Conservation, College of Forestry, Guangxi

University, Nanning, P.R. China

5Department of Molecular Biology and Bio-technology, University of Peradeniya,

Peradeniya, Sri Lanka

Corresponding author: R.S. Rajakaruna, University of Peradeniya, Peradeniya, Sri Lanka.

Tel: +9481 239 4481(Office); +9477 704 5080 (Mobile)

Email: [email protected]

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Host resistance and tolerance to parasitism: development-dependent fitness

consequences in common hourglass tree frog (Rhacophoridae: Polypedates cruciger)

tadpoles exposed to two larval trematodes

N.U.K. Pathirana1, 2, 3, M. Meegaskumbura 4, 5 and R.S. Rajakaruna1, 2

Abstract

Tolerance and resistance to parasites are defense strategies of host organisms. Here, we tested the development-dependent tolerance and resistance of Polypedates cruciger (Blyth 1852) tadpoles to trematode infection. We exposed the tadpoles at three Gosner stages: 27, 28/29 and 30/31 to two types of cercariae (furcocercous and pleurolophocercous cercariae of

Acanthostomum burminis; Bhalerao 1926)Draft under laboratory conditions. To determine tolerance – the ability of a host to limit health effects of a given parasite load – we exposed the tadpoles until all cercariae penetrated the host. As a measure of determining resistance, we exposed tadpoles to cercariae for limited time and counted the number penetrating the tadpoles. The survival of tadpoles exposed at early stages was significantly lower than for the tadpoles exposed at later stages (mixed effect model, p <0.05), suggesting an age-dependent tolerance to parasitism. Tadpoles exposed at early stages were also smaller, took longer to metamorphosis, showed lower resistance to parasitism (ANOVA, p < 0.001) and developed axial malformations. In the resistance experiment, fewer parasites penetrated later stage tadpoles than early stage tadpoles. Tadpoles of P. cruciger showed a development-dependent tolerance and resistance to parasitism, resulting in greater survival and fewer malformations when parasitism occurs at late stages.

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Key Words: Cercariae, malformation, Common hourglass tree frog; Polypedates cruciger,

resistance, survival, tolerance

Introduction

Hosts utilize two defense strategies against parasitism to maximize their fitness. They protect

themselves against parasitism by either lessening the parasite burden (resistance) by

avoiding/resisting parasite load, or by reducing the damage caused by parasites at a given

level of burden (tolerance) (Read et al. 2008; Schneider and Ayres 2008; Raberg et al. 2009)

– they either “fight the parasite” or “fight the disease”. Hence, the concept of host resistance

and tolerance has been identified as a major area of interest within the field of species

conservation, especially in .

Studies have shown that olderDraft tadpoles have higher resistance to trematode

infections than younger ones (Schotthoefer et al. 2003; Holland et al. 2007; Raffel et al. 2010;

Rohr et al. 2010). However, findings of these studies are contrary to those of Raffel et al.

(2011) who reported that intermediate-stage tadpoles show a greater resistance to

echinostome infections than early or late-stage tadpoles. The strategies may vary among

different host species and may also depend on the parasite species. A better understanding of

host resistance and tolerance to parasitism may have important conservation implications

(e.g., potential benefits to captive breeding and reintroduction efforts, Mendelson et al. 2006).

Parasites often evolve faster relative to their hosts (Rohr et al. 2010), and tolerance,

unlike resistance, is not expected to select strongly for the parasite (i.e. "counter-measures")

(Boots 2008). Understanding the trait mediated interactions between host tolerance and

resistance to parasites, provides insights into dynamic processes such as parasite-induced

mortality and the ability of hosts to develop acquired immune memory (Anderson and May

1985; Ferguson et al.1999; Farrington et al. 2001; Duerr et al. 2003).

