Process Of Ecdysis Research Articles

Application of synchrotron radiation phase-contrast microtomography with iodine staining to Rhodnius prolixus head during ecdysis period

Synchrotron radiation phase-contrast microtomography (SR-PHC-CT) has become an important tool in studies of insects, mainly Rhodinius prolixus, the insect vector of Chagas disease. A previous work has shown that SR-PHC-CT is an excellent technique in studies about the ecdysis process of R.prolixus head. The term ecdysis refers to the set of behaviors by which an insect extracts itself from an old exoskeleton. The exoskeleton formation is indispensable for the evolutionary success of insect species, so failure to complete ecdysis will, in most cases result in death, making this process an excellent target in the search for new insect pest management strategies. Understanding the behavior of the ecdysis process is fundamental for the non-proliferation of Chagas disease. Despite it has been possible to identify the moulting process in the first work, main structures of the R.prolixus head could not be identified. In this work, it was developed a staining protocol which enabled the identification of these important structures using Iodine at SYRMEP beamline of ELETTRA. In the 3D images, it was possible to segment essential structures in the process of ecdysis. These structures have never been presented previously in the moulting period with SR-PHC-CT.

The relationship of blue crab (Callinectes sapidus) size class and molt stage to disease acquisition and intensity of Hematodinium perezi infections.

In the blue crab, Callinectes sapidus, early studies suggested a relationship between smaller crabs, which molt more frequently, and higher rates of infection by the dinoflagellate parasite, Hematodinium perezi. In order to better explore the influence of size and molting on infections, blue crabs were collected from the Maryland coastal bays and screened for the presence of H. perezi in hemolymph samples using a quantitative PCR assay. Molt stage was determined by a radioimmunoassay which measured ecdysteroid concentrations in blue crab hemolymph. Differences were seen in infection prevalence between size classes, with the medium size class (crabs 61 to 90 mm carapace width) and juvenile crabs (≤ 30 mm carapace width) having the highest infection prevalence at 47.2% and 46.7%, respectively. All size classes were susceptible to infection, although fall months favored disease acquisition by juveniles, whereas mid-sized animals (31–90 mm carapace width) acquired infection predominantly in summer. Disease intensity was also most pronounced in the summer, with blue crabs > 61 mm being primary sources of proliferation. Molt status appeared to be influenced by infection, with infected crabs having significantly lower concentrations of ecdysteroids than uninfected crabs in the spring and the fall. We hypothesize that infection by H. perezi may increase molt intervals, with a delay in the spring molt cycle as an evolutionary adaptation functioning to coincide with increased host metabolism, providing optimal conditions for H. perezi propagation. Regardless of season, postmolt crabs harbored significantly higher proportions of moderate and heavy infections, suggesting that the process of ecdysis, and the postmolt recovery period, has a positive effect on parasite proliferation.

Ecdysis Triggering Hormone Signaling (ETH/ETHR-A) Is Required for the Larva-Larva Ecdysis in Bactrocera dorsalis (Diptera: Tephritidae).

Insects must undergo ecdysis for successful development and growth, and the ecdysis triggering hormone (ETH), released by the Inka cells, is a master hormone in this process. In this study, we determined the sequence of the ETH precursor and receptors in an agriculturally important pest insect, the oriental fruit fly Bactrocera dorsalis (Hendel). We identified two functionally distinct splice receptor isoforms: BdETH-R-A and BdETH-R-B, and when expressed in Chinese hamster ovary (CHO-WTA11) cells, they exhibited a high sensitivity to the two mature peptides BdETH1 and BdETH2. The BdETH transcript was detected in the tracheal tissue of the larvae. Inka cells were identified with immunohistochemical antibody staining against Drosophila melanogaster ETH1, and in situ hybridization with specific DNA probes. Selective RNA silencing of BdETH or BdETH-R-A, but not of BdETH-R-B, caused developmental failure at ecdysis. The dsRNA-treated larvae displayed tracheal defects and could not shed the old cuticle followed by death. Our results demonstrated that BdETH, via activation of BdETH-R-A but not ETH-R-B, plays an essential role in regulating the process of larva-larva ecdysis in B. dorsalis.

