Whole-body Homogenates Research Articles

Development of Liquid Chromatography-Tandem Mass Spectrometry Analytical Methods for the Study of Whole-Body, Head, and Trunk Uptake and Elimination of Methamphetamine in 5-Day Postfertilization Zebrafish Larvae Using the Quick, Easy, Cheap, Effective, Rugged, and Safe Technique.

Synthetic cathinones are drugs of abuse substituted for amphetamine-like stimulant drugs such as methamphetamine. In this study, methamphetamine was studied as a prototypical amphetamine-like drug as a first step toward establishing methods to study this entire drug class. The internal concentration of methamphetamine in zebrafish larvae was determined using matrix-matched calibration along with extraction and purification of samples using the quick, easy, cheap, effective, rugged, and safe technique in liquid chromatography-tandem mass spectrometry. Whole-body and head/trunk uptake and elimination in 5-day postfertilization zebrafish larvae were determined. A gradient method was developed using 5 mM ammonium formate with 0.1% formic acid and methanol with 0.1% formic acid as mobile phases, 10 min of total run time, and a 0.3 mL/min flow rate. The limit of quantification was 60 ng/mL, linearity with r2 = 0.9991, and recovery values from 92% to 120%. The internal concentration of methamphetamine was quantifiable in whole-body homogenates within 15 min of uptake analysis. The internal concentration increased with time, whereas a biphasic elimination pattern was shown. With increasing length of exposure, a higher accumulation of drugs was found in the head than in the trunk.

Protein from whole-body crayfish homogenate may be a high supplier of leucine or branched-chain amino acids - A call for validation on genus Procambarus sp.

Essential proteinogenic branched-chain amino acids (BCAA), particularly leucine (Leu) have been investigated for their role in enhancing human myofibrillar protein synthesis and biomedical research on tumor models. However, only a few protein sources in our current food system have high enough BCAA or Leu coefficients (% of total amino acids) to be considered as supplements for food, sport, or biomedical research. Mostly dairy-sourced proteins such as casein and whey or rarely plant source such as maize gluten are typically regarded as the gold standards. This study hypothesized that protein isolates derived from the whole-body homogenate (including the chitinous exoskeleton) of procambarid crayfish might exhibit unusually high BCAA and Leu content. The study provides open-access data on the amino acid compositions of two procambarid crayfish (Procambarus virginalis and P. clarkii), as well as a comparison with casein. The mentioned crayfish species could offer 6.36-7.39g Leu 100g-1 dry matter (at 43-48% protein only). Crayfish whole-body protein isolates exhibit a Leu coefficient (18.41±2.51% of total amino acids) and a BCAA coefficient (28.76±2.39% of total amino acids), which is comparable to or higher than of casein (Leu coefficient 8.65±0.08%; BCAA coefficient 20.03±0.73%). However, it is important to interpret these results with caution, due to the challenges associated with leucine and isoleucine separation, as well as potential interactions within the sample matrices. Hence, international validation of these findings is recommended. NOVELTY STATEMENT: Protein isolates from whole-body homogenate (including chitinous exoskeleton) of P. virginalis and/or P. clarkii are hypothesized to be dense in BCAA and Leu. For potential use in biomedical research or as additives in supplements for BCAA and Leu.

Housing Conditions Affect Adult Zebrafish (Danio rerio) Behavior but Not Their Physiological Status.

Zebrafish is a valuable model for neuroscience research, but the housing conditions to which it is exposed daily may be impairing its welfare status. The use of environmental enrichment and the refinement of methodology for cortisol measurement could reduce stress, improving its welfare and its suitability as an animal model used in stress research. Thus, this study aimed to evaluate (I) the influence of different housing conditions on zebrafish physiology and behavior, and (II) skin mucus potential for cortisol measurement in adult zebrafish. For this, AB zebrafish were raised under barren or enriched (PVC pipes and gravel image) environmental conditions. After 6 months, their behavior was assessed by different behavioral paradigms (shoaling, white-black box test, and novel tank). The physiological response was also evaluated through cortisol levels (whole-body homogenates and skin mucus) and brain oxidative stress markers. The results revealed that enriched-housed fish had an increased nearest neighbors' distance and reduced activity. However, no effect on body length or stress biomarkers was observed; whole-body and skin mucus cortisol levels had the same profile between groups. In conclusion, this study highlights the skin mucus potential as a matrix for cortisol quantification, and how housing conditions could influence the data in future studies.

