Variation in Freshwater Insect Osmoregulatory Traits: A Comparative Approach.

AbstractFreshwater salinity regimes vary naturally and are changing in response to anthropogenic activities. Few insect species tolerate saline waters, and biodiversity losses are associated with increasing salinity in freshwater. We used radiotracers (22Na, 35SO4, and 45Ca) to examine ion uptake rates across concentration gradients in mayflies (Ephemeroptera), caddis flies (Trichoptera), and mosquitoes (Diptera) and made observations for some traits in seven other taxa representing mayflies, stone flies (Plecoptera), true flies (Diptera), and true bugs (Hemiptera). We further assessed the permeability of the cuticle to 3H2O influx and 22Na efflux when faced with deionized water in these same taxa. We hypothesized a relationship between uptake rates and reported saline tolerances, but our data did not support this hypothesis, likely because acclimatory responses were not part of this experimental approach. However, we found several common physiological traits across the taxa studied, including (i) ionic uptake rates that were always positively correlated with dissolved concentrations, (ii) generally low Ca uptake rates relative to other freshwater taxa, (iii) greater Na loss than Na uptake in dilute conditions, (iv) ion uptake that was more variable in ion-rich conditions than in dilute conditions, and (v) 3H2O influx that occurs quickly (but this rapidly exchangeable pool of body water accounts for a surprisingly small percentage of the water content of species tested). There remains much to learn about the physiology of these important organisms in the face of changing salinity regimes worldwide.

Epigenetic mechanisms of osteoarthritis risk in human skeletal development.

The epigenome, including the methylation of cytosine bases at CG dinucleotides, is intrinsically linked to transcriptional regulation. The tight regulation of gene expression during skeletal development is essential, with ~1/500 individuals born with skeletal abnormalities. Furthermore, increasing evidence is emerging to link age-associated complex genetic musculoskeletal diseases, including osteoarthritis (OA), to developmental factors including joint shape. Multiple studies have shown a functional role for DNA methylation in the genetic mechanisms of OA risk using articular cartilage samples taken from aged patients. Despite this, our knowledge of temporal changes to the methylome during human cartilage development has been limited. We quantified DNA methylation at ~700,000 individual CpGs across the epigenome of developing human articular cartilage in 72 samples ranging from 7-21 post-conception weeks, a time period that includes cavitation of the developing knee joint. We identified significant changes in 8% of all CpGs, and >9400 developmental differentially methylated regions (dDMRs). The largest hypermethylated dDMRs mapped to transcriptional regulators of early skeletal patterning including MEIS1 and IRX1. Conversely, the largest hypomethylated dDMRs mapped to genes encoding extracellular matrix proteins including SPON2 and TNXB and were enriched in chondrocyte enhancers. Significant correlations were identified between the expression of these genes and methylation within the hypomethylated dDMRs. We further identified 811 CpGs at which significant dimorphism was present between the male and female samples, with the majority (68%) being hypermethylated in female samples. Following imputation, we captured the genotype of these samples at >5 million variants and performed epigenome-wide methylation quantitative trait locus (mQTL) analysis. Colocalization analysis identified 26 loci at which genetic variants exhibited shared impacts upon methylation and OA genetic risk. This included loci which have been previously reported to harbour OA-mQTLs (including GDF5 and ALDH1A2), yet the majority (73%) were novel (including those mapping to CHST3, FGF1 and TEAD1). To our knowledge, this is the first extensive study of DNA methylation across human articular cartilage development. We identify considerable methylomic plasticity within the development of knee cartilage and report active epigenomic mediators of OA risk operating in prenatal joint tissues.

Novel insights into paternity skew in a polyandrous social wasp.

