Range-wide genetic analysis of an endangered bumble bee (Bombus affinis, Hymenoptera: Apidae) reveals population structure, isolation by distance, and low colony abundance.

Declines in bumble bee species range and abundances are documented across multiple continents and have prompted the need for research to aid species recovery and conservation. The rusty patched bumble bee (Bombus affinis) is the first federally listed bumble bee species in North America. We conducted a range-wide population genetics study of B. affinis from across all extant conservation units to inform conservation efforts. To understand the species' vulnerability and help establish recovery targets, we examined population structure, patterns of genetic diversity, and population differentiation. Additionally, we conducted a site-level analysis of colony abundance to inform prioritizing areas for conservation, translocation, and other recovery actions. We find substantial evidence of population structuring along an east-to-west gradient. Putative populations show evidence of isolation by distance, high inbreeding coefficients, and a range-wide male diploidy rate of ~15%. Our results suggest the Appalachians represent a genetically distinct cluster with high levels of private alleles and substantial differentiation from the rest of the extant range. Site-level analyses suggest low colony abundance estimates for B. affinis compared to similar datasets of stable, co-occurring species. These results lend genetic support to trends from observational studies, suggesting that B. affinis has undergone a recent decline and exhibit substantial spatial structure. The low colony abundances observed here suggest caution in overinterpreting the stability of populations even where B. affinis is reliably detected interannually. These results help delineate informed management units, provide context for the potential risks of translocation programs, and help set clear recovery targets for this and other threatened bumble bee species.

Technological Advances to Address Current Issues in Entomology: 2020 Student Debates.

The 2020 Student Debates of the Entomological Society of America (ESA) were live-streamed during the Virtual Annual Meeting to debate current, prominent entomological issues of interest to members. The Student Debates Subcommittee of the National ESA Student Affairs Committee coordinated the student efforts throughout the year and hosted the live event. This year, four unbiased introductory speakers provided background for each debate topic while four multi-university teams were each assigned a debate topic under the theme 'Technological Advances to Address Current Issues in Entomology'. The two debate topics selected were as follows: 1) What is the best taxonomic approach to identify and classify insects? and 2) What is the best current technology to address the locust swarms worldwide? Unbiased introduction speakers and debate teams began preparing approximately six months before the live event. During the live event, teams shared their critical thinking and practiced communication skills by defending their positions on either taxonomical identification and classification of insects or managing the damaging outbreaks of locusts in crops.

High juvenile mortality in amphibians during overwintering related to fungal pathogen exposure.

The result of pathogen exposures may depend upon trade-offs in energetic demands for immune responses against host growth and survival. Environmental conditions may influence these trade-offs by affecting host size, or trade-offs may change across seasons, altering impacts of pathogens. We exposed northern leopard frog Lithobates pipiens tadpoles to different larval environments (low leaf litter, high density of conspecifics, atrazine, caged fish, or controls) that influenced size at metamorphosis. Subsequently, we exposed metamorphs to Batrachochytrium dendrobatidis (Bd), a fungal pathogen, just after metamorphosis and/or prior to overwintering 12 wk later. Bd exposure dramatically reduced survival during overwintering, with the strongest effects when hosts were exposed at both time points. Larval environments resulted in differences in host size. Those exposed to caged fish were 2.5 times larger than the smallest (those exposed to high density of conspecifics), but larval environment did not influence Bd effects on growth and survival. The largest frogs exposed to caged fish had greater survival through overwintering, but in the absence of Bd. We built stage-structured models to evaluate if overwinter mortality from Bd is capable of having effects on host populations. Our models suggest that Bd exposure after metamorphosis or before overwintering can reduce population growth rates. Our study demonstrates that hosts suffer little effects of Bd exposures following metamorphosis and that small body size did not hamper growth and survival. Instead, we provide evidence that winter mortality from Bd exposure is capable of reducing population sizes, providing a plausible mechanism for amphibian declines in temperate regions.

Characterization of gene expression endpoints during postembryonic development of the northern green frog (Rana clamitans melanota).

