Late Pupal Stage Research Articles

Larval host plant experience modulates both mate finding and oviposition choice in a moth

Host plant choice in polyphagous insects can be connected with costs of being naive when confronted with several potential host plants, for example through slower decision making. Utilization of earlier experience could be one way to overcome some of these limitations. We studied whether larval feeding experience influences mate finding by males and female choice of oviposition site in the moth Spodoptera littoralis. Larvae were reared on either artificial diet or the host plants, cotton, clover or alfalfa, and we recorded the behaviour of adults from each diet. In two sets of experiments, in both the laboratory and the field, we investigated the female's oviposition choice and the male's response to female pheromone with different plant odour backgrounds. We found that experience with cotton, clover or alfalfa during the larval period induced female oviposition on the corresponding plant both in the laboratory and in the field. Furthermore, males were more attracted to female sex pheromone combined with odour from a host plant species that they had experienced as larvae than to sex pheromone combined with odour from host plant species they had not experienced. The results show convergent modulation of male and female responses to plant odour depending on their larval food plant. The influence of larval experience during the first-to-fifth instars on host preference was also stronger than the influence of experience acquired in the late larval, pupal and early adult stages. Consequences for host plant choice efficiency and fitness effects for males and females are discussed.

Gene expression analysis of the endosymbiont-bearing midgut tissue during ontogeny of the carpenter ant Camponotus floridanus

Insects have frequently evolved mutualistic relationships with extracellular and/or intracellular bacterial endosymbionts. Infection with endosymbionts seems to affect several cellular functions of the host such as immune pathways, oxidative stress regulation and autophagy. Our current knowledge about specific host factors leading to endosymbiont tolerance and/or control is still scarce and is based on very few associations between insect hosts and bacteria only. Camponotus floridanus ants harbour the obligate intracellular bacterium Blochmannia floridanus within specialized midgut cells called bacteriocytes. The number of Blochmannia endosymbionts within the midgut tissue increases strongly during host development and reaches a maximum at the late pupal stage, where the entire midgut is transformed into a symbiotic organ. After eclosion of workers the number of Blochmannia strongly decreases again. We chose 15 candidate genes from C. floridanus likely to be involved in host–symbiont interactions based on their significant homology to previously investigated symbiosis-relevant genes from other insects. We determined the expression of these genes in the endosymbiont-bearing midgut tissue in comparison to the residual body tissue at different developmental stages of C. floridanus in order to reveal changes in gene expression correlating with changes in endosymbiont number per host. Strikingly, two pattern recognition receptors (amidase PGRP-LB and PGRP-SC2) were highly expressed in the midgut tissue at the pupal stage, potentially down-modulating the IMD pathway to enable endosymbiont tolerance. Moreover, we investigated the immune gene expression in response to bacterial challenge at the pupal stage. Results showed that the midgut tissue differs in expression pattern in contrast to the residual body. Our results support a key role for amidase PGRPs, especially PGRP-LB, in regulation of the immune response towards endosymbionts in C. floridanus and suggest an involvement of the lysosomal system in control of Blochmannia endosymbionts.

Irradiation for Quarantine Control of the Invasive Light Brown Apple Moth (Lepidoptera: Tortricidae) and a Generic Dose for Tortricid Eggs and Larvae

The effects of irradiation on egg, larval, and pupal development, and adult reproduction in light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae),were examined. Eggs, neonates, third instars, fifth instars, and early stage pupae were irradiated at target doses of 60, 90, 120, or 150 Gy or left untreated as controls in replicated factorial experiments and survival to the adult stage was recorded. Tolerance to radiation generally increased with increasing age and developmental stage. A radiation dose of 120 Gy applied to eggs and neonates prevented adult emergence. A dose of 150 Gy prevented adult emergence in larvae at all stages. In large-scale validation tests, a radiation dose of 150 Gy applied to fifth instars in diet, apples or peppers resulted in no survival to the adult stage in 37,947 treated individuals. Pupae were more radio tolerant than larvae, and late stage pupae were more tolerant than early stage pupae. Radiation treatment of late pupae at 350 and 400 Gy resulted in three and one fertile eggs in 4,962 and 4,205 total eggs laid by 148 and 289 mating pairs, respectively. For most commodities, the fifth instar is the most radio tolerant life stage likely to occur with the commodity; a minimum radiation dose of 150 Gy will prevent adult emergence from this stage and meets the zero tolerance requirement for market access. For traded commodities such as table grapes that may contain E. postvittana pupae, a radiation dose > 400 Gy may be necessary to completely sterilize emerging adults. After review of the literature, a generic radiation treatment of 250 Gy is proposed for tortricid eggs and larvae in regulated commodities.

