Ecdysteroids in helminths and annelids

Summary

This review focuses on recent research developments in the past three years concerning ecdysteroid biochemistry in helminths and annelids, with emphasis on results reported at the IXth Ecdysone Workshop in Paris, France, in September 1989. During the past three years, most of the research in this area has been concentrated on nematodes, in several species of which the occurrence of ecdysteroids has been demonstrated. Interesting biological effects of exogenously applied ecdysteroids have been discovered on meiotic reinitiation in oocytes of the dog heartworm, Dirofilaria immitis, and on microfilarial production by Brugia pahangi. These effects, together with previous discoveries, demonstrate the feasibility of affecting nematode physiology with exogenously applied ecdysteroids. However, experiments with four species of nematodes in three separate laboratories have failed to demonstrate that nematodes biosynthesize ecdysteroids from cholesterol. Therefore, it remains to be proven whether ecdysteroids are truly endogenous nematode hormones or are merely compounds with strong biological activity. Research with cestodes and trematodes has similarly revealed that ecdysteroids occur in these organisms and that the compounds have possible regulatory roles, but experiments demonstrating endogenous biosynthesis have yet to be performed. Annelids, which are more closely related to insects than helminths, also contain ecdysteroids; recent research has demonstrated the C-20 hydroxylation of ecdysone to 20-hydroxyecdysone, the active moulting hormone of most insects. Experiments to demonstrate this metabolic step in helminths have failed.     

Competitive interactions between entomopathogenic nematodes and parasitoid venom

Entomopathogenic nematodes and parasitoid larvae of some wasps play important roles in the natural control of the pest insects. However, it has not been excluded that competition between nematodes and wasps may in some cases reduce their efficacy in the pest control. Using caterpillars of Spodoptera littoralis, we examined interactions between the nematode Steinernema carpocapsae and the venom of the parasitoid Habrobracon hebetor. The survival of S. littoralis caterpillars was reduced in a dose-dependent manner when 5 to 500 nematodes or 0.005–0.1 venom units were applied to single caterpillars. High doses of either nematodes or the venom caused death within 1–3 days in all treated hosts. The low doses of nematodes killed caterpillars within a week, in some cases when they attempted to pupate. Caterpillars receiving low venom doses were characterized by extended survival time terminated with death due to starvation. Combined treatment of nematodes and the venom were mutually synergistic and elicited severe lethal effects. The nematodes were fully resistant to the venom and can feed and grow on the symbiotic bacteria in vitro. The venom impairs food processing and causes death of caterpillars due to starvation. Disruption of the hormonal regulation of metamorphosis by ecdysteroids and juvenile hormone could be responsible for defective moults block at different stages of the moulting process, regionally restricted moulting, moults to “intermediates” combining regions of newly secreted larval and pupal cuticles.     

Ecdysteroids in Axenically Propagated Caenorhabditis elegans and Culture Medium

Ecdysteroids (insect molting hormones) from Caenorhabditis elegans were chromatographically purified and quantified by radioimmunoassay. Nematodes from semidefined medium contained the immunoreactive equivalent of 460 pg ecdysone per gram dry weight. Culture medium, however, contained the immunoreactive equivalent of 68 times the quantity within the nematodes. In a defined medium lacking immunoreactivity, C. elegans contained 520 pg ecdysone equivalents per gram dry weight but reproduced slowly. Reproduction of C. elegans in defined medium was enhanced by formulation in agar. Propagation of C. elegans in either agar-based or aqueous defined medium supplemented with [¹⁴C]cholesterol of high specific activity failed to result in production of radiolabeled free ecdysteroids or polar or apolar ecdysteroid conjugates. Failure to demonstrate ecdysteroid biosynthesis in C. elegans raises questions about the ecdysteroids identified previously in nematodes being products of endogenous biosynthesis, a necessary condition for these compounds to be nematode hormones.     