Temperature alters the oxidative and metabolic biomarkers and expression of environmental stress-related genes in chocolate mahseer (Neolissochilus hexagonolepis).

Long-term acclimation temperature effects on biomarkers of oxidative stress, metabolic stress, expression of heat shock proteins (Hsps), and warm-temperature acclimation related 65-kDa protein (Wap65) were evaluated in the threatened chocolate mahseer (Neolissochilus hexagonolepis). Fifteen-day-old larvae were acclimated to different water temperatures (15, 19, 23-control group, 27, and 31°C) for 60days prior to the sampling for quantification of mRNA, enzyme, nitric oxide, and malondialdehyde (MDA) content. Acclimation to 31°C increased the basal mRNA level of glutathione S-transferase alpha 1 (GSTa1), and activities of catalase (CAT), glutathione reductase (GR), and GST enzymes and but downregulated the expression of superoxide dismutase 1 (SOD1) in the whole-body homogenate. Other antioxidant genes, i.e., CAT and GPx1a, were unaffected at 31°C, and nitric oxide (NO) concentration was significantly lower. In contrast, fish acclimated to 15°C showed an upregulated transcript level of all the antioxidant genes and no significant difference in the CAT, GR, and GST enzymes. Activities of the metabolic enzymes, aspartate transaminase (AST) and alanine transaminase (ALT), were significantly lower at 15°C. The expression of Hsp47 was upregulated at both 15 and 31°C groups, whereas Hsp70 was elevated at 27 and 31°C groups. Wap65-1 transcription did not show significant variation in treatment groups compared to control. Fish in the high (31°C) and low-temperature (15°C) acclimation groups were capable of maintaining oxidative stress by modulating their antioxidant transcripts, enzymes, and Hsps.

Influence of Calcium Resonance-Tuned Low-Frequency Magnetic Fields on Daphnia magna

A biophysical model for calculating the effective parameters of low-frequency magnetic fields was developed by Lednev based on summarized empirical data. According to this model, calcium ions as enzyme cofactors can be the primary target of low-frequency magnetic fields with different parameters tuned to calcium resonance. However, the effects of calcium-resonant combinations of static and alternating magnetic fields that correspond to Lednev's model and differ by order in frequency and intensity were not studied. It does not allow for confidently discussing the primary targets of low-frequency magnetic fields in terms of the magnetic influence on ions-enzyme cofactors. To clarify this issue, we examined the response of freshwater crustaceans Daphnia magna to the impact of combinations of magnetic fields targeted to calcium ions in enzymes according to Lednev's model that differ in order of magnitude. Life-history traits and biochemical parameters were evaluated. Exposure of daphnids to both combinations of magnetic fields led to a long-term delay of the first brood release, an increase in the brood size, a decrease in the number of broods, and the period between broods. The amylolytic activity, proteolytic activity, and sucrase activity significantly decreased in whole-body homogenates of crustaceans in response to both combinations of magnetic fields. The similarity in the sets of revealed effects assumes that different magnetic fields tuned to calcium ions in biomolecules can affect the same primary molecular target. The results suggest that the low-frequency magnetic fields with parameters corresponding to Lednev's model of interaction between biological molecules and ions can remain effective with a significant decrease in the static magnetic background.

Establishment of a lipid metabolism disorder model in ApoEb mutant zebrafish

Background and aimsApoEb is a zebrafish homologous to mammalian ApoE, whose deficiency would lead to lipid metabolism disorders (LMDs) like atherosclerosis. We attempted to knock out the zebrafish ApoEb, then establish a zebrafish model with LMD. MethodsApoEb was knocked out using the CRISPR/Cas9 system, and the accumulation of lipids was confirmed by Oil Red O staining, confocal imaging, and lipid measurements. The lipid-lowering effects of simvastatin (SIM), ezetimibe (EZE) and Xuezhikang (XZK), an extract derived from red yeast rice, were evaluated through in vivo imaging in zebrafish larvae. ResultsIn the ApoEb mutant, significant vascular lipid deposition occurred, and lipid measurement performed in the whole-body homogenate of larvae and adult plasma showed significantly increased lipid levels. SIM, EZE and XZK apparently relieved hyperlipidemia in ApoEb mutants, and XZK had a significant inhibitory effect on the recruitment of neutrophils and macrophages. ConclusionsIn this study, an LMD model has been established in ApoEb mutant zebrafish. We suggest that this versatile model could be applied in studying hypercholesterolemia and related vascular pathology in the context of early atherosclerosis, as well as the physiological function of ApoE.

