Insects In General Research Articles

Integrated Biological Control Using a Mixture of Two Entomopathogenic Bacteria, Bacillus thuringiensis and Xenorhabdus hominickii, against Spodoptera exigua and Other Congeners.

Simple SummaryAn entomopathogen, Bacillus thuringiensis (Bt), has been used to control insect pests. On the other hand, insect immune responses defend the bacterial pathogenicity. An idea to enhance the Bt virulence was to inhibit insect immune defense using immunosuppressant. Secondary metabolites from another entomopathogen, Xenorhabdus hominickii, are known to inhibit the insect immune responses, but they alone give little virulence against insects. The addition of the bacterial culture broth containing the secondary metabolites to Bt spores significantly enhanced the Bt virulence in laboratory and field trials. This study demonstrates an integrated biological control by ideally combining two entomopathogenic bacteria.Insect immunity defends against the virulence of various entomopathogens, including Bacillus thuringiensis (Bt). This study tested a hypothesis that any suppression of immune responses enhances Bt virulence. In a previous study, the entomopathogenic bacterium, Xenorhabdus hominickii (Xh), was shown to produce secondary metabolites to suppress insect immune responses. Indeed, the addition of Xh culture broth (XhE) significantly enhanced the insecticidal activity of Bt against S. exigua. To analyze the virulence enhanced by the addition of Xh metabolites, four bacterial secondary metabolites were individually added to the Bt treatment. Each metabolite significantly enhanced the Bt insecticidal activity, along with significant suppression of the induced immune responses. A bacterial mixture was prepared by adding freeze-dried XhE to Bt spores, and the optimal mixture ratio to kill the insects was determined. The formulated bacterial mixture was applied to S. exigua larvae infesting Welsh onions in a greenhouse and showed enhanced control efficacy compared to Bt alone. The bacterial mixture was also effective in controlling other Spodopteran species such as S. litura and S. frugiperda but not other insect genera or orders. This suggests that Bt+XhE can effectively control Spodoptera-associated pests by suppressing the immune defenses.

First record of two invasive species of Crypticerya (Hemiptera: Monophlebidae) causing outbreaks in urban green areas of Guayas Province, Ecuador

The scale insect genus Crypticerya Cockerell, 1895 (Hemiptera: Coccomorpha: Monophlebidae: Iceryini) is mainly distributed in the New World and includes numerous pest species despite being a relatively small group. Ovisacs have longitudinal flutes or grooves in those species that produce them, hence their common name “fluted scales”.” In the present study, Crypticerya multicicatrices Kondo & Unruh and Crypticerya genistae (Hempel) are reported for the first time from Ecuador. The two species of fluted scales were found to cause outbreaks in urban green areas in Guayas province on the Ecuadorian coast, attacking native and alien plants. Eighty-five and eleven new host plant species were recorded for C. multicicatrices and C. genistae, respectively. Both invasive fluted scales prefer hosts in the family Fabaceae. No significant differences in the infestation levels of C. multicicatrices and C. genistae were found between native and alien host-plant species. In general, C. multicicatrices prefers trees as hosts, whereas C. genistae prefers herbaceous plants and shrubs. Both species are considered serious pests in urban green areas, associated with sooty molds, yellowing of leaves, and, in some cases, tree dieback. Management strategies to control the fluted scales are discussed briefly.

Identification and Validation of Reference Genes for Expression Analysis Using qRT-PCR in Cimex hemipterus (Hemiptera: Cimicidae).

The tropical bed bug, Cimex hemipterus (F.) (Hemiptera: Cimicidae) is an important public-health pest that feeds on the blood of humans and some other animals. To explore the function of the target genes of C. hemipterus, it is essential to select suitable reference genes for the accurate quantification of gene expression. Here, we selected 10 frequently used reference genes in insects and evaluated their stability in C. hemipterus under various biotic (developmental stage, sex, and tissue) and abiotic (gas stimulation and temperature) conditions through RefFinder (which integrates four computational programs: geNorm, NormFinder, BestKeeper, and ∆Ct). Results indicate that the optimal combination of reference genes for each experimental condition was as follows: RPL8 and EF1α for the developmental stage (eggs, early instar nymphs, late instar nymphs, and adults), RPL8 and RPS16 for adult sex, RPL8 and RPL11 for adult tissue (head, thorax, abdomen, and legs), RPL8 and β-tubulin for gas stimulation (air and carbon dioxide), and RPL8 and NADH for temperature (0, 5, 17, 30, and 37 °C). Finally, the expression pattern of the HSP70 and GR21 genes were analyzed, and the results highlight the importance of appropriate reference-gene selection. Our results provide a comprehensive list of optimal reference genes from C. hemipterus for the first time, which will contribute to accurately analyzing the expression of target genes.

Revision of the Chinese species of Andropromachus Carl, 1913 (Phasmatodea, Lonchodidae, Necrosciinae).

A new species Andropromachus ynau sp. nov.. is described and the Chinese species of the moss-like stick insect genus Andropromachus are reviewed. An updated key to the known species of this genus is provided. Types of the new species are deposited in Yunnan Agricultural University (YNAU).

What Goes in Must Come Out? The Metabolic Profile of Plants and Caterpillars, Frass, And Adults of Asota (Erebidae: Aganainae) Feeding on Ficus (Moraceae) in New Guinea.

