Untangling the gordian knot: The intertwining interactions between developmental hormone signaling and epigenetic mechanisms in insects.

Hormones control developmental and physiological processes, often by regulating the expression of multiple genes simultaneously or sequentially. Crosstalk between hormones and epigenetics is pivotal to dynamically coordinate this process. Hormonal signals can guide the addition and removal of epigenetic marks, steering gene expression. Conversely, DNA methylation, histone modifications and non-coding RNAs can modulate regional chromatin structure and accessibility and regulate the expression of numerous (hormone-related) genes. Here, we provide a review of the interplay between the classical insect hormones, ecdysteroids and juvenile hormones, and epigenetics. We summarize the mode-of-action and roles of these hormones in post-embryonic development, and provide a general overview of epigenetic mechanisms. We then highlight recent advances on the interactions between these hormonal pathways and epigenetics, and their involvement in development. Furthermore, we give an overview of several 'omics techniques employed in the field. Finally, we discuss which questions remain unanswered and possible avenues for future research.

Post-feeding transcriptomics reveals essential genes expressed in the midgut of the desert locust.

The digestive tract constitutes an important interface between an animal's internal and external environment. In insects, available gut transcriptome studies are mostly exploratory or look at changes upon infection or upon exposure to xenobiotics, mainly performed in species belonging to holometabolan orders, such as Diptera, Lepidoptera or Coleoptera. By contrast, studies focusing on gene expression changes after food uptake and during digestion are underrepresented. We have therefore compared the gene expression profiles in the midgut of the desert locust, Schistocerca gregaria, between three different time points after feeding, i.e., 24 h (no active digestion), 10 min (the initial stage of feeding), and 2 h (active food digestion). The observed gene expression profiles were consistent with the polyphagous herbivorous lifestyle of this hemimetabolan (orthopteran) species. Our study reveals the upregulation of 576 genes 2 h post-feeding. These are mostly predicted to be associated with digestive physiology, such as genes encoding putative digestive enzymes or nutrient transporters, as well as genes putatively involved in immunity or in xenobiotic metabolism. The 10 min time point represented an intermediate condition, suggesting that the S. gregaria midgut can react rapidly at the transcriptional level to the presence of food. Additionally, our study demonstrated the critical importance of two transcripts that exhibited a significant upregulation 2 h post-feeding: the vacuolar-type H(+)-ATPase and the sterol transporter Niemann-Pick 1b protein, which upon RNAi-induced knockdown resulted in a marked increase in mortality. Their vital role and accessibility via the midgut lumen may make the encoded proteins promising insecticidal target candidates, considering that the desert locust is infamous for its huge migrating swarms that can devastate the agricultural production in large areas of Northern Africa, the Middle East, and South Asia. In conclusion, the transcriptome datasets presented here will provide a useful and promising resource for studying the midgut physiology of S. gregaria, a socio-economically important pest species.

The Study of Cell-Penetrating Peptides to Deliver dsRNA and siRNA by Feeding in the Desert Locust, Schistocerca gregaria.

RNA(i) interference is a gene silencing mechanism triggered by double-stranded (ds)RNA, which promises to contribute to species-specific insect pest control strategies. The first step toward the application of RNAi as an insecticide is to enable efficient gene silencing upon dsRNA oral delivery. The desert locust, Schistocerca gregaria is a devastating agricultural pest. While this species is responsive to dsRNA delivered by intra-hemocoelic injection, it is refractory to orally delivered dsRNA. In this study, we evaluated the capacity of five cell-penetrating peptides (CPPs) to bind long dsRNA and protect it from the locust midgut environment. We then selected the CPP EB1 for further in vivo studies. EB1:dsRNA complexes failed to induce RNAi by feeding. Interestingly, we observed that intra-hemocoelic injection of small-interfering (si)RNAs does not result in a silencing response, but that this response can be obtained by injecting EB1:siRNA complexes. EB1 also protected siRNAs from midgut degradation activity. However, EB1:siRNA complexes failed as well in triggering RNAi when fed. Our findings highlight the complexity of the dsRNA/siRNA-triggered RNAi in this species and emphasize the multifactorial nature of the RNAi response in insects. Our study also stresses the importance of in vivo studies when it comes to dsRNA/siRNA delivery systems.