Re-examination of a α-chymotrypsin-solubilized laccase in the pupal cuticle of the silkworm, Bombyx mori: Insights into the regulation system for laccase activation during the ecdysis process

The laccase in the pupal cuticle of the silkworm, Bombyx mori, is thought to accumulate as an inactive precursor that can be activated stage-dependently. In this study we isolated an 81-kDa laccase from cuticular extract of B. mori that was prepared by digestion of the pupal cuticles with α-chymotrypsin. The mass spectrometric analysis of the purified protein indicates that this 81-kDa laccase is a product of the Bombyx laccase2 gene. The purified 81-kDa laccase (α-chymotrypsin-solubilized Bombyx laccase2: Bm-clac2) has an N-terminal sequence of RNPADS that corresponds to Arg146 to Ser151 of the deduced protein sequence of Bmlaccase2 cDNA, indicating that Bm-clac2 lacks the N-terminal part upstream from residue Arg146. Bm-clac2 shows enzymatic activity, but its specific activity is increased around 17-fold after treatment with trypsin, which involves cleavage of peptide bonds at the C-terminal region. We also found that the activity of Bm-clac2 is increased in the presence of isopropanol. In previous reports, proteolytic processing has been hypothesized as a system for laccase activation in vivo, but the present result implies that this type of processing is not the only way to convert Bm-clac2 to the high-activity enzyme.

MECANISMOS DA AÇÃO LARVICIDA DO DIFLUBENZURON SOBRE Aedes aegypti EVIDENCIADOS PELAS ALTERAÇÕES ULTRAESTRUTURAIS

Dengue is a viral disease transmitted by Aedes aegypti in over 100 countries in the intertropical band in the world and the main forms of control are antivectorial actions. In this paper, ultrastructural changes caused by diflubenzuron (DFB) are presented. The experiments were performed with 3rd instar larvae of Ae. aegypti with DFB at concentrations of 0.1 and 1 ?g/mL. After 24h of exposure, larvae were collected, fixed, dehydrated, embedded, cut, contrasted with uranyl acetate and 3% lead citrate and examined under the electron microscope. Ultrastructural alterations caused by the diflubenzuron inhibitory activity were observed in the cuticle and in the midgut of Ae. aegypti larvae treated with 0.1 and 1 ?g/mL, for 24 hours. The alterations observed by scanning microscopy included higher number of bristles, which were thinner and longer than the controls and exhibited a coiling pattern in the intersegmental grooves. Transmission electron microscopy analysis revealed that the epicuticle almost came entirely off the new epicuticle and had no points of reinforcements commonly found in the control. The midgut cells of the larvae treated with diflubenzuron presented a spongy skeleton and in the ultrathin sections, the cells showed intense vacuolation and damage, however with secretion vesicles present and mitochondria preserved. These studies indicate that the diflubenzuron can also act in the midgut cells, being a promising product for use in Ae. aegypti control. This study showed that the DFB interferes with the process of ecdysis, prevents the release of the old cuticle that accumulates in the intersegmental spaces and strangle the segmental portions in a successive and cumulative process, blocked the changes, causing the death of the larva. It also acts destroying the midgut cells.

The Silkworm Coming of Age—Early

In order to grow, immature insects must periodically synthesize a new cuticle and shed the old one (a process of molting and ecdysis) until they have reached a stage permitting metamorphosis to the reproductive adult. Each molt is induced by a pulse of ecdysteroids, but the nature of the molt is determined by the juvenile hormones (JHs) [1]. These are sesquiterpenoids, synthesized from three isoprene units via the mevalonate pathway and decorated by an epoxide group on one end and a methyl ester on the other [1]. The JHs maintain larval characters, a status quo effect that is lifted when the level of JH drops. A threshold size must be attained for each molt and for metamorphosis to occur, and this size may relate to the limit of oxygen supply by the tracheal system [2], [3]. The number of molts varies among and sometimes within species; it is influenced by nutrition and by environmental and genetic signals. Yet, the mechanisms that “measure size” or “count the larval instars” are overridden by the experimental depletion of JHs. It has long been a tenet of insect endocrinology that removal of the corpora allata (CA), the endocrine glands that produce the JHs, causes precocious metamorphosis [1]. However, bringing the JH titer down experimentally is not as trivial as it may seem.

Comparative Characterization of Chitinases from Silkworm (Bombyx mori) and Bollworm (Helicoverpa armigera)

Chitinases play an important role in the degradation of the cuticular chitin during the process of ecdysis. In this study, we compared the chitinases of two insect species, Bombyx mori (silkworm) and Helicoverpa armigera (bollworm), to assess the relation between characteristics and chitinase patterns. Differences between two chitinases were observed after purification using ammonium sulfate precipitation, affinity chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) assay. Although the specific activities of the purified enzymes were different, the purification yields were similar. One band of 88 kDa was observed for B. mori, and the other band of 75 kDa was detected for H. armigera. When a range of properties was tested, it was found that the optimum temperatures of B. mori and H. armigera chitinases were 45 and 50°C, respectively; the optimum pH value was 6.0 for both chitinases. Mn(2+) played catalytic role while Cu(2+) and SDS strongly inhibited activities of both enzymes. Between two chitinases, differences in K (M) were also observed. K (M) of chitinase from silkworm and bollworm was found to be 22.3 and 41.0 μmol/l, respectively. Both the chitinases significantly inhibited the spore germination of two fungal species, Saccharomyces cerevisiae and Penicillium.

