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.

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.

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.

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.

Extracellular nutrient digestion and absorption in the insect gut.

Insects are the most abundant and diverse class of animals on the planet. One explanation for their success is their extraordinary ability to successfully consume a wide range of foods. Like all heterotrophic organisms, insects need to acquire vital nutrients from their diet. The central organ for food digestion and absorption of nutrients is the gastrointestinal tract. This organ's principal functions are mediating the efficient digestion of the diet and protecting the organism against harmful chemicals, microorganisms, and mechanical damage from the food. These functions are achieved through regional differentiation of the alimentary canal as well as highly flexible adaptations to the consumed diets, both at anatomical and molecular levels. Numerous studies describing the general gut morphology and associated digestive mechanisms of various insects exist. Nevertheless, the molecular patterns underlying digestion and nutrient uptake in insects are still poorly characterized. This review aims to provide an overview of the general strategies of extracellular macronutrient digestion and consequent nutrient absorption found among different orders of insects.

Molecular cloning and characterization of the SIFamide precursor and receptor in a hymenopteran insect, Bombus terrestris.

SIFamides (SIFa) are a family of neuropeptides that are highly conserved among arthropods. In insects, this peptide is mainly expressed in four medial interneurons in the pars intercerebralis and affects sexual behavior, sleep regulation and pupal mortality. Furthermore, an influence on the hatching rate has been observed. The first SIFa receptor (SIFR) was pharmacologically characterized in Drosophila melanogaster and is homologous to the vertebrate gonadotropin-inhibitory hormone (GnIH) receptor (NPFFR). In this study, we pharmacologically characterized the SIFR of the buff-tailed bumblebee Bombus terrestris. We demonstrated an intracellular increase in calcium ions and cyclic AMP (cAMP) upon ligand binding with an EC50 value in the picomolar and nanomolar range, respectively. In addition, we studied the agonistic properties of a range of related and modified peptides. By means of quantitative real time PCR (qPCR), we examined the relative transcript levels of Bomte-SIFa and Bomte-SIFR in a variety of tissues.

Analysis of Peptide Ligand Specificity of Different Insect Adipokinetic Hormone Receptors.

Adipokinetic hormone (AKH) is a highly researched insect neuropeptide that induces the mobilization of carbohydrates and lipids from the fat body at times of high physical activity, such as flight and locomotion. As a naturally occurring ligand, AKH has undergone quite a number of amino acid changes throughout evolution, and in some insect species multiple AKHs are present. AKH acts by binding to a rhodopsin-like G protein-coupled receptor, which is related to the vertebrate gonadotropin-releasing hormone receptors. In the current study, we have cloned AKH receptors (AKHRs) from seven different species, covering a wide phylogenetic range of insect orders: the fruit fly, Drosophila melanogaster, and the yellow fever mosquito, Aedes aegypti (Diptera); the red flour beetle, Tribolium castaneum, and the large pine weevil, Hylobius abietis (Coleoptera); the honeybee, Apis mellifera (Hymenoptera); the pea aphid, Acyrthosiphon pisum (Hemiptera); and the desert locust, Schistocerca gregaria (Orthoptera). The agonistic activity of different insect AKHs, including the respective endogenous AKHs, at these receptors was tested with a bioluminescence-based assay in Chinese hamster ovary cells. All receptors were activated by their endogenous ligand in the nanomolar range. Based on our data, we can refute the previously formulated hypothesis that a functional AKH signaling system is absent in the beneficial species, Apis mellifera. Furthermore, our data also suggest that some of the investigated AKH receptors, such as the mosquito AKHR, are more selective for the endogenous (conspecific) ligand, while others, such as the locust AKHR, are more promiscuous and can be activated by AKHs from many other insects. This information will be of high importance when further analyzing the potential use of AKHRs as targets for developing novel pest control agents.

Insulin/IGF signaling in Drosophila and other insects: factors that regulate production, release and post-release action of the insulin-like peptides.

Insulin, insulin-like growth factors (IGFs) and insulin-like peptides (ILPs) are important regulators of metabolism, growth, reproduction and lifespan, and mechanisms of insulin/IGF signaling (IIS) have been well conserved over evolution. In insects, between one and 38 ILPs have been identified in each species. Relatively few insect species have been investigated in depth with respect to ILP functions, and therefore we focus mainly on the well-studied fruitfly Drosophila melanogaster. In Drosophila eight ILPs (DILP1-8), but only two receptors (dInR and Lgr3) are known. DILP2, 3 and 5 are produced by a set of neurosecretory cells (IPCs) in the brain and their biosynthesis and release are controlled by a number of mechanisms differing between larvae and adults. Adult IPCs display cell-autonomous sensing of circulating glucose, coupled to evolutionarily conserved mechanisms for DILP release. The glucose-mediated DILP secretion is modulated by neurotransmitters and neuropeptides, as well as by factors released from the intestine and adipocytes. Larval IPCs, however, are indirectly regulated by glucose-sensing endocrine cells producing adipokinetic hormone, or by circulating factors from the intestine and fat body. Furthermore, IIS is situated within a complex physiological regulatory network that also encompasses the lipophilic hormones, 20-hydroxyecdysone and juvenile hormone. After release from IPCs, the ILP action can be modulated by circulating proteins that act either as protective carriers (binding proteins), or competitive inhibitors. Some of these proteins appear to have additional functions that are independent of ILPs. Taken together, the signaling with multiple ILPs is under complex control, ensuring tightly regulated IIS in the organism.

Critical role for protein kinase A in the acquisition of gregarious behavior in the desert locust.

