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Little is known about the metabolic basis of life-history trade-offs but lipid stores seem to play a pivotal role. During reproduction, an energetically highly costly process, animals mobilize fat reserves. Conversely, reduced or curtailed reproduction promotes lipid storage in many animals. Systemic signals from the gonad seem to be involved: Caenorhabditis elegans lacking germline stem cells display endocrine changes, have increased fat stores and are long-lived. Similarly, germline-ablated Drosophila melanogaster exhibit major somatic physiological changes, but whether and how germline loss affects lipid metabolism remains largely unclear. Here we show that germline-ablated flies have profoundly altered energy metabolism at the transcriptional level and store excess fat as compared to fertile flies. Germline activity thus constrains or represses fat accumulation, and this effect is conserved between flies and worms. More broadly, our findings confirm that lipids represent a major energetic currency in which costs of reproduction are paid., (© The Society for the Study of Evolution (SSE) and European Society for Evolutionary Biology (ESEN) 2023.)

Many bees and stinging wasps, or aculeates, exhibit striking colour patterns or conspicuous coloration, such as black and yellow stripes. Such coloration is often interpreted as an aposematic signal advertising aculeate defences: the venomous sting. Aposematism can lead to Müllerian mimicry, the convergence of signals among different species unpalatable to predators. Müllerian mimicry has been extensively studied, notably on Neotropical butterflies and poison frogs. However, although a very high number of aculeate species harbour putative aposematic signals, aculeates are under-represented in mimicry studies. Here, we review the literature on mimicry rings that include bee and stinging wasp species. We report over a hundred described mimicry rings, involving a thousand species that belong to 19 aculeate families. These mimicry rings are found all throughout the world. Most importantly, we identify remaining knowledge gaps and unanswered questions related to the study of Müllerian mimicry in aculeates. Some of these questions are specific to aculeate models, such as the impact of sociality and of sexual dimorphism in defence levels on mimicry dynamics. Our review shows that aculeates may be one of the most diverse groups of organisms engaging in Müllerian mimicry and that the diversity of aculeate Müllerian mimetic interactions is currently under-explored. Thus, aculeates represent a new and major model system to study the evolution of Müllerian mimicry. Finally, aculeates are important pollinators and the global decline of pollinating insects raises considerable concern. In this context, a better understanding of the impact of Müllerian mimicry on aculeate communities may help design strategies for pollinator conservation, thereby providing future directions for evolutionary research., (© 2023 The Authors. Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society.)

Sarracenia pitcher plants display interspecific differences in prey, so far only explained by pitcher morphology. We hypothesized that pitcher odours play a role in prey composition. We first compared odour and prey compositions among Sarracenia taxa grown together, forming a kinship gradient from S. purpurea known to capture primarily ants towards S. leucophylla known to capture many flying insects: S. purpurea, S. X mitchelliana, and S. X Juthatip soper & S. X leucophylla horticultural hybrids. We then measured several pitcher traits to disentangle the contributions of morphology and odour to prey variation. The pitcher odours were as diverse as those of generalist-pollinated flowers but with notable differences among taxa, reflecting their relatedness. VOC similarity analyses revealed taxon specificities, that mirrored those revealed by prey similarity analyses. S. X leucophylla stood out by being more specialised in flying insects like bees and moths and by releasing more monoterpenes known to attract flower visitors. S. X Juthatip soper trapped as many bees but fewer moths, sesquiterpenes contributing less to its scent. Ants and Diptera were the main prey of the other two with fatty-acid-derivative-dominated scents. Quantities of the different prey groups can be inferred 98% from quantities of the odour classes and pitcher dimensions. Two syndromes were revealed: ants associated with fatty-acid-derivatives and short pitchers; flying insects associated with monoterpenes, benzenoids and tall pitchers. In S. X leucophylla, emission rate of fatty-acid-derivatives and pitcher length explained most variation in ant captures; monoterpenes and pitcher length explained most variation in bee and moth captures; monoterpenes alone explained most variation in Diptera and wasp captures. Our results suggest that odours are key factors of the diet composition of pitcher plants. They support the hypothesis of perceptual exploitation of insect biases in carnivorous plants and provide new insights into the olfactory preferences of insect groups., Competing Interests: This work was publicly supported by the French National Research Agency (ANR-16-IDEX-0006 to LG, CV and DG. under the “Investissements d’avenir” program), which funded material, plants and chemical analyses, as well as the PhD salary of CD. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., (Copyright: © 2023 Dupont et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The more restrictive regulations of pesticides in Europe have led to an increase in conservation biological control (CBC) research. However, little attention has been paid to the main determinants of Lobesia botrana parasitism. The Douro Demarcated Region landscape offers scope for the use of CBC. The study was conducted between 2002 and 2015 aiming at: (i) identifying parasitoids associated with L. botrana and evaluating their impact as biological control agents in each generation of the pest, and (ii) evaluating the effect of both the proportion of ecological infrastructures (EI) near the vineyards, and the impact of management practices (chemical treatments and ground cover) on the parasitism of L. botrana . A total of 3226 larvae/pupae of L. botrana were collected (15% were parasitized and 485 parasitoids emerged). A complex of 16 taxa of parasitoids was identified, the majority belonging to Hymenoptera. The most abundant were Elachertus sp. (Eulophidae), Campoplex capitator Aubert (Ichneumonidae), and Brachymeria tibialis (Walker) (Chalcididae), which represented 62.5, 12.6, and 12.0% of the total assemblage of parasitoids which emerged, respectively. The percentage of parasitism ranged from 0.0 to 61.5% (first generation), from 0.0 to 36.8% (second generation), and from 0.0 to 12.1% (third generation). Importantly, it was found that the parasitism rate was higher in vineyards with ground cover. In addition, EI in the area surrounding the vineyards produced a significant increase in parasitism. These results suggest potential for CBC of L. botrana if EI around vineyards, and ground cover with native perennial plants within vineyards, are encouraged.

This second paper on the species of Diaparsis Frster occurring in Papua New Guinea comprises descriptions of ten new species and the second part of the key to the species. The following species are described as new to science: D. alpha sp. nov., D. beta sp. nov., D. delta sp. nov., D. epsilon sp. nov., D. eta sp. nov., D. gamma sp. nov., D. iota sp. nov., D. kappa sp. nov., D. theta sp. nov. and D. zeta sp. nov., raising the total number of currently known Diaparsis species in Papua New Guinea to nineteen.

