Degeneration and cell regeneration in the midgut of Podisus nigrispinus (Heteroptera: Pentatomidae) during post-embryonic development

Cell death, proliferation, and differentiation in some developmental stages of insects have been studied in the midgut of ametabolous, which undergo only continuous growth, and holometabolous, which undergo complete metamorphosis. However, in hemimetabolous insects, evolutionarily intermediate between ametabolous and holometabolous, midgut reorganization during the post-embryonic development has been poorly studied. The present study evaluates the post-embryonic development of the midgut of a hemimetabolous insect, Podisus nigrispinus, to test the hypothesis that these insects have programmed cell death and proliferation followed by differentiation of regenerative cells during midgut growth from nymphs to adult. The morphometrical data showed a 6-fold increase in midgut length from the first instar nymph to the adult, which did not result from an increase in the size of the midgut cells, suggesting that the growth of the midgut occurs by an increase in cell number. Cell death was rarely found in the midgut, whereas proliferation of regenerative cells occurred quite frequently. The growth of the midgut of P. nigrispinus appears to result from the proliferation of regenerative cells present in the epithelium; unlike ametabolous and holometabolous insects, the midgut of P. nigrispinus does not undergo extensive remodeling, as shown by the low frequency of digestive cell death.     

Ontogeny of hemocyanin in the ovoviviparous cockroach Blaptica dubia suggests an embryo-specific role in oxygen supply

For a long time it had been assumed that specific oxygen transport proteins are absent in insects. Only recently it has been demonstrated that hemocyanins occur in the hemolymph of many ametabolous and hemimetabolous insect taxa, but not in the Eumetabola (Hemiptera + Holometabola). Therefore, the loss of respiratory hemocyanin in insects is not correlated with the evolution of an efficient tracheal system. The specific contribution of hemocyanin to oxygen supply in insects, however, has remained uncertain. Here we investigate the stage-specific expression of hemocyanin in the ovoviviparous cockroach Blaptica dubia (Blattaria), which consists of two distinct subunit types (Hc1 and Hc2). Employing quantitative real-time RT-PCR and Western blotting, we showed that the expression of hemocyanin is restricted to late embryos, thus being detectable also in whole female extracts and oothecae. Hemocyanin protein is also present in 1st instar nymphs, but not in later developmental stages. The ontogeny of hemocyanin in cockroaches is distinct from that known from Zygentoma and Plecoptera, in which hemocyanin occurs in both nymphal and adult stages. Our findings suggest a specific role of hemocyanin in embryonic cockroaches, which may be related to an enhanced oxygen supply in the oothecae. For some reason, the fundamental physiological changes associated to the evolution of holometaboly have made hemocyanin unnecessary.     

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.     

METABOLISM DURING EMBRYONIC AND METAMORPHIC DEVELOPMENT OF INSECTS

1. For species of insects that deposit their eggs on foliage, as Leptinotarsa decemlineata, Crioceris asparagi, and Anasa tristis, the rates of metabolism as measured by the CO₂ output or the oxygen intake indicate, during embryonic development, a short formative period, followed by a very active extended growth. Those species that deposit their eggs in the soil, as Cotinis nitida and Popillia japonica, show that the formative period is greatly extended and the growth period similarly lengthened. 2. It has been shown from metabolism experiments that intensive histolysis occurs during the prepupal period and becomes less intensive during pupal development. 3. Metabolism experiments show a greater amount of energy change during embryonic development as compared to the energy developed during metamorphosis. This is shown by the greater CO₂ output and by the oxygen intake. 4. Low respiratory quotients, varying from 0.42 to 0.71, have been obtained during the embryonic and pupal development of insects, resembling similar low quotients obtained with hibernating forms. 5. Changes from a neutral to an acid reaction (pH 6.8 to 5.9) have been observed to take place in some species during prepupal and early pupal development. As metamorphosis is completed a converse change occurs (pH 5.9 to 6.8). Changes in pH from an acid to neutral reaction were also observed to take place in some species, as Cotinis nitida and Hylemyia cilicrura, during embryonic development.     

