The effects of commercial 2,4-D herbicide on game fish species: Natural lake water vs. laboratory system water.

Aquatic herbicides with active ingredient 2,4-dichlorophenoxyacteic acid (2,4-D) are commonly used to control and combat aquatic non-native species that cause detrimental impacts including habitat destruction, strained resources among biota, and biodiversity loss. While many (eco)toxicology studies are performed in the laboratory under highly controlled circumstances, these studies may disregard the nuances and disorder that come with the complexity of natural aquatic ecosystems. Therefore, we conducted a series of laboratory experiments using laboratory system water, different lake waters, and different water parameters to determine the effects of ecologically relevant concentrations of 2,4-D (0.00-4.00 ppm a.e.) on the development and survival of two freshwater game species (Sander vitreus and Esox lucius). For 2,4-D exposures using different water sources, there were significant main effects of 2,4-D concentration and water source on walleye embryo and larval survival, however, there was no significant interaction between 2,4-D exposure and water source. For 2,4-D exposures and pH (5-9 pH), there were significant main effects of 2,4-D concentration and pH on walleye and northern pike embryo survival and a significant interaction between 2,4-D exposure and pH. Our results indicate that 2,4-D exposures in controlled laboratory system water can predict similar outcomes as 2,4-D exposures in natural lake water. Moreover, individual water parameters, such as pH, play a significant role in the toxicity of 2,4-D. Taken together, these results suggest that highly controlled laboratory studies are a useful tool for predicting impacts on survival of non-target fish in natural waters, but it is crucial for management agencies to consider individual water sources and specific lake water parameters in herbicide risk assessments to minimize the impacts to non-target organism.

Rapid genome functional annotation pipeline anchored to the house sparrow (Passer domesticus, Linnaeus 1758) genome reannotation.

The house sparrow (Passer domesticus) is a valuable avian model for studying evolutionary genetics, development, neurobiology, physiology, behavior, and ecology, both in laboratory and field-based settings. The current annotation of the P. domesticus genome available at the Ensembl Rapid Release site is primarily focused on gene set building and lacks functional information. In this study, we present the first comprehensive functional reannotation of the P. domesticus genome using intestinal Illumina RNA sequencing (RNA-Seq) libraries. Our revised annotation provides an expanded view of the genome, encompassing 38592 transcripts compared to the current 23574 transcripts in Ensembl. We also predicted 14717 protein-coding genes, achieving 96.4% completeness for Passeriformes lineage BUSCOs. A substantial improvement in this reannotation is the accurate delineation of untranslated region (UTR) sequences. We identified 82.7% and 93.8% of the transcripts containing 5'- and 3'-UTRs, respectively. These UTR annotations are crucial for understanding post-transcriptional regulatory processes. Our findings underscore the advantages of incorporating additional specific RNA-Seq data into genome annotation, particularly when leveraging fast and efficient data processing capabilities. This functional reannotation enhances our understanding of the P. domesticus genome, providing valuable resources for future investigations in various research fields.

Opportunities Lost? Evolutionary Causes and Ecological Consequences of the Absence of Trehalose Digestion in Birds.

AbstractTrehalose is a nonreducing disaccharide that is a primary storage and energy source in prokaryotes, yeasts, fungi, and invertebrates. Vertebrates digest trehalose with the intestinal brush border membrane (BBM) enzyme trehalase. Intestinal trehalase activity is reported to be either very low or absent in several bird species. We assayed trehalase activity in 19 avian species, used proteomic analysis to quantify its abundance in the intestinal BBM, and used analyses of available genomes to detect the presence of the gene that codes for trehalase (Treh). We found no intestinal trehalase activity in birds, trehalase was absent from the proteome of their intestinal BBM, and the gene coding for trehalase was absent in their genomes. Surveys of available transcriptomes support the hypothesis that Treh is absent in birds. The trehalase gene was found in the same conserved syntenic block within the genome of all vertebrates surveyed except birds. Our analysis suggests that Treh was lost in an inversion followed by a reinsertion of a large gene block. This event appears to have taken place after the split between crocodiles and birds and dinosaurs. Birds are unable to digest a common dietary sugar like trehalose because their ancestor lost the trehalase gene. The loss of this gene seems to represent an ecological cost, as insectivorous birds seem to be unable to digest a carbohydrate present in their prey. We also speculate that the paucity of mycophagy in birds is due to the presence of large amounts of this sugar in fungal tissues.