The Abelson tyrosine kinase regulates Notch endocytosis and signaling to maintain neuronal cell fate inDrosophilaphotoreceptors

The development of a functional organ requires coordinated programs of cell fate specification, terminal differentiation and morphogenesis. Whereas signaling mechanisms that specify individual cell fates are well documented, little is known about the pathways and molecules that maintain these fates stably as normal development proceeds or how their dysregulation may contribute to altered cell states in diseases such as cancer. In Drosophila, the tyrosine kinase Abelson (Abl) interfaces with multiple signaling pathways to direct epithelial and neuronal morphogenesis during embryonic and retinal development. Here we show that Abl is required for photoreceptor cell fate maintenance, as Abl mutant photoreceptors lose neuronal markers during late pupal stages but do not re-enter a proliferative state or undergo apoptosis. Failure to maintain the differentiated state correlates with impaired trafficking of the Notch receptor and ectopic Notch signaling, and can be suppressed by reducing the genetic dose of Notch or of its downstream transcriptional effector Suppressor of Hairless. Together, these data reveal a novel mechanism for maintaining the terminally differentiated state of Drosophila photoreceptors and suggest that neuronal fates in the fly retina retain plasticity late into development. Given the general evolutionary conservation of developmental signaling mechanisms, Abl-mediated regulation of Notch could be broadly relevant to cell fate maintenance and reprogramming during normal development, regeneration and oncogenic transformation.

The TSC1/2 Complex Controls Drosophila Pigmentation through TORC1-Dependent Regulation of Catecholamine Biosynthesis

In Drosophila, the pattern of adult pigmentation is initiated during late pupal stages by the production of catecholamines DOPA and dopamine, which are converted to melanin. The pattern and degree of melanin deposition is controlled by the expression of genes such as ebony and yellow as well as by the enzymes involved in catecholamine biosynthesis. In this study, we show that the conserved TSC/TORC1 cell growth pathway controls catecholamine biosynthesis in Drosophila during pigmentation. We find that high levels of Rheb, an activator of the TORC1 complex, promote premature pigmentation in the mechanosensory bristles during pupal stages, and alter pigmentation in the cuticle of the adult fly. Disrupting either melanin synthesis by RNAi knockdown of melanogenic enzymes such as tyrosine hydroxylase (TH), or downregulating TORC1 activity by Raptor knockdown, suppresses the Rheb-dependent pigmentation phenotype in vivo. Increased Rheb activity drives pigmentation by increasing levels of TH in epidermal cells. Our findings indicate that control of pigmentation is linked to the cellular nutrient-sensing pathway by regulating levels of a critical enzyme in melanogenesis, providing further evidence that inappropriate activation of TORC1, a hallmark of the human tuberous sclerosis complex tumor syndrome disorder, can alter metabolic and differentiation pathways in unexpected ways.

Transgenic sexing system for Ceratitis capitata (Diptera: Tephritidae) based on female-specific embryonic lethality

Fruit fly pest species have been successfully controlled and managed via the Sterile Insect Technique (SIT), a control strategy that uses infertile matings of sterile males to wild females to reduce pest populations. Biological efficiency in the field is higher if only sterile males are released in SIT programs and production costs are also reduced. Sexing strains developed in the Mediterranean fruit fly Ceratitis capitata (medfly) through classical genetics are immensely beneficial to medfly SIT programs but exhibit reduced fertility and fitness. Moreover, transfer of such classical genetic systems to other tephritid species is difficult. Transgenic approaches can overcome this limitation of classical genetic sexing strains (GSSs), but had resulted so far in transgenic sexing strains (TSSs) with dominant lethality at late larval and pupal stages. Here we present a transgene-based female-specific lethality system for early embryonic sexing in medfly. The system utilizes the sex-specifically spliced transformer intron to restrict ectopic mRNA translation of the pro-apoptotic gene hidAla5 to females only. The expression of this lethal effector gene is driven by a tetracycline-repressible transactivator gene tTA that is under the control of promoters/enhancers of early-acting cellularization genes. Despite observed position effects on the sex-specific splicing, we could effectively establish this early-acting transgenic sexing system in the medfly C. capitata. After satisfactory performance in large scale tests, TSSs based on this system will offer cost-effective sexing once introduced into SIT programs. Moreover, this approach is straight forward to be developed also for other insect pest and vector species.