Diverging functions of Scr between embryonic and post-embryonic development in a hemimetabolous insect, Oncopeltus fasciatus

Hemimetabolous insects undergo an ancestral mode of development in which embryos hatch into first nymphs that resemble miniature adults. While recent studies have shown that homeotic (hox) genes establish segmental identity of first nymphs during embryogenesis, no information exists on the function of these genes during post-embryogenesis. To determine whether and to what degree hox genes influence the formation of adult morphologies, we performed a functional analysis of Sex combs reduced (Scr) during post-embryonic development in Oncopeltus fasciatus. The main effect was observed in prothorax of Scr-RNAi adults, and ranged from significant alterations in its size and shape to a near complete transformation of its posterior half toward a T2-like identity. Furthermore, while the consecutive application of Scr-RNAi at both of the final two post-embryonic stages (fourth and fifth) did result in formation of ectopic wings on T1, the individual applications at each of these stages did not. These experiments provide two new insights into evolution of wings. First, the role of Scr in wing repression appears to be conserved in both holo- and hemimetabolous insects. Second, the prolonged Scr-depletion (spanning at least two nymphal stages) is both necessary and sufficient to restart wing program. At the same time, other structures that were previously established during embryogenesis are either unaffected (T1 legs) or display only minor changes (labium) in adults. These observations reveal a temporal and spatial divergence of Scr roles during embryonic (main effect in labium) and post-embryonic (main effect in prothorax) development.     

The effect of manipulating ecdysteroid signaling on embryonic eye development in the locust<Emphasis Type="Italic"> Schistocerca americana</Emphasis>

Adult body plan differentiation in holometabolous insects depends on global induction and control by ecdysteroid hormones during the final phase of postembryogenesis. Studies in Drosophila melanogaster and Manduca sexta have shown that this pertains also to the development of the compound eye retina. It is unclear whether the hormonal control of postembryonic eye development in holometabolous insects represents evolutionary novelty or heritage from hemimetabolous insects, which develop compound eyes during embryogenesis. We therefore investigated the effect of manipulating ecdysteroid signaling in cultured embryonic eye primordia of the American desert locust Schistocerca americana, in which ecdysteroid level changes are known to induce three rounds of embryonic molt. Although at a considerably reduced rate compared to in vivo development, early differentiation and terminal maturation of the embryonic retina was observed in culture even if challenged with the ecdysteroid antagonist cucurbitacin B. Supplementing cultures with 20-hydroxyecdysone (20E) accelerated differentiation and maturation, and enhanced cell proliferation. Considering these results, and the relation between retina differentiation and ecdysteroid level changes during locust embryogenesis, we conclude that ecdysteroids are not an essential but possibly a modulatory component of embryonic retina development in S. americana. We furthermore found evidence that 20E initiated precocious epithelial morphogenesis of the posterior retinal margin indicating a more general role of ecdysteroids in insect embryogenesis.Electronic Supplementary Material Supplementary material is available in the online version of this article at Edited by C. Desplan     

The hormonal pathway controlling cell death during metamorphosis in a hemimetabolous insect

Metamorphosis in holometabolous insects is mainly based on the destruction of larval tissues. Intensive research in Drosophila melanogaster, a model of holometabolan metamorphosis, has shown that the steroid hormone 20-hydroxyecdysone (20E) signals cell death of larval tissues during metamorphosis. However, D. melanogaster shows a highly derived type of development and the mechanisms regulating apoptosis may not be representative in the insect class context. Unfortunately, no functional studies have been carried out to address whether the mechanisms controlling cell death are present in more basal hemimetabolous species. To address this, we have analyzed the apoptosis of the prothoracic gland of the cockroach Blattella germanica, which undergoes stage-specific degeneration just after the imaginal molt. Here, we first show that B. germanica has two inhibitor of apoptosis (IAP) proteins and that one of them, BgIAP1, is continuously required to ensure tissue viability, including that of the prothoracic gland, during nymphal development. Moreover, we demonstrate that the degeneration of the prothoracic gland is controlled by a complex 20E-triggered hierarchy of nuclear receptors converging in the strong activation of the death-inducer Fushi tarazu-factor 1 (BgFTZ-F1) during the nymphal-adult transition. Finally, we have also shown that prothoracic gland degeneration is effectively prevented by the presence of juvenile hormone (JH). Given the relevance of cell death in the metamorphic process, the characterization of the molecular mechanisms regulating apoptosis in hemimetabolous insects would allow to help elucidate how metamorphosis has evolved from less to more derived insect species.     

