Polycystin-2-dependent transcriptome reveals early response of autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in polycystin genes, Pkd1 and Pkd2, but the underlying pathogenic mechanisms are poorly understood. To identify genes and pathways that operate downstream of polycystin-2 (PC2), a comprehensive gene expression database was created, cataloging changes in the transcriptome immediately following PC2 protein depletion. To explore cyst initiation processes, an immortalized mouse inner medullary collecting duct line was developed with the ability to knock out the Pkd2 gene conditionally. Genome-wide transcriptome profiling was performed using RNA sequencing in the cells immediately after PC2 was depleted and compared with isogenic control cells. Differentially expressed genes were identified, and a bioinformatic analysis pipeline was implemented. Altered expression of candidate cystogenic genes was validated in Pkd2 knockout mice. The expression of nearly 900 genes changed upon PC2 depletion. Differentially expressed genes were enriched for genes encoding components of the primary cilia, the canonical Wnt pathway, and MAPK signaling. Among the PC2-dependent ciliary genes, the transcription factor Glis3 was significantly downregulated. MAPK signaling formed a key node at the epicenter of PC2-dependent signaling networks. Activation of Wnt and MAPK signaling, concomitant with the downregulation of Glis3, was corroborated in Pkd2 knockout mice. The data identify a PC2 cilia-to-nucleus signaling axis and dysregulation of the Gli-similar subfamily of transcription factors as a potential initiator of cyst formation in ADPKD. The catalog of PC2-regulated genes should provide a valuable resource for future ADPKD research and new opportunities for drug development.NEW & NOTEWORTHY Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease. Mutations in polycystin genes cause the disease, but the underlying mechanisms of cystogenesis are unknown. To help fill this knowledge gap, we created an inducible cell model of ADPKD and assembled a catalog of genes that respond in immediate proximity to polycystin-2 depletion using transcriptomic profiling. The catalog unveils a ciliary signaling-to-nucleus axis proximal to polycystin-2 dysfunction, highlighting Glis, Wnt, and MAPK signaling.

Genomic assessment of larval odyssey: self-recruitment and biased settlement in the Hawaiian surgeonfish Acanthurus triostegus sandvicensis.

The gap between spawning and settlement location of marine fishes, where the larvae occupy an oceanic phase, is a great mystery in both natural history and conservation. Recent genomic approaches provide some resolution, especially in linking parent to offspring with assays of nucleotide polymorphisms. Here, the authors applied this method to the endemic Hawaiian convict tang (Acanthurus triostegus sandvicensis), a surgeonfish with a long pelagic larval stage of c. 54-77 days. They collected 606 adults and 607 juveniles from 23 locations around the island of O'ahu, Hawai'i. Based on 399 single nucleotide polymorphisms, the authors assigned 68 of these juveniles back to a parent (11.2% assignment rate). Each side of the island showed significant population differentiation, with higher levels in the west and north. The west and north sides of the island also had little evidence of recruitment, which may be due to westerly currents in the region or an artefact of uneven sampling. In contrast, the majority of juveniles (94%) sampled along the eastern shore originated on that side of the island, primarily within semi-enclosed Kane'ohe Bay. Nearly half of the juveniles assigned to parents were found in the southern part of Kane'ohe Bay, with local settlement likely facilitated by extended water residence time. Several instances of self-recruitment, when juveniles return to their natal location, were observed along the eastern and southern shores. Cumulatively, these findings indicate that most dispersal is between adjacent regions on the eastern and southern shores. Regional management efforts for Acanthurus triostegus and possibly other reef fishes will be effective only with collaboration among adjacent coastal communities, consistent with the traditional moku system of native Hawaiian resource management.

Critical role of the mineralocorticoid receptor in aldosterone-dependent and aldosterone-independent regulation of ENaC in the distal nephron.

