Invading new climates at what cost? Ontogenetic differences in the thermal dependence of metabolic rate in an invasive amphibian.

Global warming can either promote or constrain the invasive potential of alien species. In ectotherm invaders that exhibit a complex life cycle, success is inherently dependent on the capacity of each developmental stage to cope with environmental change. This is particularly relevant for invasive anurans, which disperse on land while requiring water for reproduction. However, it remains unknown how the different life stages respond in terms of energy expenditure under different climate change scenarios. We here quantified the oxygen uptake of frogs at rest (a proxy of the standard metabolic rate) in the aquatic phase (at the tadpole and climax, i.e. during metamorphosis, stages) and in the terrestrial phase (metamorphosed stage) at three environmental temperatures. To do so, we used marsh frogs (Pelophylax ridibundus), an amphibian with the largest invasive range within the palearctic realm and for which their adaptation to global warming might be key to their invasion success. Beyond an increase of metabolic rate with temperature, our data show variation in thermal adaptation across life stages and a higher metabolic cost during metamorphosis. These results suggest that the cost to shift habitat and face changes in temperature may be a constraint on the invasive potential of species with a complex life cycle which may be particularly vulnerable during metamorphosis.

Diet influences resource allocation in chemical defence but not melanin synthesis in an aposematic moth.

For animals that synthesise their chemical compounds de novo, resources, particularly proteins, can influence investment in chemical defences and nitrogen-based wing colouration such as melanin. Competing for the same resources often leads to trade-offs in resource allocation. We manipulated protein availability in the larval diet of the wood tiger moth, Arctia plantaginis, to test how early life resource availability influences relevant life history traits, melanin production and chemical defences. We expected higher dietary protein to result in more effective chemical defences in adult moths and a higher amount of melanin in the wings. According to the resource allocation hypothesis, we also expected individuals with less melanin to have more resources to allocate to chemical defences. We found that protein-deprived moths had a slower larval development, and their chemical defences were less unpalatable for bird predators, but the expression of melanin in their wings did not differ from that of moths raised on a high-protein diet. The amount of melanin in the wings, however, unexpectedly correlated positively with chemical defences. Our findings demonstrate that the resources available in early life have an important role in the efficacy of chemical defences, but melanin-based warning colours are less sensitive to resource variability than other fitness-related traits.

Profiling the antioxidant biomarkers in marine fish larvae: a comparative assessment of different storage conditions to select the optimal strategy.

Research on antioxidant biomarkers can generate profound insights into the defense mechanisms of fish larvae against different stressors and can reveal manipulation strategies for improved growth and survival. However, the number of samples to process and unavailability of required infrastructure in larval-rearing facilities limit the immediate processing, requiring the preservation of specimens. Silver pompano (Trachinotus blochii), a potential marine aquaculture species, shows a low larval survival rate due to poorly developed antioxidant mechanism. In this context, 39 storage conditions, including three storage temperatures and different buffers, were scrutinized to select the most suitable preservation strategy for five important antioxidant biomarkers of fish larvae, viz. catalase activity, superoxide dismutase (SOD) activity, measurement of lipid peroxidation, reduced glutathione (GSH), and ascorbic acid contents. The paper proposes the optimum larval storage conditions for these five evaluated antioxidant biomarkers to generate similar results in preserved and non-preserved larval samples. Larval samples preserved in PBS at lower temperatures (- 20 °C and - 80 °C) are recommended for evaluating catalase activity and ascorbic acid content. Catalase activity can also be evaluated by preserving the larval samples at - 20 °C or - 80 °C without buffers. Larval samples held in PBS or without any buffers at - 20 °C and at - 80 °C were found to be suitable for SOD and GSH evaluation, respectively. Preservation in 50% glacial acetic acid at - 80 °C or - 20 °C was preferred for the lipid peroxidation assays. Apart from methodological perspectives, the paper provides insights into the dynamics of larval antioxidant profiles of T. blochii, for the first time.

