Melanocortin 4 receptor mutations contribute to the adaptation of cavefish to nutrient-poor conditions

Abstract

Despite recent advances in the understanding of morphological evolution, the genetic underpinnings of behavioral and physiological evolution remain largely unknown. Here, we study the metabolic changes that evolved in independently derived populations of the Mexican cavefish, Astyanax mexicanus. A hallmark of cave environments is scarcity of food. Cavefish populations rely almost entirely on sporadic food input from outside of the caves. To survive under these conditions, cavefish have evolved a range of adaptations, including starvation resistance and binge eating when food becomes available. The use of these adaptive strategies differs among independently derived cave populations. Although all cavefish populations tested lose weight more slowly than their surface conspecifics during restricted rations, only a subset of cavefish populations consume more food than their surface counterparts. A candidate gene-based screen led to the identification of coding mutations in conserved residues of the melanocortin 4 receptor (MC4R) gene, contributing to the insatiable appetite found in some populations of cavefish. Intriguingly, one of the mutated residues has been shown to be linked to obesity in humans. We demonstrate that the allele results in both reduced maximal response and reduced basal activity of the receptor in vitro. We further validate in vivo that the mutated allele contributes to elevated appetite, growth, and starvation resistance. The allele appears to be fixed in cave populations in which the overeating phenotype is present. The presence of the same allele in multiple caves appears to be due to selection from standing genetic variation present in surface populations.

Keywords: Astyanax mexicanus; MC4R; cavefish; hyperphagia; metabolic evolution.

Fig. 1.

Cavefish are adapted to nutrient-poor environments. (A) Percentage of weight loss after a 2-mo fast. Eleven to 13 fish per population were used. Each cave population was significantly different from the surface population. ***P < 0.0001 (Tukey HSD test). (B) Total triglyceride content/protein in adult (>1-y-old) fish. Each population was significantly different from one another. *P < 0.05 (Tukey HSD test). (C) Pictomicrograph of livers. (Scale bars: 2 mm.) (D) Oil red O staining of liver sections. (Scale bars: 250 μm.) Red symbols (Pachón, Tinaja, and Molino) represent cavefish, and blue symbols represent surface fish. M, Molino; P, Pachón; S, surface; T, Tinaja.

Fig. 2.

Differential appetite regulation. (A) Appetite comparison between fed and starved surface and cavefish (3 wk). Six fish were used for each condition, and population combination and the amount of worms consumed were recorded over a 24-h assay period. (B) Appetite comparison among fed Tinaja, Pachón, surface, and Tinaja/surface F1 hybrids. Twelve fish were used for each population and tested over a 36-h assay period. (C) Appetite comparison with fed Tinaja, Pachón, surface, and Pachón/Tinaja F1 hybrids. Eight fish were used for each population and tested over a 48-h period. Red symbols (Pachón, Tinaja, and Molino) represent cavefish, and blue symbols represent surface fish. P/T, Pachón/Tinaja F1 hybrid; T/s, Tinaja/surface F1 hybrid. *P < 0.05 (Tukey HSD test). K = condition factor (100 g⋅cm⁻³, cm = body length from nose to base of tail).

Fig. 3.

Hypomorphic mutations in MC4R cosegregate with hyperphagia. (A) Protein sequences of A. mexicanus MC4R [adapted after Tao (18)] shown in red are Tinaja-specific mutations. (B) Dose–response curve of Tinaja and surface alleles in vitro using 293T cells. Tinaja cavefish have a lower maximal response and basal activity than surface fish (P < 0.05). Each data point represents the mean of three wells being independently transfected and measured. (C) Appetite comparison between Pachón cavefish crosses carrying either the surface or cave allele of MC4R. C, Tinaja cave allele; S, surface allele. Twenty-two homozygous fish and 38 heterozygous fish (3-mo-old) were used, and their appetite was assayed over a 1-wk assay period. **P < 0.005 (two-tailed t test). (D) Body length comparison among Pachón cavefish MC4R genotypes. The same fish shown as in Fig. 3C, but 6 mo older. At the end of the experiment, the fish were 9 mo old. (E) Percentage of weight loss comparison among Pachón cavefish MC4R genotypes during a 3-wk fast. Same fish as in Fig. 3D were used, but they were starved after measuring length. *P < 0.05 (two-tailed t test).

Fig. S1.

Conservation of the amino acids affected by coding changes in MC4R within A. mexicanus populations among other vertebrate genomes, including the mutated allele in a case of human obesity (20). Red asterisks highlight the residue changes in the Tinaja allele.

Fig. 4.

Parallelism of hyperphagia in Astyanax mexicanus. (A) Geographic distribution of different cave populations. (B) Appetite comparison between fed young (<1-y-old) surface fish and another independently derived cave population (Molino) carrying a derived MC4R allele over a 72-h period. ***P < 0.001 (two-tailed t test).

Fig. S2.

(A) Comparison of appetite between fed old (>1-y-old) and young (<1-y-old) Tinaja fish over a 72-h period. ***P < 0.0005 (two-tailed t test). (B) Appetite comparison among MC4R genotypes of older surface/Tinaja F2 fish over a 5-d period. ns, not significant ANOVA.

Fig. S3.

(A) Liver oil red O staining of fed Pachón (>1-y-old fish) (Scale bars: 250 μm.) (B) Comparisons of body length of (>1-y-old) Pachón fish among MC4R genotypes. *P < 0.05. (C) Appetite comparison among MC4R genotypes in old (>1-y old) fed Pachón fish during a 2-wk period. MC4R genotypes have an age-dependent association with appetite, with older fish (>1-y-old) not showing a difference in appetite among genotypes.