The epithelial Na+ channel (ENaC) constitutes the rate-limiting step for Na+ absorption in the aldosterone-sensitive distal nephron (ASDN) comprising the late distal convoluted tubule (DCT2), connecting tubule (CNT), and collecting duct (CD). Previously, we demonstrated that ENaC activity in the DCT2/CNT transition zone is constitutively high and independent of aldosterone, in contrast to its aldosterone dependence in the late CNT/initial cortical CD (CCD). The mineralocorticoid receptor (MR) is expressed in the entire ASDN. Its activation by glucocorticoids is prevented through 11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2) abundantly expressed in the late but probably not early part of the ASDN. We hypothesized that ENaC function in the early part of the ASDN is aldosterone independent but may depend on MR activated by glucocorticoids due to low 11ß-HSD2 abundance. To test this hypothesis, we used doxycycline-inducible nephron-specific MR-deficient [MR knockout (KO)] mice. Whole cell ENaC currents were investigated in isolated nephron fragments from the DCT2/CNT or CNT/CCD transition zones using the patch-clamp technique. ENaC activity was detectable in the CNT/CCD of control mice but absent or barely detectable in the majority of CNT/CCD preparations from MR KO mice. Importantly, ENaC currents in the DCT2/CNT were greatly reduced in MR KO mice compared with ENaC currents in the DCT2/CNT of control mice. Immunofluorescence for 11ß-HSD2 was abundant in the CCD, less prominent in the CNT, and very low in the DCT2. We conclude that MR is critically important for maintaining aldosterone-independent ENaC activity in the DCT2/CNT. Aldosterone-independent MR activation is probably mediated by glucocorticoids due to low expression of 11ß-HSD2.NEW & NOTEWORTHY Using a mouse model with inducible nephron-specific mineralocorticoid receptor (MR) deficiency, we demonstrated that MR is not only critical for maintaining aldosterone-dependent ENaC activity in CNT/CCD but also for aldosterone-independent ENaC activity in DCT2/CNT. Furthermore, we demonstrated that cells of this latter nephron segment express little 11ß-HSD2, which probably allows glucocorticoids to stimulate MR, resulting in aldosterone-independent ENaC activity in DCT2/CNT. This site-specific ENaC regulation has physiologically relevant implications for renal sodium and potassium homeostasis.

Distal convoluted tubule sexual dimorphism revealed by advanced 3D imaging.

The thiazide-sensitive Na+-Cl- cotransporter (NCC) is more abundant in kidneys of female subjects than of male subjects. Because morphological remodeling of the distal convoluted tubule (DCT) is dependent on NCC activity, it has been generally assumed that there is a corresponding sexual dimorphism in the structure of the DCT, leading to a larger female DCT. Until now, this has never been directly examined. Here, optical clearing techniques were combined with antibody labeling of DCT segment markers, state-of-the-art high-speed volumetric imaging, and analysis tools to visualize and quantify DCT morphology in male and female mice and study the DCT remodeling response to furosemide. We found an unexpected sex difference in the structure of the DCT. Compared with the male mice, female mice had a shorter DCT, a higher cellular density of NCC, and a greater capacity to elongate in response to loop diuretics. Our study revealed a sexual dimorphism of the DCT. Female mice expressed a greater density of NCC transporters in a shorter structure to protect Na+ balance in the face of greater basal distal Na+ delivery yet have a larger reserve and structural remodeling capacity to adapt to unique physiological stresses. These observations provide insight into mechanisms that may drive sex differences in the therapeutic responses to diuretics.

Species Radiations in the Sea: What the Flock?

