Insect Postembryonic Development Research Articles

Crosstalk between the microbiota and insect postembryonic development.

Insect sequential development evolves from a simple molt towards complete metamorphosis. Like any multicellular host, insects interact with a complex microbiota. In this review, factors driving the microbiota dynamics were pointed out along their development. Special focus was put on tissue renewal, shift in insect ecology, and microbial interactions. Conversely, how the microbiota modulates its host development through nutrient acquisition, hormonal control, and cellular or tissue differentiation was exemplified. Such modifications might have long-term carry-over effects on insect physiology. Finally, remarkable microbe-driven control of insect behaviors along their life cycle was highlighted. Increasing knowledge of those interactions might offer new insights on how insects respond to their environment as well as perspectives on pest- or vector-control strategies.

Diversity, Form, and Postembryonic Development of Paleozoic Insects.

While Mesozoic, Paleogene, and Neogene insect faunas greatly resemble the modern one, the Paleozoic fauna provides unique insights into key innovations in insect evolution, such as the origin of wings and modifications of postembryonic development including holometaboly. Deep-divergence estimates suggest that the majority of contemporary insect orders originated in the Late Paleozoic, but these estimates reflect divergences between stem groups of each lineage rather than the later appearance of the crown groups. The fossil record shows the initial radiations of the extant hyperdiverse clades during the Early Permian, as well as the specialized fauna present before the End Permian mass extinction. This review summarizes the recent discoveries related to the documented diversity of Paleozoic hexapods, as well as current knowledge about what has actually been verified from fossil evidence as it relates to postembryonic development and the morphology of different body parts.

Knockdown of Ecdysone-Induced Protein 93F Causes Abnormal Pupae and Adults in the Eggplant Lady Beetle.

Ecdysone-induced protein 93F (E93) plays triple roles during post-embryonic development in insects whose juvenile instars are more than four. However, it only acts as a specifier of adult structures in Drosophila flies whose larval instars are fixed at three. In this study, we determined the functions of E93 in the eggplant lady beetle (Henosepilachna vigintioctopunctata), which has four larval instars. We uncovered that E93 was abundantly expressed at the prepupal and pupal stages. A precocious inhibition of the juvenile hormone signal by RNA interference (RNAi) of HvKr-h1 or HvHairy, two vital downstream developmental effectors, at the penultimate instar larval stage increased the expression of E93, Conversely, ingestion of JH by the third-instar larvae stimulated the expression of HvKr-h1 but repressed the transcription of either HvE93X1 or HvE93X2. However, disturbance of the JH signal neither drove premature metamorphosis nor caused supernumerary instars. In contrast, depletion of E93 at the third- and fourth-instar larval and prepupal stages severely impaired pupation and caused a larval-pupal mixed phenotype: pupal spines and larval scoli were simultaneously presented on the cuticle. RNAi of E93 at the pupal stage affected adult eclosion. When the beetles had suffered from a dsE93 injection at the fourth-instar larval and pupal stages, a few resultant adults emerged, with separated elytra, abnormally folded hindwings, a small body size and short appendages. Taken together, our results suggest the larval instars are fixed in H. vigintioctopunctata; E93 serves as a repressor of larval characters and a specifier of adult structures during the larval-pupal-adult transition.

RNAi-Mediated Silencing of Pgants Shows Core 1 O-Glycans Are Required for Pupation in Tribolium castaneum.

Protein glycosylation is one of the most common and most important post-translational modifications. Despite the growing knowledge on N-glycosylation, the research on O-glycosylation is lagging behind. This study investigates the importance of O-glycosylation in the post-embryonic development of insects using the red flour beetle, Tribolium castaneum, as a model. We identified 28 O-glycosylation-related genes (OGRGs) in the genome of the red flour beetle. 14 OGRGs were selected for functional analysis based on their involvement in the initial attachment of the carbohydrate in the different O-glycosylation pathways or the further elongation of the most abundant O-glycans and, in addition, showing severe RNAi-induced phenotypes in Drosophila melanogaster. The expression profile of these OGRGs was mapped throughout the developmental stages of the insect and in the different tissues of the pupa and adult. Subsequently, these genes were silenced using RNA interference (RNAi) to analyze their role in development. A broad spectrum of phenotypes was observed: from subtle effects and disrupted wing formation when silencing the genes involved in O-mannosylation, to blockage of pupation and high mortality after silencing of the genes involved in O-GalNAc and core 1 O-glycan (O-GalNAc-Gal) synthesis. RNAi experiments were also performed to assess the effects of blocking multiple pathways of O-glycosylation. However, the observed phenotypes induced by multiple RNAi were similar to those of the single gene RNAi experiments. The silencing of OGRGs often resulted in high mortality and wing phenotypes, indicating the importance of O-glycosylation for the survival of the insect and the formation of wings during the post-embryonic development of T. castaneum.

