The Structure-Properties-Cytotoxicity Interplay: A Crucial Pathway to Determining Graphene Oxide Biocompatibility.

Interest in graphene oxide nature and potential applications (especially nanocarriers) has resulted in numerous studies, but the results do not lead to clear conclusions. In this paper, graphene oxide is obtained by multiple synthesis methods and generally characterized. The mechanism of GO interaction with the organism is hard to summarize due to its high chemical activity and variability during the synthesis process and in biological buffers' environments. When assessing the biocompatibility of GO, it is necessary to take into account many factors derived from nanoparticles (structure, morphology, chemical composition) and the organism (species, defense mechanisms, adaptation). This research aims to determine and compare the in vivo toxicity potential of GO samples from various manufacturers. Each GO sample is analyzed in two concentrations and applied with food. The physiological reactions of an easy model Acheta domesticus (cell viability, apoptosis, oxidative defense, DNA damage) during ten-day lasting exposure were observed. This study emphasizes the variability of the GO nature and complements the biocompatibility aspect, especially in the context of various GO-based experimental models. Changes in the cell biomarkers are discussed in light of detailed physicochemical analysis.

Auditory DUM neurons in a bush-cricket: inhibited inhibitors.

Thoracic ganglia of many hearing insects house the first level of auditory processing. In bush-crickets, the largest population of local auditory neurons in the prothoracic processing centre are dorsal unpaired median (DUM) neurons. It has been suggested that DUM neurons are inhibitory using γ-aminobutyric acid (GABA) as transmitter. Immunohistochemistry reveals a population of about 35-50 GABA-positive somata in the posterior medial cluster of the prothoracic ganglion. Only very few small somata in this cluster remain unstained. At least 10 neurites from 10 neurons can be identified. Intracellularly stained auditory DUM neurons have their soma in the cluster of median GABA positive cells and most of them exhibit GABA-immunoreactivity. Responses of certain DUM neurons show obvious signs of inhibition. Application of picrotoxin (PTX), a chloride-channel blocker in insects, changes the responses of many DUM neurons. They become broader in frequency tuning and broader or narrower in temporal pattern tuning. Furthermore, inhibitory postsynaptic potentials (IPSPs) may be replaced by excitatory postsynaptic potentials. Loss of an IPSP in the rising graded potential after PTX-application leads to a significant reduction of first-spike latency. Therefore, auditory DUM neurons receive effective inhibition and are the best candidates for inhibition in DUM neurons and other auditory interneurons.

Chronic and Acute Oral Administration of Caffeine Enhance Performance of Olfactory Learning in Crickets.

Crickets were arbitrarily fed a caffeine-containing diet during their lifetime or were orally administered caffeine in solution once during the conditioning session. These chronically or acutely treated crickets were used to test whether there were positive effects of caffeine on their learning performance. The effects of chronic exposure to caffeine on crickets' growth were also examined by measuring their body weight and counting the number of adults that emerged from larvae. Chronic treatment with relatively high concentrations of caffeine (5 mg/g and 0.5 mg/g in the diet) impaired the growth of crickets. Mortality was higher with 5 mg/g caffeine, and the survivors could not attain the final molt (eclosion). Body weight gain and eclosion rate were also negatively influenced in the group fed 0.5 mg/g caffeine. The olfactory learning paradigm was used to examine the effects of chronic caffeine treatment. Of the caffeine concentrations tested (0.5 µg/g to 0.5 mg/g), significant improvement in long-term memory (LTM) formation was observed only with 50 µg/g caffeine. Acute effects on olfactory learning were examined after oral administration of 0.1 nM to 10 µM caffeine solutions during the conditioning session. Administration of caffeine ≥ 1 nM consistently resulted in a significant improvement in LTM formation. These results suggest that chronic exposure to caffeine enhances learning performance in crickets at a specific dose (50 µg/g in the diet), although it inhibits their growth at higher doses (≥ 0.5 mg/g). In contrast, acute oral administration of caffeine in naive crickets may enhance their learning performance even at a very low (nanomolar) concentration.

