The mayfly Neocloeon triangulifer senses decreasing oxygen availability (PO2) and responds by reducing ion uptake and altering gene expression.

Oxygen availability is central to the energetic budget of aquatic animals and may vary naturally and/or in response to anthropogenic activities. Yet, we know little about how oxygen availability is linked to fundamental processes such as ion transport in aquatic insects. We hypothesized and observed that ion (22Na and 35SO4) uptake would be significantly decreased at O2 partial pressures below the mean Pcrit (5.4 kPa) where metabolic rates (MO2) are compromised, and ATP production is limited. However, we were surprised to observe marked reductions in ion uptake at oxygen partial pressures well above the Pcrit, where MO2 was stable. For example, SO4 uptake decreased by 51% at 11.7kPa, and 82% at the Pcrit (5.4kPa) while Na uptake decreased by 19% at 11.7kPa, and 60% at the Pcrit. Nymphs held for longer time periods at reduced PO2 exhibited stronger reductions in ion uptake rates. Fluids from whole body homogenates exhibited a 29% decrease in osmolality in the most hypoxic condition. The differential expression of atypical guanylyl cyclase (gcy-88e) in response to changing PO2 conditions provides evidence for its potential role as an oxygen sensor. Several ion transport genes (e.g., chloride channel and sodium-potassium ATPase) and hypoxia-associated genes (e.g., ldh and egl-9) were also impacted by decreased oxygen availability. Together, our work suggests that N. triangulifer can sense decreased oxygen availability and perhaps conserves energy accordingly, even when MO2 is not impacted.

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.

Respirometry reveals major lineage-based differences in the energetics of osmoregulation in aquatic invertebrates.

All freshwater organisms are challenged to control their internal balance of water and ions in strongly hypotonic environments. We compared the influence of external salinity on the oxygen consumption rates (MO2) of three species of freshwater insects, one snail and two crustaceans. Consistent with available literature, we found a clear decrease in MO2 with increasing salinity in the snail Elimia sp. and crustaceans Hyalella azteca and Gammarus pulex (r5=-0.90, P=0.03). However, we show here for the first time that metabolic rate was unchanged by salinity in the aquatic insects, whereas ion transport rates were positively correlated with higher salinities. In contrast, when we examined the ionic influx rates in the freshwater snail and crustaceans, we found that Ca uptake rates were highest under the most dilute conditions, while Na uptake rates increased with salinity. In G. pulex exposed to a serially diluted ion matrix, Ca uptake rates were positively associated with MO2 (r5=-0.93, P=0.02). This positive association between Ca uptake rate and MO2 was also observed when conductivity was held constant but Ca concentration was manipulated (1.7-17.3 mg Ca l-1) (r5=0.94, P=0.05). This finding potentially implicates the cost of calcium uptake as a driver of increased metabolic rate under dilute conditions in organisms with calcified exoskeletons and suggests major phyletic differences in osmoregulatory physiology. Freshwater insects may be energetically challenged by higher salinities, while lower salinities may be more challenging for other freshwater taxa.

The acclimatory response of the mayfly Neocloeon triangulifer to dilute conditions is linked to the plasticity of sodium transport.

Relative to a growing body of knowledge about the negative consequences of freshwater salinization, little is known about how aquatic insects respond to progressively ion-poor conditions. Here, we examined life-history and physiological acclimation in Neocloeon triangulifer by rearing nymphs from 1-day post-egg hatch to adulthood across a gradient of decreasing Na concentrations (15, 8, 4, 2 and 1 mg l-1 Na). We found no significant changes in survival, growth, development time and whole-body Na content across these treatments. Radiotracer data revealed that nymphs acclimated to their dilute exposures by increasing their rates of Na uptake and were able to maintain a relatively narrow range of uptake rates (±s.e.m.) of 38.5 ± 4.2 µg Na g-1 h-1 across all treatments. By contrast, the Na uptake rates observed in naive nymphs were much more concentration dependent. This acclimatory response is partially explained by differences in ionocyte counts on the gills of nymphs reared under different salinities. Acclimated nymphs were surprisingly less retentive of their sodium composition when subjected to deionized water challenge. By contrasting our findings with a previous N. triangulifer salinity acclimation study, we show a physiological affinity for dilute conditions in this emerging mayfly model.

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.