The conserved Rieske oxygenase DAF-36/Neverland is a novel cholesterol-metabolizing enzyme

Steroid hormones play essential roles in a wide variety of biological processes in multicellular organisms. The principal steroid hormones in nematodes and arthropods are dafachronic acids and ecdysteroids, respectively, both of which are synthesized from cholesterol as an indispensable precursor. The first critical catalytic step in the biosynthesis of these ecdysozoan steroids is the conversion of cholesterol to 7-dehydrocholesterol. However, the enzymes responsible for cholesterol 7,8-dehydrogenation remain unclear at the molecular level. Here we report that the Rieske oxygenase DAF-36/Neverland (Nvd) is a cholesterol 7,8-dehydrogenase. The daf-36/nvd genes are evolutionarily conserved, not only in nematodes and insects but also in deuterostome species that do not produce dafachronic acids or ecdysteroids, including the sea urchin Hemicentrotus pulcherrimus, the sea squirt Ciona intestinalis, the fish Danio rerio, and the frog Xenopus laevis. An in vitro enzymatic assay system reveals that all DAF-36/Nvd proteins cloned so far have the ability to convert cholesterol to 7-dehydrocholesterol. Moreover, the lethality of loss of nvd function in the fruit fly Drosophila melanogaster is rescued by the expression of daf-36/nvd genes from the nematode Caenorhabditis elegans, the insect Bombyx mori, or the vertebrates D. rerio and X. laevis. These data suggest that daf-36/nvd genes are functionally orthologous across the bilaterian phylogeny. We propose that the daf-36/nvd family of proteins is a novel conserved player in cholesterol metabolism across the animal phyla.     

Ecdysteroids in the haemolymph and the ovaries of the firebrat Thermobia domestica (Packard) (Insecta,Thysanura): Correlations with integumental and ovarian cycles

Ecdysteroids were analysed with radioimmunoassay (RIA) and high-performance liquid chromatography (HPLC) in females of the apterygotous insect Thermobia domestica, which ahs overlapping moulting and reproductive cycles. During each moulting cycle, a peak in the haemolymph concentration of ecdysteroids occurs at day 9 (in the 11-day standard cycle), which can be correlated with apolysis and the beginning of new cuticle deposition. The ovaries show a peak of ecdysteroids at day 5 (i.e. one day before egg-laying), which suggests that these hormones are also involved in the reproductive cycle. In both cases, HPLC analysis combined with RIA suggests that the main ecdysteroid is 20-hydroxyecdysone. This duality in the function of ecdysteroids is discussed.     

Regulation of female reproduction in mites: A unifying model for the Acari

It is well established in the literature that circulating high levels of juvenile hormone (JH) are responsible for the initiation of vitellogenesis and female reproduction in most insects studied so far. Exceptions include some Diptera, Lepidoptera and Hymenoptera. The current view is that JH also regulates yolk protein (vitellogenin, Vg) synthesis and female reproduction in mites. However, there is no published evidence that mites have the common insect JHs at any stage of their development. Also, research on the effects of exogenous applications of JH and JH analogs on the reproduction of mites is contradictory. Significant information is available on the life history of mite reproduction, and new information has become available on mite storage proteins including Vg. Although initial studies suggested that ticks may respond to exogenously applied juvenile hormone or anti-JHs, current research shows that ticks cannot synthesize the common insect JHs and have no detectable levels of these hormones in their hemolymph during female reproduction. In ticks, it appears that ecdysteroids, and not JH, regulate expression of the Vg gene and the synthesis and release of Vg protein into the hemolymph. In fact within the Arthropoda, JH has been found only in insects. Methyl farnesoate and not JH regulates Vg synthesis in the Crustacea, the sister group to the insects. Based on this evidence, a new working hypothesis is proposed, i.e., that ecdysteroids and not the JHs regulate vitellogenesis in the Acari including both ticks and mites. To the present, the role of neuropeptides in the regulation of female reproduction in mites is not known.     