Pre-adult aggression and its long-term behavioural consequences in crickets.

Social experience, particularly aggression, is considered a major determinant of consistent inter-individual behavioural differences between animals of the same species and sex. We investigated the influence of pre-adult aggressive experience on future behaviour in male, last instar nymphs of the cricket Gryllus bimaculatus. We found that aggressive interactions between male nymphs are far less fierce than for adults in terms of duration and escalation. This appears to reflect immaturity of the sensory apparatus for releasing aggression, rather than the motor system controlling it. First, a comparison of the behavioural responses of nymphs and adults to mechanical antennal stimulation using freshly excised, untreated and hexane-washed antennae taken from nymphs and adults, indicate that nymphs neither respond to nor produce sex-specific cuticular semiochemicals important for releasing aggressive behaviour in adults. Second, treatment with the octopamine agonist chlordimeform could at least partially compensate for this deficit. In further contrast to adults, which become hyper-aggressive after victory, but submissive after defeat, such winner and loser effects are not apparent in nymphs. Aggressive competition between nymphs thus appears to have no consequence for future behaviour in crickets. Male nymphs are often attacked by adult males, but not by adult females. Furthermore, observations of nymphs raised in the presence, or absence of adult males, revealed that social subjugation by adult males leads to reduced aggressiveness and depressed exploratory behaviour when the nymphs become adult. We conclude that social subjugation by adults during pre-adult development of nymphs is a major determinant of consistent inter-individual behavioural differences in adult crickets.

Isolation and Characterization of Insecticidal Toxins from the Venom of the North African Scorpion, Buthacus leptochelys.

Various bioactive peptides have been identified in scorpion venom, but there are many scorpion species whose venom has not been investigated. In this study, we characterized venom components of the North African scorpion, Buthacus leptochelys, by mass spectrometric analysis and evaluated their insect toxicity. This is the first report of chemical and biological characterization of the B. leptochelys venom. LC/MS analysis detected at least 148 components in the venom. We isolated four peptides that show insect toxicity (Bl-1, Bl-2, Bl-3, and Bl-4) through bioassay-guided HPLC fractionation. These toxins were found to be similar to scorpion α- and ß-toxins based on their N-terminal sequences. Among them, the complete primary structure of Bl-1 was determined by combination of Edman degradation and MS/MS analysis. Bl-1 is composed of 67 amino acid residues and crosslinked with four disulfide bonds. Since Bl-1 shares high sequence similarity with α-like toxins, it is likely that it acts on Na+ channels of both insects and mammals.

Venom content and toxicity regeneration after venom gland depletion by electrostimulation in the scorpion Centruroides limpidus.

The scorpion venom is a cocktail of many components. Its composition can exhibit a level of plasticity in response to different behavioral and environmental factors, leading to intraspecific variation. The toxicity and specificity of scorpion venoms appear to be taxon-dependent, due to a co-evolutionary interaction with prey and predators, which shaped the composition at the molecular level. The venom regeneration by the venom glands is an asynchronous process, in which particular components are expressed at different stages and at different rates. According to this, it can be reasonably assumed that the regeneration of toxicity in the venom is also asynchronous. In this work, we studied the toxicity regeneration dynamics by the scorpion Centruroides limpidus after full venom depletion by electrical stimulation. For this, we evaluated the toxicity of venom samples extracted at different days post depletion, against insects (crickets) and mammals (humans, by assessing the venom activity on the human voltage-dependent Na+ channel Nav1.6). The regeneration of toxicity against humans lagged behind that against crickets (13 vs 10 days, respectively). Thirteen days after depletion the venom seems to be replenished. Our results show asynchrony in the regeneration of species-specific toxic activity in the venom of Centruroides limpidus. The understanding of the venom regeneration kinetics for the different scorpion species will help to design venom extraction protocols that could maximize the yield and quality of the collected venoms.

