Pupal RNA interference methods for analyzing adult development in stag beetles

Holometabolous insects dramatically change their morphology via molt, both from larva to pupa and from pupa to adult. In nonmodel insects, RNA interference (RNAi) is a strong tool for analyzing gene function during postembryonic development. In many cases, larval RNAi is effective for analyzing genes involved in morphogenesis via metamorphosis. However, RNAi of genes involved in development sometimes results in lethality before animals metamorphose to pupae and/or adults, making it impossible to analyze their function during the pupal period. In this study, we establish a pupal RNAi system in the stag beetle Dorcus rectus. We selected the genes white and scarlet for RNAi knockdown to investigate appropriate injection timing and position. Both genes are known to be involved in eye pigmentation. By using these candidate genes, we demonstrate the potential of pupal RNAi in this experimental system. This method will be useful for analyzing pupal-specific morphogenesis including fine-shaping of the enlarged male mandible in this species.     

Juvenile hormone acid synthesis and HMG-CoA reductase activity in corpora allata of Manduca sexta prepupae

Corpora allata (CA) of last instar larvae of Manduca sexta switch from juvenile hormone (JH) to JH acid secretion just before the onset of wandering behavior. JH acid secretion peaked during the prepupal period and ceased prior to pupal ecdysis. HMG-CoA reductase activity also peaked during the prepupal period and then declined. However, substantial enzyme activity was present in pupal and pharate adult glands. Removal of the brain at the wandering stage caused a reduction in JH acid secretion by prepupal CA. The profile of HMG-CoA activity in CA of debrained larvae resembled that of sham-operated larvae except that the prepupal peak was smaller than in control larvae. Addition of brain extracts to CA maintained in vitro neither stimulated not inhibited JH acid secretion and HMG-CoA reductase activity. It is suggested that the brain regulates CA activity in post-wandering stages via intact nerves.     

Some quantitative changes observed in Philosamia ricini pupal haemolymph during metamorphosis

1. The quantitative variations of the concentrations of uric acid, citrate, nucleic acids and total and acid-soluble phosphorus in the pupal haemolymph of Philosamia ricini during metamorphosis have been studied. 2. The mean value for total nitrogen in the haemolymph and total loss in weight of the insects during metamorphosis have also been recorded.     

Hormonal regulation and developmental role of Krüppel homolog 1, a repressor of metamorphosis,in the silkworm Bombyx mori

Juvenile hormone (JH) has an ability to repress the precocious metamorphosis of insects during their larval development. Krüppel homolog 1 (Kr-h1) is an early JH-inducible gene that mediates this action of JH; however, the fine hormonal regulation of Kr-h1 and the molecular mechanism underlying its antimetamorphic effect are little understood. In this study, we attempted to elucidate the hormonal regulation and developmental role of Kr-h1. We found that the expression of Kr-h1 in the epidermis of penultimate-instar larvae of the silkworm Bombyx mori was induced by JH secreted by the corpora allata (CA), whereas the CA were not involved in the transient induction of Kr-h1 at the prepupal stage. Tissue culture experiments suggested that the transient peak of Kr-h1 at the prepupal stage is likely to be induced cooperatively by JH derived from gland(s) other than the CA and the prepupal surge of ecdysteroid, although involvement of unknown factor(s) could not be ruled out. To elucidate the developmental role of Kr-h1, we generated transgenic silkworms overexpressing Kr-h1. The transgenic silkworms grew normally until the spinning stage, but their development was arrested at the prepupal stage. The transgenic silkworms from which the CA were removed in the penultimate instar did not undergo precocious pupation or larval–larval molt but fell into prepupal arrest. This result demonstrated that Kr-h1 is indeed involved in the repression of metamorphosis but that Kr-h1 alone is incapable of implementing normal larval molt. Moreover, the expression profiles and hormonal responses of early ecdysone-inducible genes (E74, E75, and Broad) in transgenic silkworms suggested that Kr-h1 is not involved in the JH-dependent modulation of these genes, which is associated with the control of metamorphosis.     

Micro-computed tomography of pupal metamorphosis in the solitary bee Megachile rotundata

Insect metamorphosis involves a complex change in form and function. In this study, we examined the development of the solitary bee, Megachile rotundata, using micro-computed tomography (μCT) and volume analysis. We describe volumetric changes of brain, tracheae, flight muscles, gut, and fat bodies in prepupal, pupal, and adult M. rotundata. We observed that individual organ systems have distinct patterns of developmental progression, which vary in their timing and duration. This has important implications for commercial management of this agriculturally relevant pollinator.     

