Cadmium and phthalate impacts developmental growth and mortality of Spodoptera littoralis, but not reproductive success.

Our environment is increasingly polluted with various molecules, some of which are considered endocrine disruptors. Metals and phthalates, originating from industrial activities, agricultural practices, or consumer products, are prominent examples of such pollutants. We experimentally investigated the impacts of the heavy metal cadmium and the phthalate DEHP on the moth Spodoptera littoralis. More specifically, larvae were reared in laboratory conditions, where they were exposed to diets contaminated with either two doses of cadmium at concentrations of 62.5 µg/g or 125 µg/g, two doses of DEHP at 100 ng/g and 10 µg/g, or a combination of both low and high doses of the two compounds, with a control group for comparison. Our findings indicate that cadmium delays the developmental transition from larva to adult. Notably, the combination of cadmium and DEHP exacerbated this delay, highlighting a synergistic effect. In contrast, DEHP alone did not affect larval development. Additionally, we observed that cadmium exposure, both alone and in combination with DEHP, led to a lower mass at all larval stages. However, cadmium-exposed individuals that reached adulthood eventually reached a similar mass to those in other groups. Interestingly, while our results did not show any effect of the treatments on hatching success, there was a higher adult mortality rate in the cadmium-treated groups. This suggests that while moths may prioritize reproductive success, their survival at the adult stage is compromised by cadmium exposure. In conclusion, our study demonstrates the impact of cadmium on the development, mass, and adult survival of moths, and reveals synergistic effects when combined with DEHP. These results confirm cadmium as an endocrine disruptor, even at low doses. These insights underscore the importance of understanding the toxicological effects of low doses of pollutants like cadmium and DEHP, both individually and in combination.

Fitness under high temperatures is overestimated when daily thermal fluctuation is ignored.

Experimental studies on the thermal biology of organisms have become crucial to investigate the impact of climate warming. However, most laboratory studies are carried out under constant temperatures and assume a negligible effect from daily fluctuating temperatures. We tested this assumption on multiple fitness traits of the moth Spodoptera littoralis, and a literature review on insects complements this study. Tests on S. littoralis focused on its optimal and maximal critical temperatures by comparing constant and daily fluctuating temperatures (±5 °C) at mean temperatures of 25, 29 and 33 °C. The nine fitness parameters investigated were influenced by mean temperature. The overall effect was a maximal multiplication rate at 29 °C and a marked decrease under the fluctuating regime at 33 °C. Effects of fluctuating temperatures differed between mean temperatures. Developmental and larval survival rates at 33 °C were lower under the fluctuating thermal regime than under a constant temperature. Our literature review also illustrates that ignoring daily fluctuations based on constant temperatures commonly leads to overestimate fitness traits at high temperatures. Overlooking the experimental bias associated with constant temperatures minimizes the expected impact of climate warming on fitness traits.

Methoprene-tolerant and Krüppel homolog 1 are actors of juvenile hormone-signaling controlling the development of male sexual behavior in the moth Agrotis ipsilon.

In insects, juvenile hormone (JH) is critical for the orchestration of male reproductive maturation. For instance, in the male moth, Agrotis ipsilon, the behavioral response and the neuronal sensitivity within the primary olfactory centers, the antennal lobes (ALs), to the female-emitted sex pheromone increase with fertility during adulthood and the coordination between these events is governed by JH. However, the molecular basis of JH action in the development of sexual behavior remains largely unknown. Here, we show that the expression of the paralogous JH receptors, Methoprene-tolerant 1 and 2 (Met1, Met2) and of the JH-inducible transcription factor, Krüppel homolog 1 (Kr-h1) within ALs raised from the third day of adult life and this dynamic is correlated with increased behavioral responsiveness to sex pheromone. Met1-, Met2- and Kr-h1-depleted sexually mature males exhibited altered sex pheromone-guided orientation flight. Moreover, injection of JH-II into young males enhanced the behavioral response to sex pheromone with increased AL Met1, Met2 and Kr-h1 mRNA levels. By contrast, JH deficiency suppressed the behavioral response to sex pheromone coupled with reduced AL Met1, Met2 and Kr-h1 mRNA levels in allatectomized old males and these inhibitions were compensated by an injection of JH-II in operated males. Our results demonstrated that JH acts through Met-Kr-h1 signaling pathway operating in ALs, to promote the pheromone information processing and consequently the display of sexual behavior in synchronization with fertility to optimize male reproductive fitness. Thus, this study provides insights into the molecular mechanisms underlying the hormonal regulation of reproductive behavior in insects.