br regulates the expression of the ecdysone biosynthesis gene npc1

The growth and metamorphosis of insects are regulated by ecdysteroid hormones produced in the ring gland. Ecdysone biosynthesis-related genes are both highly and specifically expressed in the ring gland. However, the intrinsic regulation of ecdysone biosynthesis has received little attention. Here we used the Drosophila npc1 gene to study the mechanism of ring gland-specific gene expression. npc1 is important for sterol trafficking in the ring gland during ecdysone biosynthesis. We have identified a conserved ring gland-specific cis-regulatory element (RSE) in the npc1 promoter using promoter fusion reporter analysis. Furthermore, genetic loss-of-function analysis and in vitro electrophoretic mobility shift assays revealed that the ecdysone early response gene broad complex (br) is a vital factor in the positive regulation of npc1 ring gland expression. Moreover, br also affects the ring gland expression of many other ecdysone biosynthetic genes as well as torso and InR, two key factors in the regulation of ecdysone biosynthesis. These results imply that ecdysone could potentially act through its early response gene br to achieve positive feedback regulation of ecdysone biosynthesis during development.     

Over-expression of Ultrabithorax alters embryonic body plan and wing patterns in the butterfly Bicyclus anynana

In insects, forewings and hindwings usually have different shapes, sizes, and color patterns. A variety of RNAi experiments across insect species have shown that the hox gene Ultrabithorax (Ubx) is necessary to promote hindwing identity. However, it remains unclear whether Ubx is sufficient to confer hindwing fate to forewings across insects. Here, we address this question by over-expressing Ubx in the butterfly Bicyclus anynana using a heat-shock promoter. Ubx whole-body over-expression during embryonic and larvae development led to body plan changes in larvae but to mere quantitative changes to adult morphology, respectively. Embryonic heat-shocks led to fused segments, loss of thoracic and abdominal limbs, and transformation of head limbs to larger appendages. Larval heat-shocks led to reduced eyespot size in the expected homeotic direction, but neither additional eyespots nor wing shape changes were observed in forewings as expected of a homeotic transformation. Interestingly, Ubx was found to be expressed in a novel, non-characteristic domain – in the hindwing eyespot centers. Furthermore, ectopic expression of Ubx on the pupal wing activated the eyespot-associated genes spalt and Distal-less, known to be directly repressed by Ubx in the fly?s haltere and leg primordia, respectively, and led to the differentiation of black wing scales. These results suggest that Ubx has been co-opted into a novel eyespot gene regulatory network, and that it is capable of activating black pigmentation in butterflies.     

A Baculovirus Immediate-Early Gene,ie1, Promoter Drives Efficient Expression of a Transgene in Both Drosophila melanogaster and Bombyx mori

Many promoters have been used to drive expression of heterologous transgenes in insects. One major obstacle in the study of non-model insects is the dearth of useful promoters for analysis of gene function. Here, we investigated whether the promoter of the immediate-early gene, ie1, from the Bombyx mori nucleopolyhedrovirus (BmNPV) could be used to drive efficient transgene expression in a wide variety of insects. We used a piggyBac-based vector with a 3xP3-DsRed transformation marker to generate a reporter construct; this construct was used to determine the expression patterns driven by the BmNPV ie1 promoter; we performed a detailed investigation of the promoter in transgene expression pattern in Drosophila melanogaster and in B. mori. Drosophila and Bombyx belong to different insect orders (Diptera and Lepidoptera, respectively); however, and to our surprise, ie1 promoter-driven expression was evident in several tissues (e.g., prothoracic gland, midgut, and tracheole) in both insects. Furthermore, in both species, the ie1 promoter drove expression of the reporter gene from a relatively early embryonic stage, and strong ubiquitous ie1 promoter-driven expression continued throughout the larval, pupal, and adult stages by surface observation. Therefore, we suggest that the ie1 promoter can be used as an efficient expression driver in a diverse range of insect species.     