Females of many species are polyandrous. However, polyandry can give rise to conflict among individuals within families. We examined the level of polyandry and paternity skew in the common eastern yellowjacket wasp, Vespula maculifrons, in order to gain a greater understanding of conflict in social insects. We collected 10 colonies of V. maculifrons and genotyped workers and prereproductive queens at highly variable microsatellite markers to assign each to a patriline. Genotypic data revealed evidence of significant paternity skew among patrilines. In addition, we found that patrilines contributed differentially to caste production (worker vs. queen), suggesting an important role for reproductive conflict not previously discovered. We also investigated if patterns of paternity skew and mate number varied over time. However, we found no evidence of changes in levels of polyandry when compared to historical data dating back almost 40 years. Finally, we measured a suite of morphological traits in individuals from the most common and least common patrilines in each colony to test if males that showed highly skewed reproductive success also produced offspring that differed in phenotype. Our data revealed weak correlation between paternity skew and morphological phenotype of offspring sired by different males, suggesting no evidence of evolutionary tradeoffs at the level investigated. Overall, this study is the first to report significant paternity and caste-associated skew in V. maculifrons, and to investigate the phenotypic consequences of skew in a social wasp. Our results suggest that polyandry can have important consequences on the genetic and social structure of insect societies.

Social insect transcriptomics and the molecular basis of caste diversity.

Studies of gene expression provide fundamentally important information on the molecular mechanisms underlying variation in phenotype. Recent technological advances have allowed for the robust study of gene expression through analysis of whole transcriptomes. Here, we review current advances in social insect transcriptomics and discuss their implications in understanding phenotypic diversity. Recent transcriptomic studies provide detailed inventories of the genes involved in producing distinct phenotypes in social species. These investigations have identified key genes and networks involved in producing distinct social insect castes. Nevertheless, questions concerning the evolution of gene expression patterns remain. We suggest a path forward for studying gene expression in future studies of biological systems.

Salinity-induced ionoregulatory changes in the gill proteome of the mayfly, Neocloeon triangulifer.

Ecologists have observed declines in the biodiversity of sensitive freshwater organisms in response to increasing concentrations of major ions (salinization). Yet, how changing salinities physiologically challenge aquatic organisms, such as mayflies, remains remarkably understudied. Moreover, it is not well understood the degree to which species respond and acclimate to salinity changes. Our lab is developing the Baetid mayfly, N. triangulifer, as a model organism for physiological research. We have previously described acclimatory changes in both ion flux rates and altered mRNA transcript levels in response to chronic exposures to elevated major ion concentrations at the whole animal level. In the present study, we use shotgun proteomics to identify the specific proteins associated with apical ion transport and how their abundance changes in response to chronic salinity exposures in gills. Gills were isolated from the penultimate nymphal stage of N. triangulifer reared under control culture conditions, elevated NaCl (157 mg L-1 Na), elevated CaCl2 (121 mg L-1 Ca), elevated Ca/MgSO4 (735 mg L-1 SO4). These conditions mirrored those from previously published physiological work. We also acutely exposed nymphs to dilute (50% dilution of culture water with deionized water) to explore proteomic changes in the gills in response to dilute conditions. We report 710 unique peptide sequences among treatment groups, including important apical ion transporters such as Ca-ATPase, Na/K ATPase, and V-ATPase. Treatment with elevated NaCl and Ca/MgSO4 appeared to cause more significant differential protein expression (452 and 345, respectively) compared to CaCl2 and dilute groups (134 and 17, respectively). Finally, we demonstrated the breadth of physiological functions in gills by exploring non-transport related pathways found in our dataset, including ATP synthesis, calcium signaling, and oxidative stress response. We discuss our results in the context of freshwater salinization and the challenges of working with non-model species without fully sequenced and annotated genomes.

Assessing the Pcrit in relation to temperature and the expression of hypoxia associated genes in the mayfly, Neocloeon triangulifer.

