Epithelial reorganization events during late extraembryonic development in a hemimetabolous insect

As extra-embryonic tissues, the amnion and serosa are not considered to contribute materially to the insect embryo, yet they must execute an array of morphogenetic movements before they are dispensable. In hemimetabolous insects, these movements have been known for over a century, but they have remained virtually unexamined. This study addresses late extraembryonic morphogenesis in the milkweed bug, Oncopeltus fasciatus. Cell shape changes and apoptosis profiles are used to characterize the membranes as they undergo a large repertoire of final reorganizational events that reposition the embryo (katatrepsis), and eliminate the membranes themselves in an ordered fashion (dorsal closure). A number of key features were identified. First, amnion-serosa “fusion” involves localized apoptosis in the amnion and the formation of a supracellular actin purse string at the amnion-serosa border. During katatrepsis, a ‘focus’ of serosal cells undergoes precocious columnarization and may serve as an anchor for contraction. Lastly, dorsal closure involves novel modifications of the amnion and embryonic flank that are without counterpart during the well-known process of dorsal closure in the fruit fly Drosophila melanogaster. These data also address the long-standing question of the final fate of the amnion: it undergoes apoptosis during dorsal closure and thus is likely to be solely extraembryonic.     

Morphogenesis of insect-induced plant galls: facts and questions

Insect-induced galls (‘galls’ hereafter) represent highly regulated growth manifestations on plants. They present unique geometrical forms, which are, usually, unknown in the normal plant system. Galls are the best examples for modified natural structures that arise solely because of messages from an alien organism - the insect. Galls develop as an extension of the host-plant phenotype. But how the physiological networks and signal-activated subsystems work in coordination in expressing galls that serve the nutritional and shelter needs of the inducing insect are unclear. In galls and bacteria-induced tumors, the basic developmental events are essentially similar. However, tightly regulated specific differentiation processes occur in galls, making them different from tumors. Moreover variations in differentiation patterns occur in galls induced by insects of different taxonomic groups. While providing an overview of the control of shape and structure in galls, this article identifies the unanswered questions in gall morphogenesis.By analyzing the recognizable steps in gall morphogenesis, viz., gall initiation, stimulus recognition in host plants, signal transduction in host plants, growth of galls, and qualitative differentiation in galls, I have indicated that the insect saliva flushed on the wounded plant site alters the subcellular environment of cells and thus places it in a state of chemical shock. This shock induces osmotic changes, which establishes the first recognizable stage in gall induction. To repair the wound and neutralize the osmotic-change induced stress, the plant responds by establishing from one to a few metaplasied cell(s). Localized metabolic changes spread, from these cells, not throughout the involved plant organ, but in a limited manner around the immediate site of insect occurrence. When the shock is of low intensity, the plant responds with the development of one or more metaplasied cell(s) and gall development starts; when the shock factor is of high intensity, the cells under the insect action die, rejecting the inducing insect, defending plant tissue. These changes dictate the new morphogenetic events. Insects feed on gall tissue continuously for a specific period (synchronizing with their life history) and therefore, the osmotic-change related stress prevails for that span of time, which in turn triggers a sequence of plant-mediated changes including synthesis of growth promotors. Osmotic stress affects electrical properties of the plasma membrane and impacts on IAA activity, which in turn, alters H⁺-transport systems. During the physical action of insect feeding, the host-cell wall breaks down, and the degenerated wall materials act as elicitors.Using galls (e.g., ‘cecidial shoots’ on leaves, modified vegetative buds) induced on species of south and south-east Asian Dipterocarpaceae by different Beesoniidae (Coccoidea) as model complexity in gall morphogenesis is discussed. Manipulatory experimental studies done on the regeneration of epiphyllous buds on Pteridium, Begonia, and a Helianthus hybrid indicate that insect-induced neoplasmic shoots that arise on the leaves of tropical Dipterocarpaceae fall into the morphogenetic regulation of leaf, yet maintaining their freedom of differentiation. Even though a gall is a part of the plant - a multicellular organism made of the same genetic material - organismal development generates a range of cell types with dictated functions fitting into of Waddington's epigenetic-landscape model. As of today, our knowledge stops here.Plants as living systems display different strategies to mitigate and neutralize stress. Although these strategies exist in their genetic constitution, they are mediated by complex molecular interactions. Plants have a flexible short-term strategy to respond to stress; organisms that can modify gene expression reversibly have an advantage in evolutionary terms, since they can avoid rearrangements and species diversification. Mechanisms of DNA methylation and histone modifications possibly regulate inheritance of stress ‘memories’. Inherited genetic traits also play a role in gall morphogenesis, followed by roles played by correlating morphogenetic factors. An articulated reconstruction of the developmental process commencing from either one or a group of metaplasied cells that gets transmitted through subsequent growth promoter-mediated cell expansion, until the commitment of the metaplastic cell and those in its neighbourhood enabling the start of ‘novel’ cell-cycle patterns, cell multiplication, programmed differentiation, and control is needed to explain symmetry - a morphogenetic phenomenon that makes the insect-induced galls distinct from the bacteria-induced tumors.     