Biochemical characterization of the venom of Central American scorpion Didymocentrus krausi Francke, 1978 (Diplocentridae) and its toxic effects in vivo and in vitro.

Venoms of medically important scorpions from Buthidae family have been intensively studied, in contrast to non-buthid venoms, for which knowledge is scarce. In this work, we characterized the venom of a Diplocentridae species, Didymocentrus krausi, a small fossorial scorpion that inhabits the Tropical Dry Forest of Central America. D. krausi venom soluble fraction contains proteases with enzymatic activity on gelatin and casein. Mass spectrometry and venomic analysis confirmed the presence of elastase-like, cathepsin-O-like proteases and a neprilysin-like metalloproteinase. We did not detect phospholipase A2, C or D, nor hyaluronidase activity in the venom. By homology-based venom gland transcriptomic analysis, NDBPs, a ß-KTx-like peptide, and other putative toxin transcripts were found, which, together with a p-benzoquinone compound present in the venom, could potentially explain its direct hemolytic and cytotoxic effects in several mammalian cell lines. Cytotoxicity of D. krausi venom was higher than the effect of venoms from two buthid scorpion species distributed in Costa Rica, Centruroides edwardsii and Tityus pachyurus. Even though D. krausi venom was not lethal to mice or crickets, when injected in mouse gastrocnemius muscle at high doses it induced pathological effects at 24 h, which include myonecrosis, weak hemorrhage, and inflammatory infiltration. We observed an apparent thrombotic effect in the skin blood vessels, but no in vitro fibrinogenolytic activity was detected. In crickets, D. krausi venom induced toxicity and paralysis in short periods of time.

Chemical synthesis of a two-domain scorpion toxin LaIT2 and its single-domain analogs to elucidate structural factors important for insecticidal and antimicrobial activities.

Scorpion venom contains various bioactive peptides. Among them, peptides having two different structural domains constitute a toxin family known as ß-KTx or scorpine-like peptides. These peptides consist of an α-helical structure in the N-terminal region and a cysteine-stabilized structure in the C-terminal region. This unique structure of ß-KTx peptides contributes to their diverse biological functions, but the importance of each domain for their activities is not fully understood. LaIT2 is a ß-KTx peptide isolated from the venom of the scorpion Liocheles australasiae, which shows both insecticidal and antimicrobial activities. In this study, we chemically synthesized full-length LaIT2 using a native chemical ligation technique as well as its N-terminally or C-terminally truncated single-domain analogs to evaluate structural factors important for the activities. Biological evaluation of these peptides revealed that the N-terminal α-helical domain of LaIT2 is essential for the expression of both insecticidal and antibacterial activities. This suggests that the disruption of membrane structures largely accounts for the biological activities of LaIT2.

Chronic toxicity of nanodiamonds can disturb development and reproduction of Acheta domesticus L.

The use of nanodiamonds in numerous materials designed for industry and medicine is growing rapidly. Consequently health and environmental risks associated with the exposure of humans and other biota to nanodiamonds-based materials are of the utmost importance. Scarcity of toxicological data for these particles led us to examine the potentially deleterious effects of nanodiamonds in model insect species, Acheta domesticus (Orthoptera) chronically exposed to ND in its diet. Organism-level end-point indices (lifespan, body weight, consumption, caloric value of faeces, reproduction) revealed adverse changes in the treated crickets in comparison with the control. Preliminary studies of oxidative stress level in the offspring of ND-treated crickets suggest toxicity of these particles limited to the exposed individuals. EPR analysis showing increase of radical signal in the faeces of ND-fed crickets led us to propose novel mechanism of nanodiamonds toxicity that is discussed in the light of literature data. CAPSULE: Development and reproduction of Acheta domesticus can be disturbed by the chronic exposure to nanodiamonds.

Linking organismal growth, coping styles, stress reactivity, and metabolism via responses against a selective serotonin reuptake inhibitor in an insect.