Postembryonic development of a larval tadpole into a juvenile frog involves the coordinated action of thyroid hormone (TH) across a diversity of tissues. Changes in the frog transcriptome represent a highly sensitive endpoint in the detection of developmental progression, and for the identification of environmental chemical contaminants that possess endocrine disruptive properties. Unfortunately, in contrast with their vital role as sentinels of environmental change, few gene expression tools currently exist for the majority of native North American frog species. We have isolated seven expressed gene sequences from the Northern green frog (Rana clamitans melanota) that encode proteins associated with TH-mediated postembryonic development and global stress response, and established a quantitative real-time polymerase chain reaction (qPCR) assay. We also obtained three additional species-specific gene sequences that functioned in the normalization of the expression data. Alterations in mRNA abundance profiles were identified in up to eight tissues during R. clamitans postembryonic development, and following exogenous administration of TH to premetamorphic tadpoles. Our results characterize tissue distribution and sensitivity to TH of select mRNA of a common North American frog species and support the potential use of this qPCR assay in identification of the presence of chemical agents in aquatic environments that modulate TH action.

Assessing the Impact of Chemical Algae Management Strategies on Anurans and Aquatic Communities.

Pond management with chemical and biological agents that reduce overgrowth of algae is an important means of maintaining water quality in residential ponds, yet the effects on nontarget species are not fully understood. We assessed the impact of Aquashade (a common nontoxic pond dye) and copper sulfate (a toxic algaecide) on American toad (Anaxyrus americanus), northern leopard frog (Lithobates pipiens), and Cope's gray treefrog (Hyla chrysoscelis) metamorphosis in outdoor mesocosm experiments. We also evaluated the relative impact of tadpole grazing versus chemical treatment on phytoplankton and periphyton abundance. We found no significant effects of pond management treatment on anuran metamorphosis, suggesting that addition of Aquashade and copper sulfate at tested concentrations does not significantly impact anurans under these experimental conditions. Interestingly, we found that the presence of tadpoles more strongly reduced algal abundance than Aquashade or copper sulfate by significantly decreasing phytoplankton and periphyton abundance over time. The present study suggests that anuran tadpoles may be effective at maintaining water quality, and that Aquashade and copper sulfate may have minimal effects on amphibian metamorphosis. Environ Toxicol Chem 2023;42:213-224. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Occupancy of Bombus affinis (Hymenoptera: Apidae) in Minnesota is highest in developed areas when standardized surveys are employed.

Mounting evidence of bumble bee declines and the listing of the rusty patched bumble bee (Bombus affinis Cresson) as federally endangered in the United States in 2017 and Canada in 2012 has stimulated an interest in monitoring and conservation. Understanding the influence of land use on occupancy patterns of imperiled species is crucial to successful recovery planning. Using detection data from community surveys, we assessed land use associations for 7 bumble bee species in Minnesota, USA, including B. affinis. We used multispecies occupancy models to assess the effect of 3 major land use types (developed, agricultural, and natural) within 0.5 and 1.5 km on occupancy of 7 Bombus (Hymenoptera: Apidae) species, while accounting for detection uncertainty. We found that B. affinis occupancy and detection were highest in developed landscapes and lowest in agricultural landscapes, representing an inverse relationship with the relative landcover ratios of these landscapes in Minnesota. Occupancy of 2 bumble bee species had strong positive associations with natural landscapes within 1.5 km and 2 species had strong negative associations with agricultural landscapes within 1.5 km. Our results suggest that best practices for imperiled Bombus monitoring and recovery planning depends upon the surrounding major land use patterns. We provide recommendations for urban planning to support B. affinis based on conservation success in the metropolitan areas of Minneapolis-St. Paul. We also encourage substantial survey effort be employed in agricultural and natural regions of the state historically occupied by B. affinis to determine the current occupancy state.

Evaluating the role of body size and habitat type in movement behavior in human-dominated systems: A frog's eye view.