Assessment of the perturbation induced by chitin synthesis inhibitors lufenuron, flufenoxuron and hexaflumuron in the house fly, Musca domestica vicina (Diptera: Muscidae)

BackgroundThe larvicidal and biochemical effects of chitin synthesis inhibitors (CSIs), namely lufenuron, flufenoxuron and hexaflumuron against the newly molted penultimate instar larvae of the house fly Musca domestica, were investigated.MethodsDifferent concentrations from each tested compound were applied on forty individuals of M. domestica 2nd instar larvae. Four replicates were used for each concentration.ResultsThe recorded LC25 and LC75 values were (166.11, 68.33 and 56.43 ppm) and (732.33, 283.02 and 248.45 ppm) for lufenuron, flufenoxuron and hexaflumuron, respectively. The results showed significant (P < 0.05) increase of mortality in larvae treated with different tested CSIs compounds. Mortality was greater in larvae treated by hexaflumuron than lufenuron and flufenoxuron. The main metabolites were tested in the larval whole-body tissue homogenate and findings could be summarized as follows: tested concentrations of CSIs (a) predominantly reduced the total carbohydrate, protein, lipid and cholesterol content at certain ages tested. (b) Disturbed the total carbohydrate content particularly for larvae treated with LC75 concentration of hexaflumuron. (c) Exerted the protein and lipid profiles and this effect was much more pronounced in larvae treated with hexaflumuron. (d) Reduced the quantitative cholesterol content and this reduction was found to be increased with development.ConclusionsTested CSIs in particular hexaflumuron showed remarkable larval toxicity and reduced the main metabolites content in the larval whole-body tissue homogenate of the house fly, M. domestica.

Effect of salt and brine-beet juice de-icer on osmoregulatory physiology of the freshwater amphipod Hyalella azteca (Saussure, 1858) (Amphipoda: Hyalellidae)

Abstract The anthropogenic salinization of freshwater is concerning because it can negatively impact the success and survival of freshwater animals. Road salt (NaCl) in cold climates contributes to salinization and organic based de-icers have been developed to mitigate the effects of NaCl on freshwater. One of these de-icers is sugar beet juice, and few studies have examined its effects on freshwater animals. We exposed Hyalella azteca (Saussure, 1858), a freshwater amphipod, to different concentrations of NaCl (salt-contaminated water or SCW) and a NaCl brine and beet-juice mixture used as a de-icing product (brine-beet juice de-icer, BBJD). The LC50 of NaCl on H. azteca was 12.8 g l–1 and for BBJD was 4.6% (which at that percentage contained ~ 4.2 g l–1 Na+). Sub-lethal doses of SCW elevated hemolymph Na+ and BBJD exposure resulted in elevated K+ concentration as well as acidification of the hemolymph. Both Na+/K+ ATPase (NKA) and V-type H+-ATPase (VA) were localized to the coxal gills, whereas only NKA was found in the sternal gills. There was a qualitatively apparent decrease in expression of NKA in the gills of SCW-treated amphipods. NKA and VA expression qualitatively increased with BBJD exposure in the gut. The NKA and VA activity in whole-body homogenates was lower in BBJD and SCW. Results show that Hyalella azteca responds to SCW and BBJD by altering parameters of ionoregulatory physiology in different ways.

Spatially Mapping the Baseline and Bisphenol-A Exposed Daphnia magna Lipidome Using Desorption Electrospray Ionization-Mass Spectrometry.

Untargeted lipidomics has previously been applied to the study of daphnids and the discovery of biomarkers that are indicative of toxicity. Typically, liquid chromatography—mass spectrometry is used to measure the changes in lipid abundance in whole-body homogenates of daphnids, each only ca. 3 mm in length which limits any biochemical interpretation of site-specific toxicity. Here, we applied mass spectrometry imaging of Daphnia magna to combine untargeted lipidomics with spatial resolution to map the molecular perturbations to defined anatomical regions. A desorption electrospray ionization—mass spectrometry (DESI-MS) method was optimized and applied to tissue sections of daphnids exposed to bisphenol-A (BPA) compared to unexposed controls, generating an untargeted mass spectrum at each pixel (35 µm2/pixel) within each section. First, unique lipid profiles from distinct tissue types were identified in whole-body daphnids using principal component analysis, specifically distinguishing appendages, eggs, eye, and gut. Second, changes in the lipidome were mapped over four stages of normal egg development and then the effect of BPA exposure on the egg lipidome was characterized. The primary perturbations to the lipidome were annotated as triacylglycerides and phosphatidylcholine, and the distributions of the individual lipid species within these classes were visualized in whole-body D. magna sections as ion images. Using an optimized DESI-MS workflow, the first ion images of D. magna tissue sections were generated, mapping both their baseline and BPA-perturbed lipidomes.