Species flocks are proliferations of closely-related species, usually after colonization of depauperate habitat. These radiations are abundant on oceanic islands and in ancient freshwater lakes, but rare in marine habitats. This contrast is well documented in the Hawaiian Archipelago, where terrestrial examples include the speciose silverswords (sunflower family Asteraceae), Drosophila fruit flies, and honeycreepers (passerine birds), all derived from one or a few ancestral lineages. The marine fauna of Hawai'i is also the product of rare colonization events, but these colonizations usually yield only one species. Dispersal ability is key to understanding this evolutionary inequity. While terrestrial fauna rarely colonize between oceanic islands, marine fauna with pelagic larvae can make this leap in every generation. An informative exception is the marine fauna that lack a pelagic larval stage. These low-dispersal species emulate a "terrestrial" mode of reproduction (brooding, viviparity, crawl-away larvae), yielding marine species flocks in scattered locations around the world. Elsewhere, aquatic species flocks are concentrated in specific geographic settings, including the ancient lakes of Baikal (Siberia) and Tanganyika (eastern Africa), and Antarctica. These locations host multiple species flocks across a broad taxonomic spectrum, indicating a unifying evolutionary phenomenon. Hence marine species flocks can be singular cases that arise due to restricted dispersal or other intrinsic features, or they can be geographically clustered, promoted by extrinsic ecological circumstances. Here, we review and contrast intrinsic cases of species flocks in individual taxa, and extrinsic cases of geological/ecological opportunity, to elucidate the processes of species radiations.

The sport participation legacy of major events in the UK.

This paper examines the extent to which attending major sporting events leads to subsequent changes in the sport participation behaviour of spectators. The research covered seven single-sport events of World or European level held in the UK in 2014 and was concerned with spectators (aged 16 and over) who attended one of these events. Baseline data was gathered from a sample of spectators at each event using a face-to-face survey. Follow-up data was captured using an online survey at least nine months post-event. Our analysis is based on 258 people for whom both baseline and follow-up data were available (matched pairs). Using the Transtheoretical Model (TTM), the evidence from this research points to a small (net) positive staged change in sport participation among the sample overall. Variations in the nature and scale of changes associated with events featuring different sports were observed. Progression between the TTM stages was evident for individuals who were previously in the pre-preparation, preparation and action stages. The likelihood of progression appears to be strongest where prior contemplation for behaviour change was prevalent. Event attendance emerged as an important contributor for moving individuals along the TTM continuum, alongside a range of other factors. The demonstration or trickle-down effect was the primary mechanism by which any sport participation legacy supported by these events occurred. The practical applications of the research and the wider health benefits of leveraging event-induced sport participation increases are discussed.

Life-history characteristics of an exploited bonefish Albula glossodonta population in a remote South Pacific atoll.

Bonefishes Albula spp. are important components of subsistence fisheries and lucrative sport fishing industries throughout their circumtropical distribution. In Oceania, however, Albula spp. have historically been overexploited and there is a growing need to balance the demands of competing fishing sectors, making the description of their life history a regional priority. To this aim, we collected biological samples from Albula spp. of Anaa atoll, French Polynesia, to identify the species that compose the stock and estimate their life-history parameters including age, growth, reproduction and natural mortality. Our results indicate that Albula glossodonta is the species of bonefish present, with a maximum age that is below the, 20 year longevity of the genus (8 years in males and 10 years in females). Differential growth patterns existed between the two sexes (L∞ = 58, 78 cm fork length (LF ) and K = 0.38, 0.21 for males and females, respectively). Males attained sexual maturity at 43 cm LF (c. 3 years) whereas females matured at 48 cm LF (c. 4 years) and oocyte production was significantly related to body mass, with a maximum batch fecundity of 1,133,767 oocytes in a 4406 g (70 cm LF ) female. The gonado-somatic index of harvested fishes indicated that the spawning season extends from March through September. Based on the observation of a, 20 year bonefish at the proximate Tetiaroa Atoll and several empirical models, estimates of natural mortality ranged from 0.21 to 0.68; however, an estimate of 0.21 was deemed most appropriate. This information facilitated the resurgence of a Rahui (temporary fishing closure) and community-based management to protect A. glossodonta during a critical portion of their spawning season and in this context our results provide an important demographic baseline in evaluating the recovery of this fishery.