Purification and characterization of two β-N-acetylhexosaminidases from the tobacco hornworm,Manduca sexta (L.) (Lepidoptera: Sphingidae)

β-N-Acetylhexosaminidases were detected in 10 insects including species of Lepidoptera, Coleoptera, Hemiptera, and Orthoptera. Two enzymes were purified from the tobacco hornworm, Manduca sexta (L.). EI was detected in larval and pharate pupal molting fluid, integument, and pupal hemolymph while EII was found in larval and pupal hemolymphs. They are acidic hydrolases with similar molecular weights (6.1 × 10⁴), molar extinction coefficients at 280 nm (1.9 × 10⁵ liters mol^?1 cm^?1), and pH optima (pH 6). They differ in the number of polypeptide chains per molecule (EI is a single chain and EII consists of two polypeptide chains), amino acid composition, extent of glycosylation (EII is probably a glycoprotein), isoelectric point (pI_EI = 5.9 and pI_EII ～- 5.1), tissue distribution, and reactivities toward nitrophenylated N-acetylglucosamine (k_cat,I = 328 s^?1 and k_cat,II = 103 s^?1) and N,N′-diacetylchitobiose (k_cat,I = 307 s^?1 and k_cat,II = 3 s^?1). These results suggest that EI is a chitinase and that EII may function as a hexosaminidase in vivo.     

Modulation of cyclic CO2 release in response to endogenous changes of metabolism during pupal development of Zophobas rugipes (Coleoptera: Tenebrionidae)

Understanding the mechanisms of gas exchange regulation in insects currently is a hot topic of insect physiology. Endogenous variation of metabolism during pupal development offers a great opportunity to study the regulation of respiratory patterns in insects. Here we show that metabolic rates during pupal development of the tenebrionid beetle Zophobas rugipes reveal a typical U-shaped curve and that, with the exception of 9-day-old pupae, the time between two bursts of CO₂ (interburst phase) was the only parameter of cyclic CO₂ gas exchange patterns that was adjusted to changing metabolic rates. The volume of CO₂ released in a burst was kept constant, suggesting a regulation for accumulation and release of a fixed amount of CO₂ throughout pupal development. We detected a variety of discontinuous and cyclic gas exchange patterns, which were not correlated with any periods of pupal development, suggesting a high among individual variability. An occasional occurrence of continuous CO₂ release patterns at low metabolic rates was very likely caused by single defective non-occluding spiracles.     

Implantation Serine Proteinases heterodimerize and are critical in hatching and implantation

Background

Metamorphosis is a complex, highly conserved and strictly regulated development process that involves the programmed cell death of obsolete larval organs. Here we show a novel functional role for the aspartic proteinase cathepsin D during insect metamorphosis.

Results

Cathepsin D of the silkworm Bombyx mori (BmCatD) was ecdysone-induced, differentially and spatially expressed in the larval fat body of the final instar and in the larval gut of pupal stage, and its expression led to programmed cell death. Furthermore, BmCatD was highly induced in the fat body of baculovirus-infected B. mori larvae, suggesting that this gene is involved in the induction of metamorphosis of host insects infected with baculovirus. RNA interference (RNAi)-mediated BmCatD knock-down inhibited programmed cell death of the larval fat body, resulting in the arrest of larval-pupal transformation. BmCatD RNAi also inhibited the programmed cell death of larval gut during pupal stage.

Conclusion

Based on these results, we concluded that BmCatD is critically involved in the programmed cell death of the larval fat body and larval gut in silkworm metamorphosis.     

Ecdysteroid synthesis by the ring gland of Drosophila melanogaster during late-larval,prepupal and pupal development

Radioimmunoassay has been used to determine the characteristics of ecdysteroid synthesis by ring glands and brain-ring gland preparations from late 3rd-instar larvae of Drosophila melanogaster cultured in vitro. The rate of synthesis and secretion is linear for at least 4 hr in culture. Using a 4-hr culture period, variation in the rate of ecdysteroid synthesis by brain-ring gland preparations during larval, prepupal and pupal development has been examined. The rate of synthesis and secretion is highest in late 3rd-instar larvae and decreases after puparium formation. During pupal development, at a time when the endogenous ecdysteroid titre is again increasing, the rate of ecdysteroid synthesis by brain-ring gland preparations remains low and is only 10% of that prior to puparium formation. It is, therefore, likely that the ring gland is not a major source of ecdysteroids during this period.     