Salinity affects growth performance, physiology, immune responses and temperature resistance in striped catfish (Pangasianodon hypophthalmus) during its early life stages.

In this study, striped catfish larvae were gradually exposed to the increase of different salinities, and then they reached the levels of 0, 5, 10, 15, and 20psu after 10days, followed by heat shock at 39°C to determine stress tolerance. After the 10-day experiment, the survival rate of fish exposed to the 20psu treatment was only 28.6 ± 4%, significantly lower than that of the other treatments. The results showed that the osmolality of the whole-body (WB) homogenate was gradually and significantly increased with salinity elevation, except in fish exposed to freshwater and 5psu treatments, while there were no significant changes in WB Na+/K+-ATPase activity. Digestive enzymatic activities, i.e., pepsin, α-amylase, alkaline phosphatase, and leucine alanine peptidase (leu-ala) generally increased with salinity, but not aminopeptidase and trypsin. Lysozyme and peroxidase activities increased in fish larvae exposed to 15 and 20psu. These increases proportionally improved growth performance, with the lowest and the highest final weights observed in fish reared at 0 psu (0.08 ± 0.03g/larvae) and 20 psu (0.11 ± 0.02g/larvae), respectively, although the average growth recorded at 20 psu could be biased by the high mortality in this group. Occurrence of skeleton deformities, such as in caudal vertebrae and branchiostegal rays, was significantly higher in fish exposed to the higher osmotic conditions (15.0 ± 1.2% and 10.3 ± 2.1% respectively at 0 psu vs. 31.0 ± 2.9% and 49.0 ± 5.6%, respectively at 15 psu). After the 12.5-h heat shock, survival rates significantly differed between treatments with the highest survival observed in fish submitted to 5psu (68.9%), followed by those exposed to 0 (27%) and 10 (20%) while all fish died at 15 psu. These findings suggest that the striped catfish larvae could be reared in salinity up to 5 to 10 psu with a higher survival and tolerance to thermal stress when compared to fish maintained in freshwater.

Antioxidant responses and okadaic acid accumulation in Laeonereis acuta (Annelida) exposed to the harmful dinoflagellate Prorocentrum cf. lima

We evaluated the accumulation of okadaic acid (OA), a diarrhetic toxin, and the antioxidant responses in the marine annelid Laeonereis acuta exposed to the benthic toxigenic dinoflagellate Prorocentrum cf. lima. Nontoxic Tetraselmis sp. was used as a control diet. Living cells of the two algae were supplied as food to animals kept in agar medium for 72 h. To assess the significance of the observed effects, our experimental design treated the algal species (diet), algal cell densities, and exposure time as fixed factors. Responses of the organisms were assessed through oxidative stress biomarkers (glutathione-S-transferase [GST], catalase [CAT], reduced glutathione [GSH] and lipid peroxidation [LPO]). Toxin accumulation was measured by LC-MS/MS in whole-body homogenates after 12, 24 and 72 h of exposure. Worms exposed to the toxigenic dinoflagellate gradually accumulated OA, with toxin levels directly related to the cell density of Prorocentrum cf. lima. Worms fed with Prorocentrum cf. lima exhibited decreased CAT activity, increased LPO levels – both interactively affected by algal species and time – and decreased GSH levels, which were interactively affected by algal species and cell density. Higher LPO levels, along with the inhibition of CAT and GSH, clearly indicated an oxidative stress situation in worms exposed to the toxigenic dinoflagellate. Laeonereis acuta accumulated moderate OA levels and may act as a vector of OA to food webs in estuarine areas under high Prorocentrum cf. lima abundance.

UPLC-TOF-MS Method for Simultaneous Quantification of Steroid Hormones in Tissue Homogenates of Zebrafish with Solid-Phase Extraction.