Sequential changes in the regulatory mechanism of carbohydrate digestion in larvae of the silkworm, Bombyx mori.

Nutritional signals strictly control post-embryonic development in insects. Dietary carbohydrates are hydrolyzed to monosaccharides in the gut and then transported into the hemolymph. These monosaccharides in hemolymph are rapidly taken up by tissues and utilized in glycolysis, the pentose phosphate shunt, and glycogen or trehalose synthesis. These metabolic pathways are essential for nutrient metabolism; therefore, the control of carbohydrate digestion is indispensable for maintaining energy supply during development. Carbohydrate digestion was believed to be controlled by dietary mechanisms. We previously reported that hormonal and developmental controls participate in the regulation of carbohydrate digestion during larval-pupal metamorphosis. However, it is unclear whether this regulatory mechanism also works during larval-larval molting and inter-molt feeding period. Here, we show that control mechanisms of the carbohydrate digestion show sequential changes that are controlled by different mechanisms. In the penultimate larval instar, carbohydrate hydrolysis activity changed depending on developmental progress and dietary state. Maltose- and sucrose-hydrolysis activity were suppressed by ecdysteroid, an insect steroid hormone. During the inter-molt feeding period, carbohydrate hydrolysis activities were grouped as either nutrient-sensitive or nutrient-insensitive. Although the activity in both groups was suppressed by ecdysteroid, this hormonal regulatory machinery remains in an "off-state" because ecdysteroid is scarce during the feeding period, suggesting that the carbohydrate digestion system is exclusively regulated by the dietary state during inter-molt feeding period.

Programmed cell death reshapes the central nervous system during metamorphosis in insects.

Metamorphosis is fascinating and dramatic stage of postembryonic development in insects [1]. The most prominent metamorphic changes seen in holometabolous insects involve destruction of most larval structures and concomitant generation of adult ones. Such diverse cellular events are orchestrated by ecdysone. The central nervous system (CNS) is also extensively remodeled to process new sensory inputs; to coordinate new types of locomotion; and to perform higher-order decision making [2]. Programmed cell death (PCD) is an integral part of the metamorphic development. It eliminates obsolete larval tissues and extra cells that are generated from the morphogenesis of adult tissues. In the CNS, PCD of selected neurons and glial cells as well as reshaping of persistent larval cells are essential for establishing the adult CNS. In this review, we summarize the ecdysone signaling, and then molecular and cellular events associated with PCD primarily in the metamorphosing CNS of Drosophila melanogaster.

Identification and profiling of Bactrocera dorsalis microRNAs and their potential roles in regulating the developmental transitions of egg hatching, molting, pupation and adult eclosion

MicroRNAs (miRNAs) are endogenous small noncoding RNAs (18–25 nt) that are involved in many physiological processes including development, cancer, immunity, apoptosis and host-microbe interactions through post-transcriptional regulation of gene expression. In this study, we measured the profile of small RNAs over the developmental transitions of the oriental fruit fly Bactrocera dorsalis from egg hatching, molting, and pupation to adult eclosion. We identified 250 miRNAs, including 83 known and 167 novel miRNAs, and 47 isomiRNAs. In addition, we identified the miRNAs differentially expressed over the developmental transitions. Interestingly, the miR-309 cluster, the miR-2 cluster/family and the let-7 cluster were among these differentially expressed miRNAs, suggesting a role in the regulation of egg hatching, molting and pupation/adult eclosion, respectively. Moreover, a detailed analysis of the temporal expression patterns of 14 highly expressed miRNAs in the pupal stage revealed three types of expression profiles. Furthermore, injection of a miR-100 mimic in the 3rd instar larvae resulted in a significant decrease in pupation and adult eclosion rates, whereas injection of a miR-317 antagomir resulted in a significant decrease in the pupation rate and a decrease in the pupation time, indicating that miR-100 and miR-317 are involved in the process of pupation. Finally, injection of a miR-100/miR-285 mimic or antagomir in pupae resulted in a significant decrease in the eclosion rate and a significant increase in the prevalence of a partial eclosion phenotype, implying the involvement of miR-100 and miR-285 in the process of adult eclosion. This study identified critical miRNAs involved in the transitions of this important holometabolic model and pest insect B. dorsalis from egg hatching to adult eclosion, thus providing a useful resource for exploring the regulatory role of miRNAs during insect post-embryonic development.