Effects of subchronic exposure to environmentally relevant concentrations of a commercial fluridone formulation on fathead minnows (Pimephales promelas).

Invasive aquatic plants are a widespread problem in United States' waterways, and aquatic herbicide treatments are a common tool used in their management. Fluridone is an active ingredient in aquatic herbicides used globally to control aquatic plants. In order to be effective, fluridone requires a long contact time with plants resulting in extended exposure to non-target organisms. While there has been limited studies exploring the effects of fluridone on non-target aquatic organisms, the effects of subchronic commercial fluridone exposure at concentrations representative of operational use rates for plant management on fish are poorly understood. Therefore, we conducted a series of three exposure experiments using environmentally relevant concentrations on different life stages of the fathead minnow (Pimephales promelas). We exposed fathead minnows to a commercial fluridone formulation, Spritflo®, at environmentally relevant concentrations of 0.00, 3.00, 12.00, 25.00, and 100.00 µg/L. Exposure times included subchronic periods up to 35 days and a trans-generation exposure of 65 days, which is a likely residence time of fluridone when applied for plant management. Following 30 days of fluridone exposure, adult male fish had an increased presence of nuptial tubercules, an indicator of endocrine disruption, and an enlarged liver compared to the control. Additionally, we conducted larval fish behavior experiments and found fluridone exposure negatively affected prey capture ability, locomotion, and position preference. Our findings suggest fluridone treatment concentrations used in aquatic plant management do not directly cause mortality in fathead minnows, though sub-lethal effects observed could cause a decline in biological fitness and pose potential ecological implications.

Larval Exposure to Polychlorinated Biphenyl-126 Led to a Long-Lasting Decrease in Immune Function in Postmetamorphic Juvenile Northern Leopard Frogs, Lithobates pipiens.

Amphibian populations are decreasing worldwide, and pollution is a contributing factor. Polychlorinated biphenyls (PCBs) are a class of persistent organic pollutants known to exert immunotoxicity. To assess impacts of PCBs on frogs, we exposed Lithobates pipiens tadpoles to a diet of PCB-126 (0-5 ng PCB-126/g wet food) through metamorphic climax. Postmetamorphic frogs were immunized with keyhole limpet hemocyanin (KLH); then production of KLH-specific IgY, as well as total IgY and IgM, was measured (Trial I). A second larval study (0 and 7.3 ng PCB-126/g wet food) was performed to investigate whether PCB altered antigenic responses in prometamorphic tadpoles (Gosner Stage 36-39), and to measure the innate immune response of postmetamorphic frogs (Trial II). After larval PCB-126 exposure, both KLH-specific IgY levels and complement activity were reduced. Because postmetamorphic frogs carried a body burden of PCB-126 (2.4 ng/g or less), we wanted to determine whether the effect on immune response was due to larval exposure or to the resulting body burden as frogs. To test this, we reared tadpoles under control conditions (no PCB), and limited PCB exposure to postmetamorphosis only by injecting 2-week-old frogs with 10 ng PCB-126/g (Trial III). The resulting body burden (3.4 ng/g) was similar to that of frogs in Trial I, but we no longer detected suppression of KLH-specific IgY or hemolytic activity. These results suggest life-stage-specific immune responses; however, because we administered PCB-126 differently between trials, it is premature to conclude that these differences are intrinsically life stage dependent, and further study is warranted. Regardless, our study demonstrated a long-lasting effect of larval PCB-126 exposure that persisted through metamorphosis and suppressed frog immunity. Environ Toxicol Chem 2022;41:81-94. © 2021 SETAC.

The Effects of Dietary Polybrominated Diphenyl Ether Exposure and Rearing Temperature on Tadpole Growth, Development, and Their Underlying Processes.