Muscle in the caterpillar Manduca sexta responds to an immune challenge, but at a cost, suggesting a physiological trade-off.

Although skeletal muscle is a specialized tissue that provides the motor for movement, it also participates in other functions, including the immune response. However, little is known about the effects of this multitasking on muscle. We show that muscle loses some of its capacity while it is participating in the immune response. Caterpillars (Manduca sexta) were exposed to an immune challenge, predator stress or a combination of immune challenge and predator stress. The expression of immune genes (toll-1, domeless, cactus, tube and attacin) increased in body wall muscle after exposure to an immune challenge. Muscle also showed a reduction in the amount of the energy storage molecule glycogen. During an immune challenge, the force of the defensive strike, an important anti-predator behaviour in M. sexta, was reduced. Caterpillars were also less able to defend themselves against a common enemy, the wasp Cotesia congregata, suggesting that the effect on muscle is biologically significant. Our results support the concept of an integrated defence system in which life-threatening events activate organism-wide responses. We suggest that increased mortality from predation is a non-immunological cost of infection in M. sexta. Our study also suggests that one reason non-immunological costs of infection exist is because of the participation of diverse organs, such as muscle, in immunity.

Using fly larvae to convert food waste for growing Oujiang color common carps: health risk assessment of polycyclic aromatic hydrocarbons.

The present study used Chrysomya megacephala larvae (CML) to transform food waste into safe and high-quality fish feed to substitute fish meal as a source of protein for growing Oujiang color common carps followed by a human health risk assessment of polycyclic aromatic hydrocarbons (PAHs). The results showed the ∑PAH concentration in the CML fed with food waste ranged from 50 to 370 µg kg-1, and the most abundant PAH compound in the CML was BaP, contributing 59-84% of ∑PAHs. The Pearson correlation analysis results indicated no correlation between the ∑PAH concentrations and the culture substrate ratio (p > 0.05). Concentrations of BaP in the CML decreased with the increase of breeding density (p < 0.01). The residues as organic fertilizers have no potential ecological risk for PAHs. The biotransformed larva meal was used to partially or completely replace the fish meal as supplementary protein in the experimental feeds (T0, 0%; T50, 50%; T100, 100%). No significant difference (p < 0.05) of survival rate, lipid, and protein content in Oujiang color common carp was noted among T0, T50, and T100 fish feeds. Concentrations of ∑PAHs in Oujiang color common carp fed with the CML fish feeds all met the food safety standards in the European Union (EU). Furthermore, the consumption of Oujiang color common carps fed with the CML feed does not pose any health risks of PAHs for humans.

Tetrabromobisphenol A and Diclazuril Evoke Tissue-Specific Changes of Thyroid Hormone Signaling in Male Thyroid Hormone Action Indicator Mice.

Thyroid hormone (TH) signaling is a prerequisite of normal tissue function. Environmental pollutants with the potential to disrupt endocrine functions represent an emerging threat to human health and agricultural production. We used our Thyroid Hormone Action Indicator (THAI) mouse model to study the effects of tetrabromobisphenol A (TBBPA; 150 mg/bwkg/day orally for 6 days) and diclazuril (10.0 mg/bwkg/day orally for 5 days), a known and a potential hormone disruptor, respectively, on local TH economy. Tissue-specific changes of TH action were assessed in 90-day-old THAI mice by measuring the expression of a TH-responsive luciferase reporter in tissue samples and by in vivo imaging (14-day-long treatment accompanied with imaging on day 7, 14 and 21 from the first day of treatment) in live THAI mice. This was followed by promoter assays to elucidate the mechanism of the observed effects. TBBPA and diclazuril impacted TH action differently and tissue-specifically. TBBPA disrupted TH signaling in the bone and small intestine and impaired the global TH economy by decreasing the circulating free T4 levels. In the promoter assays, TBBPA showed a direct stimulatory effect on the hdio3 promoter, indicating a potential mechanism for silencing TH action. In contrast, diclazuril acted as a stimulator of TH action in the liver, skeletal muscle and brown adipose tissue without affecting the Hypothalamo-Pituitary-Thyroid axis. Our data demonstrate distinct and tissue-specific effects of TBBPA and diclazuril on local TH action and prove that the THAI mouse is a novel mammalian model to identify TH disruptors and their tissue-specific effects.