Effects of DEHP on the ecdysteroid pathway, sexual behavior and offspring of the moth Spodoptera littoralis.

Bis(2-ethylhexyl) phthalate (DEHP) is a widely produced plasticizer that is considered to act as an endocrine-disrupting chemical in vertebrates and invertebrates. Indeed, many studies have shown that DEHP alters hormonal levels, reproduction and behavior in vertebrates. Few studies have focused on the effects of DEHP on insects, although DEHP is found almost everywhere in their natural habitats, particularly in soils and plants. Here, we investigated the effects of DEHP on the sexual behavior and physiology of a pest insect, the noctuid moth Spodoptera littoralis. In this nocturnal species, olfaction is crucial for sexual behavior, and ecdysteroids at the antennal level have been shown to modulate sex pheromone detection by males. In the present study, larvae were fed food containing different DEHP concentrations, and DEHP concentrations were then measured in the adults (males and females). Hemolymphatic ecdysteroid concentrations, the antennal expression of genes involved in the ecdysteroid pathway (nuclear receptors EcR, USP, E75, and E78 and calmodulin) and sexual behavior were then investigated in adult males. The success and latency of mating as well as the hatching success were also studied in pairs consisting of one DEHP male and one uncontaminated female or one DEHP female and one uncontaminated male. We also studied the offspring produced from pairs involving contaminated females to test the transgenerational effect of DEHP. Our results showed the general downregulation of nuclear receptors and calmodulin gene expression associated with the higher concentrations of DEHP, suggesting peripheral olfactory disruption. We found some effects on male behavior but without an alteration of the mating rate. Effects on offspring mortality and developmental rates in the N + 1 generation were also found at the higher doses of DEHP. Taken together, the results of the study show for the first time that larval exposure to DEHP can induce delayed endocrine-disruptive effects in the adults of a terrestrial insect as well as effects on the next generation. To date, our study is also the first description of an impact of endocrine disrupter on olfaction in insects.

A maternal effect influences sensitivity to chlorpyrifos pesticide in the pest moth Spodoptera littoralis.

Transgenerational effects on sensitivity to pesticides are poorly studied. This study investigated the transgenerational influences of maternal body mass in the major pest moth Spodoptera littoralis, with a focus on sensitivity to chlorpyrifos pesticide. In 147 clutches of a laboratory strain of S. littoralis, we compared larval mortality between control larvae and larvae treated with chlorpyrifos. Because of the classic positive relationships between offspring size and maternal size and between offspring size and offspring quality, sensitivity to chlorpyrifos was predicted to be lower in larvae of larger mothers. Surprisingly, we found the opposite result, with higher pesticide toxicity in larvae of larger mothers. This result is partly explained by the lack of a relationship between larval mass and larval sensitivity to chlorpyrifos. This means that another offspring characteristic linked to maternal size should have affected larval sensitivity to chlorpyrifos. More generally, knowledge of the effects of the traits and ecological environments of mothers on offspring sensitivity to pesticides remains limited. Ecotoxicologists should pay more attention to such maternal effects on sensitivity to pesticides, both in pests and non-target species.

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.

Daily temperature fluctuation interacts with the mean temperature to increase the toxicity of a pyrethroid insecticide in a moth.

Climate change complicates ecotoxicology studies because species responses to pesticides depend on temperature. Classically illustrated by the effect of constant laboratory temperatures, a recent review revealed that the toxicity of pesticides is also often increased by daily temperature fluctuations. Here, we investigated the combined effects of daily temperature fluctuation and mean temperature on the toxicity of two insecticides in the moth Spodoptera littoralis. Our study tested the toxicity of chlorpyrifos and deltamethrin on larvae of six experimental groups that crossed three treatments of daily temperature fluctuations (0, 5 or 10 °C) and two treatments of mean temperatures (25 or 33 °C). We showed that daily temperature fluctuation increased larval mortality induced by chlorpyrifos and deltamethrin. However, the response differed between the organophosphorus insecticide chlorpyrifos and the pyrethroid insecticide deltamethrin. The increase in chlorpyrifos toxicity by daily temperature fluctuation did not differ between mean temperatures of 25 and 33 °C. Remarkably, the increase in deltamethrin toxicity by daily temperature fluctuation was dependent on the crossed effects of the amplitude of daily fluctuation and mean temperature. This increase in deltamethrin toxicity occurred with a daily fluctuation of only 5 °C for larvae reared at 25 °C and a daily fluctuation of 10 °C in larvae reared at 33 °C. To confidently quantify the responses of insecticide toxicity to temperature, future ecotoxicology studies will have to evaluate the generality of the interaction between the effects of daily temperature fluctuation and mean temperature.

