Effects of bisphenol A on post-embryonic development of the cotton pest Spodoptera littoralis.

Endocrine-disrupting chemicals encompass a variety of chemicals that may interfere with the endocrine system and produce negative effects on organisms. Among them, bisphenol A is considered a major pollutant in numerous countries. The harmful effects of BPA on environmental and human health are intensely studied. However, the effects of BPA on terrestrial insects are still poorly investigated, despite that several plants can accumulate BPA in their tissues, leading to potential contamination of herbivorous insects. Here, we used the leafworm Spodoptera littoralis, a polyphagous species, to study BPA effects on post-embryonic development. We studied the effects of BPA ingestion at environmental doses (e.g., 0.01, 0.1, and 1 µg/g of BPA) and high doses (e.g., 25 µg/g) on larval weight and stage duration, pupal length and sex ratio. BPA effects were investigated in more detail during the last larval instar, a crucial period for preparing pupation and metamorphosis, which are under endocrine control. We monitored the haemolymph concentration of ecdysteroids, hormones controlling moult and metamorphosis, as well as the expression levels of several nuclear receptors involved in the ecdysteroid signalling pathway. Our integrative study showed that, upon exposure doses, BPA can induce various effects on the viability, developmental time, growth and sex ratio. These effects were correlated with a delay of the ecdysteroid peak during the last larval instar and a modification of expression of EcR, USP, E75AB, E75D and Br-c. We provide new evidence about the events that occur after BPA exposure in insect contaminated by food ingestion.

Inhibition of oxidative stress in cholinergic projection neurons fully rescues aging-associated olfactory circuit degeneration in Drosophila.

Loss of the sense of smell is among the first signs of natural aging and neurodegenerative diseases such as Alzheimer's and Parkinson's. Cellular and molecular mechanisms promoting this smell loss are not understood. Here, we show that Drosophila melanogaster also loses olfaction before vision with age. Within the olfactory circuit, cholinergic projection neurons show a reduced odor response accompanied by a defect in axonal integrity and reduction in synaptic marker proteins. Using behavioral functional screening, we pinpoint that expression of the mitochondrial reactive oxygen scavenger SOD2 in cholinergic projection neurons is necessary and sufficient to prevent smell degeneration in aging flies. Together, our data suggest that oxidative stress induced axonal degeneration in a single class of neurons drives the functional decline of an entire neural network and the behavior it controls. Given the important role of the cholinergic system in neurodegeneration, the fly olfactory system could be a useful model for the identification of drug targets.

Urinary biomarker investigation in children with Fabry disease using tandem mass spectrometry.

BACKGROUND: Fabry disease is an X-linked lysosomal storage disorder affecting both males and females with tremendous genotypic/phenotypic variability. Concentrations of globotriaosylceramide (Gb3), globotriaosylsphingosine (lyso-Gb3)/related analogues were investigated in pediatric and adult Fabry cohorts. The aims of this study were to transfer and validate an HPLC-MS/MS methodology on a UPLC-MS/MS new generation platform, using an HPLC column, for urine analysis of treated and untreated pediatric and adult Fabry patients, to establish correlations between the excretion of Fabry biomarkers with gender, treatment, types of mutations, and to evaluate the biomarker reliability for early detection of pediatric Fabry patients. METHOD: A UPLC-MS/MS was used for biomarker analysis. RESULTS: Reference values are presented for all biomarkers. Results show that gender strongly influences the excretion of each biomarker in the pediatric Fabry cohort, with females having lower urinary levels of all biomarkers. Urinary distribution of lyso-Gb3/related analogues in treated Fabry males was similar to the untreated and treated Fabry female groups in both children and adult cohorts. Children with the late-onset p.N215S mutation had normal urinary levels of Gb3, and lyso-Gb3 but abnormal levels of related analogues. CONCLUSIONS: In this study, Fabry males and most Fabry females would have been diagnosed using the urinary lyso-Gb3/related analogue profile.

Forward and feedback regulation of cyclic steroid production in Drosophila melanogaster.