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The objective of the present study is to address how tadpole developmental stage influences host tolerance and resistance to trematode parasites. Pleurolophocercous cercariae used in this study (Figure 1 A) is the larval stage of Acanthostomum burminis (Bhalerao

1926), a common trematode species of a water snake in Sri Lanka. These cercariae infect the common hourglass tree frog (Polypedates cruciger; Blyth 1852) and the Asian common toad

(Duttaphrynus melanostictus; Schneider 1799) and reduce survival, induce malformations and retard growth of the host species (Rajakaruna et al. 2008; Jayawardena et al. 2010a;

2013). Furcocercous cercariae used in this study (Figure 1 B) also infects the common hourglass tree frog (as the second intermediate host) and causes similar effects such as survival reduction and growth retardation of the tadpoles (Pathirana et al. 2017). These two cercariae types were used to determine development stage-dependent fitness consequences on

Polypedates cruciger, a common and endemicDraft Sri Lankan frog species. Here we determined the age-dependent tolerance and resistance to parasitism under laboratory conditions by exposing tadpoles of P. cruciger at three developmental (Gosner) stages: 27, 28/29 and 30/31 to two trematode larvae, pleurolophocercous cercariae of A. burminis and furcocercous cercariae. Our results suggest that tadpoles at later stages of development resist and tolerate parasitic infections better, than tadpoles at earlier stages reflected in greater rates of survival and lower rates of malformation.

Materials and Methods

Collection of frog eggs

From small ponds and pools in the Royal Botanical Gardens at Peradeniya (7°16′16″N,

80°35′44″E; May 2016) we collected foamy egg masses of Polypedates cruciger and placed them over water filled glass containers to collect the hatching tadpoles. These tadpoles were reared in glass tanks (30 x15 x 25 cm) at two densities – 10 tadpoles per tank for the

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tolerance experiment and 20 tadpoles per tank for the resistance experiment. Water in the

tanks was refreshed every week. Tadpoles in each tank were fed twice a day with flake fish-

food (~8 g). Tadpoles at 10, 20 and 30 days since hatching that translated to be

developmental stages 27, 28/29 and 30/31 (Gosner 1960) respectively, were used for all the

experiments.

Collection of cercariae from snails

From two streams in Kegalle District, we collected two species of freshwater snails:

Melanoides tuberculata (Müller 1774) from the sandy bottoms of Maha-Oya (7º15' 1242''

North 80º26'47615'' East) and Mieniplotia scabra (Müller 1774) from Hingula-Oya (7º

14'56.3ºNorth 80º28'29.7º East). Snails were transported to the lab in stream water filled glass bottles kept inside a cooler. SnailsDraft were separated and each individual was kept in a plastic vial containing 25 ml of water and exposed to sunlight or artificial light for 3-4 hours

to induce shedding of cercariae. Water quality was maintained by partial daily water changes.

Snails shedding two cercariae types: Acanthostomum burminis and furcocercous cercariae

were isolated. Cercariae were fixed using Gilson’s fixative and Borax Carmine single stain

and identified morphologically (Jayawardena et al. 2010a; Jayawardena et al. 2010b).

Tolerance of Polypedates cruciger to cercariae

We counted the cercariae using a dissecting microscope and introduced them to the tadpoles

within an hour after shedding. Tadpoles at 10, 20 and 30 days post-hatch (Gosner stages 27,

28/29 and 30/31, respectively) were each exposed to three doses (low = 16, medium = 32,

high = 48) of the two cercariae types using the protocols described in Johnson et al. (2001)

and Rajakaruna et al. (2008).

We began the exposure of tadpoles to the cercariae by isolating each tadpole in a

plastic grid plate containing 20 ml of water per tadpole (Fig 1C). The procedure was repeated

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independently for both cercariae types. A total of 630 tadpoles were used exposing 270 tadpoles to each cercariae type (90 per age, 30 per dose; as 10 tadpoles per tank). Control tanks were set upwith 90 tadpoles (30 per age as 10 tadpoles per tank) from the same clutch but without introducing the cercariae.

The resistance of Polypedates cruciger to cercariae

We counted the cercariae following one hour of shedding and used for tadpole exposures at

10, 20 and 30 days post-hatch (Gosner stages 27, 28/29 and 30/31, respectively). Each tadpole was placed in a container with 20 ml of water and was exposed to 10 cercariae over a period of one hour. Following exposure, the number of cercariae that could not penetrate the tadpole was counted. The procedure was repeated separately for both types of cercariae.