Flavonoids inhibited NADPH consumption and ecdysis processes in Oncopeltus fasciatus

Flavonoids inhibited NADPH consumption and ecdysis processes in Oncopeltus fasciatus

Knockdown of ecdysis-triggering hormone gene with a binary <italic>UAS/GAL4</italic> RNA interference system leads to lethal ecdysis deficiency in silkworm

Ecdysis-triggering hormone (ETH) is an integration factor in the ecdysis process of most insects, including Bombyx mori (silkworm). To understand the function of the ETH gene in silkworm, we developed an effective approach to knockdown the expression of ETH in vivo based on RNA interference (RNAi) and a binary UAS/GAL4 expression system that has been successfully used in other insect species. Two kinds of transgenic silkworm were established with this method: the effector strain with the ETH RNAi sequence under the control of UAS and the activator strain with the GAL4 coding sequence under the control of Bombyx mori cytoplasmic actin3. By crossing the two strains, double-positive transgenic silkworm was obtained, and their ETH expression was found to be dramatically lower than that of each single positive transgenic parent. Severe ecdysis deficiency proved lethal to the double-positive transgenic silkworm at the stage of pharate second instar larvae, while the single positive transgenic or wild-type silkworm had normal ecdysis. This UAS/GAL4 RNAi approach provides a way to study the function of endogenous silkworm genes at different development stages.

Knockdown of ecdysis-triggering hormone gene with a binaryUAS/GAL4RNA interference system leads to lethal ecdysis deficiency in silkworm

Ecdysis-triggering hormone (ETH) is an integration factor in the ecdysis process of most insects, including Bombyx mori (silkworm). To understand the function of the ETH gene in silkworm, we developed an effective approach to knockdown the expression of ETH in vivo based on RNA interference (RNAi) and a binary UAS/GAL4 expression system that has been successfully used in other insect species. Two kinds of transgenic silkworm were established with this method: the effector strain with the ETH RNAi sequence under the control of UAS and the activator strain with the GAL4 coding sequence under the control of Bombyx mori cytoplasmic actin3. By crossing the two strains, double-positive transgenic silkworm was obtained, and their ETH expression was found to be dramatically lower than that of each single positive transgenic parent. Severe ecdysis deficiency proved lethal to the double-positive transgenic silkworm at the stage of pharate second instar larvae, while the single positive transgenic or wild-type silkworm had normal ecdysis. This UAS/GAL4 RNAi approach provides a way to study the function of endogenous silkworm genes at different development stages.

DEVELOPMENT AND DIVERSIFICATION OF TRUNK PLATES OF THE LOWER CAMBRIAN LOBOPODIANS

Abstract: Isolated lobopodian plates are reported from Early Cambrian strata at five localities in southern China. A wide variety of morphologies is represented, reflecting a considerable diversification within the phylum at this time. The new taxon Microdictyon jinshaense is erected and new observations are recorded on established taxa, based on examination of more than 600 well‐preserved plates; irregular patterns of node distribution and the presence of large spines are documented on some taxa for the first time. Rare specimens in which two plates are conjoined, with a larger plate underlying a smaller one, are interpreted as showing a new sclerite emerging underneath its predecessor, which has not yet been moulted. These specimens confirm the process of ecdysis in the lobopodians and contribute to a fuller understanding of the ontogeny of these organisms. A functional hypothesis that suggests that the plates were complex visual structures is refuted; it is possible that they were sites of muscle attachment, but a protective role is regarded as more plausible.