Most flowering plants species rely on insects for pollination, a successful mutualism allowing them to reproduce over wide areas while flower-visitors are rewarded with food. This association is so conspicuous in the case of bees that other groups of potential pollinators, especially flies, have long been underestimated. However, visitors are not always pollinators. While the importance of flies in plant-visitor networks is now acknowledged, their pollination effectiveness has hardly been investigated. In this study, we assessed the pollination effectiveness of Geranium sylvaticum flower-visitors using single-visit seed set experiments, in a subalpine meadow where flies are predominant. We found that: (i) empidine dance flies were the most frequent visitors of G. sylvaticum; (ii) a single-visit by an empidine dance fly produced the same average number of seeds as a visit by a bee; (iii) large pollinators were more efficient than small pollinators irrespective of their identity. As a conclusion, large empidines were the main pollinators of G. sylvaticum. Considering the high diversity and abundance of flower-visiting fly species, such results showing their ability to be as effective pollinators as bees should encourage further studies to develop a better understanding on their role in plant-pollinator networks.

Human malaria, which remains a major public health problem, is transmitted by a subset of Anopheles mosquitoes belonging to only three out of eight subgenera: Anopheles, Cellia and Nyssorhynchus. Unlike almost every other insect species, males of some Anopheles species produce steroid hormones which are transferred to females during copulation to influence their reproduction. Steroids are consequently a potential target for malaria vector control. Here, we analysed the evolution of sexually-transferred steroids and their effects on female reproductive traits across Anopheles by using a set of 16 mosquito species (five Anopheles, eight Cellia, and three Nyssorhynchus), including malaria vector and non-vector species. We show that male steroid production and transfer are specific to the Cellia and therefore represent a synapomorphy of this subgenus. Furthermore, we show that mating-induced effects in females are variable across species and differences are not correlated with sexually-transferred steroids or with Anopheles ability to transmit human malaria. Overall, our findings highlight that Anopheles mosquitoes have evolved different reproductive strategies, independently of being a malaria vector or not.

Seven species of the genus Allophrys Förster, A. cracentis sp. nov., A. daklaka Khalaim, A. madanga sp. nov., A. miklouhomaclayi sp. nov., A. occipitata Khalaim, A. parvidentata sp. nov. and A. valorosa sp. nov., and one species of Probles Förster, P. (Euporizon) papuaensis sp. nov., have been discovered from Papua New Guinea. This is the first record of these genera, as well as the subfamily Tersilochinae, from this country. An identification key to seven species of Allophrys occurring in Papua New Guinea is provided.

The cross-pollination of most alpine plants depends on insects, whose altitudinal distribution is limited by temperature. However, although global warming is causing shifts in temporal and spatial species distribution, we are still largely unaware of how plant-pollinator interactions change with elevation and time along altitudinal gradients. This makes the detection of endangered interactions and species challenging. In this study, we aimed at providing such a reference, and tested if and how the major flower-visiting insect orders and families segregated by altitude, phenology and foraging preferences along an elevational gradient from 970 m to 2700 m in the Alps. Flies were the main potential pollinators from 1500 m, as bees and beetles decreased rapidly above that limit. Diptera, Coleoptera and Hymenoptera differed significantly in the angiosperm assemblages visited. Within Diptera, the predominant group, major families segregated by both phenology and foraging preferences along the gradient. Empidids, muscids and anthomyiids, whose role in pollination has never been investigated, dominated the upper part of the gradient. Our results thus suggest that flies and the peculiar plants they visit might be particularly at risk under global warming, and highlight the blatant lack of studies about critical components of these rich, yet fragile mountain ecosystems.

Predictive studies are of paramount importance for biological invasions, one of the biggest threats for biodiversity. To help and better prioritize management strategies, species distribution models (SDMs) are often used to predict the potential invasive range of introduced species. Yet, SDMs have been regularly criticized, due to several strong limitations, such as violating the equilibrium assumption during the invasion process. Unfortunately, validation studies-with independent data-are too scarce to assess the predictive accuracy of SDMs in invasion biology. Yet, biological invasions allow to test SDMs usefulness, by retrospectively assessing whether they would have accurately predicted the latest ranges of invasion. Here, we assess the predictive accuracy of SDMs in predicting the expansion of invasive species. We used temporal occurrence data for the Asian hornet Vespa velutina nigrithorax, a species native to China that is invading Europe with a very fast rate. Specifically, we compared occurrence data from the last stage of invasion (independent validation points) to the climate suitability distribution predicted from models calibrated with data from the early stage of invasion. Despite the invasive species not being at equilibrium yet, the predicted climate suitability of validation points was high. SDMs can thus adequately predict the spread of V. v. nigrithorax, which appears to be-at least partially-climatically driven. In the case of V. v. nigrithorax, SDMs predictive accuracy was slightly but significantly better when models were calibrated with invasive data only, excluding native data. Although more validation studies for other invasion cases are needed to generalize our results, our findings are an important step towards validating the use of SDMs in invasion biology.

A new distinctive genus Spathiophaenodus gen. nov. from the tribe Pambolini (Braconidae: Exothecinae), with type species Spathiophaenodus annae sp. nov. from Papua New Guinea, is described and illustrated. The original key to the World genera of the tribe Pambolini is provided.

Since its introduction in France 10 years ago, the yellow-legged Asian bee-hawking hornet Vespa velutina has rapidly spread to neighboring countries (Spain, Portugal, Belgium, Italy, and Germany), becoming a new threat to beekeeping activities. While introduced species often leave behind natural enemies from their original home, which benefits them in their new environment, they can also suffer local recruitment of natural enemies. Three mermithid parasitic subadults were obtained from V. velutina adults in 2012, from two French localities. However, these were the only parasitic nematodes reported up to now in Europe, in spite of the huge numbers of nests destroyed each year and the recent examination of 33,000 adult hornets. This suggests that the infection of V. velutina by these nematodes is exceptional. Morphological criteria assigned the specimens to the genus Pheromermis and molecular data (18S sequences) to the Mermithidae, due to the lack of Pheromermis spp. sequences in GenBank. The species is probably Pheromermis vesparum, a parasite of social wasps in Europe. This nematode is the second native enemy of Vespa velutina recorded in France, after a conopid fly whose larvae develop as internal parasitoids of adult wasps and bumblebees. In this paper, we provide arguments for the local origin of the nematode parasite and its limited impact on hornet colony survival. We also clarify why these parasites (mermithids and conopids) most likely could not hamper the hornet invasion nor be used in biological control programs against this invasive species.