Hypoxia is a growing concern in aquatic ecosystems. Historically, scientists have used the Pcrit (the dissolved oxygen level below which an animal can no longer oxyregulate) to infer hypoxia tolerance across species. Here, we tested the hypothesis that the Pcrit is positively correlated with temperature in the mayfly, Neocloeon triangulifer. Cross-temperature comparisons showed a modest (r = 0.47), but significant (p < 0.0001) association between temperature and Pcrit despite relatively large interindividual variability (Coefficient of Variance (CV) = 39.9% at 18 °C). We used the expression of hypoxia-responsive genes EGL-9 (an oxygen sensing gene and modulator of HIF-1a activity) and LDH (a hypoxia indicator) to test whether oxygen partial pressure near the Pcrit stimulates expression of hypoxia-responsive genes. Neither gene was upregulated at oxygen levels above the estimated Pcrit, however, at or below the Pcrit estimates, expression of both genes was stimulated (~20- and ~3-fold change for EGL-9 and LDH, respectively). Finally, we evaluated the influence of hypoxic exposure time and pretreatment conditions on the mRNA expression levels of hypoxia-responsive genes. When larvae were exposed to a gradual reduction of DO, hypoxic gene expression was more robust than during instantaneous exposure to hypoxia. Our data provide modest support for traditional interpretation of the Pcrit as a physiologically meaningful shift from aerobic to anaerobic metabolism in N. triangulifer. However, we also discuss limitations of the Pcrit as a proxy measure of hypoxia tolerance at the species level.

Physiological plasticity and acclimatory responses to salinity stress are ion-specific in the mayfly, Neocloeon triangulifer.

Freshwater salinization is a rapidly emerging ecological issue and is correlated with significant declines in aquatic biodiversity. It remains unclear how changing salinity regimes affect the physiology of sensitive aquatic insects. We used the parthenogenetic mayfly, Neocloeon triangulifer, to ask how ionic exposure history alters physiological processes and responses to subsequent major ion exposures. Using radiotracers (22Na, 35SO4, and 45Ca), we observed that mayflies chronically reared in elevated sodium or sulfate (157 mg L-1 Na or 667 mg L-1 SO4) had 2-fold (p < 0.0001) and 8-fold (p < 0.0001) lower ion uptake rates than mayflies reared in dilute control water (16 mg L-1 Na and 23 mg L-1 SO4) and subsequently transferred to elevated salinities, respectively. These acclimatory ion transport changes provided protection in 96-h toxicity bioassays for sodium, but not sulfate. Interestingly, calcium uptake was uniformly much lower and minimally influenced by exposure history, but was poorly tolerated in the toxicity bioassays. With qRT-PCR, we observed that the expression of many ion transporter genes in mayflies was influenced by elevated salinity in an ion-specific manner (general upregulation in response to sulfate, downregulation in response to calcium). Elevated sodium exposure had minimal influence on the same genes. Finally, we provide novel light microscopic evidence of histomorphological changes within the epithelium of the Malpighian tubules (insect primary excretory system) that undergoes cellular degeneration and necrosis secondary to calcium toxicity. We conclude that physiological plasticity to salinity stress is ion-specific and provide evidence for ion-specific toxicity mechanisms in N. triangulifer.

Prophylactic supplementation with selenium alters disposition of mercury in aged rats.

Mercury (Hg) is a prevalent environmental toxicant to which older individuals are particularly susceptible. Selenium (Se) has been used as an antidote following exposure to Hg. However, little is known about the effect of prophylactic supplementation with Se on the handling of Hg. The current study was designed to test the hypothesis that oral pre-treatment with Se alters the corporal disposition of Hg and reduces the risk of Hg-induced toxicity. Young and aged rats were gavaged for 10 days with sodium selenite or saline. On day 11, rats were injected intravenously with 0.5 µmol HgCl2·kg-1·2 mL-1 normal saline. After 24 h, rats were euthanized and organs and tissues were harvested for determination of Hg content. Accumulation of Hg in the kidney was reduced significantly by pre-treatment with Se in both young and aged rats. In the renal cortex, the magnitude of the reduction was greater in aged rats than in young rats but in the outer stripe of the outer medulla, the magnitude of the reduction was similar between groups of rats. Urinary excretion of Hg was also reduced in rats pre-treated with Se. In contrast, the hepatic and hematologic burden of Hg increased in rats pre-treated with Se. Fecal excretion of Hg was decreased significantly by pre-treatment with Se in young rats but not in aged rats. These data suggest that prophylactic supplementation with Se alters the corporal disposition of Hg in a way that may reduce Hg-induced toxicity in target organs.

Reaction of Cyanide with Hg0-Contaminated Gold Mining Tailings Produces Soluble Mercuric Cyanide Complexes.