Redundant ecdysis regulatory functions of three nuclear receptor HR3 isoforms in the direct-developing insect Blattella germanica

In hemimetabolous insects, the molecular basis of the 20-hydroxyecdysone (20E)-triggered genetic hierarchy is practically unknown. In the cockroach Blattella germanica, we had previously characterized one isoform of the ecdysone receptor, BgEcR-A, and two isoforms of its heterodimeric partner, BgRXR-S and BgRXR-L. One of the early-late genes of the 20E-triggered genetic hierarchy, is HR3. In the present paper, we report the discovery of three isoforms of HR3 in B. germanica, that were named BgHR3-A, BgHR3-B 1 and BgHR3-B 2. Expression studies in prothoracic gland, epidermis and fat body indicate that the expression of the three isoforms coincides with the peak of circulating ecdysteroids at each nymphal instar. Experiments in vitro with fat body tissue have shown that 20E induces the expression of BgHR3 isoforms, and that incubation with 20E and the protein inhibitor cycloheximide does not inhibit the induction, which indicates that the effect of 20E on BgHR3 activation is direct. This has been further confirmed by RNAi in vivo of BgEcR-A, which has shown that this nuclear receptor is required to fully activate the expression of BgHR3. RNAi has been also used to demonstrate the functions of BgHR3 in ecdysis. Nymphs with silenced BgHR3 completed the apolysis but were unable to ecdyse (they had duplicated and superimposed the mouth parts, the hypopharinge, the tracheal system and the cuticle layers). This indicates that BgHR3 is directly involved in ecdysis. Finally, RNAi of specific isoforms has showed that they are functionally redundant, at least regarding the ecdysis process.

Trypanosoma rangeli: Effects of physalin B on the immune reactions of the infected larvae of Rhodnius prolixus

Physalins are seco-steroids obtained from plants of the family Solanaceae. Herein, we tested Physalis angulata L purified physalin B as an immunomodulatory compound in 5th-instar larvae of Rhodnius prolixus, which were systemically infected with the H14 Trypanosoma rangeli strain protozoan. In uninfected insects, the effective concentration of physalin B, which inhibited 50% of the blood ingested (ED 50) volume, was 15.2 ± 1.6 μg/ml of the meal. Ecdysis processes and mortality in uninfected larvae, treated orally with physalin B in concentrations ranging from 1 to 10 μg/ml, was similar to that observed in insects not treated with physalin B. However, R. prolixus larvae previously fed on blood containing 1.0, 0.1, and 0.01 μg of physalin B/ml exhibited mortality rates of 78.1, 54.3, and 12.7%, respectively, 6 days after inoculation of T. rangeli (1 × 10 3 parasites/insect), whereas only 7.2% mortality was observed in the control group, injected with sterile culture medium. The insects treated with physalin B (0.1 μg/ml) and inoculated with T. rangeli did not modify the phenoloxidase (PO) activity and total hemocyte count in the hemolymph. However, physalin B treatment caused a reduction in hemocyte microaggregation and nitric oxide production and enhanced the parasitemia in the hemolymph. These results demonstrate that physalin B from P. angulata is a potent immunomodulatory substance for the bloodsucking insect, R. prolixus.

Bioefficacy and mode of action of rocaglamide from Aglaia elaeagnoidea (syn. A. roxburghiana) against gram pod borer, Helicoverpa armigera (Hübner)

Abstract: Rocaglamide, a highly substituted benzofuran, was isolated and identified as the main biologically active component in Aglaia elaeagnoidea (syn. A. roxburghiana) for gram pod borer Helicoverpa armigera (Hübner). Addition of rocaglamide to an artificial diet retarded the growth of neonate larvae in a dose‐dependent manner with EC50 values of 0.76 p.p.m. These values compared favourably with azadirachtin (EC50 = 0.23 p.p.m.). However, azadirachtin was apparently more potent than rocaglamide in inducing growth inhibition via oral administration to these first stadium larvae. The candidate compound was found to have LD50 and LD95 values of 0.40 and 1.02 μg per larva, respectively, in topical application against third instar larvae 96 h post‐treatment. However, these values for azadirachtin were 8.16 and 25.8 μg per larva for the same period. This shows that azadirachtin was less effective against third instar H. armigera larvae in inducing acute toxicity via topical treatment in comparison with rocaglamide. However, severe morphological larval deformities were observed in such azadirachtin‐treated larvae during the process of ecdysis. The cytotoxic nature of rocaglamide was established by evaluating dietary utilization and the results did not implicate any antifeedant effect but the toxicity‐mediated effect due to reduced efficiency of conversion of ingested food. It was obvious that feeding deterrence is not the primary mode of action but a centrally mediated effect, which could be due to the induced cytotoxicity at non‐specific cellular levels.

Growth Disruption in Opisina arenosella Walker by Plumbagin, A Naturally Occurring Insect Growth Regulator

—Plumbagin, a napthoquinone from the plant Plumbago zeylanica , delayed pupation when topically applied to newly moulted final instars of the leaf eating caterpillar Opisina arenosella Walker (Lepidoptera: Oecophoridae). Pupae from treated larvae emerged into non-viable adults and had abnormalities such as deformed wings and shrunken abdomens; they also died much before the control insects. The results suggest that plumbagin interferes with the process of development and ecdysis of the insect.

