Finn RN et al. (2015), Insect glycerol transporters evolved by functio...

XB-ART-53294

Nat Commun 2015 Jul 17;6:7814. doi: 10.1038/ncomms8814.

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Insect glycerol transporters evolved by functional co-option and gene replacement.

Finn RN , Chauvigné F , Stavang JA , Belles X , Cerdà J .

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Transmembrane glycerol transport is typically facilitated by aquaglyceroporins in Prokaryota and Eukaryota. In holometabolan insects however, aquaglyceroporins are absent, yet several species possess polyol permeable aquaporins. It thus remains unknown how glycerol transport evolved in the Holometabola. By combining phylogenetic and functional studies, here we show that a more efficient form of glycerol transporter related to the water-selective channel AQP4 specifically evolved and multiplied in the insect lineage, resulting in the replacement of the ancestral branch of aquaglyceroporins in holometabolan insects. To recapitulate this evolutionary process, we generate specific mutants in distantly related insect aquaporins and human AQP4 and show that a single mutation in the selectivity filter converted a water-selective channel into a glycerol transporter at the root of the crown clade of hexapod insects. Integration of phanerozoic climate models suggests that these events were associated with the emergence of complete metamorphosis and the unparalleled radiation of insects.

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Species referenced: Xenopus laevis
Genes referenced: aqp1 aqp4 ncoa6

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	Figure 1. Molecular phylogeny of arthropod orthodox aquaporins.The tree was inferred through maximum likelihood (3,002,760 heuristic rearrangements) and Bayesian analysis (15 million MCMC generations) of 219,583 nucleotide sites in a codon alignment of 269 non-redundant aquaporins, and was rooted with aqpM. The number of taxa in collapsed (triangular) nodes are indicated, with coloured circles at each node indicating posterior probabilites as defined by the key. Scale bar represents the rate of nucleotide substitution per site. Dotted lines and insect images illustrate the conducted experiments.
	Figure 2. Permeation competence of insect glycerol transporters.(a) Osmotic water permeability (Pf) of X. laevis oocytes injected with water (controls) or expressing the cockroach water channel (BgAqp), a human body louse aquaglyceroporin (PhGlp) and the sleeping chironomid Eglp (PvAqp2), in relation to the cRNA dose injected of each aquaporin. (b) Glycerol and (c) urea uptake by oocytes injected with water, or 15 ng of BgAqp, PhGlp or PvAqp2. Human AQP1 and -3 were respectively used as negative and positive controls. Data (mean±s.e.m.) are from three separate experiments (6–10 oocytes per group in each experiment). P<0.05, *P<0.01 versus water-injected controls (one-way analysis of variance).
	Figure 3. Effect of a single mutation in the ar/R selectivity filter of AQP4-related aquaporins on glycerol and urea permeability.(a) Bayesian inference (5 million MCMC generations) of 202,071 nucleotide sites and 65,196 amino acid sites of 247 non-redundant hexapod and deuterostome aquaporins. The tree is rooted with aqpM. Posterior probabilities resulting from analyses of the codon/amino acid alignments are shown at each node, with the scale bar indicating the rate of substitutions per site. (b) Extracellular view (cartoon render) of HsAQP4 (3GD8) illustrating the ar/R selectivity filter (spacefill). (c, e and g) The ar/R models of wild-type (WT) PvAqp2 (Eglp) and PvAqp2-A174H mutant (c), BgAqp-WT and BgAqp-H197S (e), and HsAQP4-WT, HsAQP4-H201A and HsAQP4-H201S (g), illustrating the effects of the mutations on the narrowing of the channel pore in silico. (d, f and h) Osmotic water permeability (Pf) and solute uptake of Xenopus laevis oocytes expressing the WT and mutant aquaporins indicated in c, e and g. Data (mean±s.e.m.) are from three separate experiments (6–10 oocytes per group in each experiment). P<0.05, *P<0.01 versus water-injected controls (one-way analysis of variance).
	Figure 4. Emergence and extinction of insect glycerol transporters.Schematic overview of aquaglyceroporin (Glp) and entomoglyceroporin (Eglp) evolution in relation to the divergence times of Arthropoda, with multiple terminal branches representing the genomic copy number. Entwined blue and magenta lines indicate incomplete lineage separation of Prip and Eglp genes, while green dotted lines indicate putative loss of Glps. Arthropod divergence times are based on molecular estimates3853, with dashed lines indicating uncertain age. Two saltatory events (vertical arrows on inferred paleoclimate) are highlighted in the early Ordovician and Carboniferous periods, when Elgps respectively evolved from Aqp4-related Prip orthologs and were subsequently positively selected in the last common ancestor of the Holometabola. The inferred paleoclimate and icehouse episodes are redrawn from 5/10 detrended running means of δ18O calcitic shells44. Np, neoproterozoic; Cm, Cambrian; Or, Ordovician; S, Silurian; De, Devonian; Ca, Carboniferous; Pe, Permian; Tr, Triassic; Ju, Jurassic; Cr, Cretaceous; Pg, Paleogene; N, Neogene. Values in the abcissa are millions of years before present.

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Abascal, Diversity and evolution of membrane intrinsic proteins. 2014, Pubmed