The quantification of steroid hormones of individual zebrafish (Danio rerio) provides perspective to understand endogenous hormone function. A UPLC–TOF–MS method was developed to provide a reproducible, sensitive, and efficient assay to determine the concentration of steroid hormones, including cortisol, testosterone, androstenedione, 11-deoxycortisol, 11-deoxycorticosterone, and 17-hydroxyprogesterone in whole-body homogenates of each zebrafish. Solid-phase extraction was used to sample matrix clean-up and acquired a recovery from 89.7% to 107.9%. The analytes were separated on an Aquity BEH C18 column using gradient elution. Mass spectrometric analysis was performed by single reaction monitoring (SRM) using positive electrospray ionization mode. The total running time was 6 min, which was greatly shortened compared with a previously reported method. The developed method exhibited excellent linearity for all the analytes, with regression coefficients higher than 0.99. The limit of detection varied between 0.1 and 0.5 ng/L and the limit of quantification was 0.5–1.7 ng/L for all analytes. The precision of the method was assessed on replicate measurements and was found to be in the ranges of 1.9 % to 6.6% and 4.3% to 8.6%, for intra- and inter-day analysis, respectively. This method was validated according to FDA guidance and applied to determine steroid hormone levels in the tissue homogenate of zebrafish acutely treated with caffeine and ethanol.

Control of Rhipicephalus annulatus resistant to deltamethrin by spraying infested cattle with synergistic eucalyptus essential oil-thymol-deltamethrin combination

The current study investigated the synergistic effect of combinations containing deltamethrin (D), Eucalyptus essential oil (E), and the thyme essential oil component thymol (T), against a field population of Rhipicephalus annulatus in Egypt that was characterized to be resistant to D. Solutions of T, E, or TE at concentrations of 1.25–5% were combined with 5% deltamethrin at different dilutions (0.25–2 mL/L). Results of the adult immersion test used to estimate the in vitro acaricidal activity of these combinations at 5% yielded LC50 values for D, E-D, T-D, and TE-D of 3.87 mL/L, 3.89 mL/L, 0.14 mL/L, and 0.05 mL/L, respectively. Biochemical analyses using whole-body homogenate of ticks from the in vitro tests revealed that the lowest acetylcholinesterase and glutathione peroxidase activity, and the maximum lipid peroxidation were recorded in ticks treated with 5% TE-D. Glutathione content significantly decreased (p ≤ 0.05) in all treated ticks. Three groups, each containing five cross breed cattle naturally infested with R. annulatus from the same area where resistance to D was detected, were sprayed twice at two-week intervals using 1 mL/L of 5% solutions of D, T-D, or TE-D. Overall efficacy of the D, T-D, and TE-D sprays by day 30 post-treatment was 21.6, 88.3, and 95 %, respectively. Ticks collected from infested cattle three days after treatment with the D spray deposited egg masses that were able to hatch, deposited small masses of eggs unable to hatch when exposed to the T-D spray, and laid few eggs that didn’t hatch when sprayed with the TE-D combination. Values for liver and kidney function parameters were comparable in cattle before and after treatment with the combination sprays tested. The TE-D spray overcame the insensitivity to D of this R. annulatus population in Egypt, which also highlighted the significant synergistic effect of thymol on the acaricidal activity of deltamethrin observed in vitro. Acaricidal activity of the TE-D combination apparently has deleterious effects on multiple tick systems involving inhibition of acetylcholinesterase, increased lipid peroxidation, and oxidative stress. These findings document that combinations of natural and synthetic products can be part of integrated management solutions to the problem with widespread resistance to pyrethroids like deltamethrin in populations of cattle ticks, including R. annulatus, around the world.

Silkworm Pupae Function as Efficient Producers of Recombinant Glycoproteins with Stable-Isotope Labeling.

Baculovirus-infected silkworms are promising bioreactors for producing recombinant glycoproteins, including antibodies. Previously, we developed a method for isotope labeling of glycoproteins for nuclear magnetic resonance (NMR) studies using silkworm larvae reared on an artificial diet containing 15N-labeled yeast crude protein extract. Here, we further develop this method by introducing a technique for the expression of isotope-labeled glycoproteins by silkworm pupae, which has several potential advantages relative to larvae-based techniques in terms of production yield, ease of handling, and storage. Here, we fed fifth instar larvae an artificial diet with an optimized composition containing [methyl-13C]methionine, leading to pupation. Nine-day-old pupae were then injected with recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid for expression of recombinant human immunoglobulin G (IgG). From the whole-body homogenates of pupae, 0.35 mg/pupa of IgG was harvested, which is a yield that is five times higher than can be obtained from larvae. Recombinant IgG, thus prepared, exhibited mainly three kinds of pauci-mannose-type oligosaccharides and had a 13C-enrichment ratio of approximately 80%. This enabled selective observation of NMR signals originating from the methionyl methyl group of IgG, confirming its conformational integrity. These data demonstrate the utility of silkworm pupae as factories for producing recombinant glycoproteins with amino-acid-selective isotope labeling.