Late extraembryonic morphogenesis and its zen-induced failure in the milkweed bug Oncopeltus fasciatus

Many insects undergo katatrepsis, essential reorganization by the extraembryonic membranes that repositions the embryo. Knockdown of the zen gene by RNA interference (RNAi) prevents katatrepsis in the milkweed bug Oncopeltus fasciatus. However, the precise morphogenetic defect has been uncertain, and katatrepsis itself has not been characterized in detail. The dynamics of wild type and zen^RNAi eggs were analyzed from time-lapse movies, supplemented by analysis of fixed specimens. These investigations identify three zen^RNAi defects. First, a reduced degree of tissue contraction implies a role for zen in baseline compression prior to katatrepsis. Subsequently, a characteristic ‘bouncing’ activity commences, leading to the initiation of katatrepsis in wild type eggs. The second zen^RNAi defect is a delay in this activity, suggesting that a temporal window of opportunity is missed after zen knockdown. Ultimately, the extraembryonic membranes fail to rupture in zen^RNAi eggs: the third defect. Nevertheless, the outer serosal membrane manages to contract, albeit in an aberrant fashion with additional phenotypic consequences for the embryo. These data identify a novel epithelial morphogenetic event – rupture of the ‘serosal window’ structure – as the ultimate site of defect. Overall, Oncopeltus zen seems to have a role in coordinating a number of pre-katatreptic events during mid embryogenesis.     

The extraembryonic serosa protects the insect egg against desiccation

Insects have been extraordinarily successful in occupying terrestrial habitats, in contrast to their mostly aquatic sister group, the crustaceans. This success is typically attributed to adult traits such as flight, whereas little attention has been paid to adaptation of the egg. An evolutionary novelty of insect eggs is the serosa, an extraembryonic membrane that enfolds the embryo and secretes a cuticle. To experimentally test the protective function of the serosa, we exploit an exceptional possibility to eliminate this membrane by zerknüllt1 RNAi in the beetle Tribolium castaneum. We analyse hatching rates of eggs under a range of humidities and find dramatically decreasing hatching rates with decreasing humidities for serosa-less eggs, but not for control eggs. Furthermore, we show serosal expression of Tc-chitin-synthase1 and demonstrate that its knock-down leads to absence of the serosal cuticle and a reduction in hatching rates at low humidities. These developmental genetic techniques in combination with ecological testing provide experimental evidence for a crucial role of the serosa in desiccation resistance. We propose that the origin of this extraembryonic membrane facilitated the spectacular radiation of insects on land, as did the origin of the amniote egg in the terrestrial invasion of vertebrates.     

The dynamic expression of extraembryonic marker genes in the beetle Tribolium castaneum reveals the complexity of serosa and amnion formation in a short germ insect

Most insect embryos develop with two distinct extraembryonic membranes, the serosa and the amnion. In the insect beetle Tribolium the early origin of the serosa within the anterior blastoderm is well established but the origin of the amnion is still debated. It is not known whether this tissue develops from a blastodermal precursor or originates de novo later from embryonic tissue during embryogenesis.We undertook an in-depth analysis of the spatio-temporal expression pattern profile of important extraembryonic membrane marker genes with emphasis on early blastoderm development in Tribolium.The amnion marker iroquois (Tc-iro) was found co-expressed with the serosa marker zerknüllt1 (Tc-zen1) during early blastoderm formation in an anterior cap domain. This domain later resolved into two adjacent domains that likely represent the precursors of the serosa and the amnion. In addition, we found the hindsight ortholog in Tribolium (Tc-hnt) to be a serosa-specific marker. Surprisingly, decapentaplegic (Tc-dpp) expression was not seen as a symmetric cap domain but detected asymmetrically first along the DV- and later also along the AP-axis. Moreover, we found a previously undescribed domain of phosphorylated MAD (pMAD) protein in anterior ventral serosal cells.This is the first study showing that the anterior-lateral part of the amnion originates from the anterior blastoderm while the precursor of the dorsal amnion develops later de novo from a dorsal-posterior region within the differentiated blastoderm.     