Nematode neuropeptides: Localization, isolation and functions

Historically, peptidergic substances (in the form of neurosecretions) were linked to moulting in nematodes. More recently, there has been a renewal of interest in nematode neurobiology, initially triggered by studies demonstrating the localization of peptide immunoreactivities to the nervous system. Here, David Brownlee, Ian Fairweather, Lindy Holden-Dye and Robert Walker will review progress on the isolation of nematode neuropeptides and efforts to unravel their physiological actions and inactivation mechanisms. Future avenues for research are suggested and the potential exploitation of peptidergic pathways in future therapeutic strategies highlighted.     

Conserving and Enhancing Biological Control of Nematodes

Conservation biological control is the modification of the environment or existing practices to protect and enhance antagonistic organisms to reduce damage from pests. This approach to biological control has received insufficient attention compared with inundative applications of microbial antagonists to control nematodes. This review provides examples of how production practices can enhance or diminish biological control of plant-parasitic nematodes and other soilborne pests. Antagonists of nematodes can be enhanced by providing supplementary food sources such as occurs when organic amendments are applied to soil. However, some organic amendments (e.g., manures and plants containing allelopathic compounds) can also be detrimental to nematode antagonists. Plant species and genotype can strongly influence the outcome of biological control. For instance, the susceptibility of the plant to the nematode can determine the effectiveness of control; good hosts will require greater levels of suppression than poor hosts. Plant genotype can also influence the degree of rhizosphere colonization and antibiotic production by antagonists, as well the expression of induced resistance by plants. Production practices such as crop rotation, fallow periods, tillage, and pesticide applications can directly disrupt populations of antagonistic organisms. These practices can also indirectly affect antagonists by reducing their primary nematode host. One of the challenges of conservation biological control is that practices intended to protect or enhance suppression of nematodes may not be effective in all field sites because they are dependent on indigenous antagonists. Ultimately, indicators will need to be identified, such as the presence of particular antagonists, which can guide decisions on where it is practical to use conservation biological control. Antagonists can also be applied to field sites in conjunction with conservation practices to improve the consistency, efficacy, and duration of biological control. In future research, greater use should be made of bioassays that measure nematode suppression because changes in abundance of particular antagonists may not affect biological control of plant parasites.     

The Future of Nematology: Integration of New and Improved Management Strategies

The potential for managing plant-parasitic nenlatodes by combining two or more control strategies in an integrated program is examined. Advantages of this approach include the use of partially effective strategies and protection of highly effective ones vulnerable from nematode adaptation or environmental risk. Strategies can be combined sequentially from season to season or applied simultaneously. Programs that have several strategies available but that are limited in the true integration of control components are used as examples of current management procedures and the potential for their improvement. These include potato cyst nematodes in northern Europe, soybean cyst nematode in North Carolina, and root-knot nematodes on vegetable and field crops in California. A simplified model of the impact of component strategies on the nematode damage function indicates the potential for combining control measures with different efficacies to give acceptable nematode population reduction and crop protection. The likelihood for additive, synergistic, or antagonistic effects from combining strategies is considered with respect to the biological target and component compatibility.     