Evidence suggests that brain serotonin (5-HT) is one of the central mediators of different types of animal personality. We tested this assumption in field crickets Gryllus integer using a selective serotonin reuptake inhibitor (SSRI). Crickets were selected for slow and rapid development and tested for their coping styles under non-stressful conditions (time spent exploring a novel object). Resting metabolic rate, maximum metabolic rate and latency to resume activity were measured under stressful conditions (stress reactivity). Measurements were taken (i) before and (ii) during the SSRI treatment. Before the SSRI treatment, a strong negative correlation was observed between coping style and stress reactivity, which suggests the existence of a behavioral syndrome. After the SSRI treatment, the syndrome was no longer evident. The results of this study show that 5-HT may be involved in regulating behavior not only along a stress reactivity gradient but also along a coping styles axis. The relationship between personality and the strength and direction of 5-HT treatment on observed behaviors indicates trait-like individual differences in 5-HT signaling. Overall, these findings do not support recent ideas arising from the pace-of-life syndrome (POLS) hypothesis, which predict higher exploration and metabolic rates in rapidly developing bold animals.

Environmental causes and transgenerational consequences of ecdysteroid hormone provisioning in Acheta domesticus.

An animal's phenotype may be shaped by its genes, but also reflects its own environment and often that of its parents. Nongenetic parental effects are often mediated by steroid hormones, and operate between parents and offspring through mechanisms that are well described in vertebrate and model systems. However, less is understood about the strength and frequency of hormone mediated nongenetic parental effects across more than one generation of descendants, and in nonmodel systems. Here we show that the concentration of active ecdysteroid hormones provided by a female house cricket (Acheta domesticus) affects the growth rate of her offspring. We also reveal that variation in the active ecdysteroid hormones provided by a female house cricket to her eggs derives primarily from the quality of nutrition available to her maternal grandmother, regardless of genetic background. This finding is in stark contrast to most previous work that documents a decline in the strength of environmentally based parental effects with each passing generation. Strong grandparental effects may be adaptive under predictable, cyclical changes in the environment. Our results also suggest that hormone-mediated grand-maternal effects represent an important potential mechanism by which organisms can respond to environmental variability, and that further study of hormone-mediated carryover effects in this context could be profitable.

Induced expression of a vestigial sexual signal.

Vestigial morphological traits are common and well known in a variety of taxa. Identification of vestigial genes has illustrated the potential for evolutionary reversals and the re-expression of atavistic traits. Here we induce expression of a behavioural sexual signal, male calling song, in a cricket species, Gryllus ovisopis, which lacks a functional calling song. We successfully used acetylcholine injections in the frontal space of the head of male crickets to activate cerebral command neurons for cricket calling, and we recorded calling songs with a temporal chirp pattern similar to that of G. ovisopis' close evolutionary relatives, G. firmus and G. pennsylvanicus, implying that the neural pattern generators that underlie cricket calling behaviour persist in a vestigial state in G. ovisopis To our knowledge, this is the first demonstration of the induced expression of a vestigial behaviour in any organism. The retention of latent neural capacity to express sexual behaviours could have important implications for rapid evolution, trait re-emergence and reproductive isolation.

Reduced fecundity and cellular changes in Acheta domesticus after multigenerational exposure to graphene oxide nanoparticles in food.

Despite the fact that the demand for graphene and its derivatives in commercial applications is still growing, many aspects of its toxicity and biocompatibility are still poorly understood. Graphene oxide, which is released into the environment (air, soil and water) as so-called nanowaste or nanopollution, is able to penetrate living organisms. It is highly probable that, due to its specific nature, it can migrate along food chains thereby causing negative consequences. Our previous studies reported that short-term exposure to graphene oxide may increase the antioxidative defense parameters, level of DNA damage, which results in numerous degenerative changes in the gut and gonads. The presented research focuses on reproductive dysfunction and cellular changes in Acheta domesticus after exposure to GO nanoparticles in food (concentrations of 20 and 200 µg·g-1 of food) throughout their entire life cycle. The results showed that long-term exposure to GO caused a significant decrease in the reproductive capabilities of the animals. Moreover, the next generation of A. domesticus had a lower cell vitality compared to their parental generation. It is possible that graphene oxide can cause multigenerational harmful effects.