Identification and Profiling of a Novel Bombyx mori latent virus Variant Acutely Infecting Helicoverpa armigera and Trichoplusia ni.

Insect cell expression systems are increasingly being used in the medical industry to develop vaccines against diseases such as COVID-19. However, viral infections are common in these systems, making it necessary to thoroughly characterize the viruses present. One such virus is Bombyx mori latent virus (BmLV), which is known to be specific to Bombyx mori and to have low pathogenicity. However, there has been little research on the tropism and virulence of BmLV. In this study, we examined the genomic diversity of BmLV and identified a variant that persistently infects Trichoplusia ni-derived High Five cells. We also assessed the pathogenicity of this variant and its effects on host responses using both in vivo and in vitro systems. Our results showed that this BmLV variant causes acute infections with strong cytopathic effects in both systems. Furthermore, we characterized the RNAi-based immune response in the T. ni cell line and in Helicoverpa armigera animals by assessing the regulation of RNAi-related genes and profiling the generated viral small RNAs. Overall, our findings shed light on the prevalence and infectious properties of BmLV. We also discuss the potential impact of virus genomic diversity on experimental outcomes, which can help interpret past and future research results.

Multiplexed neuropeptide mapping in ant brains integrating microtomography and three-dimensional mass spectrometry imaging.

Neuropeptides are important regulators of animal physiology and behavior. Hitherto the gold standard for the localization of neuropeptides have been immunohistochemical methods that require the synthesis of antibody panels, while another limiting factor has been the brain's opacity for subsequent in situ light or fluorescence microscopy. To address these limitations, we explored the integration of high-resolution mass spectrometry imaging (MSI) with microtomography for a multiplexed mapping of neuropeptides in two evolutionary distant ant species, Atta sexdens and Lasius niger. For analyzing the spatial distribution of chemically diverse peptide molecules across the brain in each species, the acquisition of serial mass spectrometry images was essential. As a result, we have comparatively mapped the three-dimensional (3D) distributions of eight conserved neuropeptides throughout the brain microanatomy. We demonstrate that integrating the 3D MSI data into high-resolution anatomy models can be critical for studying organs with high plasticity such as brains of social insects. Several peptides, like the tachykinin-related peptides (TK) 1 and 4, were widely distributed in many brain areas of both ant species, whereas others, for instance myosuppressin, were restricted to specific regions only. Also, we detected differences at the species level; many peptides were identified in the optic lobe of L. niger, but only one peptide (ITG-like) was found in this region in A. sexdens. Building upon MS imaging studies on neuropeptides in invertebrate model systems, our approach leverages correlative MSI and computed microtomography for investigating fundamental neurobiological processes by visualizing the unbiased 3D neurochemistry in its complex anatomic environment.

Sexual repurposing of juvenile aposematism in locusts.

Adaptive plasticity requires an integrated suite of functional responses to environmental variation, which can include social communication across life stages. Desert locusts (Schistocerca gregaria) exhibit an extreme example of phenotypic plasticity called phase polyphenism, in which a suite of behavioral and morphological traits differ according to local population density. Male and female juveniles developing at low population densities exhibit green- or sand-colored background-matching camouflage, while at high densities they show contrasting yellow and black aposematic patterning that deters predators. The predominant background colors of these phenotypes (green/sand/yellow) all depend on expression of the carotenoid-binding "Yellow Protein" (YP). Gregarious (high-density) adults of both sexes are initially pinkish, before a YP-mediated yellowing reoccurs upon sexual maturation. Yellow color is especially prominent in gregarious males, but the reason for this difference has been unknown since phase polyphenism was first described in 1921. Here, we use RNA interference to show that gregarious male yellowing acts as an intrasexual warning signal, which forms a multimodal signal with the antiaphrodisiac pheromone phenylacetonitrile (PAN) to prevent mistaken sexual harassment from other males during scramble mating in a swarm. Socially mediated reexpression of YP thus adaptively repurposes a juvenile signal that deters predators into an adult signal that deters undesirable mates. These findings reveal a previously underappreciated sexual dimension to locust phase polyphenism, and promote locusts as a model for investigating the relative contributions of natural versus sexual selection in the evolution of phenotypic plasticity.