The dynamic expression of extraembryonic marker genes in the beetle Tribolium castaneum reveals the complexity of serosa and amnion formation in a short germ insect

Most insect embryos develop with two distinct extraembryonic membranes, the serosa and the amnion. In the insect beetle Tribolium the early origin of the serosa within the anterior blastoderm is well established but the origin of the amnion is still debated. It is not known whether this tissue develops from a blastodermal precursor or originates de novo later from embryonic tissue during embryogenesis.We undertook an in-depth analysis of the spatio-temporal expression pattern profile of important extraembryonic membrane marker genes with emphasis on early blastoderm development in Tribolium.The amnion marker iroquois (Tc-iro) was found co-expressed with the serosa marker zerknüllt1 (Tc-zen1) during early blastoderm formation in an anterior cap domain. This domain later resolved into two adjacent domains that likely represent the precursors of the serosa and the amnion. In addition, we found the hindsight ortholog in Tribolium (Tc-hnt) to be a serosa-specific marker. Surprisingly, decapentaplegic (Tc-dpp) expression was not seen as a symmetric cap domain but detected asymmetrically first along the DV- and later also along the AP-axis. Moreover, we found a previously undescribed domain of phosphorylated MAD (pMAD) protein in anterior ventral serosal cells.This is the first study showing that the anterior-lateral part of the amnion originates from the anterior blastoderm while the precursor of the dorsal amnion develops later de novo from a dorsal-posterior region within the differentiated blastoderm.     

Concurrent effects of cold and hyperkalaemia cause insect chilling injury

Chilling injury and death are the ultimate consequence of low temperature exposure for chill susceptible insects, and low temperature tolerance is considered one of the most important factors determining insect distribution patterns. The physiological mechanisms that cause chilling injury are unknown, but chronic cold exposure that causes injury is consistently associated with elevated extracellular [K⁺], and cold tolerant insects possess a greater capacity to maintain ion balance at low temperatures. Here, we use the muscle tissue of the migratory locust (Locusta migratoria) to examine whether chill injury occurs during cold exposure or following return to benign temperature and we specifically examine if elevated extracellular [K⁺], low temperature, or a combination thereof causes cell death. We find that in vivo chill injury occurs during the cold exposure (when extracellular [K⁺] is high) and that there is limited capacity for repair immediately following the cold stress. Further, we demonstrate that that high extracellular [K⁺] causes cell death in situ, but only when experienced at low temperatures. These findings strongly suggest that that the ability to maintain ion (particularly K⁺) balance is critical to insect low temperature survival, and highlight novel routes of study in the mechanisms regulating cell death in insects in the cold.     

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel''s cartilage, nasal glands, tongue, and ear.     

Tissue Damage Disrupts Developmental Progression and Ecdysteroid Biosynthesis in Drosophila

In humans, chronic inflammation, severe injury, infection and disease can result in changes in steroid hormone titers and delayed onset of puberty; however the pathway by which this occurs remains largely unknown. Similarly, in insects injury to specific tissues can result in a global developmental delay (e.g. prolonged larval/pupal stages) often associated with decreased levels of ecdysone – a steroid hormone that regulates developmental transitions in insects. We use Drosophila melanogaster as a model to examine the pathway by which tissue injury disrupts developmental progression. Imaginal disc damage inflicted early in larval development triggers developmental delays while the effects are minimized in older larvae. We find that the switch in injury response (e.g. delay/no delay) is coincident with the mid-3rd instar transition – a developmental time-point that is characterized by widespread changes in gene expression and marks the initial steps of metamorphosis. Finally, we show that developmental delays induced by tissue damage are associated with decreased expression of genes involved in ecdysteroid synthesis and signaling.     