Renal and colonic potassium transporters in the pregnant rat.

Gestational potassium retention, most of which occurs during late pregnancy, is essential for fetal development. The purpose of this study was to examine mechanisms underlying changes in potassium handling by the kidney and colon in pregnancy. We found that potassium intake and renal excretion increased in late pregnancy while fecal potassium excretion remained unchanged and that pregnant rats exhibited net potassium retention. By quantitative PCR we found markedly increased H+-K+-ATPase type 2 (HKA2) mRNA expression in the cortex and outer medullary of late pregnant vs. virgin. Renal outer medullary potassium channel (ROMK) mRNA was unchanged in the cortex, but apical ROMK abundance (by immunofluorescence) was decreased in pregnant vs. virgin in the distal convoluted tubule (DCT) and connecting tubule (CNT). Big potassium-α (BKα) channel-α protein abundance in intercalated cells in the cortex and outer medullary collecting ducts (by immunohistochemistry) fell in late pregnancy. In the distal colon we found increased HKA2 mRNA and protein abundance (Western blot) and decreased BKα protein with no observed changes in mRNA. Therefore, the potassium retention of pregnancy is likely to be due to increased collecting duct potassium reabsorption (via increased HKA2), decreased potassium secretion (via decreased ROMK and BK), as well as increased colonic reabsorption via HKA2.

Constitutively Active SPAK Causes Hyperkalemia by Activating NCC and Remodeling Distal Tubules.

Aberrant activation of with no lysine (WNK) kinases causes familial hyperkalemic hypertension (FHHt). Thiazide diuretics treat the disease, fostering the view that hyperactivation of the thiazide-sensitive sodium-chloride cotransporter (NCC) in the distal convoluted tubule (DCT) is solely responsible. However, aberrant signaling in the aldosterone-sensitive distal nephron (ASDN) and inhibition of the potassium-excretory renal outer medullary potassium (ROMK) channel have also been implicated. To test these ideas, we introduced kinase-activating mutations after Lox-P sites in the mouse Stk39 gene, which encodes the terminal kinase in the WNK signaling pathway, Ste20-related proline-alanine-rich kinase (SPAK). Renal expression of the constitutively active (CA)-SPAK mutant was specifically targeted to the early DCT using a DCT-driven Cre recombinase. CA-SPAK mice displayed thiazide-treatable hypertension and hyperkalemia, concurrent with NCC hyperphosphorylation. However, thiazide-mediated inhibition of NCC and consequent restoration of sodium excretion did not immediately restore urinary potassium excretion in CA-SPAK mice. Notably, CA-SPAK mice exhibited ASDN remodeling, involving a reduction in connecting tubule mass and attenuation of epithelial sodium channel (ENaC) and ROMK expression and apical localization. Blocking hyperactive NCC in the DCT gradually restored ASDN structure and ENaC and ROMK expression, concurrent with the restoration of urinary potassium excretion. These findings verify that NCC hyperactivity underlies FHHt but also reveal that NCC-dependent changes in the driving force for potassium secretion are not sufficient to explain hyperkalemia. Instead, a DCT-ASDN coupling process controls potassium balance in health and becomes aberrantly activated in FHHt.

Ecological and morphological traits predict depth-generalist fishes on coral reefs.

Ecological communities that occupy similar habitats may exhibit functional convergence despite significant geographical distances and taxonomic dissimilarity. On coral reefs, steep gradients in key environmental variables (e.g. light and wave energy) restrict some species to shallow depths. We show that depth-generalist reef fishes are correlated with two species-level traits: caudal fin aspect ratio and diet. Fishes with high aspect ratio (lunate) caudal fins produce weaker vortices in the water column while swimming, and we propose that 'silent swimming' reduces the likelihood of detection and provides an advantage on deeper reefs with lower light irradiance and water motion. Significant differences in depth preference among trophic guilds reflect variations in the availability of different food sources along a depth gradient. The significance of these two traits across three geographically and taxonomically distinct assemblages suggests that deep-water habitats exert a strong environmental filter on coral reef-fish assemblages.