Knockdown of the Halloween Genes spook, shadow and shade Influences Oocyte Development, Egg Shape, Oviposition and Hatching in the Desert Locust.

Ecdysteroids are widely investigated for their role during the molting cascade in insects; however, they are also involved in the development of the female reproductive system. Ecdysteroids are synthesized from cholesterol, which is further converted via a series of enzymatic steps into the main molting hormone, 20-hydoxyecdysone. Most of these biosynthetic conversion steps involve the activity of cytochrome P450 (CYP) hydroxylases, which are encoded by the Halloween genes. Three of these genes, spook (spo), phantom (phm) and shade (shd), were previously characterized in the desert locust, Schistocerca gregaria. Based on recent sequencing data, we have now identified the sequences of disembodied (dib) and shadow (sad), for which we also analyzed spatiotemporal expression profiles using qRT-PCR. Furthermore, we investigated the possible role(s) of five different Halloween genes in the oogenesis process by means of RNA interference mediated knockdown experiments. Our results showed that depleting the expression of SchgrSpo, SchgrSad and SchgrShd had a significant impact on oocyte development, oviposition and hatching of the eggs. Moreover, the shape of the growing oocytes, as well as the deposited eggs, was very drastically altered by the experimental treatments. Consequently, it can be proposed that these three enzymes play an important role in oogenesis.

Identification and profiling of stable microRNAs in hemolymph of young and old Locusta migratoria fifth instars.

Since the discovery of the first microRNA (miRNA) in the nematode Caenorhabditis elegans, numerous novel miRNAs have been identified which can regulate presumably every biological process in a wide range of metazoan species. In accordance, several insect miRNAs have been identified and functionally characterized. While regulatory RNA pathways are traditionally described at an intracellular level, studies reporting on the presence and potential role of extracellular (small) sRNAs have been emerging in the last decade, mainly in mammalian systems. Interestingly, evidence in several species indicates the functional transfer of extracellular RNAs between donor and recipient cells, illustrating RNA-based intercellular communication. In insects, however, reports on extracellular small RNAs are emerging but the number of detailed studies is still very limited. Here, we demonstrate the presence of stable sRNAs in the hemolymph of the migratory locust, Locusta migratoria. Moreover, the levels of several extracellular miRNAs (ex-miRNAs) present in locust hemolymph differed significantly between young and old fifth nymphal instars. In addition, we performed a 'proof of principle' experiment which suggested that extracellularly delivered miRNA molecules are capable of affecting the locusts' development.

PIWI Proteins Play an Antiviral Role in Lepidopteran Cell Lines.

Insect antiviral immunity primarily relies on RNAi mechanisms. While a key role of small interfering (si)RNAs and AGO proteins has been well established in this regard, the situation for PIWI proteins and PIWI-interacting (pi)RNAs is not as clear. In the present study, we investigate whether PIWI proteins and viral piRNAs are involved in the immunity against single-stranded RNA viruses in lepidopteran cells, where two PIWIs are identified (Siwi and Ago3). Via loss- and gain-of-function studies in Bombyx mori BmN4 cells and in Trichoplusia ni High Five cells, we demonstrated an antiviral role of Siwi and Ago3. However, small RNA analysis suggests that viral piRNAs can be absent in these lepidopteran cells. Together with the current literature, our results support a functional diversification of PIWI proteins in insects.

Methods for the Cost-Effective Production of Bacteria-Derived Double-Stranded RNA for in vitro Knockdown Studies.