Depression of growth rate due to polybrominated diphenyl ethers (PBDEs) has been documented in birds, mammals, amphibians, and fish at single temperatures. However, the underlying energetic mechanism for this effect and how it might change in relation to changing environmental temperature remain unstudied. We used a simple energy budget to address hypotheses regarding effects of PBDEs on tadpole (Lithobates pipiens) growth: that reductions in growth are linked to increased respiratory costs, reductions in digestive performance, differences in body composition, reductions in food intake, or a combination of these factors. From 18 days postfertilization (dpf) until 42 dpf, tadpoles were exposed dietarily to a pentabromodiphenyl ether mixture (DE-71TM ) at a concentration of 100 ng DE-71/g wet mass under a rearing temperature of either 22 or 27 °C. After 20 days of PBDE exposure, total PBDEs in tadpoles averaged 148.4 ng/g wet mass, with no differences by rearing temperature and approximately 50% higher than in their diet; controls not fed PBDE had levels <1 ng/g. Exposure to PBDE resulted in reductions in body length, mass, and development compared to controls, independent of rearing temperature; PBDE had no effect on measures of body composition, dry matter digestibility, or oxygen consumption. A simple energy budget using data from the present study revealed that a 10% decrease in feeding rate could explain the lower mass gain of tadpoles exposed to PBDE. Growth depression by PBDE could be due to (1) direct inhibition of growth processes by PBDE that indirectly decreases total energy demand and food intake, and (2) direct inhibition of food intake. Future studies to disentangle these possible pathways of PBDE effects are warranted. Environ Toxicol Chem 2021;40:3181-3192. © 2021 SETAC.

Subchronic impacts of 2,4-D herbicide Weedestroy®AM40 on associative learning in juvenile yellow perch (Perca flavescens).

Aquatic herbicides are commonly used to control a wide variety of invasive and nuisance plants. One common active ingredient used in commercial herbicide formulations in Midwestern states is 2,4-dichlorophenoxyacetic acid (2,4-D). Due to the stability of 2,4-D in aquatic environments, many non-target aquatic species experience prolonged exposure throughout critical developmental life stages that can affect essential behaviors. However, the impacts of 2,4-D exposure on learning behaviors in juvenile fish are poorly understood. Therefore, we conducted a series of experiments using a maze environment to determine the effects of a commercial 2,4-D amine salt herbicide formulation (Weedestroy®AM40; WAM40; at 0.00, 0.50, 2.00, and 50.00 mg/L 2,4-D acid equivalent (a.e.)) exposure on juvenile yellow perch's ability to perform a feed associated learning behavior. We observed a significant decrease in the ability of yellow perch to correctly complete the feed associated learning behavior within 200 s when exposed to WAM40 at 2.00 and 50.00 mg/L 2,4-D as compared to controls (p = 0.0002; p < 0.0001, respectively) and within 600 s when exposed to WAM40 at 2.00 and 50.0 mg/L 2,4-D as compared to the controls (p = 0.0107 and p < 0.0001). These data suggest that exposure to 2,4-D in WAM40 can both increase the amount of time it takes for yellow perch to complete a feed associated learning behavior and/or obstruct the behavior altogether. Further experiments showed no significant decreases in locomotion (p > 0.05), hunger motivation (p > 0.05), and a visually guided startle response (p > 0.05), in all treatment groups tested as compared to controls. This suggests that 2,4-D in WAM40 does not inhibit feed associated learning behaviors via interaction with these mechanisms. Altogether, the results indicate that the use of 2,4-D herbicides for weed control in aquatic ecosystems could present risks to cognitive functions that control essential behaviors of yellow perch.

Warmer temperature increases toxicokinetic elimination of PCBs and PBDEs in Northern leopard frog larvae (Lithobates pipiens).

