The adaptive evolution of arthropod and vertebrate aquaporins: Deciphering the differential basis of water homeostasis

Initial studies focused on identifying the genomic repertoires of aquaporins in a broad range of prokaryotic and eukaryotic organisms in order to lay the groundwork for understanding the evolutionary history of the superfamily, and for identifying the full complement of aquaporin genes in the salmon louse and its host, the Atlantic salmon. These studies included the assembly of >8000 aquaporin fragments from >300 prokaryotic and eukaryotic genomes, which were then phylogenetically classified using Bayesian inference. These data revealed an unexpected diversity of water channel genes in Prokaryota with four separate grades of aquaporins encoded in the genomes of Archaea and Bacteria (AqpZ, AqpN, AqpM and GlpF). These discoveries led to a new understanding of how glycerol transporters (NIPs) evolved by horizontal gene transfer in plants. In addition, the combined data sets revealed that although many different forms of aquaporins are found in eukaryotic organisms, the metazoan channels can be classified into four major grades consisting of (1) Aqp4-like classical water transporting aquaporins, (2) Aqp8-like aquaammoniaporins, (3) water and glycerol transporting aquaglyceroporins (Glps) and (4) Aqp12-like unorthodox aquaporins. Thus although the vertebrate repertoire of aquaporin genes was thought to consist of 13 subfamilies (AQP0 to -12), the present studies have revealed that the genomes of non-eutherian vertebrates encode additional aquaporin subfamilies termed AQP13, -14, -15 and -16, while arthropods lack the Aqp8-related grade of aquaporins. Parallel studies examined the regulation, function, and evolution of the arthropod and vertebrate aquaporins. This included the identification and partial characterisation of 7 and 42 aquaporins in the genomes of the salmon louse and Atlantic salmon, respectively. However, although the aquaporin repertoire of Atlantic salmon represents a 6-fold redundancy compared to the louse, the functional assays revealed that the permeation properties of the different crustacean grades of aquaporin are largely conserved to the vertebrate counterparts. The data are thus the first to reveal the substrate permeability properties of aquaporins in a crustacean organism and uncover the broader genomic diversity of the superfamily in the four extant lineages of Arthropoda. Expression data showed that each of the salmon louse aquaporin is expressed throughout the life cycle, with one exception where an N-terminal splice variant of Glp1_v1 is specific to pre-adult and adult males. An important finding was the discovery that one of the splice variants (Glp3_v1) is expressed throughout the intestine in the enterocyte brush border of male and female lice, which could represent an eminent candidate for vaccination. The studies of the arthropod aquaporin superfamily further uncovered the loss of aquaglyceroporins in holometabolan insects, the most successful group of terrestrial organisms in the history of life. These investigations led to the discovery of a novel class of aquaporins, termed entomoglyceroporins (Eglps) in insects. Both mutagenesis and phylogenetic studies revealed that the Eglps evolved from mutated water channels in the oldest hexapod lineages and replaced the ancestral aquaglyceroporins in the holometabolan insects. Intriguingly, the origin of Eglps in basal hexapods and subsequent replacement of the ancestral aquaglyceroporins in holometabolan insects coincided with episodes of fluctuating temperatures that led to glaciation periods. This suggested that the emergence of the insect Eglps may have favored the unprecedented radiation of insects in all types of ecosystems. This is because a unique innovation of holometabolan insects is the sedentary pupal stage of the life cycle, in which larvae are fully transformed into the adult body pattern, such as a caterpillar to a butterfly. The pupal stage, which is particularly vulnerable to low temperatures, accumulates particularly high levels of glycerol and may thus not have arisen without the emergence of the Eglps. These novel discoveries are now published in Nature Communications and the perspectives highlighted in international science boards.

The epizootic caused by the Atlantic salmon louse, a caligid ectoparasitic crustacean, is a severe threat to wild and domestic salmonids. Novel therapeutic approaches to dealing with this pest are needed. Exposure of the louse to freshwater rapidly breaks down its semi-adaptive homeostasis highlighting the osmoregulatory systems for potential therapeutic action. During ecdysis, the louse swells almost exclusively with water, suggesting that disruption of water transport mechanisms can expose an untapped v ulnerabilty. The cellular mechanisms that underlie the growth and water homeostasis of the louse are, however, virtually unknown. Aquaporins are major targets for drug discovery in biomedical research, and have recently gained ground in the fight against malaria where channel disruption increases the host´s resistance to infection. By targeting the adaptive channel transport systems of the louse in relation to its vertebrate host, we aim to generate an avenue for differentially regulating aquaporins as a potential means of prophilaxis. Using a heterologous Xenopus oocyte system, structural and functional data will be obtained to determine channel solute selectivity of host and parasite aquaporins. Knock-down experiments in the louse using RNAi technology will be applied to decipher the phenotypic effects on its osmotic physiology, growth and differentiation. Potential chemotherapeutants will be tested to screen for differential disruptors of the louse rather than the host aquaporins. Crystallographic data will be generated to determine critical residues associated with the channel architecture and site-directed mutagenesis used to validate the structure-function relationships. We further aim to validate our hypothesis that aquaporin evolution was fundamen tally associated with animal radiation during the Devonian Period. The project thus provides an exciting combination of molecular genetics, evolution, and potential drug targeting of invertebrate aquaporins.