Looking into the puparium: Micro‐CT visualization of the internal morphological changes during metamorphosis of the blow fly,Calliphora vicina,with the first quantitative analysis of organ development in cyclorrhaphous dipterans

Metamorphosis of cyclorrhaphous flies takes place inside a barrel‐like puparium, formed by the shrinking, hardening and darkening of the third‐instar larval cuticle. The opacity of this structure hampers the visualization of the morphological changes occurring inside and therefore a full understanding of the metamorphosis process. Here, we use micro‐computed tomography (micro‐CT) to describe the internal morphological changes that occur during metamorphosis of the blow fly, Calliphora vicina Robineau‐Desvoidy 1830 (Diptera: Calliphoridae) at a greater temporal resolution than anything hitherto published. The morphological changes were documented at 10% intervals of the total intra‐puparial period, and down to 2.5% intervals during the first 20% interval, when the most dramatic morphological changes occur. Moreover, the development of an internal gas bubble, which plays an essential role during early metamorphosis, was further investigated with X‐ray images and micro‐CT virtual sections. The origin of this gas bubble has been largely unknown, but micro‐CT virtual sections show that it is connected to one of the main tracheal trunks. Micro‐CT virtual sections also provided enough resolution for determining the completion of the larval‐pupal and pupal‐adult apolyses, thus enabling an accurate timing of the different intra‐puparial life stages. The prepupal, pupal, and pharate adult stages last for 7.5%, 22.5%, and 70% of the total intra‐puparial development, respectively. Furthermore, we provide for the first time quantitative data on the development of two organ systems of the blow fly: the alimentary canal and the indirect flight muscles. There is a significant and negative correlation between the volume of the indirect flight muscles and the pre‐helicoidal region of the midgut during metamorphosis. The latter occupies a large portion of the thorax during the pupal stage but narrows progressively as the indirect flight muscles increase in volume during the development of the pharate adult.     

Different pathways of DNA synthesis are operative in the fat body of Spodoptera litura (insecta) during larval and pupal development

In Spodoptera litura, the DNA content of the fat body increases during the last larval instar. Radiolabelling studies reveal high degree of H3-thymidine incorporation in the fat cell DNA, during last larval stage of development. At prepupal stage the DNA synthesised during larval development is degraded, at least partially and the resultant nucleotides are most likely used as building blocks for the fat cell DNA synthesis during pupal development and pupal-adult metamorphosis, because very little H3 -thymidine injected into pupae is incorporated in DNA, though in fact, significant amount of DNA is synthesised during this period.     

Observations on development,larval growth and metamorphosis of four species of aplysiidae (gastropoda: Opisthobranchia) in laboratory culture

The development of simple, reliable techniques for the laboratory culture of aplysiid gastropods through their complete life cycle, has enabled us to study the larval biology, metamorphosis, and early juvenile development of these animals. Egg masses, duration of the embryonic phase, veligers, and larval growth and development are described for four species of Hawaiian Aplysiidae, namely, Aplysia dactylomela Rang, Aplysia Juliana Quoy and Gaimard, Dolabella auricularia (Lightfoot) and Stylocheilus longicauda (Quoy and Gaimard). Metamorphosis and early juvenile development of A. Juliana are described in detail with additional comments on these processes in the other three species. Length of the embryonic phase and size of the veliger at hatching are a function of the size of the uncleaved egg. All four species develop planktotrophically and have ≈ 30-day larval phases. In each species the larval phase includes a period of rapid shell growth to a species-specific size followed by a non-growth period during which other morphological developments occur to culminate in metamorphic competence. The larvae of each species metamorphose preferentially on a particular species of benthic algae. The events of metamorphosis require 2 to 4 days for completion and transform the planktonic filter-feeding larva into a benthic, radular-feeding juvenile. Postlarval development includes growth of the shell, parapodia, oral tentacles, rhinophores, anal siphon, and structures of the mantle cavity.     