We used a total of 720 tadpoles,Draft exposing 240 tadpoles to each cercariae type (80 tadpoles per age group; 20 tadpoles per tank) and 240 tadpoles (80 tadpoles per age group; 20 tadpoles per tank) as controls. A subsample of 15 tadpoles (exposed to and fully penetrated by cercariae) from each age group was randomly selected and the number of cysts and their locations in the body were recorded for five consecutive days post-exposure following the protocols in Rajakaruna et al. (2008). Each tadpole was placed in a solid watch glass with 25 ml of water and observed under a dissecting microscope (without anesthetizing them). The tadpoles were randomly selected from each age group, examined each day and were returned to the tank after observation. The tadpoles used for counting the cysts were raised separately from other tadpoles. This procedure was repeated for both cercariae types. Only the cysts in the tail region were counted since they were externally visible under a dissecting microscope.

In addition, the majority of both cercariae types penetrated in the tail region (personal observation); the cysts in the other parts of the body were not clearly visible.

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Rearing of tadpoles

Following exposure to cercariae, the tadpoles were reared in glass tanks (30 x15 x 25 cm)

with 10 tadpoles per tank from the tolerance experiment and 20 tadpoles per tank from the

resistance experiment. Mortality was noted daily. The growth of tadpoles was measured at

metamorphosis as snout to vent length (SVL; to the nearest 0.01 cm using a vernier caliper)

and body mass (to the nearest 0.001 g using an electronic balance) and time required for the

forelimb emergence for half of the number of tadpoles (TE50). Malformations were recorded

at 40 days post-hatch stage (Gosner 31/32). Malformations observed in tadpoles and

metamorphs were categorized according to Meteyer (2000). All the tadpoles/metamorphs in

the exposed and the control groups were euthanized and preserved in 5% formalin.

The protocols of this study were approved by the Ethical Review Committee of the

Postgraduate Institute of Science, UniversityDraft of Peradeniya, Sri Lanka.

Statistical analyses

The survival of the individuals at different exposure ages and the differences in growth

parameters (SVL and body mass) and TE50 were compared using linear mixed effect model in

both tolerance and resistance experiments. Age was used as the fixed effect while dose was

used as the random effect. A Chi-square test was performed to analyze the frequency of

malformations in relation to the age of tadpoles. The differences in the total number of

cercariae penetrated and number of cysts (the reduction in the number of cysts for five days

after exposure) were analyzed at different age groups using one-way ANOVA and individual

comparisons were done using Tukey Pair-wise Comparisons. MINITAB18.0 for Windows

was used for statistical analyses.

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Results

Of the 160 Melanoides tuberculata snails collected, 31 (19.4%) released A. burminis

cercariae while only ten out of 160 Mieniplotia scabra (6.3%) shed furcocercous cercariae.

Tolerance of Polypedates cruciger to cercariae

Survival

All the cercariae introduced at different doses penetrated the tadpoles of all three age groups.

The survival of the tadpoles that were exposed at early stages (at 10 and 20 days post-hatch)

was relatively lower than those exposed at late stages (30 days post-hatch; Figs 2 and 3) for

both cercariae types (A. burminis, linear mixed effect model, F(2,22)= 16.67, p< 0.001; furcocercous, linear mixed effect model, F(2,22)= 7.75, p= 0.003). The greatest mortality occurred within the first five days followingDraft exposure (Fig 3).

Growth and Developmental Period (TE50)

Tadpoles exposed to cercariae at early stages were smaller in size and took more time to

develop than tadpoles exposed at a later stage (A. burminis; linear mixed effect model; body

mass; F (2, 22) = 5.78; p< 0.05, SVL; F(2,22)= 14.22; p< 0.001, TE50; F(2,22)= 25.58; p< 0.001, furcocercous; linear mixed effect model; body mass; F (2, 22) = 4.64; p< 0.05, SVL; F(2,22)=

8.34; p<0.05,TE50; F(2,22)= 54.65; p < 0.001).

Development of Malformations in Tadpoles

The types of malformations were kyphosis (hunched back) and scoliosis (curvature of the spine; Fig 4). Development of malformations in tadpoles that were exposed was age- dependent with more malformations in individuals exposed at early stages (Table 1).

Severely malformed tadpoles with scoliosis showed difficulties in swimming with belly-up movements (Fig 4 F and G).