RNAi-Mediated Knockdown of Transcription Factor E93 in Nymphs of the Desert Locust (Schistocerca gregaria) Inhibits Adult Morphogenesis and Results in Supernumerary Juvenile Stages.

Postembryonic development of insects is coordinated by juvenile hormone (JH) together with ecdysteroids. Whereas the JH early response gene krüppel-homolog 1 (Kr-h1) plays a crucial role in the maintenance of juvenile characteristics during consecutive larval stages, the ecdysteroid-inducible early gene E93 appears to be a key factor promoting metamorphosis and adult morphogenesis. Here, we report on the developmental and molecular consequences of an RNAi-mediated knockdown of SgE93 in the desert locust, Schistocerca gregaria, a hemimetabolan species. Our experimental data show that injection of gregarious locust nymphs with a double-stranded RNA construct targeting the SgE93 transcript inhibited the process of metamorphosis and instead led to supernumerary nymphal stages. These supernumerary nymphal instars still displayed juvenile morphological features, such as a nymphal color scheme and body shape, while they reached the physical body size of the adult locusts, or even surpassed it after the next supernumerary molt. Interestingly, when compared to control locusts, the total duration of the fifth and normally final nymphal (N5) stage was shorter than normal. This appeared to correspond with temporal and quantitative changes in hemolymph ecdysteroid levels, as well as with altered expression of the rate-limiting Halloween gene, Spook (SgSpo). In addition, the levels of the ecdysone receptor (SgEcR) and retinoïd X receptor (SgRXR) transcripts were altered, indicating that silencing SgE93 affects both ecdysteroid synthesis and signaling. Upon knockdown of SgE93, a very potent upregulation of the SgKr-h1 transcript levels was observed in both head and fat body, while no significant changes were detected in the transcript levels of SgJHAMT and SgCYP15A1, the enzymes that catalyze the two final steps in JH biosynthesis. Moreover, the process of molting was disturbed in these supernumerary nymphs. While attempting ecdysis to the next stage, 50% of the N6 and all N7 nymphal instars eventually died. S. gregaria is a very harmful, swarm-forming pest species that destroys crops and threatens food security in many of the world’s poorest countries. We believe that a better knowledge of the mechanisms of postembryonic development may contribute to the discovery of novel, more selective and sustainable strategies for controlling gregarious locust populations. In this context, identification of molecular target candidates that are capable of significantly reducing the fitness of this devastating swarming pest will be of crucial importance.

Comparison of Predicted Amino Acid Sequences of Allatotropin/Allatostatin Receptors from Solitary to Eusocial Bee Species (Hymenoptera, Apoidea)

Abstract An increasingly amount of evidence supports that the evolution of eusociality is accompanies by shifts in ancient molecular and physiological pathways. The juvenile hormone, one of the most important hormones in the post-embryonic development of insects, attracts the most attention in the context of social organization. Allatoregulatory neuropeptides (Allatotropin, Allatostatin-A and Allatostatin-C) are known to regulate juvenile hormone synthesis and release in insects. In order to clarify the transitions of juvenile hormone synthesis involved in eusocial evolution, the substitutions of amino acid residues and the complexity of post-translational modifications in allatoregulatory neuropeptide receptors were characterized. Both allatotropin and allatostatin receptors are identified in all examined bee species regardless if they are solitary or eusocial. Although the amino acid sequences are highly conserved, phylogenetic results are consistent with the eusocial status. The abundance of predicted post-translational modifications correlates with social complexity except for that in allatostatin-C receptors. Even though the consequences of these specific amino acid substitutions and various post-translational modification complexity have not been studied, they likely contribute to the localizing, binding and coupling characteristics of the receptor groups.

