Space colonization by branching trachea explains the morphospace of a simple respiratory organ.

Branching morphogenesis helps increase the efficiency of gas and liquid transport in many animal organs. Studies in several model organisms have highlighted the molecular and cellular complexity behind branching morphogenesis. To understand this complexity, computational models have been developed with the goal of identifying the "major rules" that globally explain the branching patterns. These models also guide further experimental exploration of the biological processes that execute and maintain these rules. In this paper we introduce the tracheal gills of mayfly (Ephemeroptera) larvae as a model system to study the generation of branched respiratory patterns. First, we describe the gills of the mayfly Cloeon dipterum, and quantitatively characterize the geometry of its branching trachea. We next extend this characterization to those of related species to generate the morphospace of branching patterns. Then, we show how an algorithm based on the "space colonization" concept (SCA) can generate this branching morphospace via growth towards a hypothetical attractor molecule (M). SCA differs from other branch-generating algorithms in that the geometry generated depends to a great extent on its perception of the "external" space available for branching, uses few rules and, importantly, can be easily translated into a realistic "biological patterning algorithm". We identified a gene in the C. dipterum genome (Cd-bnl) that is orthologous to the fibroblast growth factor branchless (bnl), which stimulates growth and branching of embryonic trachea in Drosophila. In C. dipterum, this gene is expressed in the gill margins and areas of finer tracheolar branching from thicker trachea. Thus, Cd-bnl may perform the function of M in our model. Finally, we discuss this general mechanism in the context of other branching pattern-generating algorithms.

Can't take the heat: Temperature-enhanced toxicity in the mayfly Isonychia bicolor exposed to the neonicotinoid insecticide imidacloprid.

Neonicotinoid insecticide usage has increased globally in recent decades. Neonicotinoids, such as imidacloprid, are potent insect neurotoxicants that may pose a threat to non-target aquatic organisms, such as aquatic insects. In nature, insects typically live in thermally fluctuating conditions, which may significantly alter both contaminant exposures and affects. Here we investigate the relationship between temperature and time-to-effect for imidacloprid toxicity with the aquatic insect Isonychia bicolor, a lotic mayfly. Additionally, we examined the mechanisms driving temperature-enhanced toxicity including metabolic rate, imidacloprid uptake rate, and tissue bioconcentration. Experiments included acute toxicity tests utilizing sublethal endpoints and mortality, as well as respirometry and radiotracer assays with [(14)C] imidacloprid. Further, we conducted additional uptake experiments with a suite of aquatic invertebrates (including I. bicolor, Neocloeon triangulifer, Macaffertium modestum, Pteronarcys proteus, Acroneuria carolinensis, and Pleuroceridae sp) to confirm and contextualize our findings from initial experiments. The 96h EC50 (immobility) for I. bicolor at 15°C was 5.81µg/L which was approximately 3.2 fold lower than concentrations associated with 50% mortality. Assays examining the impact of temperature were conducted at 15, 18, 21, and 24°C and demonstrated that time-to-effect for sublethal impairment and immobility was significantly decreased with increasing temperature. Uptake experiments with [(14)C] imidacloprid revealed that initial uptake rates were significantly increased with increasing temperature for I. bicolor, as were oxygen consumption rates. Further, in the separate experiment with multiple species across temperatures 15, 20, and 25°C, we found that all the aquatic insects tested had significantly increased imidacloprid uptake with increasing temperatures, with N. triangulifer accumulating the most imidacloprid on a mass-specific basis. Our acute toxicity results highlight the importance of evaluating sublethal endpoints, as profound impairments of motor function were evident far before mortality. Further, we demonstrate that temperature is a powerful modulator of sublethal toxicity within a range of environmentally relevant temperatures, impacting both uptake rates and metabolic rates of I. bicolor. Finally, we show that temperature alters imidacloprid uptake across a range of species, highlighting the physiological variation present within aquatic invertebrate communities and the challenge associated with relying solely on surrogate species. Taken together, this research points to the need to consider the role of temperature in toxicity assessments.