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Resistance to cercariae

All ten cercariae quickly gathered around young tadpoles (10 days post-hatch) once

introduced and penetration occurred within 5–20 min. However, most of the cercariae

exposed to older tadpoles (30 days post-hatch) could not penetrate within the given time

period (1 h). A significantly smaller number of cercariae penetrated into tadpoles at 30 days

post-hatch tadpoles (A. burminis, one-way ANOVA, Tukey pair-wise comparison, F = 24.46,

p <0.001; furcocercous, F = 13.27, p<0.001; Table 2). For both types of cercariae, older

tadpoles showed higher resistance to parasite penetration compared to younger tadpoles.

Significant differences were observed between 10 days vs 30 days post-hatch exposures and

20 days vs 30 days post-hatch exposures (Tukey Pair-wise comparisons, p < 0.001) but not

between 10 days and 20 days post-hatch tadpoles. A similar pattern was seen for both

cercariae types (Tukeys Pair-wise comparisonsDraft p > 0.05; Table 2). The majority (>69 %) of

cercariae were seen penetrating the tail region (Fig 5A and B), with a few (11%- 15%)

penetrating other parts of the body such as head and eyes (number of cercariae penetrating in

the tail region; A. burminis; 10 days = 52.7%, 20 days = 47.3%, 30 days = 54.6%;

furcocercous; 10 days = 55.3%, 20 days = 46.0%, 30 days = 51.3%; Fig. 5C and D).

Parasite cysts

Most of the cysts were in the tail region of the tadpoles. Only the cysts in the tail region were

counted since they were externally visible under a dissecting microscope. In both cercariae

types, the majority penetrated the tail region. There was no difference in the number of cysts

induced by the two cercariae (one-way ANOVA, F = 0.21, p = 0.662). An increase in the

number of cysts was observed for all treatments until the second day, after which the numbers

decreased (Fig 6). The disappearance of cysts was not age dependent in either of the species

(A. burminis; one-way ANOVA, F = 1.61, p = 0.239; furcocercous; one-way ANOVA, F =

1.55, p =0.228).

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Survival in the resistance experiment

More tadpoles died when they were exposed to cercariae at early stages when compared to

those exposed at later stages (A. burminis; linear mixed effect model; F (2, 22) = 29.16; p =

<0.001, furcocercous; linear mixed effect model; F (2, 22) = 18.18; p = 0.001, Fig 7). Mortality of tadpoles caused by A. burminis was higher than that of furcocercous (A. burminis, 10 days=60.0%, 20 days=73.3%, 30 days=93.3%, furcocercous, 10 days=67.5%, 20 days=76.5%, 30 days=98.6%).

Discussion

This study shows patterns in tolerance and resistance of an larval host’s developmental stages to two types of parasitic trematodes. Effects induced by A. burminis and furcocercous cercariae were severeDraft for the P. cruciger tadpoles exposed at early stages, suggesting an age-dependent tolerance and resistance. In general, our finding conforms with other studies with Echinostoma trivolvis (Cort 1914) and Ribeiroia ondatrae (Looss 1907) where it was shown that young tadpoles at early developmental stages experienced the highest mortality and highest growth reduction (Fried et al. 1997; Schotthoefer et al. 2003;

Holland et al. 2007; Rohr et al. 2010); it has been suggested that relative body sizes between cercariae and hosts have important fitness consequences (Rohr et al. 2010).

The degree of tolerance to parasitism was reflected in the survival of hosts, growth, and development of malformations – growth (SVL, body weight) and developmental period

(TE50) of early stage tadpoles were significantly more affected by parasite exposure compared to those exposed at late stages.

The incidence of malformations was also dependent on the developmental stage of the tadpole at which it was exposed – malformations were more prevalent in early stages

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compared to late stages suggesting a development-dependent tolerance to parasitism by the

tadpoles. The parasite may gain a fitness advantage by making the tadpoles more vulnerable

to predation by the definitive host through malformations, size reduction and increased time

to metamorphosis. This also suggests tadpoles that survive even with malformations facilitate

parasite perpetuation.