Wing venation has long been used for insect identification. Lately, the characterization of venation shape using geometric morphometrics has further improved the potential of using the wing for insect identification. However, external factors inducing variation in wing shape could obscure specific differences, preventing accurate discrimination of species in heterogeneous samples. Here, we show that interspecific difference is the main source of wing shape variation within social wasps. We found that a naive clustering of wing shape data from taxonomically and geographically heterogeneous samples of workers returned groups congruent with species. We also confirmed that individuals can be reliably attributed to their genus, species and populations on the basis of their wing shape. Our results suggested that the shape variation reflects the evolutionary history with a potential influence of other factors such as body shape, climate and mimicry selective pressures. However, the high dimensionality of wing shape variation may have prevented absolute convergences between the different species. Wing venation shape is thus a taxonomically relevant marker combining the accuracy of quantitative characters with the specificity required for identification criteria. This marker may also highlight adaptive processes that could help understand the wing's influence on insect flight., (© 2014 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2014 European Society For Evolutionary Biology.)

Cytoplasmic dynein 1 (dynein) is a minus end-directed microtubule motor protein with many cellular functions, including during cell division. The role of the light intermediate chains (LICs; DYNC1LI1 and 2) within the complex is poorly understood. In this paper, we have used small interfering RNAs or morpholino oligonucleotides to deplete the LICs in human cell lines and Xenopus laevis early embryos to dissect the LICs' role in cell division. We show that although dynein lacking LICs drives microtubule gliding at normal rates, the LICs are required for the formation and maintenance of a bipolar spindle. Multipolar spindles with poles that contain single centrioles were formed in cells lacking LICs, indicating that they are needed for maintaining centrosome integrity. The formation of multipolar spindles via centrosome splitting after LIC depletion could be rescued by inhibiting Eg5. This suggests a novel role for the dynein complex, counteracted by Eg5, in the maintenance of centriole cohesion during mitosis., (© 2014 Jones et al.)

Pollination is one of the most important ecosystem services and bees the most important pollinators. As a population decline of bees has been documented in numerous regions of the world, it is crucial to develop understanding on other possible pollinators. Here, we study the potential pollination impact of Diptera, and among them Empidinae, in an alpine environment, where the abundance of bees is naturally lower. Interactions between 19 entomophilous plants and their flower visitors were recorded in a subalpine meadow in the French Alps during six weeks. Visitation frequencies were used to build the flower-visitor network. Our results show that interactions between flies and plants are dominant; flies represent more than 60% of all visitors, with 54% of them being Empidinae. We especially found that flies, Empidinae and bees are the main visitors of 11, three and one plants, respectively. When considering both bees and Syrphidae together, six plants were more visited by Empidinae; when considering bees and Syrphidae separately, 10 plants were more visited by Empidinae than by bees or Syrphidae. The results support the idea that flies widely replace bees as main flower visitors at altitude, and among them the Empidinae might play a key role in pollination., (© 2014 The Author(s) Published by the Royal Society. All rights reserved.)

Neighboring plants within a local community may be separated by many millions of years of evolutionary history, potentially reducing enemy pressure by insect herbivores. However, it is not known how the evolutionary isolation of a plant affects the fitness of an insect herbivore living on such a plant, especially the herbivore's enemy pressure. Here, we suggest that evolutionary isolation of host plants may operate similarly as spatial isolation and reduce the enemy pressure per insect herbivore. We investigated the effect of the phylogenetic isolation of host trees on the pressure exerted by specialist and generalist enemies (parasitoids and birds) on ectophagous Lepidoptera and galling Hymenoptera. We found that the phylogenetic isolation of host trees decreases pressure by specialist enemies on these insect herbivores. In Lepidoptera, decreasing enemy pressure resulted from the density dependence of enemy attack, a mechanism often observed in herbivores. In contrast, in galling Hymenoptera, enemy pressure declined with the phylogenetic isolation of host trees per se, as well as with the parallel decline in leaf damage by non-galling insects. Our results suggest that plants that leave their phylogenetic ancestral neighborhood can trigger, partly through simple density-dependency, an enemy release and fitness increase of the few insect herbivores that succeed in tracking these plants.

The preclinical development process of chemotherapeutic drugs is often carried out in two-dimensional monolayer cultures. However, a considerable amount of evidence demonstrates that two-dimensional cell culture does not accurately reflect the three-dimensional in vivo tumour microenvironment, specifically with regard to gene expression profiles, oxygen and nutrient gradients and pharmacokinetics. With this objective in mind, we have developed and established a physiologically relevant three-dimensional in vitro model of colorectal cancer based on the removal of interstitial fluid from collagen type I hydrogels. We employed the RAFT™ (Real Architecture For 3D Tissue) system for producing three-dimensional cultures to create a controlled reproducible, multiwell testing platform. Using the HT29 and HCT116 cell lines to model epidermal growth factor receptor expressing colorectal cancers, we characterized three-dimensional cell growth and morphology in addition to the anti-proliferative effects of the anti-epidermal growth factor receptor chemotherapeutic agent cetuximab in comparison to two-dimensional monolayer cultures. Cells proliferated well for 14 days in three-dimensional culture and formed well-defined cellular aggregates within the concentrated collagen matrix. Epidermal growth factor receptor expression levels revealed a twofold and threefold increase in three-dimensional cultures for both HT29 and HCT116 cells in comparison to two-dimensional monolayers, respectively (p < 0.05; p < 0.01). Cetuximab efficacy was significantly lower in HT29 three-dimensional cultures in comparison to two-dimensional monolayers, whereas HCT116 cells in both two-dimension and three-dimension were non-responsive to treatment in agreement with their KRAS mutant status. In summary, these results confirm the use of a three-dimensional in vitro cancer model as a suitable drug-screening platform for in vitro pharmacological testing.