The mechanisms that integrate genetic and environmental information to coordinate the expression of complex phenotypes are little understood. We investigated the role of two protein kinases (PKs) in the population density-dependent transition to gregarious behavior that underlies swarm formation in desert locusts: the foraging gene product, a cGMP-dependent PK (PKG) implicated in switching between alternative group-related behaviors in several animal species; and cAMP-dependent PK (PKA), a signal transduction protein with a preeminent role in different forms of learning. Solitarious locusts acquire key behavioral characters of the swarming gregarious phase within just 1 to 4 h of forced crowding. Injecting the PKA inhibitor KT5720 before crowding prevented this transition, whereas injecting KT5823, an inhibitor of PKG, did not. Neither drug altered the behavior of long-term gregarious locusts. RNAi against foraging effectively reduced its expression in the central nervous system, but this did not prevent gregarization upon crowding. By contrast, solitarious locusts with an RNAi-induced reduction in PKA catalytic subunit C1 expression behaved less gregariously after crowding, and RNAi against the inhibitory R1 subunit promoted more extensive gregarization following a brief crowding period. A central role of PKA is congruent with the recent discovery that serotonin mediates gregarization in locusts and with findings in vertebrates that similarly implicate PKA in the capacity to cope with adverse life events. Our results show that PKA has been coopted into effecting the wide-ranging transformation from solitarious to gregarious behavior, with PKA-mediated behavioral plasticity resulting in an environmentally driven reorganization of a complex phenotype.

Silencing D. melanogaster lgr1 impairs transition from larval to pupal stage.

G protein-coupled receptors (GPCRs) play key roles in a wide diversity of physiological processes and signalling pathways. The leucine-rich repeats containing GPCRs (LGRs) are a subfamily that is well-conserved throughout most metazoan phyla and have important regulatory roles in vertebrates. Here, we report on the critical role of Drosophila melanogaster LGR1, the fruit fly homologue of the vertebrate glycoprotein hormone receptors, in development as a factor involved in the regulation of pupariation. Transcript profiling revealed that lgr1 transcripts are most abundant in third instar larvae and adult flies. The tissues displaying the highest transcript levels were the hindgut, the rectum and the salivary glands. Knockdown using RNA interference (RNAi) demonstrated that white pupa formation was severely suppressed in D. melanogaster lgr1 RNAi larvae. Associated with this developmental defect was a reduced ecdysteroid titer, which is in line with significantly reduced transcript levels detected for the Halloween genes shadow (sad) and spookier (spok) in the third instar lgr1 RNAi larvae compared to the control condition.

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.

Sex peptides and MIPs can activate the same G protein-coupled receptor.

In many animal species, copulation elicits a number of physiological and behavioral changes in the female partner. In Drosophila melanogaster, the main molecular effector of these physiological responses has been identified as sex peptide (SP). The sex peptide receptor (SPR) has been characterized and recently, its activation by Drosophila myoinhibiting peptides (MIPs)-in addition to SP-has been demonstrated. The myoinhibiting peptides are members of a conserved peptide family, also known as B-type allatostatins, which generally feature the C-terminal motif -WX6Wamide.

Neuropeptidergic regulation of reproduction in insects.

Successful animal reproduction depends on multiple physiological and behavioral processes that take place in a timely and orderly manner in both mating partners. It is not only necessary that all relevant processes are well coordinated, they also need to be adjusted to external factors of abiotic and biotic nature (e.g. population density, mating partner availability). Therefore, it is not surprising that several hormonal factors play a crucial role in the regulation of animal reproductive physiology. In insects (the largest class of animals on planet Earth), lipophilic hormones, such as ecdysteroids and juvenile hormones, as well as several neuropeptides take part in this complex regulation. While some peptides can affect reproduction via an indirect action (e.g. by influencing secretion of juvenile hormone), others exert their regulatory activity by directly targeting the reproductive system. In addition to insect peptides with proven activities, several others were suggested to also play a role in the regulation of reproductive physiology. Because of the long evolutionary history of many insect orders, it is not always clear to what extent functional data obtained in a given species can be extrapolated to other insect taxa. In this paper, we will review the current knowledge concerning the neuropeptidergic regulation of insect reproduction and situate it in a more general physiological context.

Characterisation of a functional allatotropin receptor in the bumblebee, Bombus terrestris (Hymenoptera, Apidae).

Allatotropins (ATs) are multifunctional neuropeptides initially isolated from the tobacco hornworm, Manduca sexta, where they were found to stimulate juvenile hormone synthesis and release from the corpora allata. ATs have been found in a wide range of insects, but appear to be absent in Drosophila. The first AT receptor (ATR) was characterised in 2008 in the lepidopteran Bombyx mori. Since then ATRs have been characterised in Coleoptera and Diptera and in 2012, an AT precursor gene was identified in hymenopteran species. ATRs show large sequence and structural similarity to vertebrate orexin receptors (OXR). Also, AT in insects and orexin in vertebrates show some overlap in functions, including modulation of feeding behaviour and reproduction. The goal of this study was to identify a functional ATR in a hymenopteran species. We used ATRs (insect sequences) and OXRs (vertebrate sequences) to search the genome of the bumblebee, Bombus terrestris. Two receptors (XP_003402490 and XP_003394933) with resemblance to ATRs and OXRs were found. Phylogenetic analysis provided the first indication that XP_003402490 was more closely related to ATRs than XP_003394933. We investigated the transcript level distribution of both receptors and the AT precursor gene by means of quantitative real-time reverse transcriptase PCR. XP_003402490 displayed a tissue distribution comparable with ATRs in other species, with high transcript levels in the male accessory glands. After pharmacological characterisation, it appeared that XP_003402490 is indeed a functional ATR. Activation of the receptor causes an increase in intracellular calcium and cyclic AMP levels with an EC50 value in the low nanomolar to picomolar range. XP_003394933 remains an orphan receptor.

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.

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.