Elemental mercury (Hg0) contamination in artisanal and small-scale gold mining (ASGM) communities is widespread, and Hg0-contaminated tailings are often reprocessed with cyanide (-CN) to extract residual gold remaining after amalgamation. Hg0 reacts with -CN under aerobic conditions to produce Hg(CN)42- and other Hg(CN)nn-2 complexes. The production of solvated Hg(CN)nn-2 complexes increases upon agitation in the presence of synthetic and authentic Hg0-contaminated tailings that aid in dispersing the Hg0, increasing its reactive surface area. Adult rats were exposed to various concentrations of Hg(CN)2, and accumulation in organs and tissues was quantified using direct mercury analysis. The primary site of Hg(CN)2 accumulation was the kidney, although accumulation was also detected in the liver, spleen, and blood. Little accumulation was observed in the brain, suggesting that Hg(CN)2 complexes do not cross the blood-brain barrier. Renal tissue was particularly sensitive to the effects of Hg(CN)2, with pathological changes observed at low concentrations. Hg(CN)2 complexes are handled by mammalian systems in a manner similar to other inorganic species of Hg, yet appear to be more toxic to organ systems. The findings from this study are the first to show that Hg(CN)2 complexes are highly stable complexes that can lead to cellular injury and death in mammalian organ systems.

Chronic kidney disease in pregnant mothers affects maternal and fetal disposition of mercury.

Chronic kidney disease (CKD) affects over 15 % of the adults in the United States. Pregnant women with CKD present an additional challenge in that they are at increased risk for adverse events such as preterm birth. Exposure to environmental toxicants, such as methylmercury, may exacerbate maternal disease and increase the risk of adverse fetal outcomes. We hypothesized that fetuses of mothers with CKD are more susceptible to accumulation of methylmercury than fetuses of healthy mothers. The current data show that when mothers are in a state of renal insufficiency, uptake of mercury in fetal kidneys is enhanced significantly. Accumulation of Hg in fetal kidneys may be related to the flow of amniotic fluid, maternal handling of Hg, and/or underdeveloped mechanisms for cellular export and urinary excretion. The results of this study indicate that renal insufficiency in mothers leads to significant alterations in the way toxicants such as mercury are handled by maternal and fetal organs.

It's all about the fluxes: Temperature influences ion transport and toxicity in aquatic insects.

Many freshwater ecosystems are becoming saltier and/or warmer, but our understanding of how these factors interact and affect the physiology and life history outcomes of most aquatic species remain unknown. We hypothesize that temperature modulates ion transport rates. Since ion transport is energetically expensive, increases in salinity and/or temperature may influence ion flux rates and ultimately, organismal performance. Radiotracer (22Na+, 35SO4-2, and 45Ca2+) experiments with lab-reared mayflies (N. triangulifer) and other field-collected insects showed that increasing temperature generally increased ion transport rates. For example, increasing temperature from 15 °C to 25 °C, increased 22Na+ uptake rates by two-fold (p < 0.0001) and 35SO4-2 uptake rates by four-fold (p < 0.0001) in the caddisfly, Hydropsyche sparna. Smaller changes in 22Na+ and 35SO4-2 uptake rates were observed in the mayflies, Isonychia sayi and Maccaffertium sp., suggesting species-specific differences in the thermal sensitivity of ion transport. Finally, we demonstrated that the toxicity of SO4 was influenced by temperature profoundly in a 96-h bioassay. Under the saltiest conditions (1500 mg L-1 SO4), mayfly survival was 78 % at 15 °C, but only 44 % at 25 °C (p < 0.0036). Conceivably, the energetic cost of osmoregulation in warmer, saltier environments may cause significant major ion toxicity in certain freshwater insects.

Co-administration of Selenium with Inorganic Mercury Alters the Disposition of Mercuric Ions in Rats.