Postembryonic development of the auditory system of the cicada Okanagana rimosa (Say) (Homoptera: Auchenorrhyncha: Cicadidae)

Cicadas (Homoptera: Auchenorrhyncha: Cicadidae) use acoustic signalling for mate attraction and perceive auditory signals by a tympanal organ in the second abdominal segment. The main structural features of the ear are the tympanum, the sensory organ consisting of numerous scolopidial cells, and the cuticular link between sensory neurones and tympanum (tympanal ridge and apodeme). Here, a first investigation of the postembryonic development of the auditory system is presented. In insects, sensory neurones usually differentiate during embryogenesis, and sound-perceiving structures form during postembryogenesis. Cicadas have an elongated and subterranian postembryogenesis which can take several years until the final moult. The neuroanatomy and functional morphology of the auditory system of the cicada Okanagana rimosa (Say) are documented for the adult and the three last larval stages. The sensory organ and the projection of sensory afferents to the CNS are present in the earliest stages investigated. The cuticular structures of the tympanum, the tympanal frame holding the tympanum, and the tympanal ridge differentiate in the later stages of postembryogenesis. Thus, despite the different life styles of larvae and adults, the neuronal components of the cicada auditory system develop already during embryogenesis or early postembryogenesis, and sound-perceiving structures like tympana are elaborated later in postembryogenesis. The life cycle allows comparison of cicada development to other hemimetabolous insects with respect to the influence of specially adapted life cycle stages on auditory maturation. The neuronal development of the auditory system conforms to the timing in other hemimetabolous insects.     

SpolvlgA is a DDX3/PL10-related DEAD-box RNA helicase expressed in blastomeres and embryonic cells in planarian embryonic development

Planarian flatworms have an impressive regenerative power. Although their embryonic development is still poorly studied and is highly derived it still displays some simple characteristics. We have identified SpolvlgA, a Schmidtea polychroa homolog of the DDX3/PL10 DEAD-box RNA helicase DjvlgA from the planarian species Dugesia japonica. This gene has been previously described as being expressed in planarian adult stem cells (neoblasts), as well as the germ line. Here we present the expression pattern of SpolvlgA in developing embryos of S. polychroa and show that it is expressed from the first cleavage rounds in blastomere cells and blastomere-derived embryonic cells. These cells are undifferentiated cells that engage in a massive wave of differentiation during stage 5 of development. SpolvlgA expression highlights this wave of differentiation, where nearly all previous structures are substituted by blastomere-derived embryonic cells. In late stages of development SpolvlgA is expressed in most proliferating and differentiating cells. Thus, SpolvlgA is a gene expressed in planarian embryos from the first stages of development and a good marker for the zygote-derived cell lineage in these embryos. Expression in adult worms is also monitored and is found in the planarian germ line, where it is showed to be expressed in spermatogonia, spermatocytes and differentiating spermatids.     

The esterases of Drosophila. I. The anodal esterases and their possible role in eclosion

The ontogeny of esterase activity was analyzed electrophoretically throughout the development of Drosophila pseudoobscura. The major adult enzyme form, Esterase 5, and several minor components, showed no important developmental variation. A group of several rapidly migrating (anodal) esterases, in contrast, accumulated maximum activity during late pupal stages, but was completely absent in newly emerged adults. This disappearance was not the result of some rapid inactivation, for subsequent to eclosion these enzymatic forms could be fully recovered from the discarded pupal case. Of the four additional Drosophila species examined, all but one displayed a similar pattern of ontogenesis. The possible role of these pupa-specific esterases is discussed.     

On the pronymphal stage in the migratory locust (Locusta migratoria, Orthoptera, Acrididae)

The structure of the integument, somatic and visceral muscles, midgut, and Malpighian tubules were investigated at the late stages of the embryonic and early postembryonic development of the migratory locust, Locusta migratoria, to assess the organization of its pronymphal stage. In its morphogenetic features, the vermiform locust larva sometimes called the pronymph corresponds to the first nymphal instar covered with the second embryonic cuticle which has not been shed. Since the first-instar locust nymphs before and after the shedding of this embryonic cuticle differ significantly in many morphological characters, two consecutive phases of this nymphal instar can be distinguished: the first phase existing from the moment of development of the third embryonic cuticle to the shedding of the second one; the second phase existing from the shedding of the second embryonic cuticle to the formation of the cuticle of the second nymphal instar. Since the pronymphal stage should precede the nymph stage, it may be concluded that the pronymph of the locust is fully embryonized and covered with the second embryonic cuticle, which is also typical of other insects with hemimetabolous development (Konopová and Zrzavý, 2005). Therefore, it would be erroneous to refer to the vermiform first-instar nymph as the pronymph, because the two stages are separated by molting and formation of a new cuticle.