Txp40, an insecticidal toxin protein from Xenorhabdus nematophila: Purification, toxicity assessment and biophysical characterization.

Txp40 is a ubiquitous toxin from Xenorhabdus and Photorhabdus bacteria, exhibits insecticidal activity against a wide range of insect pests belonging to Lepidoptera and Diptera orders. Initially, Txp40 affects midgut of the target insect and further damages some other tissues like fat bodies but the detailed mode of action is not known. Txp40 shares no significant sequence match to any proteins with known structure or function, suggesting that it is a novel type of insecticidal toxin. Here, we report purification, toxicity and biophysical characterization of the Txp40b toxin from X. nematophila (ATCC, 19061). The recombinant Txp40b was found toxic to Galleria mellonella larvae with LD50 of 30.42 ng larva-1. Circular dichroism spectroscopy revealed that purified Txp40b is an α-helix rich protein with a relatively lower melting temperature of 45 °C. In-silico model generated suggests two domain structure of Txp40b toxin. Detailed structural analysis of Txp40b will provide new insights about the mode of action and possibly it would illustrate a new domain and/or motif in the area of insecticidal proteins.

Safety assessment of Mpp75Aa1.1, a new ETX_MTX2 protein from Brevibacillus laterosporus that controls western corn rootworm.

The recently discovered insecticidal protein Mpp75Aa1.1 from Brevibacillus laterosporus is a member of the ETX_MTX family of beta-pore forming proteins (ß-PFPs) expressed in genetically modified (GM) maize to control western corn rootworm (WCR; Diabrotica virgifera virgifera LeConte). In this manuscript, bioinformatic analysis establishes that although Mpp75Aa1.1 shares varying degrees of similarity to members of the ETX_MTX2 protein family, it is unlikely to have any allergenic, toxic, or otherwise adverse biological effects. The safety of Mpp75Aa1.1 is further supported by a weight of evidence approach including evaluation of the history of safe use (HOSU) of ETX_MTX2 proteins and Breviballus laterosporus. Comparisons between purified Mpp75Aa1.1 protein and a poly-histidine-tagged (His-tagged) variant of the Mpp75Aa1.1 protein demonstrate that both forms of the protein are heat labile at temperatures at or above 55°C, degraded by gastrointestinal proteases within 0.5 min, and have no adverse effects in acute mouse oral toxicity studies at a dose level of 1920 or 2120 mg/kg body weight. These results support the use of His-tagged proteins as suitable surrogates for assessing the safety of their non-tagged parent proteins. Taken together, we report that Mpp75Aa1.1 is the first ETX-MTX2 insecticidal protein from B. laterosporus and displays a similar safety profile as typical Cry proteins from Bacillus thuringiensis.

Purification, characterization and toxicity assessment of PirAB toxins from Photorhabdus akhurstii subsp. akhurstii K-1.

Photorhabdus insect related proteins A & B (PirA, PirB) from Photorhabdus and Xenorhabdus bacteria exhibit both oral and injectable toxicity against lepidopteran and dipteran insect pest. The pirA, pirAt (encoding 6 N-terminal truncated PirA), pirB genes, pirA-pirB (with ERIC sequences), pirA-pirB-mERIC (modified pirA-pirB with mutated ERIC sequences) and polycistronic-pirAB were cloned and expressed in Escherichia coli. However, PirA protein was expressed in insoluble form and therefore the pirA gene was modified to produce PirAt. Moreover, pirA-pirB-mERIC, polycistronic-pirAB and co-transformed pirA/pirB genes were not expressed in the studied prokaryotic expression systems. None of the single purified proteins or mixtures of the individually expressed and purified proteins were toxic to mosquito larvae of Aedes aegypti and Culex quinquefasciatus. However, PirA-PirB protein mixtures purified from pirA-pirB operon plasmid were toxic to A. aegypti and C. quinquefasciatus larvae with LC50 values of 991 and 614 ng/ml, respectively. The presence of ERIC sequences between the two orfs of the pirA-pirB operon could help to obtain the proteins in biologically active form. Further, results confirm that PirA-PirB proteins of P. akhurstii subsp. akhurstii K-1 are binary insecticidal toxins and ERIC sequences could play an important role in expression of Pir proteins. Reports of biophysical characterization of individually purified PirAt, PirB and expressed PirA-PirB toxin mixture could provide the structural insight into these proteins.