The innovation of the final moult and the origin of insect metamorphosis.

The three modes of insect postembryonic development are ametaboly, hemimetaboly and holometaboly, the latter being considered the only significant metamorphosis mode. However, the emergence of hemimetaboly, with the genuine innovation of the final moult, represents the origin of insect metamorphosis and a necessary step in the evolution of holometaboly. Hemimetaboly derives from ametaboly and might have appeared as a consequence of wing emergence in Pterygota, in the early Devonian. In extant insects, the final moult is mainly achieved through the degeneration of the prothoracic gland (PG), after the formation of the winged and reproductively competent adult stage. Metamorphosis, including the formation of the mature wings and the degeneration of the PG, is regulated by the MEKRE93 pathway, through which juvenile hormone precludes the adult morphogenesis by repressing the expression of transcription factor E93, which triggers this change. The MEKRE93 pathway appears conserved in extant metamorphosing insects, which suggest that this pathway was operative in the Pterygota last common ancestor. We propose that the final moult, and the consequent hemimetabolan metamorphosis, is a monophyletic innovation and that the role of E93 as a promoter of wing formation and the degeneration of the PG was mechanistically crucial for their emergence. This article is part of the theme issue 'The evolution of complete metamorphosis'.

Juvenile Hormone receptor Met is essential for ovarian maturation in the Desert Locust, Schistocerca gregaria

Juvenile hormones (JH) are key endocrine regulators produced by the corpora allata (CA) of insects. Together with ecdysteroids, as well as nutritional cues, JH coordinates different aspects of insect postembryonic development and reproduction. The function of the recently characterized JH receptor, Methoprene-tolerant (Met), appears to be conserved in different processes regulated by JH. However, its functional interactions with other hormonal signalling pathways seem highly dependent on the feeding habits and on the developmental and reproductive strategies employed by the insect species investigated. Here we report on the effects of RNA interference (RNAi) mediated SgMet knockdown during the first gonadotrophic cycle in female desert locusts (Schistocerca gregaria). This voracious, phytophagous pest species can form migrating swarms that devastate field crops and harvests in several of the world’s poorest countries. A better knowledge of the JH signalling pathway may contribute to the development of novel, more target-specific insecticides to combat this very harmful swarming pest. Using RNAi, we show that the JH receptor Met is essential for ovarian maturation, vitellogenesis and associated ecdysteroid biosynthesis in adult female S. gregaria. Interestingly, knockdown of SgMet also resulted in a significant decrease of insulin-related peptide (SgIRP) and increase of neuroparsin (SgNP) 3 and 4 transcript levels in the fat body, illustrating the existence of an intricate regulatory interplay between different hormonal factors. In addition, SgMet knockdown in females resulted in delayed display of copulation behaviour with virgin males, when compared with dsGFP injected control animals. Moreover, we observed an incapacity of adult dsSgMet injected female locusts to oviposit during the time of the experimental setup. As such, SgMet is an essential gene playing crucial roles in the endocrine communication necessary for successful reproduction of the desert locust.

Effects of DEHP on post-embryonic development, nuclear receptor expression, metabolite and ecdysteroid concentrations of the moth Spodoptera littoralis

Di (2-ethylhexyl) phthalate (DEHP) is recognized in vertebrates as an Endocrine Disrupting Chemical (EDC). DEHP can alter steroid hormones production, development, reproduction and behavior in vertebrates. Only few studies investigated DEHP effects on insects. However, some recent studies on aquatic insects showed that DEHP could also act as an EDC by interfering with the signaling pathways of ecdysteroids, the main hormones involved in the control of insect post-embryonic development and physiology. The aim of the study was to investigate (1) the fate of DEHP within a terrestrial insect species by exposing larvae to food containing a wide range of DEHP concentrations and (2) the effects of this chemical on their post-embryonic development and metamorphosis, by using a multi-level approach. DEHP was shown to be present both in larvae and resulting stages, with higher concentrations in chrysalises and adults than in larvae. DEHP concentrations also decreased at the end of the last larval instar, suggesting the metabolic transformation or excretion of this chemical during this time. Only the two highest DEHP doses induced higher insect mortality, whereas low and intermediate concentrations increased larval food consumption without affecting body weight. Metabolic profiles showed that in control insects, the last three days before metamorphosis correspond to a metabolic transition, but with time-dependent changes in treated insects. Interestingly, DEHP treatments also alter both hemolymphatic ecdysteroid titers and expression levels of ecdysteroid response genes. These results confirm that DEHP can alter insect post-embryonic development and metamorphosis, by interfering with ecdysteroid pathways.

