Conceived and designed the experiments: SMB JV BW. Performed the experiments: SMB JV. Analyzed the data: SMB. Wrote the paper: SMB JV BW.
Current address: Department of Biology, Emory University, Atlanta, Georgia, United States of America
Current address: Department of Biological Sciences, Florida International University, Miami, Florida, United States of America
Current address: Bio-Protection and Ecology Division, Lincoln University, Canterbury, New Zealand
The authors have declared that no competing interests exist.
Given their well-developed systems of innate and adaptive immunity, global population declines of amphibians are particularly perplexing. To investigate the role of the major histocompatibilty complex (MHC) in conferring pathogen resistance, we challenged
The major histocompatibility complex (MHC) encodes cellular mechanisms that determine immunological self/non-self recognition in vertebrates. Genetic relatives share MHC alleles, which encode T-cell repertoires, so their immune systems should recognize similar arrays of pathogens. Because MHC alleles are codominant, individuals that are heterozygous at the MHC should have a larger immunological repertoire than homozygotes
Unlike those of many other vertebrates, African clawed frog (
Amphibian populations have been declining worldwide, and pathogens may be responsible for many population declines
We examined whether MHC genotype affected the survival and growth of
We bred
Between 13:00 and 15:00 on the day of breeding, we isolated and primed females by injecting their dorsal lymph sac with 0.03 mg luteinizing hormone–releasing hormone (LH-RH; Argent Chemical Laboratories, Redmond, Washington, USA) dissolved in 150 µL of autoclaved double distilled water. We monitored the cloacae of the frogs from 5 to 8 h after priming. Once cloacae displayed swelling and redness due to increased blood flow, we injected the females with an additional 0.1 mg LH-RH dissolved in 500 µL of autoclaved double distilled water, and immediately placed them into breeding tanks. To ensure that the breeding pair would not consume the eggs, we covered the substrate of breeding tanks with a plastic mesh grid which allowed fertilized eggs to fall through to the bottom. For breeding frogs and rearing tadpoles, we used aerated, carbon-filtered Christchurch city municipal water, which is sourced from deep-water aquifers without chemical treatment.
We isolated a strain of
We bred 3 male and 3 female MHC-homozygous (
Brood | Parental MHC types | Tadpole MHC type | Exposure (cfu/ml) | Control (heat-killed bacteria) | |||
♀ | ♂ | 1.0×106 | 2.5×106 | 3.0×106 | |||
1 (early) | 8 | 8 | 8 | 8 | |||
8 | 8 | 8 | 8 | ||||
8 | 8 | 8 | 8 | ||||
2 (early) | 8 | 8 | 8 | 8 | |||
8 | 8 | 8 | 8 | ||||
8 | 8 | 8 | 8 | ||||
3 (late) | 8 | 8 | 8 | 8 | |||
8 | 8 | 8 | 8 | ||||
8 | 8 | 8 | 8 | ||||
4 (late) | 8 | 8 | 8 | 8 | |||
8 | 8 | 8 | 8 | ||||
8 | 8 | 8 | 8 |
To obtain baseline size measurements of the tadpoles prior to inoculating them with bacteria, we randomly selected 32 tadpoles from each clutch, 16 days after hatching. We photographed each in its own Petri dish from 60 cm directly above with a Nikon Coolpix 4500 digital camera. A 10 cm ruler was included in the photographs for scale. We measured body length (BL, from the tip of the snout to the vent at the base of the tail) and total length (TL, from the tip of the head to the tip of the tail) from digital images using NIH ImageJ 1.3 (National Institutes of Health, Bethesda, Maryland, USA). We then placed each tadpole into an individual 1 L polypropylene beaker (day 0).
We exposed tadpoles by pipetting an inoculum of
We first compared Kaplan-Meier survival curves with log rank tests using the survdiff procedure in R 2.3.0 (R Development Core Team, Vienna, Austria). The survival curves allow inspection of gross differences in survival over time. We then analyzed how total mortality at day 35 was affected by MHC genotype, bacterial dose, clutch (early and late), and block with a generalized linear mixed model (GLMM) using the glmmML package in R (Göran Broström, Department of Statistics, Umeå University) with binomial error distribution and logit-link function. The glmmML package fits models using maximum likelihood estimation. We treated genotype, bacterial dose, clutch and block as fixed variables, and subject (individual identity) as a random variable. We included starting body length as a covariate. We compared body and total lengths associated with the same fixed factors using repeated-measures ANOVA. We compared the lengths of control tadpoles to those of tadpoles exposed to bacteria by orthogonal contrasts. All repeated measures analyses were conducted with Statistica 6.1 (Statsoft, Tulsa, Oklahoma, USA) using Type III sums of squares.
Despite using half-siblings to limit the heritable effects of non-MHC genes, these genes still may have had effects on disease resistance. To control for non-MHC variation, we conducted within-family tests in an additional experiment, as follows.