RNA interference (RNAi) is a highly conserved pathway for the post-transcriptional regulation of gene expression. It has become a crucial tool in life science research, with promising potential for pest-management applications. To induce an RNAi response, long double-stranded RNA (dsRNA) sequences specific to the target gene must be delivered to the cells. This dsRNA substrate is then processed to small RNA (sRNA) fragments that direct the silencing response. A major obstacle to applying this technique is the need to produce sufficiently large amounts of dsRNA in a very cost-effective manner. To overcome this issue, much attention has been given to the development and optimization of biological production systems. One such system is the E. coli HT115 strain transformed with the L4440 vector. While its effectiveness at inducing knockdowns in animals through feeding of the bacteria has been demonstrated, there is only limited knowledge on the applicability of bacteria-derived dsRNA for in vitro experiments. In this paper, we describe and compare methods for the economical (43.2 €/mg) and large-scale (mg range) production of high-quality dsRNA from the HT115 bacterial system. We transformed the bacteria with constructs targeting the Helicoverpa-specific gene Dicer2 and, as a non-endogenous control, the Green Fluorescent Protein gene (GFP). First, we compared the total RNA extraction yields of four cell-lysis treatments: heating, lysozyme digestion, sonication, and a control protocol. Second, we assessed the quality and purity of these extracted dsRNAs. Third, we compared methods for the further purification of dsRNAs from crude RNA extracts. Finally, we demonstrated the efficiency of the produced dsRNAs at inducing knockdowns in a lepidopteran cell line. The insights and results from this paper will empower researchers to conduct otherwise prohibitively expensive knockdown studies, and greatly reduce the production times of routinely or large-scale utilized dsRNA substrates.

Knockdown of ecdysone receptor in male desert locusts affects relative weight of accessory glands and mating behavior.

Locusts have been known as pests of agricultural crops for thousands of years. Recently (2018-2021) the world has faced the largest swarms of desert locusts, Schistocerca gregaria, in decades and food security in large parts of Africa and Asia was under extreme pressure. There is an urgent need for the development of highly specific bio-rational pesticides to combat these pests. However, to do so, fundamental research is needed to better understand the molecular mechanisms behind key physiological processes underpinning swarm formation, such as development and reproduction. The scope of this study is to investigate the possible role(s) of the ecdysteroid receptor in the reproductive physiology of male S. gregaria. Ecdysteroids and juvenile hormones are two important classes of insect hormones and are key regulators of post-embryonic development. Ecdysteroids are best known for their role in moulting and exert their function via a heterodimer consisting of the nuclear receptors ecdysone receptor (EcR) and retinoid-X receptor (RXR). To gain insight into the role of SgEcR and/or SgRXR in the male reproductive physiology of S. gregaria we performed RNAi-induced knockdown experiments. A knockdown of SgEcR, but not SgRXR, resulted in an increased relative weight of the male accessory glands (MAG). Furthermore, the knockdown of these genes, either in combination or separately, caused a significant delay in the onset of mating behavior. Nevertheless, the MAG appeared to mature normally and the fertility of mated males was not affected. The high transcript levels of SgEcR in the fat body, especially towards the end of sexual maturation in both males and females, represent a remarkable finding since as of yet the exact role of SgEcR in this tissue in S. gregaria is unknown. Finally, our data suggest that in some cases SgEcR and SgRXR might act independently of each other. This is supported by the fact that the spatiotemporal expression profiles of SgEcR and SgRXR do not always coincide and that knockdown of SgEcR, but not SgRXR, significantly affected the relative weight of the MAG.

Schistocerca neuropeptides - An update.

We compiled a comprehensive list of 67 precursor genes encoding neuropeptides and neuropeptide-like peptides using the Schistocerca gregaria genome and several transcriptome datasets. 11 of these 67 precursor genes have alternative transcripts, bringing the total number of S. gregaria precursors identified in this study to 81. Based on this precursor information, we used different mass spectrometry approaches to identify the putative mature, bioactive peptides processed in the nervous system of S. gregaria. The thereby generated dataset for S. gregaria confirms significant conservation of the entire neuropeptidergic gene set typical of insects and also contains precursors typical of Polyneoptera only. This is in striking contrast to the substantial losses of peptidergic systems in some holometabolous species. The neuropeptidome of S. gregaria, apart from species-specific sequences within the known range of variation, is quite similar to that of Locusta migratoria and even to that of less closely related Polyneoptera. With the S. gregaria peptidomics data presented here, we have thus generated a very useful source of information that could also be relevant for the study of other polyneopteran species.

Prothoracicostatic Activity of the Ecdysis-Regulating Neuropeptide Crustacean Cardioactive Peptide (CCAP) in the Desert Locust.