Remodeling of the juvenile hormone pathway through caste-biased gene expression and positive selection along a gradient of termite eusociality.

The evolution of division of labor between sterile and fertile individuals represents one of the major transitions in biological complexity. A fascinating gradient in eusociality evolved among the ancient hemimetabolous insects, ranging from noneusocial cockroaches through the primitively social lower termites-where workers retain the ability to reproduce-to the higher termites, characterized by lifetime commitment to worker sterility. Juvenile hormone (JH) is a prime candidate for the regulation of reproductive division of labor in termites, as it plays a key role in insect postembryonic development and reproduction. We compared the expression of JH pathway genes between workers and queens in two lower termites (Zootermopsis nevadensis and Cryptotermes secundus) and a higher termite (Macrotermes natalensis) to that of analogous nymphs and adult females of the noneusocial cockroach Blattella germanica. JH biosynthesis and metabolism genes ranged from reproductive female-biased expression in the cockroach to predominantly worker-biased expression in the lower termites. Remarkably, the expression profile of JH pathway genes sets the higher termite apart from the two lower termites, as well as the cockroach, indicating that JH signaling has undergone major changes in this eusocial termite. These changes go beyond mere shifts in gene expression between the different castes, as we find evidence for positive selection in several termite JH pathway genes. Thus, remodeling of the JH pathway may have played a major role in termite social evolution, representing a striking case of convergent molecular evolution between the termites and the distantly related social hymenoptera.

CREB-binding protein plays key roles in juvenile hormone action in the red flour beetle, Tribolium Castaneum

Juvenile hormones (JH) and ecdysteroids regulate many biological and metabolic processes. CREB-binding protein (CBP) is a transcriptional co-regulator with histone acetyltransferase (HAT) activity. Therefore, CBP is involved in activation of many transcription factors that regulate expression of genes associated with postembryonic development in insects. However, the function of CBP in JH action in insects is not well understood. Hence, we studied the role of CBP in JH action in the red flour beetle, Tribolium castaneum and the Tribolium cell line. CBP knockdown caused a decrease in JH induction of genes, Kr-h1, 4EBP and G13402 in T. castaneum larvae, adults and TcA cells whereas, Trichostatin A [TSA, a histone deacetylase (HDAC) inhibitor] induced the expression of these JH-response genes. Western blot analysis with specific antibodies revealed the requirement of CBP for the acetylation of H3K18 and H3K27 in both T. castaneum and TcA cells. Chromatin immunoprecipitation (Chip) assays showed the importance of CBP-mediated acetylation of H3K27 for JH induction of Kr-h1, 4EBP, and G13402 in TcA cells. These data suggest that CBP plays an important role in JH action in the model insect, T.castaneum.

Multiple functions of CREB-binding protein during postembryonic development: identification of target genes

BackgroundJuvenile hormones (JH) and ecdysteroids control postembryonic development in insects. They serve as valuable targets for pest management. Hence, understanding the molecular mechanisms of their action is of crucial importance. CREB-binding protein (CBP) is a universal transcriptional co-regulator. It controls the expression of several genes including those from hormone signaling pathways through co-activation of many transcription factors. However, the role of CBP during postembryonic development in insects is not well understood. Therefore, we have studied the role of CBP in postembryonic development in Tribolium, a model coleopteran insect.ResultsCBP is ubiquitously expressed in the red flour beetle, Tribolium castaneum. RNA interference (RNAi) mediated knockdown of CBP resulted in a decrease in JH induction of Kr-h1 gene expression in Tribolium larvae and led to a block in their development. Moreover, the injection of CBP double-stranded RNA (dsRNA) showed lethal phenotypes within 8 days of injection. RNA-seq and subsequent differential gene expression analysis identified CBP target genes in Tribolium. Knockdown of CBP caused a decrease in the expression of 1306 genes coding for transcription factors and other proteins associated with growth and development. Depletion of CBP impaired the expression of several JH response genes (e.g., Kr-h1, Hairy, early trypsin) and ecdysone response genes (EcR, E74, E75, and broad complex). Further, GO enrichment analyses of the downregulated genes showed enrichment in different functions including developmental processes, pigmentation, anatomical structure development, regulation of biological and cellular processes, etc.ConclusionThese data suggest diverse but crucial roles for CBP during postembryonic development in the coleopteran model insect, Tribolium. It can serve as a target for RNAi mediated pest management of this stored product pest.