Mercury (Hg) is a common environmental toxicant to which humans are exposed regularly through occupational and dietary means. Although selenium supplementation has been reported to prevent the toxic effects of Hg in animals, the mechanisms for this prevention are not well understood. The purpose of the current study was to determine the effects of selenium on the disposition and toxicity of Hg. Wistar rats were injected intravenously with a non-nephrotoxic dose (0.5 µmol kg-1) or a nephrotoxic dose (2.5 µmol kg-1) of HgCl2 (containing radioactive Hg) with or without co-administration of sodium selenite (Na2SeO3). Twenty-four hours after exposure, rats were euthanized, and organs were harvested. Co-administration of SeO32- with HgCl2 reduced the renal burden of Hg and the urinary excretion of Hg while increasing the amount of Hg in blood and spleen. We propose that Hg reacts with reduced selenite in the blood to form large Hg-Se complexes that are unable to be filtered at the glomerulus. Consequently, these complexes remain in the blood and are able to accumulate in blood-rich organs. These complexes, which may have fewer toxic effects than other species of Hg, may be eliminated slowly over the course of weeks to months.

Alteration in the mRNA expression of genes associated with gastrointestinal permeability and ileal TNF-α secretion due to the exposure of silver nanoparticles in Sprague-Dawley rats.

BACKGROUND: Silver ions from silver nanoparticles (AgNP) or AgNPs themselves itself that are ingested from consumer health care products or indirectly from absorbed food contact material can interact with the gastrointestinal tract (GIT). The permeability of the GIT is strictly regulated to maintain barrier function and proper nutrient absorption. The single layer intestinal epithelium adheres and communicates actively to neighboring cells and the extracellular matrix through different cell junctions. In the current study, we hypothesized that oral exposure to AgNPs may alter the intestinal permeability and expression of genes controlling cell junctions. Changes in cell junction gene expression in the ileum of male and female rats administered different sizes of AgNP for 13-weeks were assessed using qPCR. RESULTS: The results of this study indicate that AgNPs have an altering effect on cell junctions that are known to dictate intestinal permeability. mRNA expression of genes representing tight junction (Cldn1, Cldn5, Cldn6, Cldn10 and Pecam1), focal adhesion (Cav1, Cav2, and Itgb2), adherens junction (Pvrl1, Notch1, and Notch2), and hemidesmosome (Dst) groups were upregulated significantly in females treated with 10 nm AgNP, while no change or downregulation of same genes was detected in male animals. In addition, a higher concentration of pro-inflammatory cytokine, TNF-α, was noticed in AgNP-treated female animals as compared to controls. CONCLUSIONS: This study proposes that interaction of silver with GIT could potentially initiate an inflammatory process that could lead to changes in the gastrointestinal permeability and/or nutrient deficiencies in sex-specific manner. Fully understanding the mechanistic consequences of oral AgNP exposure may lead to stricter regulation for the commercial usage of AgNPs and/or improved clinical therapy in the future.

Potential mechanisms of cellular injury following exposure to a physiologically relevant species of inorganic mercury.

Mercury is a toxic metal that is found ubiquitously in the environment. Humans are exposed to different forms of mercury via ingestion, inhalation, and/or dermal absorption. Following exposure, mercuric ions may gain access to target cells and subsequently lead to cellular intoxication. The mechanisms by which mercury accumulation leads to cellular injury and death are not understood fully. Therefore, purpose of this study was to identify the specific intracellular mechanisms that are altered by exposure to inorganic mercury (Hg2+). Normal rat kidney (NRK) cells were exposed to a physiologically relevant form of Hg2+, as a conjugate of cysteine (10 µM or 50 µM). Alterations in oxidative stress were estimated by measuring lipid peroxidation and mitochondrial oxidative stress. Alterations in actin and tubulin were measured using specific fluorescent dyes. Calcium levels were measured using Fluo-3 AM Calcium Indicator while autophagy was identified with Premo™ Autophagy Sensor LC3B-GFP. The current findings show that exposure to Hg2+ leads to enhanced oxidative stress, alterations in cytoskeletal structure, increases in intracellular calcium, and enhanced autophagy. We have established a more complete understanding of intoxication and cellular injury induced by a relevant form of Hg2+ in proximal tubule cells.

Exposure to mixtures of mercury, cadmium, lead, and arsenic alters the disposition of single metals in tissues of Wistar rats.