Ecdysteroids： the overlooked sex steroids of insects？ Males： the black box

The paradigm, still around in textbooks, that ＇in insects sex is strictly genetic, thus that they do not have sex hormones＇, is mainly based on a wrong interpretation of the ＇gynandromorph argument＇. It is no longer tenable. Given the fact that vertebrates and invertebrates probably had a common, sexually reproducing ancestor, there is no reason to assume that only vertebrates need sex hormones. The major function of sex hormones is to inform the somatoplasm about developmental changes that take place in the gonads. In contrast to juvenile hormone and neuropeptides, ecdysteroids meet all criteria to act as sex hormones, which was probably their ancient role. Their much better documented role in moulting and metamorphosis was a secondary acquisition that enabled arthropods to cope with growth problems, imposed by a rigid cuticle. Female insects use 20-hydroxyecdysone （20E）, secreted by the follicle cells of the ovary, in a similar way as females of egg-laying vertebrates use estrogens. For a variety of reasons, the possibility that ecdysteroids, in particular ecdysone （E）, might also act as sex hormones in male insects, thus as the counterpart of testosterone of vertebrates, has been very much overlooked. Thanks to the recent discovery of the molecular basis of the haploid-diploid system of sex determination in the honeybee, the characterization of Halloween genes, proteomics, RNAi and so on, it now becomes possible to verify whether in insects, as with vertebrates, males are the endocrinologically default gender form.     

Enzymes for ecdysteroid biosynthesis: their biological functions in insects and beyond

Steroid hormones are responsible for the coordinated regulation of many aspects of biological processes in multicellular organisms. Since the last century, many studies have identified and characterized steroidogenic enzymes in vertebrates, including mammals. However, much less is known about invertebrate steroidogenic enzymes. In the last 15?years, a number of steroidogenic enzymes and their functions have been characterized in ecdysozoan animals, especially in the fruit fly Drosophila melanogaster. In this review, we summarize the latest knowledge of enzymes crucial for synthesizing ecdysteroids, the principal insect steroid hormones. We also discuss the functional conservation and diversity of ecdysteroidogenic enzymes in other insects and even non-insect species, such as nematodes, vertebrates, and lower eukaryotes.     

Remodeling of an identified motoneuron during metamorphosis: hormonal influences on the growth of dendrites and axon terminals

During metamorphosis of the tobacco hawkmoth Manduca sexta, the femoral depressor motoneuron (FeDe MN) undergoes remodeling of its dendrites and motor terminals. Previous studies have established that remodeling of MNs during metamorphosis is mediated by the same hormones that control metamorphosis: the ecdysteroids and juvenile hormone (JH). During the pupal stage, the ecdysteroids promote adult-specific growth of MNs in the absence of JH, but JH or its synthetic mimics can interfere with ecdysteroid-mediated growth if applied during early sensitive periods. Hence, the application of a JH mimic (JHM) either systemically or locally to a target muscle has been used to distinguish those aspects of motor-terminal remodeling that are controlled by ecdysteroid action on the CNS from those that are influenced by ecdysteroid action on the peripheral targets. Here, we have extended the analysis of central and peripheral hormonal influences on MN remodeling by injecting JHM locally into the CNS thus altering the hormonal environment of the FeDe MN soma without altering the hormonal environment of its target muscle. Our results demonstrate that adult dendritic growth and motor-terminal growth can be experimentally uncoupled, suggesting that each is regulated independently. JHM application to the CNS perturbed dendritic growth, but had no measurable impact on motor-terminal growth. Peripheral actions of ecdysteroids, therefore, appear sufficient to promote the development of adult-specific motor terminals but not the development of an adult-specific dendritic arbor.     

Parasitism genes and host range disparities in biotrophic nematodes: the conundrum of polyphagy versus specialisation

This essay considers biotrophic cyst and root-knot nematodes in relation to their biology, host-parasite interactions and molecular genetics. These nematodes have to face the biological consequences of the physical constraints imposed by the soil environment in which they live while their hosts inhabit both above and below ground environments. The two groups of nematodes appear to have adopted radically different solutions to these problems with the result that one group is a host specialist and reproduces sexually while the other has an enormous host range and reproduces by mitotic parthenogenesis. We consider what is known about the modes of parasitism used by these nematodes and how it relates to their host range, including the surprising finding that parasitism genes in both nematode groups have been recruited from bacteria. The nuclear and mitochondrial genomes of these two nematode groups are very different and we consider how these findings relate to the biology of the organisms.     