Refinement of ectopic protein expression through the GAL4/UAS system in Bombyx mori: application to behavioral and developmental studies

Silkmoth, Bombyx mori, is one of the important model insects in which transgenic techniques and the GAL4/UAS system are applicable. However, due to cytotoxicity and low transactivation activity of GAL4, effectiveness of the GAL4/UAS system and its application in B. mori are still limited. In the present study, we refined the previously reported UAS vector by exploiting transcriptional and translational enhancers, and achieved 200-fold enhancement of reporter GFP fluorescence in the GAL4/UAS system. Enhanced protein expression of membrane-targeted GFP and calcium indicator protein (GCaMP5G) drastically improved visualization of fine neurite structures and neural activity, respectively. Also, with the refined system, we generated a transgenic strain that expresses tetanus toxin light chain (TeTxLC), which blocks synaptic transmission, under the control of GAL4. Ectopic TeTxLC expression in the sex pheromone receptor neurons inhibited male courtship behavior, proving effectiveness of TeTxLC on loss-of-function analyses of neural circuits. In addition, suppression of prothoracicotropic hormone (PTTH) or insulin-like peptide (bombyxin) secretion impaired developmental timing and growth rate, respectively. Furthermore, we revealed that larval growth is sex-differentially regulated by these peptide hormones. The present study provides important technical underpinnings of transgenic approaches in silkmoths and insights into mechanisms of postembryonic development in insects.

Dusky-like is required for epidermal pigmentation and metamorphosis in Tribolium castaneum.

Dusky-like (Dyl) is associated with the morphogenesis of embryonic denticle, adult sensory bristle and wing hair in Drosophila melanogaster. And whether Dyl involved in insect post-embryonic development and its signal transduction are poorly understood. Here, phylogenetic analysis revealed that dyl displayed one-to-one orthologous relationship among insects. In Tribolium castaneum, dyl is abundantly expressed at the late embryonic stage. Tissue-specific expression analysis at the late adult stage illustrated high expression of dyl in the fat body and ovary. Knockdown of dyl resulted in the defects in larval epidermal pigmentation and completely blocked the transitions from larval to pupal and pupal to adult stages of T. castaneum. We further discovered that dyl RNAi phenotypes were phenocopied by blimp-1 or shavenbaby (svb) silencing, and dyl was positively regulated by blimp-1 through svb in T. castaneum. These results suggest that Dyl functions downstream of Blimp-1 through Svb for larval epidermal pigmentation and metamorphosis. Moreover, ftz-f1 was down-regulated after RNA interference (RNAi) suppressing any of those three genes, indicating that Ftz-f1 works downstream of Dyl to mediate the effects of Blimp-1, Svb and Dyl on metamorphosis in T. castaneum. This study provides valuable insights into functions and signaling pathway of insect Dyl.

Molecular cloning and characterization of the putative Halloween gene Phantom from the small brown planthopper Laodelphax striatellus.

Ecdysteroid hormone 20-hydroxyecdysone plays fundamental roles in insect postembryonic development and reproduction. Several cytochrome P450 mono-oxygenases (CYPs), encoded by the Halloween genes, have been documented to be involved in ecdysteroidogenesis in representative insects in Diptera, Lepidoptera and Orthoptera. Here the putative Halloween gene Phantom (Phm, cyp306a1) from a hemipteran insect species, the small brown planthopper Laodelphax striatellus, was cloned. LsPHM shows five insect conserved P450 motifs, that is, Helix-C, Helix-I, Helix-K, PERF and heme-binding motifs. Temporal and spatial expression patterns of LsPhm were evaluated by quantitative polymerase chain reaction. Through the fourth-instar and the early fifth-instar stages, LsPhm showed two expression peaks in day 2 and days 4-5 fourth-instar nymphs, and three troughs in day 1 and 3 fourth instars and day 1 fifth instars. On day 5 of the fourth-instar nymphs, LsPhm clearly had a high transcript level in the thorax where the prothoracic glands were located. Dietary introduction of double-stranded RNA (dsRNA) of LsPhm at the nymph stage successfully knocked down the target gene, decreased expression level of ecdysone receptor (LsEcR) gene and caused a higher nymphal mortality rate and delayed development. Ingestion of 20-hydroxyecdysone on LsPhm-dsRNA-exposed nymphs did not increase LsPhm expression level, but almost completely rescued the LsEcR mRNA level, and relieved the negative effects on survival and development. Thus, our data suggest that the putative LsPhm encodes a functional 25-hydroxylase that catalyzes the biosynthesis of ecdysteroids in L. striatellus.