Coloration of stinging insects is often based on contrasted patterns of light and black pigmentations as a warning signal to predators. However, in many social wasp species, geographic variation drastically modifies this signal through melanic polymorphism potentially driven by different selective pressures. To date, surprisingly little is known about the geographic variation of coloration of social wasps in relation to aposematism and melanism and to genetic and developmental constraints. The main objectives of this study are to improve the description of the colour variation within a social wasp species and to determine which factors are driving this variation. Therefore, we explored the evolutionary history of a polymorphic hornet, Vespa velutina Lepeletier, 1836, using mitochondrial and microsatellite markers, and we analysed its melanic variation using a colour space based on a description of body parts coloration. We found two main lineages within the species and confirmed the previous synonymy of V. auraria Smith, 1852, under V. velutina, differing only by the coloration. We also found that the melanic variation of most body parts was positively correlated, with some segments forming potential colour modules. Finally, we showed that the variation of coloration between populations was not related to their molecular, geographic or climatic differences. Our observations suggest that the coloration patterns of hornets and their geographic variations are determined by genes with an influence of developmental constraints. Our results also highlight that Vespa velutina populations have experienced several convergent evolutions of the coloration, more likely influenced by constraints on aposematism and Müllerian mimicry than by abiotic pressures on melanism.

In insects, the steroid hormone 20-hydroxyecdysone (20E) coordinates major developmental transitions. While the first and the final steps of 20E biosynthesis are characterized, the pathway from 7-dehydrocholesterol to 5β-ketodiol, commonly referred as the "black box", remains hypothetical and whether there are still unidentified enzymes is unknown. The black box would include some oxidative steps, which are believed to be mediated by P450 enzymes. To identify new enzyme(s) involved in steroid synthesis, we analyzed by small-scale microarray the expression of all the genes encoding P450 enzymes of the malaria mosquito Anopheles gambiae in active steroidogenic organs of adults, ovaries from blood-fed females and male reproductive tracts, compared to inactive steroidogenic organs, ovaries from non-blood-fed females. Some genes encoding P450 enzymes were specifically overexpressed in female ovaries after a blood-meal or in male reproductive tracts but only three genes were found to be overexpressed in active steroidogenic organs of both females and males: cyp307a1, cyp4g16 and cyp6n1. Among these genes, only cyp307a1 has an expression pattern similar to other mosquito steroidogenic genes. Moreover, loss-of-function by transient RNAi targeting cyp307a1 disrupted ecdysteroid production demonstrating that this gene is required for ecdysteroid biosynthesis in Anopheles gambiae.

The resistance of mosquitoes to chemical insecticides is threatening vector control programmes worldwide. Cytochrome P450 monooxygenases (CYPs) are known to play a major role in insecticide resistance, allowing resistant insects to metabolize insecticides at a higher rate. Among them, members of the mosquito CYP6Z subfamily, like Aedes aegypti CYP6Z8 and its Anopheles gambiae orthologue CYP6Z2, have been frequently associated with pyrethroid resistance. However, their role in the pyrethroid degradation pathway remains unclear. In the present study, we created a genetically modified yeast strain overexpressing Ae. aegypti cytochrome P450 reductase and CYP6Z8, thereby producing the first mosquito P450-CPR (NADPH-cytochrome P450-reductase) complex in a yeast recombinant system. The results of the present study show that: (i) CYP6Z8 metabolizes PBAlc (3-phenoxybenzoic alcohol) and PBAld (3-phenoxybenzaldehyde), common pyrethroid metabolites produced by carboxylesterases, producing PBA (3-phenoxybenzoic acid); (ii) CYP6Z8 transcription is induced by PBAlc, PBAld and PBA; (iii) An. gambiae CYP6Z2 metabolizes PBAlc and PBAld in the same way; (iv) PBA is the major metabolite produced in vivo and is excreted without further modification; and (v) in silico modelling of substrate-enzyme interactions supports a similar role of other mosquito CYP6Zs in pyrethroid degradation. By playing a pivotal role in the degradation of pyrethroid insecticides, mosquito CYP6Zs thus represent good targets for mosquito-resistance management strategies.

Mosquitoes are vectors of several major human diseases and their control is mainly based on the use of chemical insecticides. Resistance of mosquitoes to organochlorines, organophosphates, carbamates and pyrethroids led to a regain of interest for the use of neonicotinoid insecticides in vector control. The present study investigated the molecular basis of neonicotinoid resistance in the mosquito Aedes aegypti. A strain susceptible to insecticides was selected at the larval stage with imidacloprid. After eight generations of selection, larvae of the selected strain (Imida-R) showed a 5.4-fold increased tolerance to imidacloprid while adult tolerance level remained low. Imida-R larvae showed significant cross-tolerance to other neonicotinoids but not to pyrethroids, organophosphates and carbamates. Transcriptome profiling identified 344 and 108 genes differentially transcribed in larvae and adults of the Imida-R strain compared to the parental strain. Most of these genes encode detoxification enzymes, cuticle proteins, hexamerins as well as other proteins involved in cell metabolism. Among detoxification enzymes, cytochrome P450 monooxygenases (CYPs) and glucosyl/glucuronosyl transferases (UDPGTs) were over-represented. Bioassays with enzyme inhibitors and biochemical assays confirmed the contribution of P450s with an increased capacity of the Imida-R microsomes to metabolize imidacloprid in presence of NADPH. Comparison of substrate recognition sites and imidacloprid docking models of six CYP6s over-transcribed in the Imida-R strain together with Bemisia tabaci CYP6CM1vQ and Drosophila melanogaster CYP6G1, both able to metabolize imidacloprid, suggested that CYP6BB2 and CYP6N12 are good candidates for imidacloprid metabolism in Ae. aegypti. The present study revealed that imidacloprid tolerance in mosquitoes can arise after few generations of selection at the larval stage but does not lead to a significant tolerance of adults. As in other insects, P450-mediated insecticide metabolism appears to play a major role in imidacloprid tolerance in mosquitoes., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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.