We studied the temperature dependence of accumulation and elimination of two polychlorinated biphenyls (PCBs; PCB-70 and PCB-126) and a commercial mixture of congeners of polybrominated diphenyl ethers (PBDEs; DE-71™)) in Northern leopard frog (Lithobates pipiens) tadpoles. We reared tadpoles at 18, 23, or 27 °C for 5.3 or up to 13.6 weeks (longer at cooler temperature where development is slower) on diets containing the toxicants, each at several different toxicant concentrations, and compared tissue concentrations as a function of food concentration and rearing temperature. Following > 1 month of accumulation, tissue concentrations of all three toxicants in exposed tadpoles were linearly related to dietary concentrations as expected for first order kinetics, with no significant effect of rearing temperature.We also raised free-swimming L. pipiens tadpoles for 14 days on foods containing either toxicant at 18 or 27 °C during an accumulation phase, and then during depuration (declining toxicant) phase of 14 days we provided food without toxicants and measured the decline of toxicants in tadpole tissue. All the congeners were eliminated faster at warmer rearing temperature, as expected. Using Arrhenius' equation, we calculated that the apparent activation energy for elimination of both PCB congeners by tadpoles was 1.21 eV (95% confidence interval 0.6-1.8 eV). We discuss how this value was within the range of estimates for metabolic reactions generally (range 0.2 - 1.2 eV), which might include metabolic pathways for biotransformation and elimination of PCBs. Furthermore, we discuss how the lack of an effect of rearing temperature on tadpole near-steady-state tissue residue levels suggests that faster elimination at the warmer temperature was balanced by faster uptake, which is plausible considering the similar temperature sensitivities (i.e., activation energies) of all these processes. Although interactions between toxicants and temperature can be complex and likely toxicant-dependent, it is plausible that patterns observed in tadpoles might apply to other aquatic organisms. Published data on depuration in 11 fish species eliminating 8 other organic toxicants indicated that they also had similar apparent activation energy for elimination (0.82 ± 0.12 eV; 95% confidence interval 0.56 - 1.08 eV), even though none of those studied toxicants were PCBs or PBDEs. Additional research on toxicant-temperature interactions can help improve our ability to predict toxicant bioaccumulation in warming climate scenarios.

Impacts of subchronic exposure to a commercial 2,4-D herbicide on developmental stages of multiple freshwater fish species.

Invasive, nuisance aquatic species such as Eurasian watermilfoil (Myriophyllum spicatum) are rapidly spreading across the United States. One common active ingredient used to control this invasive species is 2,4-Dichlorophenoxyacetic acid (2,4-D). Application of 2,4-D to aquatic environments typically occurs while many freshwater fish are spawning and due to 2,4-D stability in aquatic environments, many non-target species experience prolonged exposure throughout embryogenesis and larval development. The impacts of 2,4-D exposure on phylogenetically distant fish species is poorly understood. Herein, we investigated the impacts of the 2,4-D commercial herbicide DMA4®IVM on nine freshwater fish species from six different families (four orders) at different points during ontogeny. Each species was exposed to ecologically relevant concentrations of a commercial 2,4-D herbicide (0.05, 0.50, and 2.00 ppm or mg/L 2,4-D a. e.), and effects on morphology, survival, and growth were evaluated. Our results demonstrate that exposure of embryonic and larval fish to ecologically relevant concentrations of a commercial 2,4-D herbicide reduced survival in early developmental stages of six freshwater species that spanned five phylogenetic families and three phylogenetic orders; however, sensitivity to 2,4-D exposure did not show correlation with phylogenetic proximity. Altogether, our results indicate that the use of 2,4-D herbicides in aquatic ecosystems at current recommended concentrations (≤2 ppm whole-lake treatment) could present risk to multiple freshwater fish species survival during early development.

Adaptation of intestinal epithelial hydrolysis and absorption of dietary carbohydrate and protein in mammals and birds.

The small intestine of mammals and birds exhibits fascinating variation across taxa, body size, and life history features such as locomotion and diet. In the intestine's brush border membrane (BBM), hydrolases are more abundant than transporters in both mammals and birds, but there are differences among the groups in abundance of certain hydrolases and possibly in transporters. For example, mammals express two α-glucosidases, sucrase-isomaltase (SI) and maltase glucoamylase (MGAM), whereas songbirds we studied have only SI, and the chicken expresses SI plus another α-glucosidase that functions similarly to MGAM but is not a true ortholog. For intestinal absorption of sugars and amino acids, small fliers rely on a paracellular pathway to a greater extent than do nonflying mammals, which rely more on transporters. Possibly having evolved in fliers as compensation for lower intestinal nominal surface area (NSA), the fliers' reliance on paracellular absorption is supported by their greater villous surface enlargement that leads to more (per cm2 NSA) tight junctions and greater clearance of passively absorbed compounds. To match digestive capacity to nutrient load, a positive relationship is often observed between dietary intake of macronutrients and intestinal activity of the enzymes and transporters of their respective constituents. In enterocytes, rapid, fine-tuned adjustment to high dietary carbohydrate and protein involves rapid, specific correlated increase in activity and abundance of hydrolases and transporters in the BBM and increases in their mRNA.

Rapid and parallel changes in activity and mRNA of intestinal peptidase to match altered dietary protein levels in juvenile house sparrows (Passer domesticus).