Effect of Cold Acclimation on Selected Metabolic Enzymes During Diapause in The European Corn Borer Ostrinia nubilalis (Hbn.)

The European corn borer, Ostrinia nubilalis Hbn., is a pest Lepidopteran species whose larvae overwinter by entering diapause, gradually becoming cold-hardy. To investigate metabolic changes during cold hardening, activities of four metabolic enzymes – citrate synthase (CS), lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured in whole-body homogenates of pupae, non-diapausing and diapausing larvae acclimated to 5 °C, −3 °C and −16 °C. The highest CS activity was detected in non-diapausing larvae, reflecting active development, while the highest in vitro LDH activity was recorded in diapausing larvae at temperatures close to 0 °C, evidencing a metabolic switch towards anaerobic metabolism. However, in-gel LDH activity showed that production of pyruvate from lactate is triggered by sub-zero temperatures. The activities of both aminotransferases were highest in non-diapausing larvae. Our findings suggest that during diapause and cold hardening the aminotransferases catalyse production of L-alanine, an important cryoprotectant, and L-aspartate, which is closely tied to both transamination reactions and Krebs cycle. The results of this study indicate that, during diapause, the activity of metabolic enzymes is synchronized with exogenous factors, such as temperatures close to 0 °C. These findings support the notion that diapause is metabolically plastic and vibrant, rather than simply a passive, resting state.

Organophosphate contaminants in North Atlantic fin whales

Pollution of the marine environment by litter composed of plastics is a growing concern. Chemical additives such as organophosphate flame retardants (OPFRs), which are added to plastics to improve their qualities, are in focus because they allegedly cause adverse effects on marine fauna. Here we analyse OPFR levels in the muscle of fin whales because, as a mysticete, this cetacean obtains its food by filter-feeding and is thus highly vulnerable to marine litter. Moreover, the fin whale performs long-range migrations from low-latitude areas in winter to high-latitude areas in summer, a trait that makes it a potentially good large-scale biomonitor of pollution. We also analyse OPFR levels in its main prey, the krill Meganyctiphanes norvegica, to assess transfer through diet. The samples analysed consisted of muscle tissue from 20 fin whales and whole-body homogenates of 10 krill samples, all collected off West Iceland. From the 19 OPFRs analysed, we detected 7 in the fin whale and 5 in the krill samples. Tri-n-butyl phosphate (TNBP), Isopropylated triphenyl phosphate (IPPP) and Triphenylphosphine oxide (TPPO) were the most abundant compounds found in both species. Mean ∑OPFR concentration, expressed on a lipid weight basis, was 985 (SD = 2239) ng g−1 in fin whale muscle, and 949 (SD = 1090) ng g−1 in krill homogenates. These results constitute the first evidence of the presence of OPFRs in the tissues of fin whales. Furthermore, they seem to support the non-significance of bioaccumulation of OPFRs through lifespan and of biomagnification trough the food web.

Sugar, amino acid and inorganic ion profiling of the honeydew from different hemipteran species feeding on Abies alba and Picea abies.

Several hemipteran species feed on the phloem sap of plants and produce large amounts of honeydew that is collected by bees to produce honeydew honey. Therefore, it is important to know whether it is predominantly the hemipteran species or the host plant to influence the honeydew composition. This is particularly relevant for those botanical and zoological species from which the majority of honeydew honey originates. To investigate this issue, honeydew from two Cinara species located on Abies alba as well as from two Cinara and two Physokermes species located on Picea abies were collected. Phloem exudates of the host plants were also analyzed. Honeydew of all species contained different proportions of hexoses, sucrose, melezitose, erlose, and further di- and trisaccharides, whereas the phloem exudates of the host trees contained no trisaccharides. Moreover, the proportions of sugars differed significantly between hemipteran species feeding on the same tree species. Sucrose hydrolysis and oligosaccharide formation was shown in whole-body homogenates of aphids. The type of the produced oligosaccharides in the aphid-extracts correlated with the oligosaccharide composition in the honeydew of the different aphid species. The total contents of amino acids and inorganic ions in the honeydew were much lower than the sugar content. Glutamine and glutamate were predominant amino acids in the honeydew of all six hemipteran species and also in the phloem exudates of both tree species. Potassium was the dominant inorganic ion in all honeydew samples and also in the phloem exudate. Statistical analyses reveal that the sugar composition of honeydew is determined more by the hemipteran species than by the host plant. Consequently, it can be assumed that the sugar composition of honeydew honey is also more influenced by the hemipteran species than by the host tree.