Postgastrular zen expression is required to develop distinct amniotic and serosal epithelia in the scuttle fly Megaselia

The amnioserosa is an extraembryonic epithelium that evolved in higher cyclorrhaphan flies from distinct serosal and amniotic epithelia. The underlying genetic mechanism of this evolutionary transition is unknown. Amnioserosa development of Drosophila correlates with novel expression characteristics of the homeobox gene zerknüllt (zen), including a broad zen expression domain in the syncytial blastoderm and the complete absence of postgastrular zen expression. Here we examine the functional significance of these features by altering the activity profile of zen in Megaselia (a lower cyclorrhaphan fly with distinct serosal and amniotic epithelia) and Drosophila, and by examining in Megaselia the function of u-shaped group (ush-group) genes, which in Drosophila maintain the amnioserosa after gastrulation when zen is no longer expressed. In Megaselia, loss of postgastrular zen expression abrogates serosa development but allows amnion development. Ectopic expression of zen in early Megaselia embryos allows serosa formation but perturbs amnion development. Megaselia homologues of u-shaped group genes are not essential for serosa formation but mediate germband retraction and dorsal closure. Finally, ectopic postgastrular zen expression in Drosophila causes an enlargement of amnioserosa cells and interferes with the morphogenetic functions of the amnioserosa. Our results suggest that the origin of the amnioserosa involved the loss of postgastrular zen expression from extraembryonic tissue, that the early broad expression domain of Drosophila zen evolved afterwards, and that the ush-group genes ancestrally played a role in morphogenetic functions of the amnion.     

Developmental O-glycan profile analysis shows pentasaccharide mucin-type O-glycans are linked with pupation of Tribolium castaneum

Eukaryotic cells can decorate their proteins with carbohydrate structures or glycans, significantly affecting the properties and activities of these proteins. Despite the importance of protein glycosylation in numerous biological processes, our knowledge of this modification in insects is far from complete. While N-glycosylation is the most studied, the study of O-glycans in insects is still very fragmentary and these studies are limited to a specific developmental stage or a specific tissue. In this article, matrix-assisted laser desorption/ionization (MALDI)-Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) technology was used to analyze the O-glycan profile for the different developmental stages of egg, larva, pupa, and adult of the red flour beetle Tribolium castaneum, an important insect model and pest worldwide. The results on the O-glycan profile showed that the mucin-type glycans dominate the O-glycome of the red flour beetle. Interestingly, some of the more complex mucin-type O-glycans, such as a tetra- (O-GalNAcGalGlcAGalNAc) and pentasaccharide O-glycan (O-GalNAc(GalGlcA)GalNAcGlcA), were highly abundant during the pupa stage, the intermediate stage between larval and adult stage in holometabolous insects, demonstrating that insect metamorphosis is accompanied with a change in the insect O-glycan profile. Together with the N-glycan profile, the current data are a foundation to better understand the role of protein glycosylation in the development of insects.     

Glycogen and Glucose Metabolism Are Essential for Early Embryonic Development of the Red Flour Beetle Tribolium castaneum

Control of energy metabolism is an essential process for life. In insects, egg formation (oogenesis) and embryogenesis is dependent on stored molecules deposited by the mother or transcribed later by the zygote. In oviparous insects the egg becomes an isolated system after egg laying with all energy conversion taking place during embryogenesis. Previous studies in a few vector species showed a strong correlation of key morphogenetic events and changes in glucose metabolism. Here, we investigate glycogen and glucose metabolism in the red flour beetle Tribolium castaneum, an insect amenable to functional genomic studies. To examine the role of the key enzymes on glycogen and glucose regulation we cloned and analyzed the function of glycogen synthase kinase 3 (GSK-3) and hexokinase (HexA) genes during T. castaneum embryogenesis. Expression analysis via in situ hybridization shows that both genes are expressed only in the embryonic tissue, suggesting that embryonic and extra-embryonic cells display different metabolic activities. dsRNA adult female injection (parental RNAi) of both genes lead a reduction in egg laying and to embryonic lethality. Morphological analysis via DAPI stainings indicates that early development is impaired in Tc-GSK-3 and Tc-HexA1 RNAi embryos. Importantly, glycogen levels are upregulated after Tc-GSK-3 RNAi and glucose levels are upregulated after Tc-HexA1 RNAi, indicating that both genes control metabolism during embryogenesis and oogenesis, respectively. Altogether our results show that T. castaneum embryogenesis depends on the proper control of glucose and glycogen.     