Accurate control of innate behaviors associated with developmental transitions requires functional integration of hormonal and neural signals. Insect molting is regulated by a set of neuropeptides, which trigger periodic pulses in ecdysteroid hormone titers and coordinate shedding of the old cuticle during ecdysis. In the current study, we demonstrate that crustacean cardioactive peptide (CCAP), a structurally conserved neuropeptide described to induce the ecdysis motor program, also exhibits a previously unknown prothoracicostatic activity to regulate ecdysteroid production in the desert locust, Schistocerca gregaria. We identified the locust genes encoding the CCAP precursor and three G protein-coupled receptors that are activated by CCAP with EC50 values in the (sub)nanomolar range. Spatiotemporal expression profiles of the receptors revealed expression in the prothoracic glands, the endocrine organs where ecdysteroidogenesis occurs. RNAi-mediated knockdown of CCAP precursor or receptors resulted in significantly elevated transcript levels of several Halloween genes, which encode ecdysteroid biosynthesis enzymes, and in elevated ecdysteroid levels one day prior to ecdysis. Moreover, prothoracic gland explants exhibited decreased secretion of ecdysteroids in the presence of CCAP. Our results unequivocally identify CCAP as the first prothoracicostatic peptide discovered in a hemimetabolan species and reveal the existence of an intricate interplay between CCAP signaling and ecdysteroidogenesis.

Accelerated delivery of dsRNA in lepidopteran midgut cells by a Galanthus nivalis lectin (GNA)-dsRNA-binding domain fusion protein.

Lepidopteran insects are highly refractory to oral RNA interference (RNAi). Degradation, impaired cellular uptake and intracellular transport of double-stranded RNA (dsRNA) are considered the major factors responsible for the reduced RNAi efficiency in these insects. In this study, the potential of lectins to improve dsRNA delivery and RNAi efficacy was evaluated. First, a fusion protein consisting of the Galanthus nivalis agglutinin (GNA) and a dsRNA binding domain was developed, further referred to as GNA:dsRBD (GNAF). Then, its ability to increase dsRNA uptake and transfection efficiency in lepidopteran midgut cells was evaluated, as well as its ability to protect and promote the RNAi response in the beet armyworm Spodoptera exigua. Confocal microscopy analysis showed that GNAF-complexed dsRNA was internalized faster in Choristoneura fumiferana midgut CF1 cells (1 min) compared to naked dsRNA (>1 h). The faster uptake was also correlated with an increased RNAi efficiency in these CF1 cells. In vivo feeding bioassays with GNAF-complexed dsRNA led to an increased mortality in S. exigua compared to the controls. By targeting the essential gene V-ATPase A, we observed that the mortality increased to 48% in the GNAF-dsRNA treatment compared to only 8.3% and 6.6% in the control treatments with the naked dsRNA and the GNAF, respectively.

RNAs on the Go: Extracellular Transfer in Insects with Promising Prospects for Pest Management.

RNA-mediated pathways form an important regulatory layer of myriad biological processes. In the last decade, the potential of RNA molecules to contribute to the control of agricultural pests has not been disregarded, specifically via the RNA interference (RNAi) mechanism. In fact, several proofs-of-concept have been made in this scope. Furthermore, a novel research field regarding extracellular RNAs and RNA-based intercellular/interorganismal communication is booming. In this article, we review key discoveries concerning extracellular RNAs in insects, insect RNA-based cell-to-cell communication, and plant-insect transfer of RNA. In addition, we overview the molecular mechanisms implicated in this form of communication and discuss future biotechnological prospects, namely from the insect pest-control perspective.

Crucial Role of Juvenile Hormone Receptor Components Methoprene-Tolerant and Taiman in Sexual Maturation of Adult Male Desert Locusts.