Caste shape dimorphism (CShD) has previously been studied in wasps through comparison of different body parts, originating from different imaginal discs. Using geometric morphometrics with a new protocol for measuring wings of pinned specimens from natural history collections, we tested CShD of three hornet species in an organ developed from a single imaginal disc: the forewing. Gaussian mixture models retrieved most castes and species levels, confirming that caste is an important component of wing variations in females of these hornets. Size and allometry - the influence of size on shape - contribution to wing dimorphism between castes was major, but failed to explain the entire shape dimorphism. This deviation from simple allometric scaling was not similar in the three species: in Vespa tropica, allometric directions in the shape space differed between castes, whereas in V. crabro and V. velutina, they were similar but a significant part of CShD resulted from lateral transpositions. These results clearly indicate that queens are not just enlarged workers. They also support that the different patterns of CShD may result from different developmental mechanisms. Finally, they highlight that even in a highly social group like hornets, there is still variation in caste dimorphism among species., (© 2012 The Authors. Journal of Evolutionary Biology © 2012 European Society For Evolutionary Biology.)

Ecdysteroids exert many pharmacological effects in mammals (including humans), most of which appear beneficial, but their mechanism of action is far from understood. Whether they act directly and/or after the formation of metabolites is still an open question. The need to investigate this question has gained extra impetus because of the recent development of ecdysteroid-based gene-therapy systems for mammals. In order to investigate the metabolic fate of ecdysteroids in mice, [1α,2α-(3)H]20-hydroxyecdysone was prepared and injected intraperitoneally to mice. Their excretory products (urine+faeces) were collected and the different tritiated metabolites were isolated and identified. The pattern of ecdysteroid metabolites is very complex, but no conjugates were found, in contrast to the classical fate of the (less polar) endogenous vertebrate steroid hormones. Primary reactions involve dehydroxylation at C-14 and side-chain cleavage between C-20 and C-22, thereby yielding 14-deoxy-20-hydroxyecdysone, poststerone and 14-deoxypoststerone. These metabolites then undergo several reactions of reduction involving, in particular, the 6-keto-group. A novel major metabolite has been identified as 2β,3β,6α,22R,25-pentahydroxy-5β-cholest-8(14)-ene. The formation of this and the other major metabolites is discussed in relation to the various effects of ecdysteroids already demonstrated on vertebrates., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2010 Elsevier Inc. All rights reserved.)

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., (Copyright 2010 Elsevier B.V. All rights reserved.)

Steroid hormones regulate many physiological processes in vertebrates, nematodes, and arthropods through binding to nuclear receptors (NR), a metazoan-specific family of ligand-activated transcription factors. The main steps controlling the diversification of this family are now well-understood. In contrast, the origin and evolution of steroid ligands remain mysterious, although this is crucial for understanding the emergence of modern endocrine systems. Using a comparative genomic approach, we analyzed complete metazoan genomes to provide a comprehensive view of the evolution of major enzymatic players implicated in steroidogenesis at the whole metazoan scale. Our analysis reveals that steroidogenesis has been independently elaborated in the 3 main bilaterian lineages, and that steroidogenic cytochrome P450 enzymes descended from those that detoxify xenobiotics.

In female insects, the steroid hormone 20-hydroxyecdysone (20E) plays a major role in activating vitellogenesis, a process required for egg development. By contrast with vertebrates, production of large amounts of hormonal steroids has not been reported in adult male insects. In the present study, we analyzed steroidogenesis in both male and female adult of the malaria mosquito Anopheles gambiae and we found that A. gambiae male mosquitoes produce high amounts of the steroid hormone 20E. Importantly, we found that male accessory glands, but not testes, are the source of 20E. Moreover, this steroid hormone is stored in male accessory glands and delivered to females during mating. These findings suggest that male 20E may not act as a true male sex steroid, but more likely as an allohormone. Our results give new insights into species-specific physiological processes that govern the reproductive success of the malaria mosquito. This could thus lead to the identification of new target genes for manipulating male and/or female reproductive success, a promising way to reduce or eliminate mosquito population and therefore to control malaria transmission.

In insects, control of body size is intimately linked to nutritional quality as well as environmental and genetic cues that regulate the timing of developmental transitions. Prothoracicotropic hormone (PTTH) has been proposed to play an essential role in regulating the production and/or release of ecdysone, a steroid hormone that stimulates molting and metamorphosis. In this report, we examine the consequences on Drosophila development of ablating the PTTH-producing neurons. Surprisingly, PTTH production is not essential for molting or metamorphosis. Instead, loss of PTTH results in delayed larval development and eclosion of larger flies with more cells. Prolonged feeding, without changing the rate of growth, causes the overgrowth and is a consequence of low ecdysteroid titers. These results indicate that final body size in insects is determined by a balance between growth-rate regulators such as insulin and developmental timing cues such as PTTH that set the duration of the feeding interval.

Alterations of hepatopancreatic multi-transcript expression patterns, related to induced moult cycle, were identified in male Cherax quadricarinatus through cDNA microarray hybridizations of hepatopancreatic transcript populations. Moult was induced by X-organ sinus gland extirpation or by repeated injections of 20-hydroxyecdysone. Manipulated males were sacrificed at premoult or early postmoult, and a reference population was sacrificed at intermoult. Differentially expressed genes among the four combinations of two induction methods and two moult stages were identified. Biologically interesting clusters revealing concurrently changing transcript expressions across treatments were selected, characterized by a general shift of expression throughout premoult and early postmoult vs. intermoult, or by different premoult vs. postmoult expressions. A number of genes were differentially expressed in 20-hydroxyecdysone-injected crayfish vs. X-organ sinus gland extirpated males.