Although dietary flexibility in digestive enzyme activity (i.e. reaction rate) is widespread in vertebrates, mechanisms are poorly understood. When laboratory rats are switched to a higher protein diet, the activities of apical intestinal peptidases increase within 15 h, in some cases by rapid increase in enzyme transcription followed by rapid translation and translocation to the intestine's apical, brush-border membrane (BBM). Focusing on aminopeptidase-N (APN), we studied intestinal digestive enzyme flexibility in birds, relying on activity and mRNA data from the same animals. Our model was nestling house sparrows (Passer domesticus), already known to modulate intestinal peptidase activity when switching between lower and higher protein diets. Twenty-four hours after a switch from an adequate, lower protein diet to a higher protein diet, APN activity was increased in both whole intestinal tissue homogenates and in isolated BBM, but not at 12 h post-diet switch. Twenty-four hours after a reverse switch back to the lower protein diet, APN activity was decreased, but not at 12 h post-diet switch. Changes in APN activity in both diet switch experiments were associated with parallel changes in APN mRNA. Although transcriptional changes seem to be an important mechanism underlying dietary modulation of intestinal peptidase in both nestling house sparrows and laboratory rodents, the time course for modulation in nestlings seemed slower (taking approximately twice as long) compared with laboratory rodents. It may be ecologically advantageous if nestlings biochemically restructure their gut in response to a sustained increase in insects and protein intake rather than one or a few lucky insect meals.

Dietary adaptation to high starch involves increased relative abundance of sucrase-isomaltase and its mRNA in nestling house sparrows.

Dietary flexibility in digestive enzyme activity is widespread in vertebrates but mechanisms are poorly understood. When laboratory rats are switched to a higher carbohydrate diet, the activities of the apical intestinal α-glucosidases (AGs) increase within 6-12 h, mainly by rapid increase in enzyme transcription, followed by rapid translation and translocation to the intestine's apical, brush-border membrane (BBM). We performed the first unified study of the overall process in birds, relying on activity, proteomic, and transcriptomic data from the same animals. Our avian model was nestling house sparrows (Passer domesticus), which switch naturally from a low-starch insect diet to a higher starch seed diet and in whom the protein sucrase-isomaltase (SI) is responsible for all maltase and sucrase intestinal activities. Twenty-four hours after the switch to a high-starch diet, SI activity was increased but not at 12 h post diet switch. SI was the only hydrolase increased in the BBM, and its relative abundance and activity were positively correlated. Twenty-four hours after a reverse switch back to the lower starch diet, SI activity was decreased but not at 12 h post diet switch. Parallel changes in SI mRNA relative abundance were associated with the changes in SI activity in both diet-switch experiments, but our data also revealed an apparent diurnal rhythm in SI mRNA. This is the first demonstration that birds may rely on rapid increase in abundance of SI and its mRNA when adjusting to high-starch diet. Although the mechanisms underlying dietary induction of intestinal enzymes seem similar in nestling house sparrows and laboratory rodents, the time course for modulation in nestlings seemed half as fast compared with laboratory rodents. Before undertaking modulation, an opportunistic forager facing limited resources might rely on more extensive or prolonged environmental sampling, because the redesign of the intestine's hydrolytic capacity shortly after just one or a few meals of a new substrate might be a costly mistake.

Seasonal variation in body composition in an Afrotropical passerine bird: increases in pectoral muscle mass are, unexpectedly, associated with lower thermogenic capacity.

Phenotypic flexibility in avian metabolic rates and body composition have been well-studied in high-latitude species, which typically increase basal metabolic rate (BMR) and summit metabolism (Msum) when acclimatized to winter conditions. Patterns of seasonal metabolic acclimatization are more variable in lower-latitude birds that experience milder winters, with fewer studies investigating adjustments in avian organ and muscle masses in the context of metabolic flexibility in these regions. We quantified seasonal variation (summer vs winter) in the masses of organs and muscles frequently associated with changes in BMR (gizzard, intestines and liver) and Msum (heart and pectoral muscles), in white-browed sparrow-weavers (Plocepasser mahali). We also measured pectoral muscle thickness using a portable ultrasound system to determine whether we could non-lethally estimate muscle size. A concurrent study measured seasonal changes in BMR and Msum in the same population of sparrow-weavers, but different individuals. There was no seasonal variation in the dry masses of the gizzard, intestines or liver of sparrow-weavers, and during the same period, BMR did not vary seasonally. We found significantly higher heart (~ 18% higher) and pectoral muscle (~ 9% higher) dry mass during winter, although ultrasound measurements did not detect seasonal changes in pectoral muscle size. Despite winter increases in pectoral muscle mass, Msum was ~ 26% lower in winter compared to summer. To the best of our knowledge, this is the first study to report an increase in avian pectoral muscle mass but a concomitant decrease in thermogenic capacity.