The putative Halloween gene phantom involved in ecdysteroidogenesis in the white-backed planthopper Sogatella furcifera.

Postembryonic development of insects is highly dependent on ecdysteroid hormones ecdysone (E) and 20-hydroxyecdysone (20E). A cytochrome P450 monooxygenase CYP306A1, the product of the Halloween gene phantom (phm), is involved in the ecdysteroidogenesis in representative insects in Diptera, Lepidoptera and Orthoptera. In the present paper, Sfphm was cloned from a hemipteran insect species, the white-backed planthopper Sogatella furcifera. SfPHM has five insect conserved P450 motifs, i.e., Helix-C, Helix-I, Helix-K, PERF and heme-binding motifs. Temporal and spatial expression patterns of Sfphm were evaluated by q-PCR. Sfphm showed three expression peaks in late second-, third- and fourth-instar stages. In contrast, the expression levels were lower and formed three troughs in the newly-molted second-, third- and fourth-instar nymphs. The relative 20E levels exhibited similar temporal patterns to Sfphm expression levels. On day 3 of the fourth-instar nymphs, Sfphm clearly had a high transcript level in the thorax where PGs were located. Dietary introduction of double-stranded RNA of Sfphm into the second instars successfully knocked down the target gene, and greatly reduced 20E level and ecdysone receptor (EcR) expression level. Moreover, knockdown of Sfphm caused lethality and slowed down ecdysis during nymphal stages. Furthermore, ingestion of 20-hydroxyecdysone did not alter Sfphm expression level, but almost completely rescued SfEcR expression level, and relieved the negative effects on nymphal survival and ecdysis in Sfphm-dsRNA-exposed planthoppers. Thus, our results suggest that Sfphm plays a critical role in ecdysteroidogenesis in S. furcifera.

RNA interference‐mediated silencing of a Halloween gene spookier affects nymph performance in the small brown planthopper Laodelphax striatellus

Post-embryonic development of insects is highly dependent on ecdysteroid hormone 20-hydroxyecdysone. Halloween gene spookier (spok, cyp307a2) has been documented to be involved in ecdysteroidogenesis in Drosophila melanogaster and Bombyx mori. We describe here the cloning and characterization of Halloween gene spookier (Lsspok, Lscyp307a2) in the small brown planthopper Laodelphax striatellus, a hemipteran insect species. LsSPOK has three insect-conserved P450 motifs, that is, Helix-K, PERF motif and heme-binding domain. Temporal and spatial expression patterns of Lsspok were evaluated by quantitative polymerase chain reaction. Through the fouth-instar and the early fifth-instar stages, Lsspok showed two expression peaks in the second- and fifth-day fourth-instar nymphs, and two troughs in the first-day fourth and fifth instars. On day 5 of the fourth-instar nymphs, Lsspok clearly had a high transcript level in the thorax where prothoracic glands were located. Dietary introduction of double-stranded RNA of Lsspok in the nymph stage successfully knocked down the target gene, decreased expression level of ecdysone receptor (LsEcR) gene, caused nymphal lethality and delayed development. Ingestion of 20-hydroxyecdysone in Lsspok-dsRNA-exposed nymphs did not increase Lsspok expression level, but almost completely rescued the LsEcR mRNA level and relieved the negative effects on survival and development. Thus, our data suggest that the ecdysteroidogenic pathway is conserved in insects and LsSPOK is responsible for specific steps in ecdysteroidogenesis in L. striatellus.