In most animals, steroid hormones are crucial regulators of physiology and developmental life transitions. Steroid synthesis depends on extrinsic parameters and autoregulatory processes to fine-tune the dynamics of hormone production. In Drosophila, transient increases of the steroid prohormone ecdysone, produced at each larval stage, are necessary to trigger moulting and metamorphosis. Binding of the active ecdysone (20-hydroxyecdysone) to its receptor (EcR) is followed by the sequential expression of the nuclear receptors E75, DHR3 and ßFtz-f1, representing a model for steroid hormone signalling. Here, we have combined genetic and imaging approaches to investigate the precise role of this signalling cascade within theprothoracic gland (PG), where ecdysone synthesis takes place. We show that these receptors operate through an apparent unconventional hierarchy in the PG to control ecdysone biosynthesis. At metamorphosis onset, DHR3 emerges as the downstream component that represses steroidogenic enzymes and requires an early effect of EcR for this repression. To avoid premature repression of steroidogenesis, E75 counteracts DHR3 activity, whereas EcR and ßFtz-f1 act early in development through a forward process to moderate DHR3 levels. Our findings suggest that within the steroidogenic tissue, a given 20-hydroxyecdysone peak induces autoregulatory processes to sharpen ecdysone production and to confer competence for ecdysteroid biosynthesis at the next developmental phase, providing novel insights into steroid hormone kinetics.

Is the rapid post-mating inhibition of pheromone response triggered by ecdysteroids or other factors from the sex accessory glands in the male moth Agrotis ipsilon?

In many animals, male copulation is dependent on the detection and processing of female-produced sex pheromones, which is generally followed by a sexual refractory post-ejaculatory interval (PEI). In the male moth, Agrotis ipsilon, this PEI is characterized by a transient post-mating inhibition of behavioral and central nervous responses to sex pheromone, which prevents males from re-mating until they have refilled their reproductive tracts for a potential new ejaculate. However, the timing and possible factors inducing this rapid olfactory switch-off are still unknown. Here, we determined the initial time delay and duration of the PEI. Moreover, we tested the hypothesis that the brain, the testis and/or the sex accessory glands (SAGs) could produce a factor inducing the PEI. Lastly, we investigated the possible involvement of ecdysteroids, hormones essential for development and reproduction in insects, in this olfactory plasticity. Using brain and SAG cross-injections in virgin and newly-mated males, surgical treatments, wind tunnel behavioral experiments and EIA quantifications of ecdysteroids, we show that the PEI starts very shortly after the onset of copulation, and that SAGs contain a factor, which is produced/accumulated after copulation to induce the PEI. Moreover, SAGs were found to be the main source of ecdysteroids, whose concentration decreased after mating, whereas it increased in the haemolymph. 20-Hydroxyecdysone (20E) was identified as the major ecdysteroid in SAGs of A. ipsilon males. Finally, 20E injections did not reduce the behavioral pheromone response of virgin males. Altogether our data indicate that 20E is probably not involved in the PEI.

Study on ecdysteroid levels and gene expression of enzymes related to ecdysteroid biosynthesis in the larval testis of Spodoptera littoralis.

We investigated here the ecdysteroid titers and the expression of six genes coding for known enzymes of the ecdysteroid biosynthesis in the testes of last instar larvae of the pest cotton leafworm, Spodoptera littoralis. We showed that the timing of the ecdysteroid profile was the same in testes and in hemolymph, with a small peak at day 2 and a large one at day 4 after ecdysis. Ecdysone and 20-hydroxyecdysone (20E) were detected in both tissues. 20E was the major ecdysteroid in testes and in hemolymph from day 4. Interestingly, the gene expression of the steroidogenetic enzymes, Neverland, and the five cytochrome P450 enzymes encoded by the Halloween genes was confirmed in the testes, and varied during the instar. However, from the data obtained so far, we cannot conclude that the measured ecdysteroids in the testes result from the activity of the genes under study. Indeed, it is suggested that the ecdysone produced centrally in the prothoracic glands, could have been transformed into 20E in the testes, where Sl-shade is well expressed.

Mutations in the neverland gene turned Drosophila pachea into an obligate specialist species.