Repeated exposure of fluazinam fungicides affects gene expression profiles yet carries no costs on a nontarget pest.

Fungicides are used to control pathogenic fungi of crop species, but they have also been shown to alter behavioral, life history and fitness related traits of nontarget insects. Here, we tested the fungicide effects on feeding behavior, survival and physiology of the nontarget pest insect, the Colorado potato beetle (CPB) (Leptinotarsa decemlineata). Feeding behavior was studied by a choice test of adult beetles, which were allowed to choose between a control and a fungicide (fluazinam) treated potato leaf. Larval survival was recorded after 24 and 72 h exposure to control and fungicide-treated leaves with 2 different concentrations. The adults did not show fungicide avoidance behavior. Similarly, survival of the larvae was not affected by the exposure to fungicides. Finally, to understand the effects of fungicides at the physiological level (gene expression), we tested whether the larval exposure to fungicide alter the expression of 5 metabolic pathway and stress associated genes. Highest concentration and 72-h exposure caused upregulation of 1 cytochrome P450 (CYP9Z14v2) and 1 insecticide resistance gene (Ldace1), whereas metabolic detoxification gene (Ugt1) was downregulated. At 24-h exposure, highest concentration caused downregulation of another common detoxification gene (Gs), while both exposure times to lowest concentration caused upregulation of the Hsp70 stress tolerance gene. Despite these overall effects, there was a considerable amount of variation among different families in the gene expression levels. Even though the behavioral effects of the fungicide treatments were minor, the expression level differences of the studied genes indicate changes on the metabolic detoxifications and stress-related pathways.

Rapid and cost-effective process based on insect larvae for scale-up production of SARS-COV-2 spike protein for serological COVID-19 testing.

Serology testing for COVID-19 is important in evaluating active immune response against SARS-CoV-2, studying the antibody kinetics, and monitoring reinfections with genetic variants and new virus strains, in particular, the duration of antibodies in virus-exposed individuals and vaccine-mediated immunity. In this study, recombinant S protein of SARS-CoV-2 was expressed in Rachiplusia nu, an important agronomic plague. One gram of insect larvae produces an amount of S protein sufficient for 150 determinations in the ELISA method herein developed. We established a rapid production process for SARS-CoV-2 S protein that showed immunoreactivity for anti-SARS-CoV-2 antibodies and was used as a single antigen for developing the ELISA method with high sensitivity (96.2%) and specificity (98.8%). Our findings provide an efficient and cost-effective platform for large-scale S protein production, and the scale-up is linear, thus avoiding the use of complex equipment like bioreactors.

Lead toxicity in Lucilia cuprina and electrochemical analysis: a simple and low-cost alternative for forensic investigation.