Developmentally Regulated Zinc Metalloproteinases from Third- and Fourth-Stage Larvae of the Ovine Nematode Haemonchus contortus

Parasitic third-stage larvae of the sheep abomasal nematode Haemonchus contortus develop and molt in vitro to the fourth stage (L4) in 48-72 hr, at which time they begin feeding. Coincident with the third molt, larvae begin to secrete significant amounts of protein into culture fluids, including a zinc metalloproteinase. This culture-derived zinc metalloproteinase differs from a previously described metalloproteinase from infective third-stage larvae (L3[2M]), which mediates the ecdysis process. These differences include time of expression, molecular mass, and substrate specificity. The purified proteinase, from cultures of L4, has a molecular weight of approximately 46 kDa, functions as an endopeptidase, and digests several native proteins of host origin including fibrinogen and fibronectin.

Effects of erythrocyte component diets on ecdysteroid production and ecdysis of Rhodnius prolixus larvae

Blood meal components in addition to the abdominal distension observed after feeding, are important in producing ecdysteroids and establishing the ecdysis processes in fourth-instar larvae of Rhodnius prolixus. This hypothesis is supported by the following results: (i) numbers undergoing ecdysis were proportional to the percentage haemoglobin added to the plasma upon which they feed; (ii) plasma alone induced only about 20% ecdysis; (iii) the haemoglobin content rather than the red cell membrane component stimulates ecdysis; (iv) ecdysteroid production was dependent upon the presence of haemoglobin or of erythrocytes but not plasma or erythrocyte membranes in the meal. The significance of these data is discussed in relation to the effects of haemoglobin or other erythrocyte components on ecdysis processes in R. prolixus larvae.

Light and Scanning Electron Microscopy of the Ecdysis of Haemonchus contortus Infective Larvae

During the second ecdysis of ruminant trichostrongyles, a region of the second molt cuticle is digested by a 44-kDa Zn-metalloprotease. We have examined this digestion process by light and scanning electron microscopy (SEM). The substrate region of the cuticle appeared, during the ecdysis process, as an indented ring at the 20th cuticular annulus coincident with the anterior terminus of the lateral alae. Continued digestion of the cuticle resulted in holes in the ring region that expanded until they became continuous and separation occurred between the anterior and posterior portions of the cuticle. Mechanical movements of the L3 forced aside the cuticle cap that generally remained attached on one side to the posterior portion as the larva escaped from the sheath. The site of secretion of the 44-kDa ecdysing enzyme causing cuticle digestion was not clear from morphological observations; however, existing evidence strongly points to the release of enzyme from the esophageal (pharyngeal) glands through the mouth.

SEXUAL REPRODUCTION AND MEIOSIS IN PERIDINIUM INCONSPICUUM LEMMERMANN (DINOPHYCEAE)

In Peridinium inconspicuum Lemmermann, sexual reproduction occurs in both nitrogen‐enriched and nitrogen‐deficient media. In this homothallic strain, protoplasmic fusion begins between two thecate gametes; but zygote formation is completed in a space outside the fusing pair. This diploid cell can form a plated theca which is shed as the cell enlarges. This spherical zygote then forms a new non‐plated theca. The process of ecdysis and the formation of a new non‐plated theca is repeated several times. During this process the zygote gradually elongates and by cytoplasmic infurrowing becomes peanut‐shaped. Eventually two cells are formed.The first and second meiotic divisions are greatly separated in time. The first meiotic division occurs in the spherical non‐thecate zygote. The second meiotic division can occur in the peanut‐shaped zygote before it completes cytokinesis. This meiotic division may not be synchronous, occasionally resulting in a trinucleate stage. Eventually four flagellated, haploid products are produced.

Setal development, moult-staging and ecdysis in the banana prawn Penaeus merguiensis

Specimens of juvenile Penaeus merguiensis de Man (1888) were captured by seine netting from the shores of Cleveland Bay, Townsville, Queensland Australia between February 1980 and November 1981. Accurate determination of moult cycle stage can be achieved rapidly by microsopical examination of setal development in an excised whole pleopod. Four major stages and 5 substages within premoult were clearly defined. The rate of setogenesis varies along the length of a pleopod, precluding the examination of only one specific location on the pleopod for accurate moult cycle staging. The process of ecdysis lasts for no more than 40 s.