We crossed three pairs of MHC-identical heterozygous frogs (
Cross | Genotype | Exposed | Control (heat-killed bacteria) | Control (clean media) |
10 | 10 | - | ||
10 | 10 | 11 | ||
10 | 10 | - | ||
14 | 14 | - | ||
16 | 16 | 16 | ||
16 | 16 | - | ||
15 | 15 | - | ||
16 | 16 | 16 | ||
13 | 13 | - |
Two weeks after hatching, we genotyped 150 tadpoles from each clutch for MHC type
The numbers of each genotype that were produced in the spawn limited the sample sizes (
The initial dose of bacteria failed to induce mortality so we increased the exposure dose. On day 5, we exposed the tadpoles to 4.0×107 cfu/ml of
We compared Kaplan-Meier survival curves with log rank tests using the survdiff procedure as before. We analyzed how mortality, at the end of the experiment (day 28), was affected by MHC genotype, family nested within genotype, and bacterial exposure using a generalized linear mixed model (GLMM, R 2.3.0) with binomial error distribution and logit-link function. Family, corresponding to the breeding regimen (
Tadpole mortality was affected by exposure to
(A) Percent mortality of tadpoles exposed to the control (3.0×106 cfu/ml heat-killed), low (1.0×106 cfu/ml), medium (2.5×106 cfu/ml), and high (3.0×106 cfu/ml) doses of
Kaplan-Meier plots showing the survival of (A) tadpoles exposed to the control (3.0×106 cfu/ml heat-killed), low (1.0×106 cfu/ml), medium (2.5×106 cfu/ml), and high (3.0×106 cfu/ml) doses of
Some MHC genotypes suffered less mortality than others (
Tadpoles from earlier clutches were more likely to die (8.9%) than their full siblings from later clutches (3.4%;
Tadpoles significantly differed in length as a function of their MHC genotype (
Body length (
Tadpoles that developed from clutches laid earlier in the evening were significantly smaller than those laid later in the same evening. On day 25, tadpoles from the later clutches were 4% larger than tadpoles from earlier clutches (BL: early 8.61±0.07 mm, late 8.81±0.07 mm,
Control tadpoles were significantly larger (TL: 22.36±0.34 mm) than all those exposed (21.19±0.22 mm) at day 25 (BL:
Tadpoles died in higher numbers when exposed to live rather than heat-killed
(A) Percent mortality of tadpoles exposed to live (exposed) and heat-killed (control)
Kaplan-Meier plots showing the survival of (A) tadpoles exposed to live (exposed) or heat-killed (control)
Surviving tadpoles that had been exposed to live
Body length (
(A) Total and (B) body length (
We have shown (1) that exposure of
In both experiments,
Differences in resistance conferred by MHC alleles have been documented in many vertebrates including fishes
Immune responses protect individuals against pathogens and parasites, but can incur fitness costs
Unlike in the first experiment, however, almost all genotypes in the second experiment grew more rapidly when exposed to the pathogen. This difference in response to the bacterial challenge might be due to our isolation of subjects into beakers earlier in the second experiment, which was necessary to genotype individuals. Consequently, subjects' growth, and probably their development, accelerated to a point at which they may have been less susceptible to the pathogen
Although exposed tadpoles in the second experiment grew larger, resistant tadpoles in the first experiment appear to have allocated less of their energy resources toward growth than did susceptible tadpoles. MHC class II molecules initiate immune responses to extracellular pathogens such as bacteria, and these class II molecules are expressed in high concentrations in the intestines of
Risk of infection likely depends on the MHC and kinship composition of schooling tadpoles, pathogen pressure, and developmental stage. Association preferences appear to be labile in terms of MHC and kin composition within a school
Tadpoles that developed from eggs that had been laid earlier in the evening were smaller and more likely to die than those from the same parents that had been deposited later in the evening. The ecological significance of ovum size variability in growth and survival in
Although our results suggest that differential susceptibility to the pathogen reflects genetic variation in resistance conferred by different MHC alleles, we did not assay pathogen load. Differences in growth and survival may have resulted from variation in tolerance of pathogen load rather than resistance to infection
Despite having a comprehensive system of innate immunity that includes an extensive and exceptionally effective suite of antimicrobial peptides present in the skin
We have presented evidence for specific MHC haplotype-based resistance to, or tolerance of, a common, if opportunistic, amphibian pathogen. Knowledge of specific resistances conferred by different genotypes may be critical to the success of captive rearing programs
We thank Louis Du Pasquier for supplying us with the frogs that he selectively bred over many generations at the Basel Institute for Immunology; Nicole Gerardo, Marie Hale, Koji Mochida, and Akira Mori for reading drafts of the manuscript; Dave Kelly, Koji Mochida, and Daisuki Muramatsu for statistical advice; Rebecca McCurdy, Tia Neha, and Toby Win for their assistance; and two anonymous referees whose comments greatly improved the paper. All protocols involving animals were approved by the University of Canterbury Animal Ethics Committee.