Comparative sodium transport patterns provide clues for understanding salinity and metal responses in aquatic insects.

The importance of insects in freshwater ecosystems has led to their extensive use in ecological monitoring programs. As freshwater systems are increasingly challenged by salinization and metal contamination, it is important to understand fundamental aspects of aquatic insect physiology (e.g., osmoregulatory processes) that contribute to insect responses to these stressors. Here we compared the uptake dynamics of Na as NaCl, NaHCO3 and Na2SO4 in the caddisfly Hydropsyche betteni across a range of Na concentrations (0.06-15.22 mM) encompassing the vast majority of North American freshwater ecosystems. Sulfate as the major anion resulted in decreased Na uptake rates relative to the chloride and bicarbonate salts. A comparison of Na (as NaHCO3) turnover rates in the caddisfly Hydropsyche sparna and the mayfly Maccaffertium sp. revealed different patterns in the 2 species. Both species appeared to tightly regulate their whole body sodium concentrations (at ∼47±1.8 µmol/g wet wt) across a range of Na concentrations (0.06-15.22 mM) over 7 days. However, at the highest Na concentration (15.22 mM), Na uptake rates in H. sparna (419.1 µM Na g(-1) hr(-1) wet wt) appeared close to saturation while Na uptake rates in Maccaffertium sp. were considerably faster (715 g µM Na g(-1) hr(-1) wet wt) and appeared to not be close to saturation. Na efflux studies in H. sparna revealed that loss rates are commensurate with uptake rates and are responsive to changes in water Na concentrations. A comparison of Na uptake rates (at 0.57 mM Na) across 9 species representing 4 major orders (Ephemeroptera, Plecoptera, Trichoptera and Diptera) demonstrated profound physiological differences across species after accounting for the influence of body weight. Faster Na uptake rates were associated with species described as being sensitive to salinization in field studies. The metals silver (Ag) and copper (Cu), known to be antagonistic to Na uptake in other aquatic taxa did not generally exhibit this effect in aquatic insects. Ag only reduced Na uptake at extremely high concentrations, while Cu generally stimulated Na uptake in aquatic insects, rather than suppress it. These results help explain the lack of insect responses to dissolved metal exposures in traditional toxicity testing and highlight the need to better understand fundamental physiological processes in this ecologically important faunal group.

Dietary (periphyton) and aqueous Zn bioaccumulation dynamics in the mayfly Centroptilum triangulifer.

Diet is often the predominant route of trace metal exposure in aquatic insects. In freshwater ecosystems, periphyton serves as a primary source of food to many aquatic insects and is a major sink for trace metals. We investigated the bioconcentration of the essential metal Zn by periphyton using (65)Zn as a radiotracer. At relatively low dissolved concentrations (2-20 µg L(-1)), non steady state Zn bioconcentration by periphyton averaged 6,099 ± 2,430-fold, with much of the variability determined by loading regime (number of renewals and duration of exposures). Labeled periphyton was used as a food source for dietary accumulation studies with the mayfly Centroptilum triangulifer. After 29 days, larvae concentrated Zn 19-, 16- and 17-fold relative to dietary Zn concentrations of 8.1, 43.2 and 82.3 µg g(-1) (dry weight), respectively. Adults from that same cohort only concentrated Zn 8-, 3- and 3- fold relative to those same dietary concentrations, revealing that mayflies lose significant Zn prior to reaching adulthood. Anecdotal evidence suggests that this loss occurs prior to emergence to the subimago, as negligible Zn was found in the subimago to imago exuvium. Across a range of adult tissue concentrations, maternal transfer consistently averaged 26.7 %. Uptake (k(u), 0.26 L g(-1 )d(-1)) and efflux rate constants (k(e), 0.001-0.007 d(-1)) were measured and assimilation efficiencies from dietary Zn concentrations of 4.9 and 59.7 µg Zn g(-1) were estimated to be 88 ± 4 % and 64 ± 15 %, respectively. Both life cycle and biodynamic modeling approaches point towards diet being the primary route of Zn bioaccumulation in this mayfly.

