Introduction
Melanic pigmentation is widespread in nature, and plays a crucial role in
species fitness. The eastern mosquitofish (Gambusia holbrooki) melanic male morph is found in natural populations at frequencies typically maintained below 0.05 of the male population (Horth and Travis 2002; Horth et al 2010). A single albino western mosquitofish (Gambusia affinis) was found in nature and bred in captivity. To elucidate the genetic mechanisms that contribute to these two very rare phenotypes the genes encoding the melanocortin-1 receptor (MC1R) and enzyme tyrosinase (TYR) were analyzed for nucleotide sequence variation and gene expression differences (Horth 2006). Sequence analyses of both genes revealed non-synonymous mutations in melanics and albinos as compared to the wild-types. Expression of both genes was higher in melanics compared to wild-types, and lower in albinos for both genes. These results demonstrate a correlation between gene expression differences and unique genotypes in the melanin biosynthesis pathway.
Results
� We analyzed the nucleotide sequence for 45 mosquitofish, 30 silver
and 15 black-spotted, from 9 different populations for MC1R and TYR in both morphs of G. holbrooki. Analyses of MC1R open reading frame (ORF) revealed synonymous heterozygous single nucleotide polymorphisms (SNPs) in melanics and wild-types at pos.9 A>T and pos.53 A>T. The following synonymous SNPs were found in 4 melanic males from Alligator Point (Franklin Co., FL) and McBride slough (Wakulla Co., FL): pos.488 C>T, pos.548 G>A, and pos. 614 G>A. Analysis of the MC1R gene amplified from cDNA revealed a non-synonymous SNP (pos.426 T>C) that resulted in an amino acid change Ile142Thr in a melanic male from Miami.
When compared with wild-type G. affinis and G. holbrooki males,
there was a point mutation (pos.677 A>T) in albino G. affinis males that resulted in an amino substitution at a potential phosphorylation site in one of the intracellular loops of MC1R. Quantitative PCR assays for MC1R expression revealed increased expression of MC1R in melanic G. holbrooki compared with wild-types, showing a 3-4 fold increase. Albino G. affinis demonstrated an expression level of MC1R 4-5 fold less than the wild-type.
Quantitative PCR assays for TYR expression revealed an average
3-4 fold increase in melanic G. holbrooki males when compared to wild-type males. Of the 15 melanic males tested, 7 were from the Miami Everglades, 7 were from Newport Springs, and 1 was from Lake Panasoffkee. Within melanic fish, TYR expression was higher in individuals that produced more melanin and displayed darker phenotypes. Albino G. affinis showed lower expression levels of TYR by 1-2 fold compared with G. affinis and G. holbrooki wild-types. There was no variation in expression among 4 albino individuals from the same mutant line.
Hampus Engstroem and Lisa Horth
Department of Biological Sciences
Old Dominion University, Norfolk, Virginia
Point Mutations That Drive Pigmentation Differences in Mosquitofish
Figure 1. (a) The rare, melanic (mottled-black) eastern mosquitofish (Gambusia holbrooki) genotype.
(b) The rare albino western mosquitofish (Gambusia affinis).
(c) The common, silvery-gray eastern mosquitofish genotype.
Materials and methods
Several mosquitofish were collected and stored at -80°C. Using the Qiagen DNeasy Blood & Tissue kit genomic DNA were isolated from the fish. The MC1R gene was amplified using primers XiphMC1R-F1 (5′ATGGATTTCAACAACAGCTCGCTGC-3’) and XiphMC1R-R1 (5’-CTACATGCCAAAGCACCAGGAG-3’) under the following PCR conditions: 94 °C for 2 min. followed by 40 cycles of 94 °C or 30 sec., 54 °C or 30 sec., 72 °C for 1 min., and a final extension at 72 °C for 7 min. PCR fragments were visualized on a SYBR Green-stained aragose gel, extracted and purified using the QIAquick Gel Extraction Kit (Qiagen). Purified PCR products were cloned into the pGEM-T Easy Vector (Promega). Cycle sequencing was performed using the BigDye Terminatior v3.1 Cycle Sequencing Kit (Applied Biosystems) and M13 primer. DNA sequences were manually corrected and aligned using Sequencher 4.8 (Gene Codes).
Conclusions
The melanic phenotype exhibited in a very small percentage
male G. holbrooki may be linked to a mutation in the mRNA transcript as a result of splicing variants. The Ile142Thr variant in melanics would be positioned in the second transmembrane (TM2) region of the receptor when compared to the predicted protein sequence of a closely related fish, the southern platyfish Xiphophorus maculatus. The reduced expression of MC1R in albinos may be related to an amino acid substitution (Thr226Ser) located at a potential phosphorylation site on the third intracellular loop (see Fig. 3). The third intracellular loop has been demonstrated to be important in G-protein coupling and receptor function, with mutations in this loop in a marked decrease in the functional response of MC1R.
Literature cited
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(b)
(c)
Figure 2. Chromatogram showing a mutation site in the MC1R gene of albino G. affinis.
Figure 3. The predicted protein structure of MC1R in Xiphophorus maculatus. The arrow points to a threonine residue thought to be a possible site for MC1R phosphorylation. This residue is replaced by a serine in albino G. affinis, which may play a role in decreased or no receptor function.
Horth, L. & J. Travis. 2002. Frequency-dependent numerical dynamics in mosquitofish. Proceedings of the Royal Society. 269: 2239-2247.
Horth, L., Binckley, C. Wilk, R., Reddy, P., & Reddy, A. 2010 Color, body-size and genitalia size are correlated traits in eastern mosquitofish. Copeia 2010 (2): 196-202
Horth, L. 2006. A sex-linked allele, autosomal modifiers and temperature-dependence appear to regulate melanism in male mosquitofish. Journal Experimental Biology. 209: 4938-4945.
Acknowledgements
Thanks to National Science Foundation (DEB 1113004 to Lisa Horth)
Jeffress Memorial Trust
John Hicks