Regal phylogeography: Range-wide survey of the marine angelfish Pygoplites diacanthus reveals evolutionary partitions between the Red Sea, Indian Ocean, and Pacific Ocean.

The regal angelfish (Pygoplites diacanthus; family Pomacanthidae) occurs on reefs from the Red Sea to the central Pacific, with an Indian Ocean/Rea Sea color morph distinct from a Pacific Ocean morph. To assess population differentiation and evaluate the possibility of cryptic evolutionary partitions in this monotypic genus, we surveyed mtDNA cytochrome b and two nuclear introns (S7 and RAG2) in 547 individuals from 15 locations. Phylogeographic analyses revealed four mtDNA lineages (d=0.006-0.015) corresponding to the Pacific Ocean, the Red Sea, and two admixed lineages in the Indian Ocean, a pattern consistent with known biogeographic barriers. Christmas Island in the eastern Indian Ocean had both Indian and Pacific lineages. Both S7 and RAG2 showed strong population-level differentiation between the Red Sea, Indian Ocean, and Pacific Ocean (ΦST=0.066-0.512). The only consistent population sub-structure within these three regions was at the Society Islands (French Polynesia), where surrounding oceanographic conditions may reinforce isolation. Coalescence analyses indicate the Pacific (1.7Ma) as the oldest extant lineage followed by the Red Sea lineage (1.4Ma). Results from a median-joining network suggest radiations of two lineages from the Red Sea that currently occupy the Indian Ocean (0.7-0.9Ma). Persistence of a Red Sea lineage through Pleistocene glacial cycles suggests a long-term refuge in this region. The affiliation of Pacific and Red Sea populations, apparent in cytochrome b and S7 (but equivocal in RAG2) raises the hypothesis that the Indian Ocean was recolonized from the Red Sea, possibly more than once. Assessing the genetic architecture of this widespread monotypic genus reveals cryptic evolutionary diversity that merits subspecific recognition. We recommend P.d. diacanthus and P.d. flavescens for the Pacific and Indian Ocean/Red Sea forms.

SPAK-mediated NCC regulation in response to low-K+ diet.

The NaCl cotransporter (NCC) of the renal distal convoluted tubule is stimulated by low-K(+) diet by an unknown mechanism. Since recent work has shown that the STE20/SPS-1-related proline-alanine-rich protein kinase (SPAK) can function to stimulate NCC by phosphorylation of specific N-terminal sites, we investigated whether the NCC response to low-K(+) diet is mediated by SPAK. Using phospho-specific antibodies in Western blot and immunolocalization studies of wild-type and SPAK knockout (SPAK(-/-)) mice fed a low-K(+) or control diet for 4 days, we found that low-K(+) diet strongly increased total NCC expression and phosphorylation of NCC. This was associated with an increase in total SPAK expression in cortical homogenates and an increase in phosphorylation of SPAK at the S383 activation site. The increased pNCC in response to low-K(+) diet was blunted but not completely inhibited in SPAK(-/-) mice. These findings reveal that SPAK is an important mediator of the increased NCC activation by phosphorylation that occurs in the distal convoluted tubule in response to a low-K(+) diet, but other low-potassium-activated kinases are likely to be involved.

Genomic signatures of geographic isolation and natural selection in coral reef fishes.