The effects of bifenthrin and temperature on the endocrinology of juvenile Chinook salmon.

The San Francisco Bay delta (USA) is experiencing seasonally warmer waters attributable to climate change and receives rainstorm runoff containing pyrethroid pesticides. Chinook salmon (Oncorhynchus tshawytscha) inhabit the affected waterways from hatch through smoltification, and thus juvenile fish may experience both pyrethroid and warmer water exposures. The effects of higher temperatures and pesticide exposure on presmolt Chinook are unknown. To improve understanding of the potential interaction between temperature and pesticide exposure on salmonid development, juvenile alevin and fry were reared in 11, 16.4, and 19 °C freshwater for 11 d and 2 wk, respectively, and exposed to nominal concentrations of 0, 0.15, and 1.5 µg/L bifenthrin for the final 96 h of rearing. Estradiol-17β (E2), testosterone, triiodothyronine, and thyroxine levels were measured in whole-body homogenates using hormone-specific enzyme-linked immunosorbent assays. Brain gonadotropin-releasing hormone receptor (GnRH2), dopamine receptor 2A, and growth hormone 1 (GH1) mRNA levels were measured using quantitative PCR. Results showed significantly decreased survival and condition factors observed with increasing temperature in alevin. Alevin thyroid hormones increased significantly with temperature, but fry thyroid hormones trended toward a decrease at lower temperatures with increasing bifenthrin exposure. There were significant reductions in fry testosterone and E2 at 11 °C with increasing bifenthrin treatments and significant changes in GnRH2 and GH1 gene expression in both alevin and fry, indicating potential disruption of hormonal and signaling pathways. Environ Toxicol Chem 2019;38:852-861. © 2019 SETAC.

Sub-lethal Responses of the Polychaete Armandia agilis in Whole-sediment Toxicity Testing.

The present study assessed biochemical responses as sublethal endpoints in the polychaete Armandia agilis exposed to contaminated sediments to in order to assess its potential use as a test organism. Sediment samples from several locations at a dredging site were obtained and used in whole-sediment exposures. Samples were tested with A. agilis to determine the 10-day toxicity of the 100% sample and the enzymatic activity of catalase (CAT), glutathione-S-transferase (GST) and acetylcholinesterase (AChE) biochemical measurements made in whole-body homogenates of a subset of the surviving organisms. Biochemical responses reported in A. agilis were not statistically different from the reference site sediment, however, the integrated analysis demonstrated that contaminants bound to sediment samples influenced the sublethal effects.

Effect of hydration status on pesticide uptake in anurans following exposure to contaminated soils.

In this study, the impact of hydration status on dermal uptake of pesticides in two species of amphibians is examined. Absorption of pesticides in anurans occurs primarily through a highly vascularized dermal seat patch; however, pesticides can also enter through the superficial dermis following exposure. Despite the growing body of literature on dermal exposure in amphibians, little is known on how hydration status influences uptake. Thus, the objective of this study was to investigate the influence of hydration status on absorption of pesticides (atrazine, triadimefon, metolachlor, chlorothalonil, and imidacloprid) in southern leopard frogs (Lithobates sphenocephala) and Fowler's toads (Anaxyrus fowleri). Amphibian treatments included dehydration periods of 0, 2, 4, 6, 8, or 10h prior to exposure to pesticide-contaminated soils for 8h. Following exposure, soil and whole-body homogenates were extracted and analyzed by LC-MS/MS. Dehydration time was then regressed against post-exposure concentrations to infer the impact of dehydration on dermal pesticide uptake. Increased dehydration time resulted in significantly lowered pesticide concentrations in both species (F6, 293 = 67.66, p = 0.007) for the five pesticides studied. This phenomenon could be due to an energy and/or dilution effect.