Effects of Microbial and Other Antagonistic Organism and Competition on Entomopathogenic Nematodes

Antagonistic factors, broadly identified as antibiosis, competition and natural enemies, impact on entomopathogenic nematodes. Antibiosis can occur through the release of plant chemicals from the roots into the soil, which may adversely affect the host-finding behavior of the infective stage nematode, or the presence of these chemicals in the host insect may negatively affect nematode reproduction. In laboratory studies, intra-specific and inter-specific competition reduces nematode fitness, and inter-specific competition can cause local extinction of a nematode species. For example, after concomitant infection of a host, a steinernematid species usually excludes a heterorhabditid species. The mechanism for the steinernematid superiority has been postulated to be a bacteriocin(s) produced by Xenorhabdus, the symbiotic bacterium of the steinernematid, which prevents Photorhabdus, the symbiotic bacterium of the heterorhabditid, from multiplying. Inter-specific competition between two steinernematid species shows that both can co-exist in a host, but one species will eventually prevail in the environment. By having different foraging strategies, however, both steinermatid species may co-exist in the same habitat. An important issue is whether the introduction of an exotic entomopathogenic nematode species will competitively displace an indigenous nematode species. Although the environmental risks are small, the recommended policy is that the introduction of exotic nematodes be regulated. With other pathogens, entomopathogenic nematodes can out-compete entomopathogenic fungi, but not Bacillus thuringiensis, for the same host individual when both the nematode and entomopathogen are applied simultaneously. The best studied natural enemy is the nematophagous fungus, Hirsutella rhossiliensis, which causes higher mortality in Steinernema glaseri compared with Heterorhabditis bacteriorphora. Differential susceptibility to the fungus may be associated with the retention of the second-stage cuticle by H. bacteriophora. Invertebrate predators including mites and collembolans feed on entomopathogenic nematodes. Although a number of studies have been conducted with antagonists, there is a dearth of field data. We suggest that long-term research plots be established where natural populations of entomopathogenic nematodes occur and include antagonists as a component of such studies.     

Review of Pasteuria penetrans: Biology,Ecology, and Biological Control Potential

Pasteuria penetrans is a mycelial, endospore-forming, bacterial parasite that has shown great potential as a biological control agent of root-knot nematodes. Considerable progress has been made during the last 10 years in understanding its biology and importance as an agent capable of effectively suppressing root-knot nematodes in field soil. The objective of this review is to summarize the current knowledge of the biology, ecology, and biological control potential of P. penetrans and other Pasteuria members. Pasteuria spp. are distributed worldwide and have been reported from 323 nematode species belonging to 116 genera of free-living, predatory, plant-parasitic, and entomopathogenic nematodes. Artificial cultivation of P. penetrans has met with limited success; large-scale production of endospores depends on in vivo cultivation. Temperature affects endospore attachment, germination, pathogenesis, and completion of the life cycle in the nematode pseudocoelom. The biological control potential of Pasteuria spp. have been demonstrated on 20 crops; host nematodes include Belonolaimus longicaudatus, Heterodera spp., Meloidogyne spp., and Xiphinema diversicaudatum. Pasteuria penetrans plays an important role in some suppressive soils. The efficacy of the bacterium as a biological control agent has been examined. Approximately 100,000 endospores/g of soil provided immediate control of the peanut root-knot nematode, whereas 1,000 and 5,000 endospores/g of soil each amplified in the host nematode and became suppressive after 3 years.     