Perception of insect feeding by plants

The recognition of phytophagous insects by plants induces a set of very specific responses aimed at deterring tissue consumption and reprogramming metabolism and development of the plant to tolerate the herbivore. The recognition of insects by plants requires the plant’s ability to perceive chemical cues generated by the insects and to distinguish a particular pattern of tissue disruption. Relatively little is known about the molecular basis of insect perception by plants and the signalling mechanisms directly associated with this perception. Importantly, the insect feeding behaviour (piercing‐sucking versus chewing) is a decisive determinant of the plant’s defence response, and the mechanisms used to perceive insects from different feeding guilds may be distinct. During insect feeding, components of the saliva of chewing or piercing‐sucking insects come into contact with plant cells, and elicitors or effectors present in this insect‐derived fluid are perceived by plant cells to initiate the activation of specific signalling cascades. Although receptor–ligand interactions controlling insect perception have yet not been molecularly described, a significant number of regulatory components acting downstream of receptors and involved in the activation of defence responses against insects has been reported. Some of these regulators mediate changes in the phytohormone network, while others directly control gene expression or the redox state of the cell. These processes are central in the orchestration of plant defence responses against insects.     

First report of insulin receptor in thysanoptera and its expression variation with development of western flower thrips,Frankliniella occidentalis

Insulin signaling is required for insect development. Insulin-like peptides and their signalings have been extensively assessed in various insects. However, little is known about them in Thysanoptera. This study identified the first insulin receptor in Thysanoptera using western flower thrips, Frankliniella occidentalis. A single Fo-InR (F. occidentalis insulin receptor) gene is encoded in its genome. A predicted amino acid sequence (1,645 residues) was highly (>70% homologies) matched to known insect InRs. However, it did not cluster with other known insect taxonomic groups. Domain analysis suggested an intracellular protein kinase and an extracellular ligand-binding regions. All developmental stages from larva to adult expressed Fo-InR. Starvation up-regulated its expression levels at all developmental stages except pupal stage. Different diet conditions resulting in different developmental rates altered expression levels of Fo-InR. There was a negative correlation between developmental rate and Fo-InR expression levels, especially at adult stage. Results of this study suggests a physiological role of insulin signaling in F. occidentalis development.     

Down syndrome cell adhesion molecule is important for early development in Xenopus tropicalis

The Down syndrome cell adhesion molecule (DSCAM) is an Ig containing cell adhesion molecule with remarkable structural conservation throughout metazoans. In insects, DSCAM has 38,000 potential isoforms that convey axon guidance, fasciculation, and dendrite morphogenesis during neurodevelopment. In vertebrates, DSCAM is expressed throughout the nervous system and seems to also mediate proper axonal guidance and synaptogenesis without the isoform diversity found in insects. Differences in DSCAM function among several vertebrate species complicate the understanding of an evolutionarily conserved role during embryogenesis. We take advantage of the frog developmental model Xenopus tropicalis to study DSCAM function in early development by expression analysis and morpholino‐mediated knockdown. Our results indicate that DSCAM is expressed early in development and restricted to the head and nervous system. Knockdown of protein expression results in early morphogenetic phenotypes characterized by failed gastrulation and improper posterior neural tube closure. Our results reveal a specific, fundamental role of DSCAM in early morphogenetic movements, presumably through its well‐known role in homophilic cell adhesion. genesis 52:849–857, 2014. © 2014 Wiley Periodicals, Inc.     

Regressive evolution of the arthropod tritocerebral segment linked to functional divergence of the Hox gene labial

The intercalary segment is a limbless version of the tritocerebral segment and is present in the head of all insects, whereas other extant arthropods have retained limbs on their tritocerebral segment (e.g. the pedipalp limbs in spiders). The evolutionary origin of limb loss on the intercalary segment has puzzled zoologists for over a century. Here we show that an intercalary segment-like phenotype can be created in spiders by interfering with the function of the Hox gene labial. This links the origin of the intercalary segment to a functional change in labial. We show that in the spider Parasteatoda tepidariorum the labial gene has two functions: one function in head tissue maintenance that is conserved between spiders and insects, and a second function in pedipalp limb promotion and specification, which is only present in spiders. These results imply that labial was originally crucial for limb formation on the tritocerebral segment, but that it has lost this particular subfunction in the insect ancestor, resulting in limb loss on the intercalary segment. Such loss of a subfunction is a way to avoid adverse pleiotropic effects normally associated with mutations in developmental genes, and may thus be a common mechanism to accelerate regressive evolution.