Most living species exploit a limited range of resources. However, little is known about how tight associations build up during evolution between such specialist species and the hosts they use. We examined the dependence of Drosophila pachea on its single host, the senita cactus. Several amino acid changes in the Neverland oxygenase rendered D. pachea unable to transform cholesterol into 7-dehydrocholesterol (the first reaction in the steroid hormone biosynthetic pathway in insects) and thus made D. pachea dependent on the uncommon sterols of its host plant. The neverland mutations increase survival on the cactus's unusual sterols and are in a genomic region that faced recent positive selection. This study illustrates how relatively few genetic changes in a single gene may restrict the ecological niche of a species.

Juvenile development, ecdysteroids and hemolymph level of metabolites in the spider Brachypelma albopilosum (Theraphosidae).

In the present work, juvenile development and physiological state of mygalomorph Brachypelma albopilosum were investigated by means of individual rearing under controlled conditions. Males required 4-5 years for development from first juvenile instar to adulthood, passing through 8 to 12 juvenile molts. Females developed to adults in 5-6 years with a variable juvenile molt number from 9 to 13. The development and growth of males and females took place in a similar way until the last juvenile molt leading to subadults. Ecdysteroids, total lipid, cholesterol, and protein concentrations increased along with the different development instars in both males and females. After the last juvenile molt, spiders presented morphological and biochemical sex differences. Subadult and adulthood males were smaller in size and weight than females; hemolymph levels of ecdysteroids, total lipids, cholesterol, and glucose were higher in males. These physiological and biochemical differences can be correlated to the different sexual development between males and females.

The genome of Tetranychus urticae reveals herbivorous pest adaptations.

The spider mite Tetranychus urticae is a cosmopolitan agricultural pest with an extensive host plant range and an extreme record of pesticide resistance. Here we present the completely sequenced and annotated spider mite genome, representing the first complete chelicerate genome. At 90 megabases T. urticae has the smallest sequenced arthropod genome. Compared with other arthropods, the spider mite genome shows unique changes in the hormonal environment and organization of the Hox complex, and also reveals evolutionary innovation of silk production. We find strong signatures of polyphagy and detoxification in gene families associated with feeding on different hosts and in new gene families acquired by lateral gene transfer. Deep transcriptome analysis of mites feeding on different plants shows how this pest responds to a changing host environment. The T. urticae genome thus offers new insights into arthropod evolution and plant-herbivore interactions, and provides unique opportunities for developing novel plant protection strategies.

CYP18A1, a key enzyme of Drosophila steroid hormone inactivation, is essential for metamorphosis.

Ecdysteroids are steroid hormones, which coordinate major developmental transitions in insects. Both the rises and falls in circulating levels of active hormones are important for coordinating molting and metamorphosis, making both ecdysteroid biosynthesis and inactivation of physiological relevance. We demonstrate that Drosophila melanogaster Cyp18a1 encodes a cytochrome P450 enzyme (CYP) with 26-hydroxylase activity, a prominent step in ecdysteroid catabolism. A clear ortholog of Cyp18a1 exists in most insects and crustaceans. When Cyp18a1 is transfected in Drosophila S2 cells, extensive conversion of 20-hydroxyecdysone (20E) into 20-hydroxyecdysonoic acid is observed. This is a multi-step process, which involves the formation of 20,26-dihydroxyecdysone as an intermediate. In Drosophila larvae, Cyp18a1 is expressed in many target tissues of 20E. We examined the consequences of Cyp18a1 inactivation on Drosophila development. Null alleles generated by excision of a P element and RNAi knockdown of Cyp18a1 both result in pupal lethality, possibly as a consequence of impaired ecdysteroid degradation. Our data suggest that the inactivation of 20E is essential for proper development and that CYP18A1 is a key enzyme in this process.

Characterization of ecdysteroids in Drosophila melanogaster by enzyme immunoassay and nano-liquid chromatography-tandem mass spectrometry.