INSECT HORMONES IN DEVELOPMENT

Advances in studies of prothoracicotropic hormone (ecdysiotropin), ecdysteroids and juvenile hormones in the past decade are considered:
1. Until recently there has been little progress with prothoracicotropic hormone. The development of a sensitive bioassay for the hormone promises to produce rapid advances.
2. Current methods of hormone analysis are described, with detection limits. The application of these methods in studies of hormones at different stages and in different tissues of insects have revealed a far greater complexity in hormone titres than was predicted from classical studies.
3. Very few studies employ chemical characterization of hormones and some assays do not distinguish biologically inactive metabolites of the hormones from the active hormones. Many studies have thus failed to reveal the numerous rapid fluctuations in hormone titre necessary for insect development.
4. While ecdysteroids act, via a receptor, on specific chromosome sites, the cellular mode of action of juvenile hormone in larval development is still unknown. Recent evidence suggests that juvenile hormone acts prior to the time at which its effects are realized by ecdysteroids.
5. Insect hormones produce dramatic changes in gene activity and their co-ordinate control of specific protein synthesis has been the basis for a number of 'model systems' of gene control in higher eukaryotes.     

Influence of Nematodes on Resource Utilization by Bacteria—an in vitro Study

The positive influence of bacterial feeding nematodes on bacterial mediated processes such as organic matter mineralization and nutrient cycling is widely accepted, but the mechanisms of these interactions are not always apparent. Both transport of bacteria by nematodes, and nutritional effects caused by nematode N excretion are thought to be involved, but their relative importance is not known because of the difficulties in studying these interactions in soil. We developed a simple in vitro assay to study complex nematode/bacterial interactions and used it to conduct a series of experiments to determine the potential influence of nematode movement and nutritional effects on bacterial resource use. The system used bacterial feeding and nonfeeding insect parasitic nematodes, and luminescent bacteria marked with metabolic reporter genes. Both nutritional enhancement of bacterial activity and bacterial transport were observed and we hypothesize that in nature, the relative importance of transport is likely to be greater in bulk soil, whereas nematode excretion may have greater impact in the rhizosphere. In both cases, the ability of nematodes to enhance bacterial resource utilization has implications for soil components of biogeochemical cycling.     

线虫耐寒性研究进展

对于分布在温带和寒带的线虫,它们只有战胜冬季寒冷的挑战,才能有利于种群的存在与发展。因此,耐寒性是线虫生物学研究中不可忽视的内容。综述了关于线虫在低温胁迫下的耐寒性测定方法、耐寒对策及耐寒机制等方面的研究进展。线虫的耐寒性和昆虫一样,可通过过冷却点和低温存活率两种指标进行评价,但在具体的实验方法上,线虫耐寒性研究有其不同之处。线虫的耐寒对策和耐寒机制具有多样化。耐寒对策主要有耐冻和避冻,二者能共同渗透于线虫的耐寒过程中。耐寒机制包括特殊发育阶段的形成、低温驯化作用、低分子量抗冻物质的聚集、以及高分子量抗冻蛋白和热休克蛋白的产生,等等。此外,还强调应从多个角度研究线虫的耐寒性,如寒冷敏感型线虫的研究、寄生线虫的耐寒对策研究以及交叉胁迫的研究。     

Diethylcarbamazine Increases Activation of Voltage-Activated Potassium (SLO-1) Currents in Ascaris suum and Potentiates Effects of Emodepside

Diethylcarbamazine is a drug that is used for the treatment of filariasis in humans and animals; it also has effects on intestinal nematodes, but its mechanism of action remains unclear. Emodepside is a resistance-busting anthelmintic approved for treating intestinal parasitic nematodes in animals. The novel mode of action and resistance-breaking properties of emodepside has led to its use against intestinal nematodes of animals, and as a candidate drug for treating filarial parasites. We have previously demonstrated effects of emodepside on SLO-1 K⁺-like currents in Ascaris suum. Here, we demonstrate that diethylcarbamazine, which has been proposed to work through host mediated effects, has direct effects on a nematode parasite, Ascaris suum. It increases activation of SLO-1 K⁺ currents and potentiates effects of emodepside. Our results suggest consideration of the combination of emodepside and diethylcarbamazine for therapy, which is predicted to be synergistic. The mode of action of diethylcarbamazine may involve effects on parasite signaling pathways (including nitric oxide) as well as effects mediated by host inflammatory mediators.