Tadpoles exposed to cercariae also showed resistance to parasitism – not all the

cercariae manage to penetrate the tadpoles during the limited exposure time. Even when they

did, some encysted metacercariae were absorbed by the tadpole within two days following

exposure. Resistance to parasitism was also dependent on the development stage in P.

cruciger. The number of cercariae penetrating during a given time period was fewer in older tadpoles compared to the younger tadpoles.Draft This result also agrees with previous studies in other tadpole-trematode systems (Schotthoefer et al. 2003; Holland et al. 2007; Raffel et al.

2010).

Tadpoles also showed anti-parasitic defensive behaviors like rapid swimming,

twisting and wiggling soon after cercariae were introduced to the plastic cups containing

tadpoles. Similar antiparasitic defense behaviors have been observed in tadpoles of D.

melanostictus upon exposure to cercariae (Jayawardena et al. 2013). Despite a greater surface

area available for penetration, the stronger movements of the larger tadpoles seem to

discourage intensity of infection by parasites.

The intensity magnitude of the effects induced by the two parasites on the tolerance

and resistance of host across developmental stages was different. A possible explanation for

these results may be the variations of the response towards different parasite species from the

same host as recently showed by Pathirana and Rajakaruna (2018).

Conclusion:

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Our work facilitated an understanding of host defensive mechanism systems to two types of trematode larvae in an endemic amphibian species in Sri Lanka. The results suggests that tadpoles show a development-dependent tolerance and resistance to parasitism, resulting in greater rates of survival and lower rates of malformation in late stage tadpoles in the presence of parasites. However, additional host and parasite species should be tested to assess whether the effects observe here generalize to other systems.

Acknowledgment

Financial assistance from the National Science Foundation Sri Lanka (Grant No

RG/2014/EB/02) is acknowledged.

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Figure Captions

Fig 1 A) Pleurolophocercous cercariae of Acanthostomum burminis isolated from Melanoides

tuberculata; B) Furcocercous cercariae isolated from Mieniplotia scabra C) Exposure of

tadpoles to cercariae was conducted in small plastic grid plate, where tadpoles were kept

individually.

Fig 2 Percentage survival of Polypedates cruciger tadpoles exposed to pleurolophocercous

cercariae of Acanthostomum burminis and furcocercous cercariae at 10, 20 and 30 post-hatch

(Gosner 27, Gosner 28/29, Gosner 30/31, respectively) in a dose-dependent manner

(control=0, low=16, medium=32, high=48) Note; for an example low (A) =Acanthostomum

burminis in low dose, low (F) =furcocercousDraft in low dose.

Fig 3 Survivorship curves displaying cumulative survival of tadpoles of Polypedates cruciger

from the initial date of exposure to (A) Pleurolophocercous cercariae of Acanthostomum

burminis (B) furcocercous cercariae until the10th day of post exposure. Note; For an

example, 10 days low means 10 days post exposure and low dose (16 cercariae per tadpole)

In A, 30 days post hatch low, 30 days post hatch medium, 30 days post hatch high, and

control have the same cumulative survival with each other (i.e., lines overlap). In B, 30 days

post hatch low and 30 days post hatch high have the same cumulative survival with each

other (i.e., lines overlap), and Control and 30 days post hatch medium also have the same

cumulative survival with each other (i.e., lines overlap). Survival of the control is 100%.

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Fig 4 Infected tadpoles and metamorphs of Polypedates cruciger exposed to cercariae with kyphosis (A, B and C) and scoliosis (D and E) Severely malformed tadpole with difficulties in swimming (F, G) Note; tadpole A; 28 days post hatch with spinal curvature in the ventral plane, malformation represents in a froglet as a curvature of the spine, Froglet E; 115 days post hatch with a lateral curvature of the absorbing tail, the malformation is absent in the adult as the tail disappears.

Fig 5 Penetration of cercariae into the tail region of the Polypedates cruciger tadpole,(A) furcocercous cercariae (B)Pleurolophocercous cercariae of Acanthostomum burminis(C) exposed tadpole with cysts in the tail region (D) Enlarged cyst in the tail region. Draft

Fig 6 Number of cysts in the tail region per Polypedates cruciger tadpole for five consecutive dayspost-exposure in the experimental and control groups. No cysts were observed in the tadpoles in the control group. The experiment was done for the tadpoles of Gosner 27,

Gosner 28/29 and Gosner 30/31 stages Note; Cercariae A denotes pleurolophocercous cercariae of Acanthostomum burminis, Cercariae F denotes furcocercous cercariae, Values were obtained by dividing the number of cysts from the initial number of tadpoles in each subsample (15) collected. Note; the numbers on the plot next to each line are the mean number of cysts ± SD.