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.

Steroid hormone signaling is involved in the age-dependent behavioral response to sex pheromone in the adult male moth Agrotis ipsilon.

In most animals, including insects, male reproduction depends on the detection and processing of female-produced sex pheromones. In the male moth, Agrotis ipsilon, both behavioral response and neuronal sensitivity in the primary olfactory center, the antennal lobe (AL), to female sex pheromone are age- and hormone-dependent. In many animal species, steroids are known to act at the brain level to modulate the responsiveness to sexually relevant chemical cues. We aimed to address the hypothesis that the steroidal system and in particular 20-hydroxyecdysone (20E), the main insect steroid hormone, might also be involved in this olfactory plasticity. Therefore, we first cloned the nuclear ecdysteroid receptor EcR (AipsEcR) and its partner Ultraspiracle (AipsUSP) of A. ipsilon, the expression of which increased concomitantly with age in ALs. Injection of 20E into young sexually immature males led to an increase in both responsiveness to sex pheromone and amount of AipsEcR and AipsUSP in their ALs. Conversely, the behavioral response decreased in older, sexually mature males after injection of cucurbitacin B (CurB), an antagonist of the 20E/EcR/USP complex. Also, the amount of AipsEcR and AipsUSP significantly declined after treatment with CurB. These results suggest that 20E is involved in the expression of sexual behavior via the EcR/USP signaling pathway, probably acting on central pheromone processing in A. ipsilon.

Effects of phthalate and bisphenol plasticizers on the activity of glycolytic enzymes of the moth Spodoptera littoralis.

Environmental plastic pollution has significantly increased in the recent decades, and severely impacts economies, human and biodiversity health. Plastics are made of several chemical additives, including bisphenol and phthalate plasticizers such as bisphenol A (BPA) and Di(2-ethylhexyl)phthalate (DEHP). In some animal species, both BPA and DEHP are known as endocrine disruptor compounds, and can alter physiological and metabolic homeostasis, reproduction, development and/or behavior. To date, the impacts of BPA and DEHP have mainly focused on vertebrates, and to a lesser extent, on aquatic invertebrates. Yet, the few studies which examined the effects of DEHP on terrestrial insects also revealed the impacts this pollutant can have on development, hormone titrations, and metabolic profiles. In particular, it has been hypothesized in the Egyptian cotton leafworm Spodoptera littoralis that the observed metabolic alterations could result from the energetic costs necessary for DEHP detoxification or to the dysregulation of hormonally-controlled enzymatic activities. To get additional insights into the physiological effects of bisphenol and phthalate plasticizers on the moth S. littoralis, larvae were fed with food contaminated by BPA, DEHP, or the mixture of both compounds. Then, activities of four glycolytic enzymes, hexokinase, phosphoglucose isomerase, phosphofructokinase, and pyruvate kinase were measured. BPA and/or DEHP had no effects on the activities of phosphofructokinase and pyruvate kinase. Conversely, BPA-contaminated larvae were characterized by a 1.9-fold increase in phosphoglucose isomerase activity, and BPA + DEHP-fed larvae had highly variable hexokinase activity. Overall, since no disruption of glycolytic enzyme was observed in DEHP-contaminated larvae, our work tended to demonstrate that exposure to bisphenol and DEHP increased the amount of oxidative stress experienced.

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 maspardin, responsible for human Mast syndrome, in an insect species and analysis of its evolution in metazoans.

Mast syndrome is a complicated form of human hereditary spastic paraplegias, caused by a mutation in the gene acid cluster protein 33, which encodes a protein designated as "maspardin." Maspardin presents similarity to the α/ß-hydrolase superfamily, but might lack enzymatic activity and rather be involved in protein-protein interactions. Association with the vesicles of the endosomal network also suggested that maspardin may be involved in the sorting and/or trafficking of molecules in the endosomal pathway, a crucial process for maintenance of neuron health. Despite a high conservation in living organisms, studies of maspardin in other animal species than mammals were lacking. In the cotton armyworm Spodoptera littoralis, an insect pest model, analysis of an expressed sequence tag collection from antenna, the olfactory organ, has allowed identifying a maspardin homolog (SlMasp). We have investigated SlMasp tissue distribution and temporal expression by PCR and in situ hybridization techniques. Noteworthy, we found that maspardin was highly expressed in antennae and associated with the structures specialized in odorant detection. We have, in addition, identified maspardin sequences in numerous "nonmammalian" species and described here their phylogenetic analysis in the context of metazoan diversity. We observed a strong conservation of maspardin in metazoans, with surprisingly two independent losses of this gene in two relatively distant ecdysozoan taxa that include major model organisms, i.e., dipterans and nematodes.