Adipokinetic hormone signaling determines dietary fatty acid preference through maintenance of hemolymph fatty acid composition in the cricket Gryllus bimaculatus.

Adipokinetic hormone (AKH), an analog of mammalian glucagon, functions in supplying the required energy by releasing lipids and carbohydrates from the fat body into the hemolymph. Our previous study showed that AKH receptor (AKHR) knockdown in the two-spotted cricket Gryllus bimaculatus decreased hemolymph lipid levels and increased its feeding frequency. To reveal underlying mechanisms by which AKH signaling modulates lipid homeostasis, we analyzed the fatty acid composition as the lipid structure in the crickets. AKH administration significantly increased the proportion of unsaturated fatty acids (USFAs) to total fatty acids with decrease of the saturated fatty acids (SFAs) in hemolymph, while these proportions were inversely changed in RNA interference-mediated AKHR-knockdowned (AKHRRNAi) crickets. Interestingly, knockdown of hormone-sensitive lipase (Hsl) by RNAi (HslRNAi) affected the proportion of USFAs and SFAs in a similar manner to that observed in AKHRRNAi crickets. AKH administration in HslRNAi crickets did not change hemolymph fatty acid composition, indicating that AKH signaling critically altered fatty acid composition in the hemolymph through Hsl. In addition, a choice assay revealed that AKHRRNAi significantly increases the preference of USFAs. These data indicate that hemolymph lipid level and composition were modulated by AKH signaling with a complementary feeding behavior toward USFAs.

Pyrethroid residue dynamics in insects depends on the circadian clock.

Many factors may affect pesticide effectiveness against pests. One of the factors that should be considered is circadian rhythmicity. In this study, we evaluated daily variations in pyrethroid susceptibility in the house cricket, Acheta domesticus L. Crickets were exposed to a standard dose of ß-cyfluthrin at different times of a day, and pesticide residue levels were evaluated using gas chromatography. Results demonstrate that the time of pyrethroid disappearance is correlated with the circadian clock, with the highest decomposition rate at night. Furthermore, crickets also showed the highest resistance to the insecticide at night, expressed as a high survival rate. Moreover, ß-cyfluthrin induced significant changes in thermal preferences of intoxicated crickets. This is the first report showing that pyrethroid residue levels in the crickets' body depend on its circadian clock.

Behavioral tracing demonstrates dietary nutrient discrimination in two-spotted crickets Gryllus bimaculatus.

Animals select appropriate diets to meet their nutritional requirements. Here, we demonstrate the availability for analysis of feeding preference using an orthopteran, the two-spotted cricket Gryllus bimaculatus. A time-course study of these insects, involving continuous recording and tracing behavior for 9 h, allowed us to monitor discrimination of diet that contained various nutrients.

Weathering in soil increases nanoparticle CuO bioaccumulation within a terrestrial food chain.