Transcription profiling of guanine nucleotide binding proteins during developmental regulation and pesticide response in Solenopsis invicta (Hymenoptera: Formicidae)

Correspondence: Liming Zhao BCPRU-MSA-ARS USDA, Insect Physiology, 59 Lee Road, Stoneville, MS 38776 USA Tel +66 268 630 67 Fax +66 268 659 22 Email liming.zhao@ars.usda.gov Abstract: Guanine nucleotide binding proteins (GNBP), glycoproteins anchored on the cytoplasmic cell membrane, are mediators for many cellular processes. Complete cDNA of the GNBP gene β-subunit (SiGNBP) was cloned and sequenced from Solenopsis invicta Buren (Hymenoptera: Formicidae) worker ants. To understand whether SiGNBP is developmentally regulated in S. invicta, its expression levels in different developmental stages were examined using quantitative real-time polymerase chain reaction. SiGNBP was expressed in each developmental stage, and was especially highly expressed in the late larval and early pupal stages, as well among the dealate females (queens) ∼10 days post nuptial flight. Quantitative real-time polymerase chain reaction also showed that mRNA transcription levels of SiGNBP in S. invicta workers were regulated during the duration of the study in response to heat shock, ultraviolet light, and boric acid treatment. These results suggest that the SiGNBP gene may play an important role in the development of S. invicta, and this has potential for use as a target in new insecticides for the control of fire ants.

Spatio‐temporal pattern of programmed cell death in the developing Drosophila optic lobe

A large number of cells die via programmed cell death during the normal development of the Drosophila optic lobe. In this study, we report the precise spatial and temporal pattern of cell death in this organ. Cell death in the developing optic lobe occurs in two distinct phases. The first phase extends from the start of metamorphosis to the mid-pupal stage. During this phase, a large number of cells die in the optic lobe as a whole, with a peak of cell death at an early pupal stage in the lamina and medulla cortices and the region of the T2/T3/C neurons, and a smaller number of dead cells observed in the lobula plate cortex. The second phase extends from the mid-pupal stage to eclosion. Throughout this period, a small number of dying cells can be observed, with a small peak at a late pupal stage. Most of the dying cells are neurons. During the first phase, dying cells are distributed in specific patterns in cortices. The lamina cortex contains two distinct clusters of dying cells; the medulla cortex, four clusters; the lobula plate cortex, one cluster; and the region of the T2/T3/C neurons, one cluster. Many of the clusters maintain their distinct positions in the optic lobe but others extend the region they cover during development. The presence of distinct clusters of dying cells at different phases suggests that distinct mechanisms control cell death during different stages of optic lobe development in Drosophila.

Determinants of the Drosophila Odorant Receptor Pattern

In most olfactory systems studied to date, neurons that express the same odorant receptor (Or) gene are scattered across sensory epithelia, intermingled with neurons that express different Or genes. In Drosophila, olfactory sensilla that express the same Or gene are dispersed on the antenna and the maxillary palp. Here we show that Or identity is specified in a spatially stereotyped pattern by the cell-autonomous activity of the transcriptional regulators Engrailed and Dachshund. Olfactory sensilla then become highly motile and disperse beneath the epidermis. Thus, positional information and cell motility underlie the dispersed patterns of Drosophila Or gene expression.