Entomotoxicology allows the detection and analysis of substances such as poisons, drugs, and metals in necrophagous insects using analytical protocols. In a forensic situation related to death by gunshot, the gunshot residue (GSR) is dispersed at the crime scene and may be consumed by necrophagous insects. Lead (Pb) is the most abundant metal in GSR samples and it can be determined using non-portable methods. However, the toxicity effects of GSR samples on Lucilia cuprina (Diptera: Calliphoridae) and the detection of Pb via portable electrochemical methods have not been investigated. This study describes for the first time the toxicity analysis of Pb on immature L. cuprina through their survival rate and influence of Pb on immature development. In addition, the bioaccumulation of Pb in the larvae samples was determined based on square wave anodic stripping voltammetry (SWASV) measurements. The results revealed a low limit of detection to Pb (6.5 µg L-1) and the analytical performance was satisfactory because it measures Pb levels in larvae exposed to a diet containing 50 µg Pb g-1. Furthermore, the levels of Pb influenced the survival rate and development time of the immature L. cuprina. Larvae exposed to a high concentration of the metal (50 µg Pb g -1) showed statistically significant changes (p < 0.05). The presence of Pb in immature L. cuprina can be used to estimate the post-mortem interval; thus, the present study provides important information in forensic entomology.

Transgenic Cry1Ac/CpTI cotton assessment finds no detrimental effects on the insect predator Chrysoperla sinica.

The widespread commercialization of genetically modified (GM) cotton makes it important to assess the potential impact of this recombinant crop on non-target organisms. As important natural enemies of cotton field predators, green lacewing Chrysoperla sinica larvae are exposed to Bt insecticidal proteins expressed by GM cotton by feeding on herbivorous pests, and adults are directly exposed to Bt proteins by cotton pollen consumption. However, potential impacts of transgenic Bt cotton on C. sinica remain unclear. In this study, we evaluated the effects of two transgenic cotton varieties, CCRI41 and CCRI45, which express Cry1Ac (Bt toxin) and CpTI (Cowpea Trypsin Inhibitor), on C. sinica larvae and adults. After being fed with cotton aphids Aphis gossypii reared on transgenic cotton, the survival rate, developmental duration, pupation rate, and emergence rate of larvae were not adversely affected. After being fed two types of transgenic cotton pollen, the 7-day weight of adults and the preoviposition period and the cumulative oviposition of females were not significantly different from control specimen. Taken together, these results indicate that the potential risks of the two tested GM cotton varieties for the predator C. sinica are negligible. CAPSULE: Our study indicated that GM cotton varieties CCRI41 and CCRI45 have no adverse effects on insect predator C. sinica.

Expression and function assessment of two serpin-type serine protease inhibitors from Haemaphysalis doenitzi.

Serine protease inhibitors (serpins) in ticks are implicated in the modulation of the vertebrate host response to the tick bite. Experimentally, it has been demonstrated that serpins interfere with tick-borne pathogen transmission. However, knowledge on serpins in the tick Haemaphysalis doenitzi is lacking. In this study, the expression of two serpin genes, named HDS1 and HDS2, were assessed in H. doenitzi, and their roles in immune regulation were further investigated. The expression of HDS1 and HDS2 showed no tissue specificity, with maximum expression levels detected in the hemolymph and salivary gland, respectively. Among the developmental stages, the highest expression of HDS1 and HDS2 were detected in larvae and adults, respectively. The recombinant protein rHDS1 displayed obvious inhibitory effects on trypsin and thrombin, whereas rHDS2 clearly inhibited thrombin only. In addition, rHDS1 and rHDS2 showed certain inhibitory activities against bacteria and fungi. The female engorgement body weight, female engorgement rate, and egg hatchability were significantly decreased after injection of double-stranded RNA (dsRNA) of HDS1 gene, whereas no significant effects were observed concerning the feeding period or attachment rate at 24 h after introduction via rabbit ears. When injected with dsRNA of HDS2 gene, no significant effect was observed on the attachment rate at 24 h after introduction into the rabbit ears, but the engorgement body weight and engorgement rate of female ticks were significantly decreased, and no egg hatchment occurred. The above results contribute to better understanding the function of serpins in the development and innate immunity of H. doenitzi.

The extremely low energy cost of biosynthesis in holometabolous insect larvae.

