One prediction of optimal digestion theory is that organisms will increase the relative length of their digestive tracts when food resources become limited. We used theory of optimal digestion to test whether tadpoles can adjust the relative length of their intestines when challenged with the fungal pathogen Batrachochytrium dendrobatidis (Bd). The degree of tadpole mouthpart damage, a symptom of Bd infections that reduces food consumption, was associated positively with the length of tadpole intestines relative to their body size, consistent with optimal digestion theory. After controlling for mouthpart damage, tadpoles exposed to Bd had shorter intestines relative to their body size, opposite to the predictions of optimal digestion theory. One explanation of why tadpoles with higher Bd loads have shorter relative intestinal lengths is that they divert energy from maintaining intestinal and overall growth towards anti-parasite defences.
Defences against parasites can use a substantial amount of an organism's energy budget . To meet the increased energy demands associated with parasite resistance, some host species can offset the costs of resistance by choosing diets that optimize their immune defences . However, if hosts do not have access to high quantity or quality food, they might use alternative strategies to meet the high costs associated with anti-parasite defences.
One such way for an organism to improve energy intake without increasing food consumption is to increase its digestive efficiency by increasing the relative length of its intestine. Optimal digestion theory predicts that digestive tract length should be inversely correlated with food consumption (or food quality), because longer intestines can improve digestive efficiency by increasing food transit time . Although optimal digestion theory has traditionally been used to predict phenotypic changes in digestive tract length as a function of changes in food quantity/quality (reviewed in ), there is increasing interest surrounding other ecological contexts that result in intestinal plasticity. For example, Relyea & Auld  combined theory on optimal digestion and phenotypic plasticity to show that tadpoles raised with a high density of competitors developed long intestines relative to their body size, whereas tadpoles raised in predator environments developed relatively short intestines.
We conducted a laboratory study using southern leopard frog (Lithobates sphenocephalus) tadpoles and tested whether tadpoles can adjust the relative length of their intestine when challenged with the pathogenic chytrid fungus (Batrachochytrium dendrobatidis; hereafter ‘Bd’). Bd infects the keratinized mouthparts of tadpoles and results in tooth row loss , and reduced feeding efficiency  and growth . Thus, changes in intestinal length relative to body size could be influenced by a number of mechanisms, such as reduced food intake from mouthpart damage or from trade-offs with anti-parasite defences. We used path analyses to test how Bd exposure/infection, mouthpart damage and tadpole size influence relative intestinal length. Borrowing from theory on optimal digestion, we predicted that, when controlling for mouthpart damage, tadpoles that were exposed to Bd or those that were infected but relatively resistant to Bd (i.e. those with lower Bd abundance) would have long intestines relative to their body size to meet the energetic demands of Bd resistance. We also predicted that tadpoles with more mouthpart damage, a pathology that reduces food consumption, would be positively associated with intestinal length relative to body size.
2. Material and methods
(a) Bd inoculation and tadpole husbandry
Bd was maintained according to standard protocols . We placed 28 southern leopard frog (L. sphenocephalus) tadpoles of similar developmental stages in 75 ml water baths and haphazardly assigned them to receive Bd or not. Tadpoles in the Bd-exposed treatment were inoculated with 1.1 × 107 Bd zoospores. We then re-exposed the same tadpoles to 4.5 × 106 zoospores 72 h after the first exposure. Tadpoles from the non-exposed treatment were given a similar volume inoculate without Bd zoospores. Twenty-four hours after the final inoculation, we transferred the tadpoles to plastic containers filled with 1 l of aged tap water and raised in the laboratory at 19°C. Tadpoles were fed every 3 days, and we changed the water in each container once per week. Tadpoles were euthanized 46 days after the second dose of Bd. See the electronic supplementary material for more details on tadpole husbandry.
(b) Morphological traits
We measured the size (snout–vent length; hereafter ‘SVL’) of each tadpole with digital callipers (to the nearest 0.1 mm) after euthanasia. We then dissected the mouthparts and the alimentary tract (excluding the foregut and the colon; hereafter ‘intestine’) of each tadpole. We straightened the intestine on a dissection pan and measured the length of each intestine with digital callipers (to the nearest 0.1 mm). Last, for each tadpole, we calculated a mouthpart deformation score, which was adapted from Venesky et al. , based on the percentage of teeth missing from each side (left and right) of the five labial tooth rows. See the electronic supplementary material for more details on the deformation score.
(c) Quantitative PCR analysis
We used quantitative PCR (qPCR) to quantify the amount of Bd on the mouthparts of each tadpole. Our DNA extractions and qPCR analysis followed the methods of Boyle et al.  and Hyatt et al. . DNA extractions were diluted 1 : 100 and processed in triplicate in a step-one real-time PCR system (Applied Biosystems).
(d) Statistical analyses
We used path analysis with maximum-likelihood estimation to examine the relationship between Bd, mouthpart damage, tadpole size (SVL) and relative intestinal length (by controlling for differences in SVL on changes in intestinal length). We ran three separate path analyses that differed only in whether we treated Bd as a categorical predictor (exposed/not exposed) or as a continuous predictor (abundance; the amount of Bd in infected and non-infected tadpoles). When considering Bd abundance as a predictor, we ran two path models; the first included the Bd abundance of all tadpoles (exposed and non-exposed) and second included only the tadpoles exposed to Bd. The results from these models were consistent with each other (table 1), and we thus refer to the model using the categorical predictor Bd exposure in the main text.