Background: Nasopharyngeal carcinomas (NPC) are consistently associated with the Epstein-Barr virus (EBV). Their malignant epithelial cells contain the viral genome and express several antigenic viral proteins. However, the mechanisms of immune escape in NPCs are still poorly understood. EBV-transformed B-cells have been reported to release exosomes carrying the EBV-encoded latent membrane protein 1 (LMP1) which has T-cell inhibitory activity. Although this report suggested that NPC cells could also produce exosomes carrying immunosuppressive proteins, this hypothesis has remained so far untested., Methods: Malignant epithelial cells derived from NPC xenografts--LMP1-positive (C15) or negative (C17)--were used to prepare conditioned culture medium. Various microparticles and vesicles released in the culture medium were collected and fractionated by differential centrifugation. Exosomes collected in the last centrifugation step were further purified by immunomagnetic capture on beads carrying antibody directed to HLA class II molecules. Purified exosomes were visualized by electron microscopy and analysed by western blotting. The T-cell inhibitory activities of recombinant LMP1 and galectin 9 were assessed on peripheral blood mononuclear cells activated by CD3/CD28 cross-linking., Results: HLA-class II-positive exosomes purified from C15 and C17 cell supernatants were containing either LMP1 and galectin 9 (C15) or galectin 9 only (C17). Recombinant LMP1 induced a strong inhibition of T-cell proliferation (IC50 = 0.17 nM). In contrast recombinant galectin 9 had a weaker inhibitory effect (IC50 = 46 nM) with no synergy with LMP1., Conclusion: This study provides the proof of concept that NPC cells can release HLA class-II positive exosomes containing galectin 9 and/or LMP1. It confirms that the LMP1 molecule has intrinsic T-cell inhibitory activity. These findings will encourage investigations of tumor exosomes in the blood of NPC patients and assessment of their effects on various types of target cells.

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.

Nedd4 family ubiquitin ligases regulate trafficking and degradation of numerous target substrates in different cellular compartments, including at the plasma membrane, in endosomes, in the secretory pathway and in the nucleus. WWP1 is a Nedd4 family protein closely related to mouse Itch and Drosophila Su(dx), both of which have been shown to regulate the Notch receptor. To investigate the possibility that WWP1 is also associated with Notch signalling we coexpressed human Notch1 and WWP1 in mouse myoblast cells. We found that WWP1 could localize to both the nucleus and cytoplasm in a context dependent manner. Coexpression of human Notch1 (hN1) depleted WWP1 from the nucleus to colocalise with hN1 in early endosomes, dependent on the presence of the C-terminal HECT domain. Furthermore we found that full-length expressed WWP1 could interact in vitro with the cytoplasmic domain of human Notch1. The Notch receptor has multiple roles in development, mediating a short-range signal that controls cell fate and pattern formation. The canonical Notch signal involves proteolytic release of the soluble Notch intracellular domain and the activation by the latter of the transcription factor Suppressor of Hairless/CBF-1 in the nucleus. This pathway does not however account for all of the activity of Notch. The ability of Notch to regulate the nuclear localization of WWP1 suggests a possible alternative mechanism by which Notch may communicate a signal to the nucleus. Drosophila Notch similarly regulated the nuclear localization of the Drosophila Nedd4 family protein, Suppressor of deltex, implying conservation of this mechanism during evolution.

The response of mosquito larvae to plant toxins found in their breeding sites was investigated by using Aedes aegypti larvae and toxic arborescent leaf litter as experimental models. The relation between larval tolerance to toxic leaf litter and cytochrome P450 monooxygenases (P450s) was examined at the toxicological, biochemical and molecular levels. Larvae pre-exposed to toxic leaf litter show a higher tolerance to those xenobiotics together with a strong increase in P450 activity levels. This enzymatic response is both time- and dose-dependent. The use of degenerate primers from various P450 genes (CYPs) allowed us to isolate 16 new CYP genes belonging to CYP4, CYP6 and CYP9 families. Expression studies revealed a 2.3-fold over-expression of 1 CYP gene (CYP6AL1) after larval pre-exposure to toxic leaf litter, this gene being expressed at a high level in late larval and pupal stages and in fat bodies and midgut. The CYP6AL1 protein has a high level of identity with other insect's CYPs involved in xenobiotic detoxification. The role of CYP genes in tolerance to natural xenobiotics and the importance of such adaptive responses in the capacity of mosquitoes to colonize new habitats and to develop insecticide resistance mechanisms are discussed.

All animals coordinate growth and maturation to reach their final size and shape. In insects, insulin family molecules control growth and metabolism, whereas pulses of the steroid 20-hydroxyecdysone (20E) initiate major developmental transitions. We show that 20E signaling also negatively controls animal growth rates by impeding general insulin signaling involving localization of the transcription factor dFOXO and transcription of the translation inhibitor 4E-BP. We also demonstrate that the larval fat body, equivalent to the vertebrate liver, is a key relay element for ecdysone-dependent growth inhibition. Hence, ecdysone counteracts the growth-promoting action of insulins, thus forming a humoral regulatory loop that determines organismal size.

Variations in ecdysteroid titers play crucial roles in arthropods by initiating and regulating molting and metamorphosis. The recent identification of genes coding for cytochrome P450 enzymes involved in Drosophila ecdysteroidogenesis provides new molecular tools to investigate the regulation of insect hormone production. In the present study, we used an enzyme immunoassay to show that the molting hormone titer is strictly correlated with the steroidogenic capacity of the ring gland. A temporal correlation between dynamics of ecdysone production and expression of genes encoding steroidogenic enzymes was observed during the third instar, suggesting that the timing of hormone production depends on transcriptional regulation of the biosynthetic enzymes. Using clonal analysis, levels of two steroidogenic enzymes, Phantom (PHM) and Disembodied (DIB), were shown to be very reduced in ftz transcription factor 1 (ftz-f1) mutant ring gland cells whereas there was no effect of the without children (woc) mutation, suggesting that FTZ-F1 regulates phm and dib expression. Since betaFTZ-F1 is the homolog of the vertebrate steroidogenic factor 1 (SF1), which plays a key role in the differentiation of vertebrate steroidogenic organs through transcriptional regulation of steroidogenic enzymes, this study emphasizes the strong parallels between insects and vertebrates with respect to the regulatory mechanisms of steroidogenesis.