The drivers of speciation remain among the most controversial topics in evolutionary biology. Initially, Darwin emphasized natural selection as a primary mechanism of speciation, but the architects of the modern synthesis largely abandoned that view in favour of divergence by geographic isolation. The balance between selection and isolation is still at the forefront of the evolutionary debate, especially for the world's tropical oceans where biodiversity is high, but isolating barriers are few. Here, we identify the drivers of speciation in Pacific reef fishes of the genus Acanthurus by comparative genome scans of two peripheral populations that split from a large Central-West Pacific lineage at roughly the same time. Mitochondrial sequences indicate that populations in the Hawaiian Archipelago and the Marquesas Islands became isolated approximately 0.5 Ma. The Hawaiian lineage is morphologically indistinguishable from the widespread Pacific form, but the Marquesan form is recognized as a distinct species that occupies an unusual tropical ecosystem characterized by upwelling, turbidity, temperature fluctuations, algal blooms and little coral cover. An analysis of 3737 SNPs reveals a strong signal of selection at the Marquesas, with 59 loci under disruptive selection including an opsin Rh2 locus. While both the Hawaiian and Marquesan populations indicate signals of drift, the former shows a weak signal of selection that is comparable with populations in the Central-West Pacific. This contrast between closely related lineages reveals one population diverging due primarily to geographic isolation and genetic drift, and the other achieving taxonomic species status under the influence of selection.

Large-scale introduction of the Indo-Pacific damselfish Abudefduf vaigiensis into Hawai'i promotes genetic swamping of the endemic congener A. abdominalis.

Hybridization in the ocean was once considered rare, a process prohibited by the rapid evolution of intrinsic reproductive barriers in a high-dispersal medium. However, recent genetic surveys have prompted a reappraisal of marine hybridization as an important demographic and evolutionary process. The Hawaiian Archipelago offers an unusual case history in this arena, due to the recent arrival of the widely distributed Indo-Pacific sergeant (Abudefduf vaigiensis), which is hybridizing with the endemic congener, A. abdominalis. Surveys of mtDNA and three nuclear loci across Hawai'i (N = 396, Abudefduf abdominalis and N = 314, A. vaigiensis) reveal that hybridization is significantly higher in the human-perturbed southeast archipelago (19.8%), tapering off to 5.9% in the pristine northwest archipelago. While densities of the two species varied throughout Hawai'i, hybridization was highest in regions with similar species densities, contradicting the generalization that the rarity of one species promotes interspecific mating. Our finding of later generation hybrids throughout the archipelago invokes the possibility of genetic swamping of the endemic species. Exaptation, an adaptation with unintended consequences, may explain these findings: the endemic species has transient yellow coloration during reproduction, whereas the introduced species has yellow coloration continuously as adults, in effect a permanent signal of reproductive receptivity. Haplotype diversity is higher in Hawaiian A. vaigiensis than in our samples from the native range, indicating large-scale colonization almost certainly facilitated by the historically recent surge of marine debris. In this chain of events, marine debris promotes colonization, exaptation promotes hybridization, and introgression invokes the possible collapse of an endemic species.

SPAK isoforms and OSR1 regulate sodium-chloride co-transporters in a nephron-specific manner.

STE20/SPS-1-related proline-alanine-rich protein kinase (SPAK) and oxidative stress-related kinase (OSR1) activate the potassium-dependent sodium-chloride co-transporter, NKCC2, and thiazide-sensitive sodium-chloride cotransporter, NCC, in vitro, and both co-localize with a kinase regulatory molecule, Cab39/MO25α, at the apical membrane of the thick ascending limb (TAL) and distal convoluted tubule (DCT). Yet genetic ablation of SPAK in mice causes a selective loss of NCC function, whereas NKCC2 becomes hyperphosphorylated. Here, we explore the underlying mechanisms in wild-type and SPAK-null mice. Unlike in the DCT, OSR1 remains at the TAL apical membrane of KO mice where it is accompanied by an increase in the active, phosphorylated form of AMP-activated kinase. We found an alterative SPAK isoform (putative SPAK2 form), which modestly inhibits co-transporter activity in vitro, is more abundant in the medulla than the cortex. Thus, enhanced NKCC2 phosphorylation in the SPAK knock-out may be explained by removal of inhibitory SPAK2, sustained activity of OSR1, and activation of other kinases. By contrast, the OSR1/SPAK/M025α signaling apparatus is disrupted in the DCT. OSR1 becomes largely inactive and displaced from M025α and NCC at the apical membrane, and redistributes to dense punctate structures, containing WNK1, within the cytoplasm. These changes are paralleled by a decrease in NCC phosphorylation and a decrease in the mass of the distal convoluted tubule, exclusive to DCT1. As a result of the dependent nature of OSR1 on SPAK in the DCT, NCC is unable to be activated. Consequently, SPAK(-/-) mice are highly sensitive to dietary salt restriction, displaying prolonged negative sodium balance and hypotension.