Ecdysteroids are polyhydroxylated steroids that function as molting hormones in insects. 20-Hydroxyecdysone (a 27C-ecdysteroid) is classically considered as the major steroid hormone of Drosophilamelanogaster, but this insect also contains 28C-ecdysteroids. This arises from both the use of several dietary sterols as precursors for the synthesis of its steroid hormones, and its inability to dealkylate the 28C-phytosterols to produce cholesterol. The nature of Drosophila ecdysteroids has been re-investigated using both high-performance liquid chromatography coupled to enzyme immunoassay and a particularly sensitive nano-liquid chromatography-mass spectrometry methodology, while taking advantage of recently available ecdysteroid standards isolated from plants. In vitro incubations of the larval steroidogenic organ, the ring-gland, reveals the synthesis of ecdysone, 20-deoxy-makisterone A and a third less polar compound identified as the 24-epimer of the latter, while wandering larvae contain the three corresponding 20-hydroxylated ecdysteroids. This pattern results from the simultaneous use of higher plant sterols (from maize) and fungal sterols (from yeast). The physiological relevance of all these ecdysteroids, which display different affinities to the ecdysteroid receptors, is still a matter of debate.

Study of steroidogenesis in pupae of the forensically important blow fly Protophormia terraenovae (Robineau-Desvoidy) (Diptera: Calliphoridae).

Protophormia terraenovae is a forensically important fly whose development time is studied by forensic entomologists to establish the time elapsed since death (post-mortem interval, PMI). Quantity and nature of ecdysteroid hormones present in P. terraenovae pupae were analysed in order to determine if they could be correlated to the age of pupae found on corpses and thereby could give information on the PMI. Ecdysteroid levels were quantified during the pupal-adult development of synchronised animals using enzyme immunoassay (EIA), a sensitive method allowing acurate quantification in one pupa. Two types of pupae were compared: "fresh" pupae, kept frozen until analysis and "experimentally dried" pupae, which were left for several weeks at ambient temperature. A peak of ecdysteroids was detected between 36 and 96 h after pupariation in fresh animals. It was not observed in "experimentally dried" pupae. High-pressure liquid chromatography (HPLC) analyses combined with EIA showed that 20-hydroxyecdysone (20E) was the major free ecdysteroid at various pupal ages. Enzymatic hydrolysis experiments revealed the presence of apolar conjugates at all ages tested. However, neither qualitative nor quantitative difference was detected between early and late pupae. This study gives precise information on the nature and quantity of ecdysteroids in the course of pupal development of a calliphorid fly. The limits of using ecdysteroid measurement as a tool in forensic entomology are discussed.

Molting cycle-dependent expression of CYP4C15, a cytochrome P450 enzyme putatively involved in ecdysteroidogenesis in the crayfish, Orconectes limosus.

A cytochrome P450 enzyme cDNA (CYP4C15) has been previously cloned from a cDNA library of crayfish steroidogenic glands (Y-organs). The conceptual translation of the CYP4C15 cDNA sequence was analyzed for regions of putative high antigenicity and a mixture of two synthetic peptides was chosen for the production of a specific polyclonal antibody. Western blot analysis on Y-organ subcellular fractions indicated an endoplasmic reticulum location of CYP4C15, in agreement with the structural feature of the predicted protein, i.e. the presence of a hydrophobic N-terminal segment. The protein is only expressed in Y-organs, thus showing a similar distribution to the corresponding mRNA. From this tissue specific expression, it has been postulated that CYP4C15 would play a role in ecdysteroid biosynthesis rather than detoxification and the variations of its expression during a molt cycle were carefully examined. CYP4C15 is not detectable in intermolt animals, expression levels are maximal during early premolt and decrease during late premolt. The results are discussed in relation to the variations of hemolymphatic ecdysteroid titers and steroidogenic capacities of the Y-organs during the molt cycle.

In vitro differentiation ofPieris brassicae imaginal wing discs: Effects and metabolism of ecdysone and ecdysterone.

Imaginal wing discs ofPieris brassicae can be cultured in vitro for extended periods. Their ultrastructural development was investigated after culture in the presence of various concentrations of ecdysone and ecdysterone. When ecdysone or low concentrations of ecdysterone (2×10-7 M) were added to culture media, larval discs secreted a pupal cuticle and they subsequently differentiated scales; prepupal discs completed their imaginal development. With a higher concentration of ecdysterone (2×10-6 M), all discs produced abundant but fragmentary cuticular material.Prepupal discs were able to metabolize both hormones in vitro. Ecdysterone was mainly converted into a polar compound detectable after a short period of incubation. Ecdysone was transformed, at a slower rate, forming a polar compound and 26-hydroxyecdysone but no ecdysterone.