We have reported recently the identification and characterization of the last three mitochondrial cytochrome P450 enzymes (CYP) controlling the biosynthesis of 20-hydroxyecdysone, the molting hormone of insects. These are encoded by the following genes: disembodied (dib, Cyp302a1, the 22-hydroxylase); shadow (sad, Cyp315a1, the 2-hydroxylase); and shade (shd, Cyp314a1, the 20-hydroxylase). Employing similar gene identification and transfection techniques and subsequent biochemical analysis of the expressed enzymatic activity, we report the identity of the Drosophila gene phantom (phm), located at 17D1 of the X chromosome, as encoding the microsomal 25-hydroxylase (Cyp306a1). Similar analysis following differential display-based gene identification has also resulted in the characterization of the corresponding 25-hydroxylase gene in Bombyx mori. Confirmation of 2,22,25-trideoxyecdysone (3beta,5beta-ketodiol) conversion to 2,22-dideoxyecdysone (3beta,5beta-ketotriol) mediated by either Phm enzyme employed LC, MS and definitive NMR analysis. In situ developmental gene analysis, in addition to northern, western and RT-PCR techniques during Drosophila embryonic, larval and adult development, are consistent with this identification. That is, strong expression of phm is restricted to the prothoracic gland cells of the Drosophila larval ring gland, where it undergoes dramatic changes in expression, and in the adult ovary, but also in the embryonic epidermis. During the last larval-larval transition in Bombyx, a similar expression pattern in the prothoracic gland is observed, but as in Drosophila, slight expression is also present in other tissues, suggesting a possible additional role for the phantom enzyme., (Copyright 2004 Elsevier Ltd.)

The steroid 20-hydroxyecdysone (20E) is the primary regulatory hormone that mediates developmental transitions in insects and other arthropods. 20E is produced from ecdysone (E) by the action of a P450 monooxygenase that hydroxylates E at carbon 20. The gene coding for this key enzyme of ecdysteroidogenesis has not been identified definitively in any insect. We show here that the Drosophila E-20-monooxygenase (E20MO) is the product of the shade (shd) locus (cytochrome p450, CYP314a1). When shd is transfected into Drosophila S2 cells, extensive conversion of E to 20E is observed, whereas in sorted homozygous shd embryos, no E20MO activity is apparent either in vivo or in vitro. Mutations in shd lead to severe disruptions in late embryonic morphogenesis and exhibit phenotypes identical to those seen in disembodied (dib) and shadow (sad) mutants, two other genes of the Halloween class that code for P450 enzymes that catalyze the final two steps in the synthesis of E from 2,22-dideoxyecdysone. Unlike dib and sad, shd is not expressed in the ring gland but is expressed in peripheral tissues such as the epidermis, midgut, Malpighian tubules, and fat body, i.e., tissues known to be major sites of E20MO activity in a variety of insects. However, the tissue in which shd is expressed does not appear to be important for developmental function because misexpression of shd in the embryonic mesoderm instead of the epidermis, the normal embryonic tissue in which shd is expressed, rescues embryonic lethality.

In order to examine the usefulness of detoxifying genes as molecular markers in different chemical environments, isolation of cytochrome P450 genes (CYPs) belonging to the CYP4 family was performed in different samples from two subalpine populations of Daphnia pulex. The use of degenerate primers allowed us to isolate seven cDNAs. Four of them were assigned to the CYP4C subfamily, and were closely related to previously isolated crustacean CYP4s while the others were assigned to new CYP4AN and CYP4AP subfamilies. Expression studies, using semiquantitative polymerase chain reaction (PCR) followed by Southern blot hybridization with specific probes revealed differences in CYP4C32 and CYP4AP1 expressions between the two populations, which differ in the polyphenol richness of the vegetation surrounding their aquatic habitat. Further exposure to toxic dietary polyphenols showed different CYP induction patterns. Taken together, these preliminary results suggest a possible involvement of CYP4s in the ecological differentiation of subalpine D. pulex populations related to the polyphenol richness of the environmental vegetation. CYP4s may thus be considered as possible molecular markers in aquatic environmental bioreporting.

Five different enzymatic activities, catalyzed by both microsomal and mitochondrial cytochrome P450 monooxygenases (CYPs), are strongly implicated in the biosynthesis of ecdysone (E) from cholesterol. However, none of these enzymes have been characterized completely. The present data show that the wild-type genes of two members of the Halloween family of embryonic lethals, disembodied (dib) and shadow (sad), code for mitochondrial cytochromes P450 that mediate the last two hydroxylation reactions in the ecdysteroidogenic pathway in Drosophila, namely the C22- and C2-hydroxylases. When sad (CYP315A1) is transfected into Drosophila S2 cells, the cells metabolize 2-deoxyecdysone (2dE) to E and the [3H]ketotriol (2,22-dideoxyecdysone) to 22-deoxyecdysone. In contrast, dib (CYP302A1) is responsible for the conversion of the [3H]ketotriol to [3H]2dE. When cells are transfected with both dib and sad, they metabolize the [3H]ketotriol to [3H]E in high yield. The expression of sad and dib is concentrated within the individual segments of the developing epidermis when there is a surge of ecdysteroid midway through embryogenesis. This result occurs before the ring gland has developed and suggests that the embryonic epidermis is a site of ecdysteroid biosynthesis. This pattern then diminishes, and, during late embryogenesis, expression of both genes is concentrated in the prothoracic gland cells of the developing ring gland. Expression of dib and sad continues to be localized in this endocrine compartment during larval development, being maximal in both the late second and third instar larvae, about the time of the premolt peaks in the ecdysteroid titer.

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.

Biochemical studies on ecdysteroid metabolism in arthropods suggest that aldoketoreductase enzymes (AKRs) may be involved in this pathway, but very few molecular data are available on these oxidoreductases in invertebrates. Looking for such enzymes in the crayfish Orconectes limosus, we have used a PCR strategy with primers deduced from a recent insect 3beta-reductase sequence, and from mammalian 5beta-reductase sequences. A full-length cDNA, corresponding to a putative AKR, was isolated from crayfish antennal gland. This cDNA contains an open-reading frame of 1008 bp, encoding a predicted protein of 336 amino acids. Northern blots indicated a restricted expression of the transcript in the antennal glands, quite constant during the molting cycle, and in situ hybridization demonstrated a strong expression of the transcript in the labyrinth. This is to date the first member of the AKRs superfamily characterized in a crustacean species, and the putative function of the corresponding enzyme is discussed., (Copyright 2001 Academic Press.)

A novel cytochrome P450 was isolated from Drosophila melanogaster by PCR strategy with primers deduced from the crayfish Orconectes limosus CYP4C15 sequence, which is supposed to be involved in ecdysteroid biosynthesis. The full-length cDNA contains a 1980 bp open reading frame encoding a predicted protein of 574 amino acids and was designated CYP4G15. The corresponding gene is located at 10C1 on the X chromosome. The presence of a N-terminal segment mainly hydrophobic indicated that the corresponding enzyme is probably microsomal. In situ hybridization demonstrated predominant expression of CYP4G15 in the brain of third larval instar and Northern-blots showed no overexpression in insecticide resistant strain. This is the first indication of a specific P450 expressed in the central nervous system of Drosophila, and the putative function of the corresponding enzyme is discussed., (Copyright 2000 Academic Press.)