A Fast and Accurate Method to Identify and Quantify Enzymes in Brush-Border Membranes: In Situ Hydrolysis Followed by Nano LC-MS/MS.

A simple method for the identification of brush-border membrane α-glucosidases is described. The proteins were first solubilized and separated in a gel under native, non-denaturing, conditions. The gel was then incubated in substrate solutions (maltose or sucrose), and the product (glucose) exposed in situ by the oxidation of o-dianisidine, which yields a brown-orange color. Nano-liquid chromatography coupled to mass spectrometry analyses of proteins (nano LC-MS/MS) present in the colored bands excised from the gels, was used to confirm the presence of the enzymes. The stain is inexpensive and the procedure permits testing several substrates in the same gel. Once enzymes are identified, their abundance, relative to that of other proteins in the brush border, can be semi-quantified using nano LC-MS/MS.

Duplications and Functional Convergence of Intestinal Carbohydrate-Digesting Enzymes.

Vertebrate diets and digestive physiologies vary tremendously. Although the contribution of ecological and behavioral features to such diversity is well documented, the roles and identities of individual intestinal enzymes shaping digestive traits remain largely unexplored. Here, we show that the sucrase-isomaltase (SI)/maltase-glucoamylase (MGAM) dual enzyme system long assumed to be the conserved disaccharide and starch digestion framework in all vertebrates is absent in many lineages. Our analyses indicate that independent duplications of an ancestral SI gave rise to the mammalian-specific MGAM, as well as to other duplicates in fish and birds. Strikingly, the duplicated avian enzyme exhibits similar activities to MGAM, revealing an unexpected case of functional convergence. Our results highlight digestive enzyme variation as a key uncharacterized component of dietary diversity in vertebrates.

Morphological bases for intestinal paracellular absorption in bats and rodents.

Flying mammals present unique intestinal adaptations, such as lower intestinal surface area than nonflying mammals, and they compensate for this with higher paracellular absorption of glucose. There is no consensus about the mechanistic bases for this physiological phenomenon. The surface area of the small intestine is a key determinant of the absorptive capacity by both the transcellular and the paracellular pathways; thus, information about intestinal surface area and micro-anatomical structure can help explain differences among species in absorptive capacity. In order to elucidate a possible mechanism for the high paracellular nutrient absorption in bats, we performed a comparative analysis of intestinal villi architecture and enterocyte size and number in microchiropterans and rodents. We collected data from intestines of six bat species and five rodent species using hematoxylin and eosin staining and histological measurements. For the analysis we added measurements from published studies employing similar methodology, making in total a comparison of nine species each of rodents and bats. Bats presented shorter intestines than rodents. After correction for body size differences, bats had ~41% less nominal surface area (NSA) than rodents. Villous enhancement of surface area (SEF) was ~64% greater in bats than in rodents, mainly because of longer villi and a greater density of villi in bat intestines. Both taxa exhibited similar enterocyte diameter. Bats exceeded rodents by ~103% in enterocyte density per cm2 NSA, but they do not significantly differ in total number of enterocytes per whole animal. In addition, there is a correlation between SEF and clearance per cm2 NSA of L-arabinose, a nonactively transported paracellular probe. We infer that an increased enterocyte density per cm2 NSA corresponds to increased density of tight junctions per cm2 NSA, which provides a partial mechanistic explanation for understanding the high paracellular absorption observed in bats compared to nonflying mammals.

2,4-Dichlorophenoxyacetic acid containing herbicide impairs essential visually guided behaviors of larval fish.