Fig 7 Survival of the tadpoles of Polypedates cruciger for five consecutive days post- exposure in the experimental groups. The survival of control was 95% Note; Cercariae A denotes pleurolophocercous cercariae of Acanthostomum burminis, Cercariae F denotes

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furcocercous cercariae. The exposure was done for the tadpoles at Gosner 27, Gosner 28/29

and Gosner 30/31 stages.

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Table 1 Comparisons of malformation types and overall percentage of malformations of Polypedates cruciger tadpoles exposed to pleurolophocercous cercariae of Acanthostomum burminis and furcocercous cercariae at 10, 20 and 30 post-hatch in a dose dependent manner (control=0, low=16, medium=32, high=48) at 40 days post hatch stage (Gosner stage 31/32) among age groups (10 days post hatch, 20 days post hatch, 30 days post hatch)

Treatment with Acanthostomum burminis Treatment with furcocercous cercariae

Cumulative survival Cumulative Treatment Cercariae dose 10 days 20 days 30 days 10 days 20 days 30 days post-hatch post-hatch post-hatch post-hatch post-hatch post-hatch

Malformation Type Scoliosis Scoliosis Scoliosis Scoliosis Scoliosis Scoliosis Kyphosis Kyphosis Kyphosis Kyphosis Kyphosis Kyphosis Low 15 3 13 6 5 4 11 10 14 7 5 3 Medium 9 6 9 5 3 2 10 8 10 9 3 4 High 6 1 6 8 Draft 3 3 5 1 8 3 4 1 Overall percentage of malformations (%) 95.3 78.8 17.0 95.7 78.4 23.2 Comparison with 10 days post hatch χ2 - 11.317 123.458 - 14.324 110.572 P - <0.001* <0.001* - <0.001* <0.001* Comparison with 20 days post hatch χ2 - - 77.003 - - 60.506 P - - <0.001* - - <0.001*

Note; The overall percentages of malformations were taken by dividing the number of malformed individuals from the live individuals in a treatment exposed to a given cercariae dose. Note; *denotes the significant differences at p < 0.001 analyzed using Chi-Square Test.

Days (from exposure to metamorphosis) https://mc06.manuscriptcentral.com/cjz-pubs

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Table 2 Comparisons of the number of cercariae penetrated into Polypedates cruciger tadpoles at 10, 20 and 30 post-hatch exposed to pleurolophocercous cercariae of Acanthostomum burminis and furcocercous cercariae within one-hour period of time

Tadpoles exposed to Tadpoles exposed to Acanthostomum burminis furcocercous cercariae No. of cercariae 10 days 20 days 30 days 10 days 20 days post 30 days post penetrated post-hatch post-hatch post-hatch post hatch hatch hatch 1 1 0 4 2 1 5 2 0 2 6 1 2 4 3 1 1 10 1 2 7 4 1 3 7 3 5 9 5 4 11 10 4 9 9 6 2 4 7 2 6 7 7 2 3 5 3 5 6 8 8 3 5 Draft7 4 4 9 11 9 7 12 7 8 *10 50 44 19 45 39 21 Total No. of cercariae 717±2.06 666±2.42 494±3.01 691±2.33 652±2.60 543±3.00 penetrated F 24.46 13.27 p <0.001† <0.001‡ Note *denotes the sub sample (15 from each group) of tadpoles taken to count the parasitic cysts over five consecutive days, † denotes 10 days vs 20 days p=0.250; 10 days vs 30 days and 20 days vs 30 days p < 0.001, ‡ denotes 10 days vs 20 days p=0.312; 10 days vs 30 days and 20 days vs 30 days p < 0.001 analyzed using One way ANOVA and Tukey Pair-wise Comparisons test for individual comparisons, the rest were not significant

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