Brain development in an insect with extensive maternal care, the tsetse fly Glossina morsitans (Diptera: Glossinidae)

Brain development in an insect with extensive maternal care, the tsetse fly Glossina morsitans (Diptera: Glossinidae)

Molecular cloning and expression patterns of FK506‐binding protein 12, an immunophilin from the cabbage butterfly, Pieris rapae

FK506‐binding protein (FK506BP) class belonging to immunophilin protein family has been known to play key roles in modulating T‐cell activation, regulation of cell cycle and protein folding. However, little is known about the involvement of FK506BP during viral pathogenesis in insect host. In this study, an attempt has been made to focus on the involvement of FK506BP in antiviral innate immunity, by cloning the full‐length cDNA of FK506BP12 (PrFK506BP12) from the cabbage butterfly, Pieris rapae. It comprised of 532 bp (excluding poly‐A tail) with a longest open reading frame (ORF) of 327 bp encoding 108 amino acids. In silico analysis of PrFK506BP12 ORF revealed a highly conserved FK506‐binding domain (FKBD). As expected, it showed high homology to other FK506BPs identified from Bombyx mori (92%), Manduca sexta (91%), Suberites domuncula (82%), Tribolium castaneum (81%) and Aedes aegypti (74%). Expression of PrFK506BP12 was observed during developmental stages of P. rapae, but was pronounced in late pupal and adult stage. In addition, spatial expression pattern analysis indicated its high expression in the head and fat body. Furthermore, PrFK506BP12 mRNA was induced 12 h after LTA, Poly I:C treatment and 3h after Pieris rapae granulovirus (PrGV) treatment in carcass. It suggests that PrFK506BP12 appears to be involved in immune responses and also play an important role in the fat body, although it remains to be clarified about their precise role in response to granulovirus.

Overexpression of Dsk2/dUbqln results in severe developmental defects and lethality inDrosophila melanogasterthat can be rescued by overexpression of the p54/Rpn10/S5a proteasomal subunit

Polyubiquitin receptors execute the targeting of polyubiquitylated proteins to the 26S proteasome. In vitro studies indicate that disturbance of the physiological balance among different receptor proteins impairs the proteasomal degradation of polyubiquitylated proteins. To study the physiological consequences of shifting the in vivo equilibrium between the p54/Rpn10 proteasomal and the Dsk2/dUbqln extraproteasomal polyubiquitin receptors, transgenic Drosophila lines were constructed in which the overexpression or RNA interference-mediated silencing of these receptors can be induced. Flies overexpressing Flag-p54 were viable and fertile, without any detectable morphological abnormalities, although detectable accumulation of polyubiquitylated proteins demonstrated a certain level of proteolytic disturbance. Flag-p54 was assembled into the 26S proteasome and could fully complement the lethal phenotype of a p54 null mutant Drosophila line. The overexpression of Dsk2 caused severe morphological abnormalities in the late pupal stages, leading to pharate adult lethality, accompanied by a huge accumulation of highly polyubiquitylated proteins. The lethal phenotype of Dsk2 overexpression could be rescued in a double transgenic line coexpressing Flag-Dsk2 and Flag-p54. Although the double transgenic line was viable and fertile, it did not restore the proteolytic defects; the accumulation of the highly polyubiquitylated proteins was even more severe in the double transgenic line. Significant differences were found in the Dsk2-26S proteasome interaction in Drosophila melanogaster as compared with Saccharomyces cerevisiae. In yeast, Dsk2 can interact only with ΔRpn10 proteasomes and not with the wild-type one. In Drosophila, Dsk2 does not interact with Δp54 proteasomes, but the interaction can be fully restored by complementing the Δp54 deletion with Flag-p54.

Energetics of metamorphosis in Drosophila melanogaster

We measured the energetic cost of metamorphosis in the fruitfly, Drosophila melanogaster. Metabolic rates decreased rapidly in the first 24 h and remained low until shortly before eclosion, when the rates increased rapidly, thus creating a U-shaped metabolic curve. The primary fuel used during metamorphosis was lipid, which accounted for >80% of total metabolism. The total energy consumed during metamorphosis was lowest at 25 °C, compared to 18 and 29 °C, due to differences in metabolic rates and the length of pupal development. Temperature differentially affected metabolic rates during different stages of metamorphosis. Prepupal and late pupal stages exhibited typical increases in metabolic rate at high temperatures, whereas metabolic rates were independent of temperature during the first 2/3 of pupal development. We tested two hypotheses for the underlying cause of the U-shaped metabolic curve. The first hypothesis was that pupae become oxygen restricted as a result of remodeling of the larval tracheal system. We tested this hypothesis by exposing pupae to hypoxic and hyperoxic atmospheres, and by measuring lactic acid production during normoxic development. No evidence for oxygen limitation was observed. We also tested the hypothesis that the U-shaped metabolic curve follows changes in metabolically active tissue, such that the early decrease in metabolic rates reflects the histolysis of larval tissues, and the later increase in metabolic rates is associated with organogenesis and terminal differentiation of adult tissues. We assayed the activity of a mitochondrial indicator enzyme, citrate synthase, and correlated it with tissue-specific developmental events during metamorphosis. Citrate synthase activity exhibited a U-shaped curve, suggesting that the pattern of metabolic activity is related to changes in the amount of potentially active aerobic tissue.