The transcription factor Krüppel homolog 1 is linked to the juvenile hormone-dependent maturation of sexual behavior in the male moth, Agrotis ipsilon.

In the male moth, Agrotis ipsilon, the behavioral response and neuronal sensitivity in the primary olfactory center, the antennal lobe (AL), to sex pheromone increase with age and juvenile hormone (JH) biosynthesis. Although JH has been shown to control this age-dependent plasticity, the underlying signaling pathway remains obscure. In this context, we cloned a full cDNA encoding the Krüppel homolog 1 transcription factor (AipsKr-h1) of A. ipsilon, which was found to be predominantly expressed in ALs, where its amount increased concomitantly with age and sex pheromone responses. Conversely, the expression of AipsKr-h1 protein in the antenna was age-independent. Moreover, the administration of JH in immature males or fluvastatin, an inhibitor of JH biosynthesis, in mature males induced an increase or a decline of the AipsKr-h1 protein level in ALs, respectively. This effect was suppressed with a combined injection of fluvastatin and JH. Our results showed that Aipskr-h1 is a JH-upregulated gene that might mediate JH action on central pheromone processing, modulating sexual behavior in A. ipsilon.

The impact of temperature on insecticide sensitivity depends on transgenerational effects.

The combined effects of insecticides and temperature are increasingly being studied because species are expected to change their responses to insecticides with climate warming. As recently highlighted, the impact of temperature on insecticide sensitivity might be influenced by the environment experienced by the previous generation. However, a pioneering study that showed this transgenerational effect in the mosquito Culex pipiens needs to be confirmed because two other studies did not show similar results. Here, we performed an experiment on the moth Spodoptera littoralis to test this hypothesis. We analysed reaction norms among experimental families to test transgenerational effects, i.e., the variation in the response of families to the combined effects of the insecticide chlorpyrifos and developmental temperature. Reaction norm analyses revealed that the responses of the families to chlorpyrifos and temperature differed for developmental time and larval survival, two key parameters in S. littoralis. Crucially, for larval survival, a family effect influenced the impact of temperature on chlorpyrifos sensitivity. This finding confirms the pioneering study on C. pipiens that showed transgenerational effects on the combined effects of insecticides and temperature. This result also highlights that transgenerational plasticity can be important to consider in ecotoxicology.

Anopheles gambiae males produce and transfer the vitellogenic steroid hormone 20-hydroxyecdysone to females during mating.

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.

Combined influences of transgenerational effects, temperature and insecticide on the moth Spodoptera littoralis.

Climate warming is expected to impact the response of species to insecticides. Recent studies show that this interaction between insecticides and temperature can depend on other factors. Here, we tested for the influence of transgenerational effects on the Insecticide × Temperature interaction in the crop pest moth Spodoptera littoralis. Specifically, we analysed reaction norms among experimental clutches based on a split-plot design crossing the factors temperature, insecticide and clutch. The study was performed on 2280 larvae reared at four temperatures (23, 25, 27 and 29 °C), and their response to the insecticide deltamethrin (three concentrations and a control group) was tested. Temperature had a global influence with effects on larval survival, duration of development, pupal body mass, and significant reaction norms of the clutches for temperature variations of only 2 °C. In addition to the expected effect of deltamethrin on mortality, the insecticide slightly delayed the development of S. littoralis, and the effects on mortality and development differed among the clutches. Projection models integrating all the observed responses illustrated the additive effects of deltamethrin and temperature on the population multiplication rate. Variation in the response of the clutches showed that transgenerational effects influenced the impact of insecticide and temperature. Although no evidence indicated that the Insecticide × Temperature interaction depended on transgenerational effects, the studies on the dependence of the Insecticide × Temperature interaction on other factors continue to be crucial to confidently predict the combined effects of insecticides and climate warming.

Involvement of Methoprene-tolerant and Krüppel homolog 1 in juvenile hormone-signaling regulating the maturation of male accessory glands in the moth Agrotis ipsilon.