Food rationing affects dietary selenium bioaccumulation and life cycle performance in the mayfly Centroptilum triangulifer.

Selenium effects in nature are mediated by the relatively large bioconcentration of aqueous Se by primary producers and smaller, yet critical, dietary transfers to primary consumers. These basal processes are then propagated through food webs to higher trophic levels. Here we quantified the movement of dissolved Se (as selenite) to periphyton, and used the resultant periphyton as a food source for conducting full life-cycle dietary Se exposures to the mayfly Centroptilum triangulifer. Periphyton bioconcentrated Se ~2,200-fold from solution in a log-linear fashion over dissolved Se concentrations ranging from 1.1 to 23.1 µg L(-1). We examined the influence of two feeding ration levels (1x and 2x) on trophic transfer, tissue Se concentrations, maternal transfer, and functional endpoints of mayfly performance. Mayflies fed a lesser ration (1x) displayed greater trophic transfer factors (mean TTF, 2.8 ± 0.4) than mayflies fed 2x rations (mean TTF, 1.1 ± 0.3). In 1x exposures, mayflies exhibited significant (p < 0.05) reductions in survivorship and total body mass at dietary [Se] ≥ 11.9 µg g(-1), reduced total fecundity at ≥ 4.2 µg g(-1), and delayed development at ≥ 27.2 µg g(-1). Mayflies fed a greater ration (2x) displayed reduced tissue Se concentrations (apparently via growth dilution) relative to 1x mayflies, with no significant effects on performance. These results suggest that the influence of Se on mayfly performance in nature may be tied to food resource availability and quality. Furthermore, nutritional status is an important consideration when applying laboratory derived estimates of toxicity to risk assessments for wild populations.

Using biodynamic models to reconcile differences between laboratory toxicity tests and field biomonitoring with aquatic insects.

Aquatic insects often dominate lotic ecosystems, yet these organisms are under-represented in trace metal toxicity databases. Furthermore, toxicity data for aquatic insects do not appear to reflect their actual sensitivities to metals in nature, because the concentrations required to elicit toxicity in the laboratory are considerably higher than those found to impact insect communities in the field. New approaches are therefore needed to better understand how and why insects are differentially susceptible to metal exposures. Biodynamic modeling is a powerful tool for understanding interspecific differences in trace metal bioaccumulation. Because bioaccumulation alone does not necessarily correlate with toxicity, we combined biokinetic parameters associated with dissolved cadmium exposures with studies of the subcellular compartmentalization of accumulated Cd. This combination of physiological traits allowed us to make predictions of susceptibility differences to dissolved Cd in three aquatic insect taxa: Ephemerella excrucians, Rhithrogena morrisoni, and Rhyacophila sp. We compared these predictions with long-term field monitoring data and toxicity tests with closely related taxa: Ephemerella infrequens, Rhithrogena hageni, and Rhyacophila brunea. Kinetic parameters allowed us to estimate steady-state concentrations, the time required to reach steady state, and the concentrations of Cd projected to be in potentially toxic compartments for different species. Species-specific physiological traits identified using biodynamic models provided a means for better understanding why toxicity assays with insects have failed to provide meaningful estimates for metal concentrations that would be expected to be protective in nature.

Roles of uptake, biotransformation, and target site sensitivity in determining the differential toxicity of chlorpyrifos to second to fourth instar Chironomous riparius (Meigen).