Animal movement is a key process that connects and maintains populations on the landscape, yet for most species, we do not understand how intrinsic and extrinsic factors interact to influence individual movement behavior.Land-use/land-cover changes highlight that connectivity among populations will depend upon an individual's ability to traverse habitats, which may vary as a result of habitat permeability, individual condition, or a combination of these factors.We examined the effects of intrinsic (body size) and extrinsic (habitat type) factors on desiccation tolerance, movement, and orientation in three anuran species (American toads, Anaxyrus americanus; northern leopard frogs, Lithobates pipiens; and Blanchard's cricket frogs, Acris blanchardi) using laboratory and field studies to connect the effects of susceptibility to desiccation, size, and movement behavior in single-habitat types and at habitat edges.Smaller anurans were more vulnerable to desiccation, particularly for species that metamorphose at relatively small sizes. Habitat type had the strongest effect on movement, while body size had more situational and species-specific effects on movement. We found that individuals moved the farthest in habitat types that, when given the choice, they oriented away from, suggesting that these habitats are less favorable and could represent barriers to movement.Overall, our work demonstrated that differences in habitat type had strong impacts on individual movement behavior and influenced choices at habitat edges. By integrating intrinsic and extrinsic factors into our study, we provided evidence that population connectivity may be influenced not only by the habitat matrix but also by the condition of the individuals leaving the habitat patch.

Can predators stabilize host-parasite interactions? Changes in aquatic predator identity alter amphibian responses and parasite abundance across life stages.

The role of parasites can change depending on the food web community. Predators, for instance, can amplify or dilute parasite effects on their hosts. Likewise, exposure to parasites or predators at one life stage can have long-term consequences on individual performance and survival, which can influence population and disease dynamics. To understand how predators affect amphibian parasite infections across life stages, we manipulated exposure of northern leopard frog (Rana pipiens) tadpoles to three predators (crayfish [Orconectes rusticus], bluegill [Lepomis macrochirus], or mosquitofish [Gambusia affinis]) and to trematode parasites (Echinostoma spp.) in mesocosms and followed juveniles in outdoor terrestrial enclosures through overwintering. Parasites and predators both had strong impacts on metamorphosis with bluegill and parasites individually reducing metamorph survival. However, when fish were present, the negative effects of parasites on survival was not apparent, likely because fish altered community composition via increased algal food resources. Bluegill also reduced snail abundance, which could explain reduced abundance of parasites in surviving metamorphs. Bluegill and parasite exposure increased mass at metamorphosis, which increased metamorph jumping, swimming, and feeding performance, suggesting that larger frogs would experience better terrestrial survival. Effects on size at metamorphosis persisted in the terrestrial environment but did not influence overwintering survival. Based on our results, we constructed stage-structured population models to evaluate the lethal and sublethal effects of bluegill and parasites on population dynamics. Our models suggested that positive effects of bluegill and parasites on body size may have greater effects on population growth than the direct effects of mortality. This study illustrates how predators can alter the outcome of parasitic infections and highlights the need for long-term experiments that investigate how changes in host-parasite systems alter population dynamics. We show that some predators reduce parasite effects and have indirect positive effects on surviving individuals potentially increasing host population persistence.

Is overwintering mortality driving enigmatic declines? Evaluating the impacts of trematodes and the amphibian chytrid fungus on an anuran from hatching through overwintering.

Emerging infectious diseases are increasing globally and are an additional challenge to species dealing with native parasites and pathogens. Therefore, understanding the combined effects of infectious agents on hosts is important for species' conservation and population management. Amphibians are hosts to many parasites and pathogens, including endemic trematode flatworms (e.g., Echinostoma spp.) and the novel pathogenic amphibian chytrid fungus (Batrachochytrium dendrobatidis [Bd]). Our study examined how exposure to trematodes during larval development influenced the consequences of Bd pathogen exposure through critical life events. We found that prior exposure to trematode parasites negatively impacted metamorphosis but did not influence the effect of Bd infection on terrestrial growth and survival. Bd infection alone, however, resulted in significant mortality during overwintering-an annual occurrence for most temperate amphibians. The results of our study indicated overwintering mortality from Bd could provide an explanation for enigmatic declines and highlights the importance of examining the long-term consequences of novel parasite exposure.

Regulation of AQP2 localization by S256 and S261 phosphorylation and ubiquitination.