Effects of a protein synthesis inhibitor on the hormonally mediated regression and death of motoneurons in the tobacco hornworm,Manduca sexta

The larval–pupal transformation of Manduca sexta is accompanied by the loss of the abdominal prolegs. The proleg muscles degenerate, the dendritic arbors of proleg motoneurons regress, and a subset of the proleg motoneurons dies. The regression and death of proleg motoneurons are triggered by the prepupal peak of ecdysteroids in the hemolymph. To investigate the possible involvement of protein synthesis in these events, we gave insects repeated injections of the protein synthesis inhibitor, cycloheximide (CHX), during the prepupal peak. Examination of insects 3–5 days following CHX treatment showed that CHX inhibited the death of proleg motoneurons and the production of pupal cuticle in a dose-dependent fashion. When insects were allowed to survive for 10 days after the final CHX injection, motoneuron death and pupal cuticle production sometimes occurred belatedly, apparently in response to the ecdysteroid rise that normally triggers adult development. CHX treatments that inhibited motoneuron death were less effective in inhibiting dendritic regression in the same neurons. In another set of experiments, abdomens were isolated from the ecdysteroid-secreting glands prior to the prepupal peak, and infused with 20-hydroxyecdysone (20-HE). Single injections of CHX delivered just prior to the start of the 20-HE infusion inhibited motoneuron death and pupal cuticle production, but in the range of doses tested, did not prevent dendritic regression. Our findings suggest that protein synthesis is a required step in the steroid-mediated death of proleg motoneurons, and that dendritic regression is less susceptible to inhibition by CHX than is motoneuron death. © 1993 John Wiley & Sons, Inc.     

Constancy and variation of the serotonin-like immunoreactive neurons in the metamorphosing ventral nerve cord of the meal beetle,Tenebrio molitor L. (Coleoptera : Tenebrionidae)

Serotonin-like immunoreactive neurons were mapped in the larval, prepupal, pupal, and adult ventral nerve cord (VNC) of the beetle, Tenebrio molitor L. (Coleoptera: Tenebrionidae). The alterations of the shape of these neurons during metamorphosis were analysed. The stage-specific interindividual variability of the examined serotonin-like immunoreactive neurons is low. Serotonin-like immunoreactive neurons of the abdominal and thoracic ganglia behave differently during metamorphosis. Only in thoracic ganglia was an obvious change in the pattern of serotonin-like immunoreactive neurons observed. The shape of the dendritic trees of serotonin-like immunoreactive neurons varies in thoracic., but not in abdominal ganglia. During postlarval development, new emerging neurons that react with the anti-serotonin antibody are found only in the thoracic ganglia. Serotonin-like immunoreactive neurons are serially homologous in the larval ventral nerve cord. The basic organization of the serotonin-like immunoreactive neurons is maintained up to the adult stage. Some aspects of the metamorphosis of the nervous system are discussed with respect to the transformation of the set of immunoreactive neurons from larval to adult stage. The results are compared to those obtained in the study of serotonin-immunoreactive neurons in cockroaches, dipterans and locusts.     

Unexpected Mechanism of Symbiont-Induced Reversal of Insect Sex: Feminizing Wolbachia Continuously Acts on the Butterfly Eurema hecabe during Larval Development

When the butterfly Eurema hecabe is infected with two different strains (wHecCI2 and wHecFem2) of the bacterial endosymbiont Wolbachia, genetic males are transformed into functional females, resulting in production of all-female broods. In an attempt to understand how and when the Wolbachia endosymbiont feminizes genetically male insects, larval insects were fed an antibiotic-containing diet beginning at different developmental stages until pupation. When the adult insects emerged, strikingly, many of them exhibited sexually intermediate traits in their wings, reproductive organs, and genitalia. The expression of intersexual phenotypes was strong in the insects treated from first instar, moderate in the insects treated from third instar, and weak in the insects treated from fourth instar. The insects treated from early larval instar grew and pupated normally but frequently failed to emerge and died in the pupal case. The dead insects in the pupal case contained lower densities of the feminizing Wolbachia endosymbiont than the successfully emerged insects, although none of them were completely cured of the symbiont infection. These results suggest the following: (i) the antibiotic treatment suppressed the population of feminizing Wolbachia endosymbionts; (ii) the suppression probably resulted in attenuated feminizing activity of the symbiont, leading to expression of intersexual host traits; (iii) many of the insects suffered pupal mortality, possibly due to either intersexual defects or Wolbachia-mediated addiction; and hence (iv) the feminizing Wolbachia endosymbiont continuously acts on the host insects during larval development for expression of female phenotypes under a male genotype. Our finding may prompt reconsideration of the notion that Wolbachia-induced reproductive manipulations are already complete before the early embryonic stage and provide insights into the mechanism underlying the symbiont-induced reversal of insect sex.