Male accessory glands (MAGs) produce seminal fluid proteins that are essential for the fertility and also influence the reproductive physiology and behavior of mated females. In many insect species, and especially in the moth Agrotis ipsilon, juvenile hormone (JH) promotes the maturation of the MAGs but the underlying molecular mechanisms in this hormonal regulation are not yet well identified. Here, we examined the role of the JH receptor, Methoprene-tolerant (Met) and the JH-inducible transcription factor, Krüppel homolog 1 (Kr-h1) in transmitting the JH signal that upregulates the growth and synthetic activity of the MAGs in A. ipsilon. We cloned two full length cDNAs encoding Met1 and Met2 which are co-expressed with Kr-h1 in the MAGs where their expression levels increase with age in parallel with the length and protein content of the MAGs. RNAi-mediated knockdown of either Met1, Met2, or Kr-h1 resulted in reduced MAG length and protein amount. Moreover, injection of JH-II into newly emerged adult males induced the transcription of Met1, Met2 and Kr-h1 associated to an increase in the length and protein content of the MAGs. By contrast, JH deficiency decreased Met1, Met2 and Kr-h1 mRNA levels as well as the length and protein reserves of the MAGs of allatectomized old males and these declines were partly compensated by a combined injection of JH-II in operated males. Taken together, our results highlighted an involvement of the JH-Met-Kr-h1 signaling pathway in the development and secretory activity of the MAGs in A. ipsilon.

Effects of low concentrations of deltamethrin are dependent on developmental stages and sexes in the pest moth Spodoptera littoralis.

Effects of low concentrations of pesticides, with no or moderate mortality of targeted species, are poorly studied even though these low concentrations are common under natural conditions. Studying their effects is critical because they can induce positive hormetic responses, possibly leading to greater pest multiplication and promoting the evolution of pest resistance. Here, we investigated the responses of the pest moth Spodoptera littoralis to low concentrations of deltamethrin, and tested for variation in effects of the pesticide between developmental stages and sexes. Indeed, we show that a given concentration of deltamethrin has different effects between stages, and even between sexes. Two experimental concentrations led to very high mortality early in S. littoralis development (4th larval instar), but only to low mortality rates in adults. Moreover, our highest experimental concentration had only detrimental effects in adult females, but improved the reproductive success of adult males. Model projections showed that the lethality from treatments at the 4th larval instar was the predominant effect. Because of the high multiplication rate of S. littoralis, it was also found that treatments with very similar effects on larval mortality can lead to either population extinction or rapid pest resurgence.

Ecdysteroids from Cyanotis longifolia Benth. (Commelinaceae).

Cyanotis longifolia Benth. (Commelinaceae) contains ecdysteroids, which are highly concentrated in the roots and flowers, whereas leaves contain only very low amounts and stems intermediate amounts. 20-Hydroxyecdysone is the major component found in all tissues, but roots also contain large amounts of 20-hydroxyecdysone 3-acetate and ajugasterone C. A preparative experiment has shown that roots contain a complex ecdysteroid mixture, and the analysis of minor components has allowed the isolation of several already known ecdysteroids (polypodine B, 2-deoxy-20,26-dihydroxyecdysone, isovitexirone, poststerone) together with five new (ajugasterone C 3-acetate, 5beta-hydroxy-poststerone, poststerone 2-acetate, 14(15)-dehydro-poststerone 2-acetate, 24-epi-atrotosterone A [=24-methyl-ajugasterone C]) ecdysteroids that have been fully characterized. A preliminary investigation of 55 species belonging to 15 different genera of the Commelinaceae has shown that several of them contain significant concentrations of ecdysteroids, among which some previously uninvestigated ones appear to be very promising sources of ecdysteroids.

Phytoecdysteroid C2-hydroxylase is microsomal in spinach, Spinacia oleracea L.

An enzyme involved in the biosynthesis of phytoecdysteroids, the C2-hydroxylase, has been investigated in spinach, Spinacia oleracea. This enzyme is microsomal and its K(m) has been determined using 2-deoxy-20-hydroxyecdysone as substrate (K(m)=3.72 microM). It is much more efficient with 2-deoxy-20-hydroxyecdysone than with 2-deoxyecdysone and, conversely, the C20-hydroxylase is more active on 2-deoxyecdysone than on ecdysone. These data support the conclusion that C20-hydroxylation precedes C2-hydroxylation. The C2-hydroxylase is inhibited by high concentrations of 20E. Substrate specificity and subcellular localization of C2-hydroxylase differ between plants and insects, and these data, as well as those previously reported on other biosynthetic steps, show the great difference between plant and insect ecdysteroid biosynthetic pathways and suggest an independent origin for the pathways in both kingdoms.