The metabolic cost of growth, which quantifies the amount of energy required to synthesize a unit of biomass, is an important component of an animal's ontogenetic energy budget. Here we investigated this quantity as well as other energy budget variables of the larvae of a holometabolous insect species, Vanessa cardui (painted lady). We found that the high growth rate of this caterpillar cannot be explained by its metabolic rate and the percentage of the metabolic energy allocated to growth; the key to understanding its fast growth is the extremely low cost of growth, 336 Joules/gram of dry mass. The metabolic cost of growth in caterpillars is 15-65 times lower than that of the endothermic and ectothermic species investigated in previous studies. Our results suggest that the low cost cannot be attributed to its body composition, diet composition, or body size. To explain the "cheap price" of growth in caterpillars, we assumed that a high metabolic cost for biosynthesis resulted in a high "quality" of cells, which have fewer errors during biosynthesis and higher resistance to stressors. Considering the life history of the caterpillars, i.e., tissue disintegration during metamorphosis and a short developmental period and lifespan, we hypothesized that an energy budget that allocates a large amount of energy to biosynthesizing high quality cells would be selected against in this species. As a preliminary test of this hypothesis, we estimated the metabolic cost of growth in larvae of Manduca sexta (tobacco hornworm) and nymphs of Blatta lateralis (Turkestan cockroach). The preliminary data supported our hypothesis.

Development of reduced cost diets for the mass rearing of false codling moth (Thaumatotibia leucotreta).

BACKGROUND: Four trials were conducted; the first established optimal cooking time for false codling moth diets using a microwave oven while the other three trials aimed at determining nutrient requirements, testing these, and evaluating alternative cheaper raw materials. RESULTS: Microwave preparation for 8 min, using a 1000 W microwave oven was found to be the optimal cooking time for all treatment diets at 200 g kg-1 moisture. The minimum specification (MS) diet yielded more larvae per jar (mean ± standard deviation) (759 ± 29) than the control (CON) (652 ± 32) diet and the ideal amino acid profile (IAAP) diet (596 ± 46). The third trial subsequently utilized diets formulated according to the MS diet nutrient specifications but using novel and cheaper raw materials (D1 and D2). Production characteristics of diets D1 and D2 was compared to that of CON. Diets D1 and D2 yielded more larvae per jar (D1 = 911 ± 40; D2 = 830 ± 40) than that of CON (428 ± 40) with the added advantage that the newly formulated diets were cheaper. CONCLUSION: Reduced cost feeds were formulated, mixed and tested which resulted in substantial saving in the mass rearing of the species for integrated pest management. This method shows potential for diet development of other insect species as well. © 2019 Society of Chemical Industry.

Proactive coping style in early emerging rainbow trout carries a metabolic cost with no apparent return.

The timing with which salmonid larvae emerge from their gravel nests is thought to be correlated with a particular suite of behavioural and physiological traits that correspond to the stress coping style of the individual. Among these traits, aggressiveness, dominance and resilience to stress, are potentially interesting to exploit in aquaculture production. In the present study a series of experiments were performed, with the purpose of characterising behavioural, metabolic and production related traits in rainbow trout juveniles from different emergence fractions. Newly hatched rainbow trout were sorted according to their emergence time from an artificial redd. The early, middle, and late fractions were retained and assessed for their physiological response to stress, growth performance, metabolism, fasting tolerance, and potential for compensatory growth. The early emerging fraction showed proactive behavioural traits; they were faster to reappear following startling, showed a reduced cortisol response following stress, and a reduced metabolic cost of recovery. Emergence time was not correlated with any differences in standard or maximum metabolic rates, but was however, correlated with higher routine metabolic rates, as demonstrated by significantly bigger weight losses during fasting in the early emerging group. Growth rates and feed conversion efficiencies were not significantly different when fish were co-habitated under a restrictive feeding regime, suggesting that early emerging fish are not able to monopolise food resources. The intermediate emerging group, which makes up the bulk of a population and is often ignored, appears to possess the best growth performance traits, possibly because they do not expend excessive energy on dominance behaviour such as the early emerging group, while they are also not overly timid or stress prone such as the late emerging group.