Vasopressin-induced water reabsorption coincides with phosphorylation of aquaporin-2 (AQP2) at S256 (pS256), dephosphorylation at S261, and its translocation to the apical membrane, whereas treatment with the phorbol ester 12-tetradecanoylphorbol-13-acetate (TPA) induces AQP2 ubiquitination at K270, its internalization, and lysosomal degradation. In this study we investigated the relationship between S256 and S261 phosphorylation in AQP2 and its ubiquitination and trafficking in MDCK cells. Forskolin stimulation associated with increased pS256 and decreased pS261 AQP2, indicating that MDCK cells are a good model. After forskolin stimulation, TPA-induced ubiquitination of AQP2 preceded phosphorylation of AQP2 at S261, which in the first instance occurred predominantly on ubiquitinated AQP2. Forskolin-induced changes in pS261 were also observed for AQP2-S256A and AQP2-S256D, which constitutively localize in vesicles and the apical membrane, respectively. Although pS261 varies with forskolin as with wild-type AQP2, AQP2-S256A is not increased in its ubiquitination. Our data reveal that pS261 occurred independently of AQP2 localization and suggest that pS261 follows ubiquitination and endocytosis and may stabilize AQP2 ubiquitination and intracellular localization. The absence of increased ubiquitination of AQP2-S256A indicates that its intracellular location is due to the lack of pS256. Furthermore, AQP2-S261A and AQP2-S261D localized to vesicles, which was due to their increased ubiquitination, because changing K270 into Arg in both mutants resulted in their localization in the apical membrane. Although still increased in its ubiquitination, AQP2-S256D-S261D localized in the apical membrane. AQP2-S256D-K270R-Ub, however, localized to intracellular vesicles. Although our localization of AQP2-S261A/D is different from that of others, these data indicate that constitutive S256 phosphorylation counterbalances S261D-induced ubiquitination and internalization or changes its structure to allow distribution to the apical membrane. The vesicular localization of AQP2-S256D-K270R-Ub, however, indicates that the dominant apical sorting of S256D can again be overruled by constitutive ubiquitination. These data indicate that the membrane localization of AQP2 is determined by the balance of the extents of phosphorylation and ubiquitination.

Counteracting vasopressin-mediated water reabsorption by ATP, dopamine, and phorbol esters: mechanisms of action.

Water homeostasis is regulated by a wide variety of hormones. When in need for water conservation, vasopressin, released from the brain, binds renal principal cells and initiates a signaling cascade resulting in the insertion of aquaporin-2 (AQP2) water channels in the apical membrane and water reabsorption. Conversely, hormones, including extracellular purines and dopamine, antagonize AVP-induced water permeability, but their mechanism of action is largely unknown, which was investigated here. Addition of these hormones to mpkCCD cells decreased total and plasma membrane abundance of AVP-induced AQP2, partly by increasing its internalization to vesicles and lysosomal degradation. This internalization was ubiquitin dependent, because the hormones increased AQP2 ubiquitination, and the plasma membrane localization of AQP2-K270R, which cannot be monoubiquitinated, was unaffected by these hormones. Both hormones also increased AQP2 phosphorylation at S261, which followed ubiquitination, but was not essential for hormone-induced AQP2 degradation. A similar process occurs in vivo, as incubation of dDAVP-treated kidney slices with both hormones also resulted in the internalization and S261 phosphorylation of AQP2. Both hormones also reduced cAMP and AQP2 mRNA levels, suggesting an additional effect on AQP2 gene transcription. Interestingly, phorbol esters only reduced AQP2 through the first pathway. Together, our results indicate that ATP and dopamine counteract AVP-induced water permeability by increasing AQP2 degradation in lysosomes, preceded by ubiquitin-dependent internalization, and by decreasing AQP2 gene transcription by reducing the AVP-induced cAMP levels.

Insecticide has asymmetric effects on two tadpole species despite priority effects.

Priority effects are defined as asymmetric responses of different species colonizing the same habitat in different sequences and are important in structuring communities, particularly for ephemeral systems that are colonized annually. Amphibians often use ephemeral ponds and have been shown to experience priority effects from interspecific competition. Pesticide exposure can impact amphibian species interactions like competition, and has been linked to declines. We investigated whether insecticide exposure interacted with colonization history to impact priority effects in Bufo americanus and Rana pipiens. We predicted that exposure to the insecticide carbaryl and colonization history would affect tadpole metamorphosis independently and interactively. Our results indicated that Bufo time to metamorphosis experienced priority effects: Bufo introduced early reached metamorphosis sooner when Rana were absent, but Bufo introduced late reached metamorphosis sooner when Rana were also late. Insecticide exposure eliminated differences in Bufo time to metamorphosis due to Rana colonization history. Rana survival and mass at metamorphosis were affected by intraspecific colonization history. Bufo had significantly fewer survivors and Rana had more survivors in insecticide-exposed treatments. Bufo did better when reared without Rana, but Rana was unaffected by Bufo presence. The effects on anuran metamorphosis occurred without any effects on periphyton abundance. We suggest that the effects of insecticide exposure on amphibian metamorphosis are not related to colonization history, although each factor alone can have substantial impacts. These results preliminarily suggest that managing pesticide applications with sensitivity to amphibians may be less complex than implied by traditional food web theory, although more research is needed.