Humans throughout the world are exposed regularly to mixtures of environmental toxicants. Four of the most common heavy metal toxicants in the environment are mercury (Hg), cadmium (Cd), lead (Pb), and arsenic (As). Numerous studies have assessed the effects and disposition of individual metals in organ systems; however, humans are usually exposed to mixtures of toxicants or metals rather than to a single toxicant. Therefore, the purpose of the current study was to test the hypothesis that exposure to a mixture of toxic heavy metals alters the disposition of single metals in target organs. Wistar rats (Rattus norvegicus) were exposed to Hg, Cd, Pb, or As as a single metal or as a mixture of metals. Rats were injected intravenously for three days, following which kidneys, liver, brain, and blood were harvested. Samples were analyzed for content of Hg, Cd, Pb, and As via inductively coupled plasma mass spectrometry. In general, exposure to a mixture of metals reduced accumulation of single metals in target organs. Interestingly, exposure to mixtures of metals with Pb and/or As increased the concentration of these metals specifically in the liver. The findings from this study indicate that exposure to mixtures of toxic heavy metals may alter significantly the distribution and accumulation of these metals in target organs and tissues.

Disposition of methylmercury over time in a 75% nephrectomized rat model.

Chronic kidney disease (CKD) is a highly relevant clinical condition that is characterized by the permanent loss of functional nephrons. Individuals with CKD may exhibit impaired renal clearance, which may alter corporal handling of metabolites and xenobiotics. Methylmercury (MeHg) is an important environmental toxicant to which humans are exposed to on a regular basis. Given the prevalence of CKD and ubiquitous presence of MeHg in the environment, it is important to understand how mercuric ions are handled in patients with CKD. Therefore, the purpose of the current study was to characterize the disposition of MeHg over time in a rat model of CKD (i.e., 75% nephrectomized (NPX) rats). Control and NPX rats were exposed intravenously (iv) to a non-nephrotoxic dose of MeHg (5 mg/kg) once daily for1, 2, or 3 d and the amount of MeHg in organs, blood, urine, and feces determined. The accumulation of MeHg in kidneys and blood of controls was significantly greater than that of NPX animals. In contrast, MeHg levels in brain and liver of controls were not markedly different from corresponding NPX rats. In all organs examined, accumulation of MeHg increased over the course of exposure, suggesting that urinary and fecal elimination are not sufficient to fully eliminate all mercuric ions. The current findings are important in that the disposition of mercuric ions in rats with normal renal function versus renal insufficiency following exposure to MeHg for a prolonged period differ and need to be taken into account with respect to therapeutic management.

Pregnancy Alters Renal and Blood Burden of Mercury in Females.

Methylmercury (CH3Hg+), a common environmental toxicant, has serious detrimental effects in numerous organ systems. We hypothesize that a significant physiological change, like pregnancy, can alter the disposition and accumulation of mercury. To test this hypothesis, pregnant and non-pregnant female Wistar rats were exposed orally to CH3Hg+. The amount of mercury in blood and total renal mass was significantly lower in pregnant rats than in non-pregnant rats. This finding may be due to expansion of plasma volume in pregnant rats and dilution of mercury, leading to lower levels of mercury in maternal blood and kidneys.

Chronic Kidney Disease and Exposure to Nephrotoxic Metals.

Chronic kidney disease (CKD) is a common progressive disease that is typically characterized by the permanent loss of functional nephrons. As injured nephrons become sclerotic and die, the remaining healthy nephrons undergo numerous structural, molecular, and functional changes in an attempt to compensate for the loss of diseased nephrons. These compensatory changes enable the kidney to maintain fluid and solute homeostasis until approximately 75% of nephrons are lost. As CKD continues to progress, glomerular filtration rate decreases, and remaining nephrons are unable to effectively eliminate metabolic wastes and environmental toxicants from the body. This inability may enhance mortality and/or morbidity of an individual. Environmental toxicants of particular concern are arsenic, cadmium, lead, and mercury. Since these metals are present throughout the environment and exposure to one or more of these metals is unavoidable, it is important that the way in which these metals are handled by target organs in normal and disease states is understood completely.