Aquatic herbicides are used worldwide to eradicate nuisance and invasive plants despite limited knowledge of their toxicity to non-target organisms. 2,4-Dichlorophenoxyacetic acid (2,4-D) is a common active ingredient in commercial herbicide formulations, which triggers plant cell death by mimicking the plant-specific hormone auxin. Application practices of 2,4-D commercial herbicides typically coincide with yearly freshwater fish spawning periods. This practice exposes fish to xenobiotics at their vulnerable larval stages. The full impacts of 2,4-D on larval fish remains poorly understood, and hence, whether it may alter larval survival, larval behavior, fish populations, and ecosystem dynamics. In the present study, we exposed embryonic and larval zebrafish (Danio rerio) to the active ingredient 2,4-D (pure 2,4-D) or a 2,4-D containing commercial herbicide DMA4®IVM (DMA4) and evaluated morphology, survival, behavior, and nervous system function. At 2,4-D concentrations producing no overt morphological defects during embryonic or early larval stages, we observed reduced survival throughout a 21-day larval assay (4-8 ppm DMA4 and 0.75-4 ppm pure 2,4-D). Notably, prey capture, a behavior essential to survival, was reduced in 2,4-D-exposed larval zebrafish (4-8 ppm DMA4 and 0.75-4 ppm pure 2,4-D) and yellow perch (Perca flavescens) (4-20 ppm DMA4). In zebrafish, 8 ppm DMA4 exposure reduced prey capture when exposure was restricted to the period of visual system development. Consistent with these results, larval zebrafish exposed to 8 ppm DMA4 showed reduced neural activity within the optic tectum following prey exposure. Together, our results suggest that 2,4-D alters the development and function of neural circuits underlying vision of larval fish, and thereby reduces visually guided behaviors required for survival.

Physiological and Immune Responses of Free-Living Temperate Birds Provided a Gradient of Food Supplementation.

Food availability might sometimes be unpredictable for wild birds. To alleviate this possible food limitation, millions of households in North America provide food supplementation to bird populations. However, the ecoimmunological impacts of this supplementation on free-living birds are largely unclear. Therefore, we compared immune function and body composition of three groups of free-living black-capped chickadees (Poecile atricapillus) that were provided either constant food supplementation ("supplemented"), interrupted food supplementation ("interrupted"), or no food supplementation ("unsupplemented"). At capture, all three groups had similar body mass and fat scores. All three groups also had similar levels of circulating immunoglobulin Y antibodies and complement lysis ability, two measures of constitutive immune function. Supplemented and interrupted groups mounted a somewhat similar body mass and temperature response to injection with lipopolysaccharide; however, the supplemented group had a higher haptoglobin (acute-phase protein) response to lipopolysaccharide injection compared to the interrupted group. This study demonstrates that birds maintained similar levels of fat despite their level of food supplementation; however, sudden removal of supplemental food might elicit a short-term decline in aspects of immunity. Future studies should investigate how food supplementation might impact induced or adaptive aspects of immune function to add to our understanding of immunology in free-living animals.

Small intestinal epithelial permeability to water-soluble nutrients higher in passerine birds than in rodents.

In the small intestine transcellular and paracellular pathways are implicated in water-soluble nutrient absorption. In small birds the paracellular pathway is quantitatively important while transcellular pathway is much more important in terrestrial mammals. However, there is not a clear understanding of the mechanistic underpinnings of the differences among taxa. This study was aimed to test the hypothesis that paracellular permeability in perfused intestinal segments is higher in passerine birds than rodents. We performed in situ intestinal perfusions on individuals of three species of passerine birds (Passer domesticus, Taeniopygia guttata and Furnarius rufus) and two species of rodents (Mus musculus and Meriones ungiculatus). Using radio-labelled molecules, we measured the uptake of two nutrients absorbed by paracellular and transcellular pathways (L-proline and 3-O-methyl-D-glucose) and one carbohydrate that has no mediated transport (L-arabinose). Birds exhibited ~2 to ~3 times higher L-arabinose clearance per cm2 epithelium than rodents. Moreover, paracellular absorption accounted for proportionally more of 3-O-methyl-D-glucose and L-proline absorption in birds than in rodents. These differences could be explained by differences in intestinal permeability and not by other factors such as increased retention time or higher intestinal nominal surface area. Furthermore, analysis of our results and all other existing data on birds, bats and rodents shows that insectivorous species (one bird, two bats and a rodent) had only 30% of the clearance of L-arabinose of non-insectivorous species. This result may be explained by weaker natural selection for high paracellular permeability in animal- than in plant-consumers. Animal-consumers absorb less sugar and more amino acids, whose smaller molecular size allow them to traverse the paracellular pathway more extensively and faster than glucose.