Mutations in PTF1A cause pancreatic and cerebellar agenesis.

Individuals with permanent neonatal diabetes mellitus usually present within the first three months of life and require insulin treatment. We recently identified a locus on chromosome 10p13-p12.1 involved in permanent neonatal diabetes mellitus associated with pancreatic and cerebellar agenesis in a genome-wide linkage search of a consanguineous Pakistani family. Here we report the further linkage analysis of this family and a second family of Northern European descent segregating an identical phenotype. Positional cloning identified the mutations 705insG and C886T in the gene PTF1A, encoding pancreas transcription factor 1alpha, as disease-causing sequence changes. Both mutations cause truncation of the expressed PTF1A protein C-terminal to the basic-helix-loop-helix domain. Reporter-gene studies using a minimal PTF1A deletion mutant indicate that the deleted region defines a new domain that is crucial for the function of this protein. PTF1A is known to have a role in mammalian pancreatic development, and the clinical phenotype of the affected individuals implicated the protein as a key regulator of cerebellar neurogenesis. The essential role of PTF1A in normal cerebellar development was confirmed by detailed neuropathological analysis of Ptf1a(-/-) mice.

The complete mitochondrial genome of the Reef Manta Ray, Mobula alfredi, from Hawaii.

We provide the complete mitochondrial genome of the reef manta ray, Mobula alfredi, using an ezRAD approach. The total length of the mitogenome was 18,166 bp and contained 13 protein-coding genes, 22 transfer RNAs genes, two ribosomal RNA genes, and one non-coding control region. The gene organization and length are similar to other Mobula species. This reference mitogenome that includes the control region is expected to be a valuable resource for molecular-based species identification, population genomics, and phylogeography.

Genomic evidence indicates small island-resident populations and sex-biased behaviors of Hawaiian reef Manta Rays.

BACKGROUND: Reef manta rays (Mobula alfredi) are globally distributed in tropical and subtropical seas. Their life history traits (slow growth, late maturity, low reproductive output) make them vulnerable to perturbations and therefore require informed management strategies. Previous studies have reported wide-spread genetic connectivity along continental shelves suggesting high gene flow along continuous habitats spanning hundreds of kilometers. However, in the Hawaiian Islands, tagging and photo-identification evidence suggest island populations are isolated despite proximity, a hypothesis that has not yet been evaluated with genetic data. RESULTS: This island-resident hypothesis was tested by analyzing whole mitogenome haplotypes and 2048 nuclear single nucleotide polymorphisms (SNPs) between M. alfredi (n = 38) on Hawai'i Island and Maui Nui (the 4-island complex of Maui, Moloka'i, Lana'i and Kaho'olawe). Strong divergence in the mitogenome (ΦST = 0.488) relative to nuclear genome-wide SNPs (neutral FST = 0.003; outlier FST = 0.186), and clustering of mitochondrial haplotypes among islands provides robust evidence that female reef manta rays are strongly philopatric and do not migrate between these two island groups. Combined with restricted male-mediated migration, equivalent to a single male moving between islands every 2.2 generations (~ 64 years), we provide evidence these populations are significantly demographically isolated. Estimates of contemporary effective population size (Ne) are 104 (95% CI: 99-110) in Hawai'i Island and 129 (95% CI: 122-136) in Maui Nui. CONCLUSIONS: Concordant with evidence from photo identification and tagging studies, these genetic results indicate reef manta rays in Hawai'i have small, genetically-isolated resident island populations. We hypothesize that due to the Island Mass Effect, large islands provide sufficient resources to support resident populations, thereby making crossing deep channels separating island groups unnecessary. Small effective population size, low genetic diversity, and k-selected life history traits make these isolated populations vulnerable to region-specific anthropogenic threats, which include entanglement, boat strikes, and habitat degradation. The long-term persistence of reef manta rays in the Hawaiian Islands will require island-specific management strategies.