Effects of technical-grade active ingredient vs. commercial formulation of seven pesticides in the presence or absence of UV radiation on survival of green frog tadpoles.

Commercial formulations of pesticides contain both active and other ingredients. In some instances, the other ingredients have detrimental effects on nontarget species. Other factors such as UV radiation and predator cues have been shown to modify the toxicity of pesticides. In a laboratory study we compared the effects of technical-grade active ingredients to commercial formulations of seven common pesticides in the presence or absence of UV radiation on the survival of Rana clamitans (green frog) tadpoles over 96 h. We found a significant difference in the survival of tadpoles in technical-grade active ingredients versus commercial formulations in all of the pesticides tested. We also found that either the presence or the absence of UV radiation affected the survival of tadpoles in five of the seven pesticides tested. These results suggest that there is a need to test the effects of both active ingredients and commercial formulations of pesticides and, also, to include relevant abiotic factors like UV radiation treatments in the testing of pesticides because they can have a dramatic impact on the toxicity of some chemicals.

Investigating the Effects of Pesticides on Ramshorn Snails (Planorbella [Helisoma] trivolvis) Infected with Echinostoma spp.

Globally, parasite-induced diseases in humans and wildlife are on the rise, and pesticide pollution may be a contributing factor. Echinostoma spp. trematode parasites are prominent in North America, and they use ramshorn snails (Planorbella [Helisoma] trivolvis) as intermediate hosts. We investigated the impact of chronic exposure to 1 of 5 pesticide treatments (control, or 50 µg/L of atrazine, glyphosate, carbaryl, or malathion) on uninfected and Echinostoma-infected snails for 41 d in the laboratory. We recorded snail mortality, the number of egg masses laid, change in mass, and behavior. Chronic exposure to atrazine, carbaryl, and malathion significantly decreased snail survival, whereas parasite infection status or exposure to glyphosate did not. Pesticide and parasite treatments did not influence growth or behavior, but parasite infection caused complete reproductive failure in snail hosts. Our results indicated that the direct effects of pesticides could threaten snail populations in natural environments and disrupt host-parasite dynamics.  Environ Toxicol Chem 2021;40:2755-2763. © 2021 SETAC.

Larval development and survival of pond-breeding anurans in an agricultural landscape impacted more by phytoplankton than surrounding habitat.

The destruction of freshwater habitat is a major contributor to biodiversity loss in aquatic ecosystems. However, created or restored wetlands could partially mitigate aquatic biodiversity loss by increasing the amount of available habitat across a landscape. We investigated the impact of surrounding terrestrial habitat and water quality variables on suitability for two species of pond-breeding amphibians (bullfrogs [Lithobates catesbeianus] and Blanchard's cricket frogs [Acris blanchardi]) in created permanent wetlands located on an agricultural landscape. We examined tadpole growth and survival in field enclosures placed in ponds surrounded by agricultural, forested, or grassland habitats. We also evaluated the potential for carryover effects of the aquatic environment on terrestrial growth and overwinter survival of cricket frog metamorphs. We found that habitat adjacent to ponds did not predict tadpole growth or survival. Rather, phytoplankton abundance, which showed high variability among ponds within habitat type, was the only predictor of tadpole growth. Cricket frogs emerged larger and earlier from ponds with higher phytoplankton abundance; bullfrogs were also larger and at a more advanced developmental stage in ponds with higher levels of phytoplankton. Overwinter survival of cricket frogs was explained by size at metamorphosis and there were no apparent carryover effects of land use or pond-of-origin on overwinter growth and survival. Our results demonstrate that created ponds in human-dominated landscapes can provide suitable habitat for some anurans, independent of the adjacent terrestrial habitat.

A Vasopressin-Induced Change in Prostaglandin Receptor Subtype Expression Explains the Differential Effect of PGE2 on AQP2 Expression.