The effect of developmental stage on the sensitivity of cell and body size to hypoxia inDrosophila melanogaster

Animals reared in hypoxic environments frequently exhibit smaller body sizes than when reared under normal atmospheric oxygen concentrations. The mechanisms responsible for this widely documented pattern of body size plasticity are poorly known. We studied the ontogeny of responses of Drosophila melanogaster adult body size to hypoxic exposure. We hypothesized that there may be critical oxygen-sensitive periods during D. melanogaster development that are primarily responsive to body size regulation. Instead, our results showed that exposure to hypoxia (an atmospheric partial pressure of oxygen of 10 kPa) during any developmental stage (embryo, larvae and pupae) leads to smaller adult size. However, short hypoxic exposures during the late larval and early pupal stages had the greatest effects on adult size. We then investigated whether the observed reductions in size induced by hypoxia at various developmental stages were the result of a decrease in cell size or cell number. Abdominal epithelial cells of flies reared continuously in hypoxia were smaller in mean diameter and were size-limited compared with cells of flies reared in normoxia. Flies reared in hypoxia during the embryonic, larval or pupal stage, or during their entire development, had smaller wing areas than flies reared in normoxia. Flies reared during the pupal stage, or throughout development in hypoxia had smaller wing cells, even after controlling for the effect of wing size. These results suggest that hypoxia effects on the body size of D. melanogaster probably occur by multiple mechanisms operating at various developmental stages.

Global expression profile of silkworm genes from larval to pupal stages: Toward a comprehensive understanding of sexual differences

Sexual dimorphism is a widespread phenomenon in many higher animals. The genes and gene networks that underlie sex differences are poorly understood. Using microarray data we analyzed sex-related differences in the global expression profiles of silkworm genes from larval to pupal stages. Sex-biased genes could be divided into three clusters. Cluster 1 contained 932 genes that showed a female-biased expression trend at first and a male-biased trend afterward. Cluster 2 included 283 male-biased genes. Cluster 3 was comprised of 497 female-biased genes that were expressed during the late pupal stage. Cluster 1 genes were found to be related closely to cuticle proteins, hormones, binding proteins, enzyme regulators, structural proteins, transcription regulators and so on. Several genes in clusters 2 and 3 were associated with spermatogenesis and oogenesis, respectively. The chromosomal distribution of sex-biased genes showed evidence of chromosomal enrichment. In particular a large number of the silkworms’ male-biased genes are located on the Z chromosome. These results provide new insights into the molecular differences that dictate sexual dimorphism in the silkworm.

Protein Crosslinking by Transglutaminase Controls Cuticle Morphogenesis in Drosophila

Transglutaminase (TG) plays important and diverse roles in mammals, such as blood coagulation and formation of the skin barrier, by catalyzing protein crosslinking. In invertebrates, TG is known to be involved in immobilization of invading pathogens at sites of injury. Here we demonstrate that Drosophila TG is an important enzyme for cuticle morphogenesis. Although TG activity was undetectable before the second instar larval stage, it dramatically increased in the third instar larval stage. RNA interference (RNAi) of the TG gene caused a pupal semi-lethal phenotype and abnormal morphology. Furthermore, TG-RNAi flies showed a significantly shorter life span than their counterparts, and approximately 90% of flies died within 30 days after eclosion. Stage-specific TG-RNAi before the third instar larval stage resulted in cuticle abnormality, but the TG-RNAi after the late pupal stage did not, indicating that TG plays a key role at or before the early pupal stage. Immediately following eclosion, acid-extractable protein from wild-type wings was nearly all converted to non-extractable protein due to wing maturation, whereas several proteins remained acid-extractable in the mature wings of TG-RNAi flies. We identified four proteins—two cuticular chitin-binding proteins, larval serum protein 2, and a putative C-type lectin—as TG substrates. RNAi of their corresponding genes caused a lethal phenotype or cuticle abnormality. Our results indicate that TG-dependent protein crosslinking in Drosophila plays a key role in cuticle morphogenesis and sclerotization.