Currently (2020), Africa and Asia are experiencing the worst desert locust (Schistocerca gregaria) plague in decades. Exceptionally high rainfall in different regions caused favorable environmental conditions for very successful reproduction and population growth. To better understand the molecular mechanisms responsible for this remarkable reproductive capacity, as well as to fill existing knowledge gaps regarding the regulation of male reproductive physiology, we investigated the role of methoprene-tolerant (Scg-Met) and Taiman (Scg-Tai), responsible for transducing the juvenile hormone (JH) signal, in adult male locusts. We demonstrated that knockdown of these components by RNA interference strongly inhibits male sexual maturation, severely disrupting reproduction. This was evidenced by the inability to show mating behavior, the absence of a yellow-colored cuticle, the reduction of relative testes weight, and the drastically reduced phenylacetonitrile (PAN) pheromone levels of the treated males. We also observed a reduced relative weight, as well as relative protein content, of the male accessory glands in Scg-Met knockdown locusts. Interestingly, in these animals the size of the corpora allata (CA), the endocrine glands where JH is synthesized, was significantly increased, as well as the transcript level of JH acid methyltransferase (JHAMT), a rate-limiting enzyme in the JH biosynthesis pathway. Moreover, other endocrine pathways appeared to be affected by the knockdown, as evidenced by changes in the expression levels of the insulin-related peptide and two neuroparsins in the fat body. Our results demonstrate that JH signaling pathway components play a crucial role in male reproductive physiology, illustrating their potential as molecular targets for pest control.

Affordable Processing of Edible Orthopterans Provides a Highly Nutritive Source of Food Ingredients.

Edible orthopterans (grasshoppers, crickets, and locusts) are major delicacies, especially across sub-Saharan Africa. Their promotion as food ingredients is increasingly gaining momentum. This study evaluates the nutritional profiles of three widely consumed orthopterans: Gryllus bimaculatus, Locusta migratoria, and Schistocerca gregaria after blanching and oven-drying. All three species had high protein (65.3, 54.2, and 61.4% on a dry matter (DM) basis for G. bimaculatus, L. migratoria, and S. gregaria, respectively) and fat contents. Oleic (22.9-40.8%) and palmitic (26.1-43.0%) were the two most abundant fatty acids. All essential amino acids (in mg/100 g protein) were present, with glutamic acid (120-131), alanine (90.2-123), and leucine (82.3-84.6) being the most abundant. The minerals (in mg/100 g dry matter) potassium (796-1309) and phosphorus (697-968) were moderately high, and iron (4.60-7.31), zinc (12.7-24.9), manganese (0.40-7.15), and copper (1.20-4.86) were also observed in the samples. Vitamin B12 contents were high (0.22-1.35 µg/100 g dry matter). Our findings demonstrate that the excellent nutritional profile of the three processed insects could serve as promising alternative ingredients for improving food and nutritional security.

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.

Precocious Downregulation of Krüppel-Homolog 1 in the Migratory Locust, Locusta migratoria, Gives Rise to An Adultoid Phenotype with Accelerated Ovarian Development but Disturbed Mating and Oviposition.

Krüppel-homolog 1 (Kr-h1) is a zinc finger transcription factor maintaining the status quo in immature insect stages and promoting reproduction in adult insects through the transduction of the Juvenile Hormone (JH) signal. Knockdown studies have shown that precocious silencing of Kr-h1 in the immature stages results in the premature development of adult features. However, the molecular characteristics and reproductive potential of these premature adult insect stages are still poorly understood. Here we report on an adult-like or 'adultoid' phenotype of the migratory locust, Locusta migratoria, obtained after a premature metamorphosis induced by the silencing of LmKr-h1 in the penultimate instar. The freshly molted adultoid shows precocious development of adult features, corresponding with increased transcript levels of the adult specifier gene LmE93. Furthermore, accelerated ovarian maturation and vitellogenesis were observed in female adultoids, coinciding with elevated expression of LmCYP15A1 in corpora allata (CA) and LmKr-h1 and vitellogenin genes (LmVg) in fat body, whereas LmE93 and Methoprene-tolerant (LmMet) transcript levels decreased in fat body. In adultoid ovaries, expression of the Halloween genes, Spook (LmSpo) and Phantom (LmPhm), was elevated as well. In addition, the processes of mating and oviposition were severely disturbed in these females. L. migratoria is a well-known, swarm-forming pest insect that can destroy crops and harvests in some of the world's poorest countries. As such, a better understanding of factors that are capable of significantly reducing the reproductive potential of this pest may be of crucial importance for the development of novel locust control strategies.