Differential regulation of ROMK (Kir1.1) in distal nephron segments by dietary potassium.

ROMK channels are well-known to play a central role in renal K secretion, but the absence of highly specific and avid-ROMK antibodies has presented significant roadblocks toward mapping the extent of expression along the entire distal nephron and determining whether surface density of these channels is regulated in response to physiological stimuli. Here, we prepared new ROMK antibodies verified to be highly specific, using ROMK knockout mice as a control. Characterization with segmental markers revealed a more extensive pattern of ROMK expression along the entire distal nephron than previously thought, localizing to distal convoluted tubule regions, DCT1 and DCT2; the connecting tubule (CNT); and cortical collecting duct (CD). ROMK was diffusely distributed in intracellular compartments and at the apical membrane of each tubular region. Apical labeling was significantly increased by high-K diet in DCT2, CNT1, CNT2, and CD (P < 0.05) but not in DCT1. Consistent with the large increase in apical ROMK, dramatically increased mature glycosylation was observed following dietary potassium augmentation. We conclude 1) our new antibody provides a unique tool to characterize ROMK channel localization and expression and 2) high-K diet causes a large increase in apical expression of ROMK in DCT2, CNT, and CD but not in DCT1, indicating that different regulatory mechanisms are involved in K diet-regulated ROMK channel functions in the distal nephron.

Role of NKCC in BK channel-mediated net K⁺ secretion in the CCD.

Apical SK/ROMK and BK channels mediate baseline and flow-induced K secretion (FIKS), respectively, in the cortical collecting duct (CCD). BK channels are detected in acid-base transporting intercalated (IC) and Na-absorbing principal (PC) cells. Although the density of BK channels is greater in IC than PC, Na-K-ATPase activity in IC is considered inadequate to sustain high rates of urinary K secretion. To test the hypothesis that basolateral NKCC in the CCD contributes to BK channel-mediated FIKS, we measured net K secretion (J(K)) and Na absorption (J(Na)) at slow (∼1) and fast (∼5 nl·min(-1)·mm(-1)) flow rates in rabbit CCDs microperfused in vitro in the absence and presence of bumetanide, an inhibitor of NKCC, added to the bath. Bumetanide inhibited FIKS but not basal J(K), J(Na), or the flow-induced [Ca(2+)](i) transient necessary for BK channel activation. Addition of luminal iberiotoxin, a BK channel inhibitor, to bumetanide-treated CCDs did not further reduce J(K). Basolateral Cl removal reversibly inhibited FIKS but not basal J(K) or J(Na). Quantitative PCR performed on single CCD samples using NKCC1- and 18S-specific primers and probes and the TaqMan assay confirmed the presence of the transcript in this nephron segment. To identify the specific cell type to which basolateral NKCC is localized, we exploited the ability of NKCC to accept NH(4)(+) at its K-binding site to monitor the rate of bumetanide-sensitive cytosolic acidification after NH(4)(+) addition to the bath in CCDs loaded with the pH indicator dye BCECF. Both IC and PC were found to have a basolateral bumetanide-sensitive NH(4)(+) entry step and NKCC1-specific antibodies labeled the basolateral surfaces of both cell types in CCDs. These results suggest that BK channel-mediated FIKS is dependent on a basolateral bumetanide-sensitive, Cl-dependent transport pathway, proposed to be NKCC1, in both IC and PC in the CCD.