Arginine vasopressin (AVP) stimulates the concentration of renal urine by increasing the principal cell expression of aquaporin-2 (AQP2) water channels. Prostaglandin E2 (PGE2) and prostaglandin2α (PGF2α) increase the water absorption of the principal cell without AVP, but PGE2 decreases it in the presence of AVP. The underlying mechanism of this paradoxical response was investigated here. Mouse cortical collecting duct (mkpCCDc14) cells mimic principal cells as they endogenously express AQP2 in response to AVP. PGE2 increased AQP2 abundance without desmopressin (dDAVP), while in the presence of dDAVP, PGE2, and PGF2α reduced AQP2 abundance. dDAVP increased the cellular PGD2 and PGE2 release and decreased the PGF2α release. MpkCCD cells expressed mRNAs for the receptors of PGE2 (EP1/EP4), PGF2 (FP), and TxB2 (TP). Incubation with dDAVP increased the expression of EP1 and FP but decreased the expression of EP4. In the absence of dDAVP, incubation of mpkCCD cells with an EP4, but not EP1/3, agonist increased AQP2 abundance, and the PGE2-induced increase in AQP2 was blocked with an EP4 antagonist. Moreover, in the presence of dDAVP, an EP1/3, but not EP4, agonist decreased the AQP2 abundance, and the addition of EP1 antagonists prevented the PGE2-mediated downregulation of AQP2. Our study shows that in mpkCCDc14 cells, reduced EP4 receptor and increased EP1/FP receptor expression by dDAVP explains the differential effects of PGE2 and PGF2α on AQP2 abundance with or without dDAVP. As the V2R and EP4 receptor, but not the EP1 and FP receptor, can couple to Gs and stimulate the cyclic adenosine monophosphate (cAMP) pathway, our data support a view that cells can desensitize themselves for receptors activating the same pathway and sensitize themselves for receptors of alternative pathways.

Effect of the cGMP pathway on AQP2 expression and translocation: potential implications for nephrogenic diabetes insipidus.

BACKGROUND: Arginine vasopressin (AVP) binding to the V2 receptor (V2R) in renal collecting duct principal cells induces a cAMP signalling cascade resulting in the activation of protein kinase A (PKA), translocation of aquaporin-2 (AQP2) to the apical membrane and an increase in AQP2 expression. Consequently, concentration of urine is initiated. X-linked nephrogenic diabetes insipidus (NDI), characterized by the inability to concentrate urine in response to AVP, is caused by mutations in the V2R gene. Initiation of AQP2 translocation, while circumventing the V2R-cAMP-PKA pathway has been suggested as a putative therapy for these patients. In this respect, the activation of a cAMP-independent and cGMP-dependent pathway for AQP2 membrane insertion by different cyclic guanosine monophosphate (cGMP) pathway activators, such as atrial natriuretic peptide (ANP), l-arginine and 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP), has been put forward. However, it is unclear whether they can increase AQP2 expression. METHODS: Mouse cortical collecting duct (mpkCCD) cells were incubated with ANP, l-arginine and 8-Br-cGMP for 2 h and subjected to immunocytochemistry and cell surface biotinylation assays to examine their effect on AQP2 translocation. To test the effect of cGMP pathway activators on AQP2 expression, the mpkCCD cells were treated with dDAVP, ANP and l-arginine for 4 days, or with 8-Br-cGMP for the last day. AQP2 protein levels were determined by immunoblotting. RESULTS: ANP, l-arginine and 8-Br-cGMP induced the translocation of AQP2 in the mpkCCD cells. However, in contrast to dDAVP, ANP, l-arginine and 8-Br-cGMP did not increase the expression of AQP2. CONCLUSIONS: Our results suggest that while activators of the cGMP pathway are likely beneficial in the treatment of X-linked NDI, their ability to relieve NDI in the patients may be improved when combined with agents stimulating AQP2 expression.

LIP5 interacts with aquaporin 2 and facilitates its lysosomal degradation.