Early life stages of aquatic organisms tend to be more sensitive to various chemical contaminants than later life stages. This research attempted to identify the key biological factors that determined sensitivity differences among life stages of the aquatic insect Chironomous riparius. Specifically, second to fourth instar larvae were exposed in vivo to both low and high waterborne concentrations of chlorpyrifos to examine differences in accumulation rates, chlorpyrifos biotransformation, and overall sensitivity among instars. In vitro acetylcholinesterase (AChE) assays were performed with chlorpyrifos and the metabolite, chlorpyrifos-oxon, to investigate potential target site sensitivity differences among instars. Earlier instars accumulated chlorpyrifos more rapidly than later instars. There were no major differences among instars in the biotransformation rates of chlorpyrifos to the more polar metabolites, chlorpyrifos-oxon, and chlorpyridinol (TCP). Homogenate AChE activities from second to fourth instar larvae were refractory to chlorpyrifos, even at high concentrations. In contrast, homogenate AChE activities were responsive in a dose-dependent manner to chlorpyrifos-oxon. In general, it appeared that chlorpyrifos sensitivity differences among second to fourth instar C. riparius were largely determined by differences in uptake rates. In terms of AChE depression, fourth instar homogenates were more sensitive to chlorpyrifos and chlorpyrifos-oxon than earlier instars. However, basal AChE activity in fourth instar larvae was significantly higher than basal AChE activity in second to third instar larvae, which could potentially offset the apparent increased sensitivity to the oxon.

Phase III interlaboratory study of FETAX, Part 2: interlaboratory validation of an exogenous metabolic activation system for frog embryo teratogenesis assay--Xenopus (FETAX).

Interlaboratory validation of an exogenous metabolic activation system (MAS) developed for the alternative, short-term developmental toxicity bioassay, Frog Embryo Teratogenesis Assay-Xenopus (FETAX) was performed with cyclophosphamide and caffeine. Seven study groups within six separate laboratories participated in the study in which three definitive concentration-response experiments were performed with and without the MAS in a side-by-side format for each chemical. Since both chemicals had been previously tested in FETAX, the test concentrations were provided to each laboratory prior to testing. Interlaboratory coefficient of variation (CV) values for unactivated cyclophosphamide (no MAS) were 15%, 15%, 29%, and 25% for the 96-hr LC50, 96-hr EC50 (malformation), Minimum Concentration to Inhibit Growth (MCIG), and Teratogenic Index (TI) values, respectively. Addition of the MAS increased the CV values of each endpoint at least 3.9-fold. Interlaboratory CV values for unactivated caffeine were 31%, 18%, 31%, and 46% for the 96-hr LC50, 96-hr EC50 (malformation), MCIG, and TI values, respectively. Addition of the MAS decreased the CV values of each respective endpoint by at least 1.6-fold. Results indicated that bioactivated toxicants may be prone to greater variability in response amongst laboratories than compounds, which are detoxified. Even though more variability was noted with activated cyclophosphamide, results were within interlaboratory variation expected for other aquatic-based bioassays. Thus, results from these studies warrant the continued use and further refinement of FETAX for alternative developmental toxicity assessment.

Initial interlaboratory validation study of FETAX: phase I testing.

An interlaboratory validation study was undertaken to evaluate the repeatability and reliability of the Frog Embryo Teratogenesis Assay-Xenopus (FETAX), which is a whole embryo developmental toxicity screening assay. A three-phase experimental program with seven participants was carried out. Phase I was a training and protocol evaluation phase where the identity of the three test materials was known. Hydroxyurea, isoniazid and 6-aminonicotinamide were tested in Phase I. Because the chemicals has been tested previously in FETAX, the same concentrations needed to establish the 96-h median lethal concentration (LC50) and the concentration inducing malformations in 50% of the surviving embryos (EC50) were used by all laboratories. The results of Phase I are presented in this report, and FETAX has proved to be as repeatable and reliable as many other bioassays. Some excess variation was observed in individual laboratories. Some of this variation may have been due to training difficulties. One change in protocol design necessitated by this study was the use of 6-aminonicotinamide as a reference toxicant. While 6-aminonicotinamide provided excellent concentration-response data in most laboratories, the protocol was written too strictly based on historical FETAX data. Phases II and III are currently in progress.