We first tested the hypothesis that SVL and mouthpart damage were influenced by Bd exposure. We predicted that tadpoles exposed to Bd would be smaller and would have more mouthpart damage compared with non-infected tadpoles. We then tested the hypothesis that relative intestinal length is influenced by Bd exposure. Our model included both direct (e.g. Bd exposure to intestinal length) and indirect (e.g. Bd exposure to mouthpart damage to intestinal length) pathways by which Bd could influence relative intestinal length. If defences towards Bd infection use energy resources, then the path from Bd exposure to intestinal length should be significant and have a positive coefficient. Additionally, if the relative length of the intestine is influenced by mouthpart deformities , then the path to intestinal length from mouthpart damage should be significant and have positive coefficients.
All path analyses were conducted using the ‘lavaan’ package in R. We assessed the relative strength of each path by comparing their standardized coefficients, where higher absolute values indicate a more parsimonious path; indirect paths in our model were determined by multiplying the standardized coefficients . We used two-tailed tests to evaluate significance in all paths with the exception of the path from Bd exposure to mouthpart damage (because Bd infections can only reduce the number of teeth, not increase them).
Tadpoles exposed to Bd were smaller (p < 0.001) and tended to have more mouthpart damage (p = 0.059) compared with non-exposed tadpoles (see figure 1 and electronic supplementary material). Mouthpart damage, however, was not a significant predictor of tadpole size (p = 0.130). Contrary to our predictions, Bd exposure was a significant negative predictor of intestinal length relative to body size (p = 0.014; figure 1). Bd abundance was also a significant negative predictor of intestinal length relative to body size (see table 1 and electronic supplementary material, figures S1 and S2) and the relative intestinal length of tadpoles that were Bd+ was approxiamately 33 per cent shorter than those that were resistant to Bd (i.e. exposed but not infected). However, the relative intestinal length of resistant tadpoles did not differ from non-exposed tadpoles, suggesting that tadpoles do not increase the relative length of their intestines to meet the energetic demands of Bd resistance. Instead, tadpoles with high Bd abundance had shorter intestines relative to their body size than tadpoles with low or no Bd (electronic supplementary material, figure S3).
The direct path between Bd exposure and intestinal length was the strongest path in our model (path coefficient =−0.407), followed by the indirect paths from Bd abundance to SVL to intestinal length (path coefficient =−0.243), and Bd abundance to mouthpart damage to intestinal length (path coefficient = 0.092). Our results were consistent with each other regardless of the predictor that we used (table 1).
Our results show that Bd-infected tadpoles of L. sphenocephalus were significantly smaller and tended to have more mouthpart deformities compared with non-infected tadpoles, consistent with previous findings [8,12]. Additionally, we found that tadpoles can adjust the length of their intestines relative to their body size. Even after controlling for the effects of Bd exposure and differences in body size, tadpoles with a higher proportion of mouthpart deformities had significantly longer intestines (figure 1). This anatomic change probably occurs as a response to reduced food intake, because tadpoles with more mouthpart deformities, even in the absence of Bd infection, cannot feed as effectively as tadpoles with fewer missing teeth . This increase in intestinal length relative to their body size is probably a plastic response to increase nutrient absorption and maintain or increase growth rates, consistent with the predictions from optimal digestion theory .
Based on optimal digestion theory, one might also predict that tadpoles exposed to Bd should develop a longer intestine relative to their body size to increase nutrient absorption to meet the energetic costs of increased resistance. Following that logic, tadpoles that are the most resistant (i.e. tadpoles exposed to Bd with zero or low Bd abundance) should have longer relative gut length compared with non-exposed tadpoles. Contrary to our predictions, we found that, even after controlling for mouthpart damage, tadpoles exposed to Bd had relatively shorter intestines than non-exposed tadpoles. Moreover, tadpoles with higher Bd abundance (i.e. those that were less resistant) had relatively shorter intestines than tadpoles with low Bd abundance (see the electronic supplementary material, figures S1 and S2), and the relative gut length of tadpoles that were most resistant did not differ from those not exposed to Bd (see the electronic supplementary material, figure S3). These results, coupled with the fact that the strongest path in our path analyses was always the direct path from Bd to intestinal length, does not support the hypothesis that optimal digestion theory predicts the effects of Bd on intestinal plasticity; instead, our results suggest that the phenotypic change might be pathogen-induced.
In conclusion, our results suggest that tadpoles are capable of increasing the relative length of their intestines when resources are limited, presumably to increase digestive efficiency. Opposite to the prediction from optimal digestion theory, when tadpoles are challenged with Bd, the relative lengths of their intestines decrease. Bd infections can reduce tadpole food consumption  and tadpoles that are resource limited have reduced defences against Bd . Thus, one explanation for our results is that tadpoles divert energy resources from maintaining intestinal and overall growth towards deploying anti-parasite defences (e.g. antimicrobial peptide secretions; reviewed in Rollins-Smith et al. ), similar to the patterns observed by Relyea & Auld , who found that predator-induced tadpoles had shorter intestines relative to their body size. Alternatively, Bd might somehow directly reduce the relative intestinal length of tadpoles. Future research using optimal digestion theory and phenotypic plasticity are needed to fully understand the costs associated with parasite resistance.
We thank members of the Rohr Laboratory, L. Martin, R. Wassersug and T. Wilcoxen for comments on earlier drafts of this manuscript.
- Received January 14, 2013.
- Accepted February 5, 2013.
- © 2013 The Author(s) Published by the Royal Society. All rights reserved.