Biosynthesis of ecdysteroids, arthropod steroid molting hormones, proceeds from dietary cholesterol through a complex and still incompletely elucidated pathway. Most of the known steps are catalyzed by cytochrome P450 enzymes (CYPs) but none of their genes has yet been identified. We have established a cDNA library of crayfish steroidogenic glands (Y organs). A full length CYP-cDNA was characterized containing a 1539 bp open reading frame encoding a predicted protein of 513 amino acid residues. This novel CYP was assigned to the CYP4 family and designated CYP4C15. Northern blots demonstrated predominant expression of this gene in the active molting glands, suggesting a role in ecdysteroid biosynthesis rather than detoxification., (Copyright 1999 Academic Press.)

A cDNA homologue of the large unit rat ribosomal protein L15 was cloned from an epidermal cDNA library of the crayfish Orconectes limosus, being the first crustacean ribosomal protein gene cloned to date. It contains 204 amino acids, deduced from the nucleotide sequence, and reveals 70-76% sequence identity with other arthropod and vertebrate ribosomal L15s. A single, abundant 0.7-kb mRNA transcript was constitutively expressed and revealed similar expression levels, among various adult crayfish tissues.

In crustaceans, ecdysteroid synthesis in the Y-organs is negatively regulated by the molt-inhibiting hormone (MIH). Reduction or cessation of MIH release from the sinus gland in the eyestalk, probably due to environmental cues, is one of possibly several signals for an increase of edysteroid production and subsequently enhancement of 20-hydroxyecdysone (20E) levels in the hemolymph. The present study asks the question whether the 20E peak in premoult stages D2/D3 is explained solely bythe cessation of MIH release or whether positive feedback mechanisms are also involved. Ecdysteroid production by the Y-organ of the crayfish Orconectes limosus was found to be under negative feedback control by circulating ecdysteroids. Exogenous 20-hydroxyecdysone (20E) as well as RH-5849, a non-steroidal ecdysteroid agonist, reduced ecdysteroid synthesis significantly when injected into intermoult animals. A direct, short loop inhibitory feedback effect was demonstrated by in vitro incubations of Y-organs with RH-5849. Thus, the results presented here do not point to a stimulatory effect of 20E on Y-organ activity but suggest that during intermolt a negative feedback by ecdysteroids plays a role in addition to MIH. Arch. Copyright 1999 Wiley-Liss, Inc.

Ecdysteroid biosynthesis was analyzed in vitro using dissociated Y-organ cells from the shore crab Carcinus maenas. 3-Dehydroecdysone (3DE) was detected as a minor secretory product, in addition to the formerly identified end-products 25-deoxyecdysone and ecdysone (E). In conversion studies, 3DE was formed from tritiated 5beta-ketodiol (2,22,25-trideoxyecdysone), 2,22-deoxyecdysone and 2-deoxyecdysone but not from E. Further experiments were performed in order to understand the interconversions between 3-oxo and 3beta-OH compounds in the crab Y-organ. The enzyme involved in 3beta-dehydrogenation was not ecdysone oxidase, a soluble enzyme found in peripheral tissues of many arthropods but it presented strong similarities with 3beta-hydroxysteroid dehydrogenase enzymes from vertebrates: it was membrane-bound and NAD+-dependent. Moreover, a NADH-dependent 3beta-reduction of several 3-oxo-ecdysteroids was obtained using the same microsomal fraction (100,000 x g pellet) of Y-organs, indicating that the reaction might be reversible. As this activity was specific of molting glands, we hypothesize that there is at least one 3beta-hydroxysteroid dehydrogenase enzyme involved in the biosynthetic pathway of ecdysteroids.

Although the involvement of 3-oxo-delta 4 compounds as intermediates in arthropod ecdysteroid biosynthesis has been postulated for a long time, it has not yet been directly demonstrated. In the present study, 3-oxo-delta 4-steroids have been synthesized and incubated in vitro with dissociated moulting gland cells from the crab Carcinus maenas. The tritiated compounds were converted into 3-dehydroecdysone, ecdysone and/or 25-deoxyecdysone, i.e. final ecdysteroids. This means that the 3-oxo-delta 4 compounds had undergone a 5 beta-reduction, to give the 5 beta-conformation of ecdysteroids. Our results suggest that the 3-oxo-delta 4-steroid 4,7-cholestadien-14 alpha-ol-3,6-dione may be an intermediate in the biosynthetic pathway. The 5 beta-reduction reaction involves a cytosolic enzyme which requires NADPH as electron donor and seems specific for 3-oxo-delta 4 substrates. This reaction was the most active in crab Y-organs, as compared with other tissues. The characteristics of the 5 beta-reductase (subcellular localization, substrate and cofactor requirements) appear similar to those of the vertebrate 3-oxo-delta 4-steroid 5 beta-reductase involved in steroid hormone catabolism and bile acid biosynthesis.

The involvement of continuous protein synthesis in the mechanisms of crustacean steroidogenesis was investigated using crayfish molting glands (Y-organs). During intermolt, Y-organ steroidogenic activity is low. Eyestalk ablation initiates premolt which is characterized by a rapid increase in the production of ecdysteroids. In vitro incorporation of [14C]leucine into TCA-precipitable proteins was measured in Y-organs. A significant increase of de novo protein synthesis within 2 h and simultaneously led to a strong inhibition of the ecdysteroid synthesis. Sinus gland extracts (containing molt inhibiting hormone) also induced both a limited but reproducible inhibition of Y-organ protein synthesis and a pronounced inhibition of ecdysteroid production within 2 h. The results suggest a functional link between protein synthesis in the Y-organ and sustained ecdysteroid production. The analysis of autoradiographs from one-dimensional gel electrophoreses revealed an overall increase in de novo synthesis of glandular proteins in early premolt but also a more specific effect on distinct proteins (increase of 150, 140, 50-60, 22 and 15-18 kDa proteins) which may be more directly involved in the regulation of ecdysteroidogenesis.