This study evaluates the bioaccumulation of unweathered (U) and weathered (W) CuO in NP, bulk and ionic form (0-400 mg/kg) by lettuce exposed for 70 d in soil co-contaminated with field incurred chlordane. To evaluate CuO trophic transfer, leaves were fed to crickets (Acheta domestica) for 15 d, followed by insect feeding to lizards (Anolis carolinensis). Upon weathering, the root Cu content of the NP treatment increased 214% (327 ± 59.1 mg/kg) over unaged treatment. Cu root content decreased in bulk and ionic treatments from 70-130 mg/kg to 13-26 mg/kg upon aging in soil. Micro X-ray fluorescence (µ-XRF) analysis of W-NP-exposed roots showed a homogenous distribution of Cu (and Ca) in the tissues. Additionally, micro X-ray absorption near-edge (µ-XANES) analysis of W-NP-exposed roots showed near complete transformation of CuO to Cu (I)-sulfur and oxide complexes in the tissues, whereas in unweathered treatment, most root Cu remained as CuO. The expression level of nine genes involved in Cu transport shows that the mechanisms of CuO NPs (and bulk) response/accumulation are different than ionic Cu. The chlordane accumulation by lettuce upon co-exposure to CuO NPs significantly increased upon weathering. Conversely, bulk and ionic exposures decreased pesticide accumulation by plant upon weathering. The Cu cricket fecal content from U-NP-exposed insects was significantly greater than the bulk or ion treatments, suggesting a higher initial NP accumulation followed by significantly greater elimination during depuration. In the lizard, Cu content in the intestine, body and head did not differ as a function of weathering. This study demonstrates that CuO NPs may undergo transformation processes in soil upon weathering that subsequently impact NPs availability in terrestrial food chains.

Effects of Caffeine on Olfactory Learning in Crickets.

Caffeine is a plant-derived alkaloid that is generally known as a central nervous system (CNS) stimulant. In order to examine the effects of caffeine on higher CNS functions in insects, we used an appetitive olfactory learning paradigm for the cricket Gryllus bimaculatus. Crickets can form significant long-term memories (LTMs) after repetitive training sessions, during which they associate a conditioned stimulus (CS: odor) with an unconditioned stimulus (US: reward). Administration of hemolymphal injections of caffeine established LTM after only single-trial conditioning over a wide range of caffeine dosages (1.6 µµg/kg to 39 mg/kg). We investigated the physiological mechanisms underlying this enhancement of olfactory learning performance pharmacologically, focusing on three major physiological roles of caffeine: 1) inhibition of phosphodiesterase (PDE), 2) agonism of ryanodine receptors, and 3) antagonism of adenosine receptors. Application of drugs relevant to these actions resulted in significant effects on LTM formation. These results suggest that externally applied caffeine enhances LTM formation in insect olfactory learning via multiple cellular mechanisms.

Characterization of Three Venom Peptides from the Spitting Spider Scytodes thoracica.

We present the solution-state NMR structures and preliminary functional characterizations of three venom peptides identified from the spitting spider Scytodes thoracica. Despite little sequence identity to other venom peptides, structural characterization reveals that these peptides contain an inhibitor cystine knot motif common to many venom peptides. These are the first structures for any peptide or protein from spiders of the Scytodidae family. Many venom peptides target neuronal ion channels or receptors. However, we have not been able to determine the target of these Scytodes peptides so we can only state with certainty the channels and receptors that they do not target.

Oxidative stress and genotoxic effects of diamond nanoparticles.

Due to the unique and useful properties of nanodiamonds (ND), their production and use is rapidly increasing. Thus, more of these particles will be released into the environment and organisms will inevitably be exposed to them. The current knowledge about the toxicity of ND, especially in vivo toxicity, is fragmentary. In this study, the toxicity of nanodiamonds was assessed in Acheta domesticus following chronic exposure to different nominal concentrations of ND (20 and 200µgg(-1) food) administrated in food for the entire lifespan. The activity of oxidative stress enzymes (catalase, glutathione peroxidase), total antioxidant capacity, as well as the level of heat shock protein were determined. A significant increase in all of the measured parameters was observed after seven weeks of exposure in individuals exposed to higher concentrations of ND (200µgg(-1) food). In animals exposed to lower concentrations of ND (20µgg(-1) food), there were few significant changes to these parameters. Analysis of DNA damage performed after fourteen weeks using the comet assay revealed DNA instabilities in the insects, especially the ones that had been exposed to the higher doses of ND. These findings may suggest that the toxicity of ND is concentration dependent. While high doses interact in a toxic manner, trace amounts, which are more likely in the environment, might be safe for organisms. Extreme caution should be taken when handling nanodiamonds.