Spermatogenic cyst and organ culture in Drosophila pseudoobscura

Spermatogenesis is a complex series of processes that involves (1) the maintenance of a renewable pool of diploid stem cells within a niche, (2) the mitotic expansion of a subpopulation of stem cells committed to the spermatogenic pathway, and (3) the differentiation of diploid cells into highly specialized, haploid spermatozoa through meiotic and post-meiotic cellular transformations. Drosophila melanogaster is a desirable model for studying spermatogenesis, as similarities exist between mammalian and fly spermatogenesis. Like mammals, flies maintain a spermatogenic stem cell niche; the steps involved in mammalian spermatogenesis are mimicked in flies, with the main difference being that fly sperm develop within cysts rather than an epithelial cell layer. Here, we report a reliable robust system for culturing whole testes and individual spermatogenic cysts obtained from mid- to late-pupal stages of Drosophila pseudoobscura. D. pseudoobscura testes can be easily distinguished in later pupal stages because of their intense red pigmentation and are easily handled because of their simple ellipsoidal morphology. Cultured cysts are comparable in length to cysts obtained from adult flies, and motility is consistently achieved in vitro. This system not only offers a method for dissecting the mechanisms involved in meiotic and post-meiotic cellular transformations, but also can be used for the study of signaling during spermatogenesis.

Maternal influence on diapause induction in Trichogramma (Hymenoptera: Trichogrammatidae): the dynamics of photosensitivity

It is known that the prepupal diapause in Trichogramma is dependent on temperature conditions of embryonic and larval development and on the photoperiodic conditions of preimaginal development of maternal females. However, the relative photosensitivity of different preimaginal stages has been never investigated. We studied the position of the photosensitive period over the preimaginal development of maternal females in T. embryophagum Htg. and T. principium Sug. et Sor. by transferring preimaginal stages between diapause-averting ‘long’ day (L : D = 20 : 4) and diapause-inducing ‘short’ day (L : D = 12 : 12). Results showed that the influence of maternal photoperiod on progeny diapause was determined during the late pupal stage (last days of preimaginal development at 20°C). During this time, the critical duration of the photoperiodic induction was extremely small: one short or long day caused almost the same effect as the permanent development under these conditions. As a whole, photoperiodic response in Trichogramma was found to be unusually rapid, labile and easily reversible which is probably explained by extremely small size and fast development of these egg parasitoids. The results of this study could be used for elaboration of optimal methods for Trichogramma mass rearing and storage and for prediction of its seasonal cycles under natural conditions.

Dynamic Filopodia Transmit Intermittent Delta-Notch Signaling to Drive Pattern Refinement during Lateral Inhibition

The organization of bristles on the Drosophila notum has long served as a popular model of robust tissue patterning. During this process, membrane-tethered Delta activates intracellular Notch signaling in neighboring epithelial cells, which inhibits Delta expression. This induces lateral inhibition, yielding a pattern in which each Delta-expressing mechanosensory organ precursor cell in the epithelium is surrounded on all sides by cells with active Notch signaling. Here, we show that conventional models of Delta-Notch signaling cannot account for bristle spacing or the gradual refinement of this pattern. Instead, the pattern refinement we observe using live imaging is dependent upon dynamic, basal actin-based filopodia and can be quantitatively reproduced by simulations of lateral inhibition incorporating Delta-Notch signaling by transient filopodial contacts between nonneighboring cells. Significantly, the intermittent signaling induced by these filopodial dynamics generates a type of structured noise that is uniquely suited to the generation of well-ordered, tissue-wide epithelial patterns.

Relationship of larval desiccation to Anopheles gambiae Giles and An. arabiensis Patton survival

Relationship of larval desiccation to Anopheles gambiae Giles and An. arabiensis Patton survival