Vasopressin binding to the V2 receptor in renal principal cells leads to activation of protein kinase A, phosphorylation of aquaporin 2 (AQP2) at Ser256, and the translocation of AQP2 to the apical membrane, resulting in concentration of the urine. In contrast, phorbol ester-induced activation of protein kinase C pathway leads to ubiquitination of AQP2 at Lys270 and its internalization to multivesicular bodies, where it is targeted for lysosomal degradation or stored for recycling. Because little is known about the regulation of AQP2 trafficking, we used the carboxy-terminal tail of constitutively nonphosphorylated AQP2 (S256A) as a bait for interacting proteins in a yeast two-hybrid assay. We isolated lysosomal trafficking regulator-interacting protein 5 (LIP5) and found that LIP5 interacted with the proximal carboxy-terminal tail (L230-D243) of AQP2 in vitro but not with AQP3 or AQP4, which are also expressed in principal cells. Immunohistochemistry revealed that LIP5 co-localized with AQP2 in principal cells. LIP5 binding occurred independent of the state of Ser256 phosphorylation or Lys270 ubiquitination. LIP5 has been shown to facilitate degradation of the EGF receptor; here, LIP5 seemed to bind this receptor. Knockdown of LIP5 in mouse renal cells (mpkCCD) reduced the phorbol ester-induced degradation of AQP2 approximately two-fold. In summary, LIP5 binds cargo proteins and, considering the role of LIP5 in protein sorting to multivesicular bodies, plays a role in the degradation of AQP2, possibly by reducing the formation of late endosomes.

Nucleotides downregulate aquaporin 2 via activation of apical P2 receptors.

Vasopressin regulates water reabsorption in the collecting duct, but extracellular nucleotides modulate this regulation through incompletely understood mechanisms. We investigated these mechanisms using immortalized mouse collecting duct (mpkCCD) cells. Basolateral exposure to dDAVP induced AQP2 localization to the apical membrane, but co-treatment with ATP internalized AQP2. Because plasma membrane-bound P2 receptors (P2R) mediate the effects of extracellular nucleotides, we examined the abundance and localization of P2R in mpkCCD cells. In the absence of dDAVP, P2Y(1) and P2Y(4) receptors localized to the apical membrane, whereas P2X(2), P2X(4), P2X(5), P2X(7), P2Y(2), P2Y(11), and P2Y(12) receptors localized to the cytoplasm. dDAVP induced gene expression of P2X(1), which localized to the apical domain, and led to translocation of P2X(2) and P2Y(2) to the apical and basolateral membranes, respectively. In co-expression experiments, P2R activation decreased membrane AQP2 and AQP2-mediated water permeability in Xenopus oocytes expressing P2X(2), P2Y(2,) or P2Y(4) receptors, but not in oocytes expressing other P2R subtypes. In summary, these data suggest that AQP2-mediated water transport is downregulated not only by basolateral nucleotides, mediated by P2Y(2) receptors, but also by luminal nucleotides, mediated by P2X(2) and/or P2Y(4) receptors.

LETHAL AND SUBLETHAL AMPHIBIAN HOST RESPONSES TO BATRACHOCHYTRIUM DENDROBATIDIS EXPOSURE ARE DETERMINED BY THE ADDITIVE INFLUENCE OF HOST RESOURCE AVAILABILITY.

Host species may differ in their responses to pathogen exposures based on host energy reserves, which could be important for long-term trends in host population growth. Batrachochytrium dendrobatidis (BD) is a pathogen associated with amphibian population declines but also occurs without causing mass mortalities. The impact of BD in populations without associated declines is not well understood, and food abundance could play a role in determining the magnitude of its effects. We exposed American toad (Anaxyrus americanus), northern leopard frog (Lithobates pipiens), and cricket frog (Acris blanchardi) metamorphs to BD under low or high food treatments. Overall, anuran species responded differently to BD exposure and the combined effect of BD exposure and food abundance was additive. American toad survival was lowered by BD exposure and low food availability. Based on these results, we developed a population model for American toads to estimate how reductions in survival could influence population growth. We found that BD could reduce population growth by 14% with high food availability and 21% with low food availability. In contrast, survival of northern leopard frogs was high across all treatments, but their growth was negatively impacted by the additive effects of BD exposure and low food availability. Cricket frog growth and survival were unaffected by BD exposure, suggesting that this species is not sensitive to the effects of this pathogen in terms of growth and survival across environments of different quality in the time period examined. Our results showed that low food availability additively increased the species-specific lethal and sublethal impacts of BD on hosts, which could have implications for long-term host population dynamics.