Immunofluorescence/fluorescence assessment of brain-derived neurotrophic factor, c-Fos activation, and apoptosis in the brain of zebrafish (Danio rerio) larvae exposed to glufosinate.

In this study, we investigated the potential neuro-toxicological mechanism of the glufosinate in the brain of zebrafish larvae in terms of BDNF and c-Fos proteins by evaluating apoptosis, immunofluorescence BDNF, and c-FOS activation. We also measured survival rate, hatching rate, and body malformations during 96 h exposure time. For this purpose, zebrafish embryos were treated with graded concentrations of dosing solutions (0.5, 1, 3, and 5 ppm) of glufosinate. End of the treatment, acridine orange staining was used to detect apoptotic cells in the brain of zebrafish larvae at 96 hpf. Texas Red and FITC/GFP labeled protein-specific antibodies were used in immunofluorescence assay for BDNF and c-FOS, respectively. The results have indicated that exposure to glufosinate caused to embryonic death, hatching delay, induction of apoptosis, increasing of c-FOS activity and the level of BDNF in a dose-dependent manner. As a conclusion, we suggested that c-Fos might play a role in the regulation of BDNF which responses to prevent the cell from apoptosis even in case of unsuccessful in zebrafish larvae exposed to glufosinate.

Elementary budget of stag beetle larvae associated with selective utilization of nitrogen in decaying wood.

Wood degradation by insects plays important roles for the forest matter cycling. Since wood is deficient in nitrogen compared to the insect body, wood-feeding insects need to assimilate the nitrogen selectively and discard an excess carbon. Such a stoichiometric imbalance between food and body will cause high metabolic cost; therefore, wood-feeding insects may somehow alleviate the stoichiometric imbalance. Here, we investigated the carbon and nitrogen budgets of the larvae of stag beetle, Dorcus rectus, which feed on decaying wood. Assimilation efficiency of ingested wood was 22%, and those values based on the carbon and nitrogen were 27 and 45%, respectively, suggesting the selective digestion of nitrogen in wood. Element-based gross growth efficiency was much higher for nitrogen (45%) than for carbon (3%). As a result, the larvae released 24% of the ingested carbon as volatile, whereas almost no gaseous exchange was observed for nitrogen. Moreover, solubility-based elementary analysis revealed that the larvae mainly utilized alkaline-soluble-water-insoluble fraction of wood, which is rich in nitrogen. Actually, the midgut of the larvae was highly alkaline (pH 10.3). Stag beetle larvae are known to exhibit coprophagy, and here we also confirmed that alkaline-soluble-water-insoluble nitrogen increased again from fresh feces to old feces in the field. Stable isotope analysis suggested the utilization of aerial nitrogen by larvae; however, its actual contribution is still disputable. Those results suggest that D. rectus larvae selectively utilize alkaline-soluble nitrogenous substrates by using their highly alkaline midgut, and perhaps associate with microbes that enhance the nitrogen recycling in feces.

Toxicological assessment of PEGylated single-walled carbon nanotubes in early developing zebrafish.

Functionalization of single-walled carbon nanotubes (SWCNT) with polyethylene glycol (PEG) is among the most promising strategies to avoid SWCNT aggregation in aqueous media, improving its interactions with biological systems. However, the best molecular PEG weight and functionalization strategy remain under investigation. In this work we assessed the toxicological effects of SWCNT functionalized with PEG at 600 Da in zebrafish embryos. Embryos were exposed to SWCNT at 0.01, 0.1 and 1 mg/L from 3 to 96 h post-fertilization (hpf). At the highest concentration, SWCNT led to toxic effects at several endpoints, including mortality, delayed hatching, malformations, reduced body length, increased ROS production and DNA damage. Even with these effects, SWCNT could not be detected within the bodily tissues of the larvae. Our results give evidence that the tested PEGylation approach was unsuitable to avoid SWCNT aggregation in aqueous media, and that SWCNT can induce toxicity even without being absorbed by the organism by obstructing the chorion pores.