Insect herbivores have evolved a broad spectrum of adaptations in response to the diversity of chemical defences employed by plants. Here we focus on two species of New Guinean Asota and determine how these specialist moths deal with the leaf alkaloids of their fig (Ficus)hosts. As each focal Asota species is restricted to one of three chemically distinct species of Ficus, we also test whether these specialized interactions lead to similar alkaloid profiles in both Asota species. We reared Asota caterpillars on their respective Ficus hosts in natural conditions and analyzed the alkaloid profiles of leaf, frass, caterpillar, and adult moth samples using UHPLC-MS/MS analyses. We identified 43 alkaloids in our samples. Leaf alkaloids showed various fates. Some were excreted in frass or found in caterpillars and adult moths. We also found two apparently novel indole alkaloids-likely synthesized de novo by the moths or their microbiota-in both caterpillar and adult tissue but not in leaves or frass. Overall, alkaloids unique or largely restricted to insect tissue were shared across moth species despite feeding on different hosts. This indicates that a limited number of plant compounds have a direct ecological function that is conserved among the studied species. Our results provide evidence for the importance of phytochemistry and metabolic strategies in the formation of plant-insect interactions and food webs in general. Furthermore, we provide a new potential example of insects acquiring chemicals for their benefit in an ecologically relevant insect genus.

The IMD pathway in Hemipteran: A comparative analysis and discussion

The evolutionary patterns of the genes in the IMD pathway in hemipterans were characterized and compared. The hemipteran insects were clustered into two groups. One group that encompasses whitefly, plant lice, and scale insect partially lacks the IMD pathway and all antimicrobial peptide (AMP) genes, with the vast majority of IMD pathway and all AMP genes being absent in aphids. The reasons for the absence of the IMD pathway and AMP genes in hemipterans were analyzed based on aphids, in terms of fitness costs. In case of limited resources, aphids have to make a trade-off between the necessary costs such as clean food sources, the essential amino acids supplied by primary bacterial symbionts for survival, nutrients and/or protections against stress provided by secondary symbionts, and the high reproductive capacity, and the costs that do not increase the fitness. Obviously, aphids have to abandon the strong immune system, especially the AMPs and IMD pathway which is mainly against Gram-negative bacteria. The common ground shared with aphids may be the reason for the absence of the IMD pathway and AMP genes in other hemipteran insects.

Application progress of plant-mediated RNAi in pest control.

RNA interference (RNAi)-based biopesticides are novel biologic products, developed using RNAi principles. They are engineered to target genes of agricultural diseases, insects, and weeds, interfering with their target gene expression so as to hinder their growth and alleviate their damaging effects on crops. RNAi-based biopesticides are broadly classified into resistant plant-based plant-incorporated protectants (PIPs) and non-plant-incorporated protectants. PIP RNAi-based biopesticides are novel biopesticides that combine the advantages of RNAi and resistant transgenic crops. Such RNAi-based biopesticides are developed through nuclear or plastid transformation to breed resistant plants, i.e., dsRNA-expressing transgenic plants. The dsRNA of target genes is expressed in the plant cell, with pest and disease control being achieved through plant-target organism interactions. Here, we review the action mechanism and strategies of RNAi for pest management, the development of RNAi-based transgenic plant, and the current status and advantages of deploying these products for pest control, as well as the future research directions and problems in production and commercialization. Overall, this study aims to elucidate the current development status of RNAi-based biopesticides and provide guidelines for future research.

Characterization and expression profiling of G protein-coupled receptors (GPCRs) in Spodoptera litura (Lepidoptera: Noctuidae)

Spodoptera litura is a highly destructive omnivorous pest, and they caused serious damage to various crops. G protein-coupled receptors (GPCRs) mediate dozens of physiological processes including reproduction, development, life span and behaviors, but the information of these receptors has been lacking in S. litura. Here, we methodically identified 122 GPCRs in S. litura and made an assay of their expression patterns in different tissues. Comparing the identified GPCRs with homologous genes of other insects, it is obvious that the subfamily A2 (biogenic amine receptors) and the subfamily A3 (neuropeptide and protein hormone receptors) of S. litura have expanded to a certain extent, which may be related to the omnivorous nature and drought environment resistance of S. litura. Besides, the large Methuselah (Mth)/Methuselah-like (Mthl) subfamily of S. litura may be involved in many physiological functions such as longevity and stress response. Apart from duplicate receptors, the loss of parathyroid hormone receptor (PTHR) and the bride of sevenless (Boss) receptor in the lepidopteran insects may imply a new pattern of wing formation and energy metabolism in lepidopteran insects. In addition, the high expression level of GPCRs in different tissues reflects the functional diversity of GPCRs regulating. Systemic identification and initial characterization of GPCRs in S. litura provide a basis for further studies to reveal the functions of these receptors in regulating physiology and behavior.

Mosaic Evolution of Molecular Pathways for Sex Pheromone Communication in a Butterfly.

Unraveling the origin of molecular pathways underlying the evolution of adaptive traits is essential for understanding how new lineages emerge, including the relative contribution of conserved ancestral traits and newly evolved derived traits. Here, we investigated the evolutionary divergence of sex pheromone communication from moths (mostly nocturnal) to butterflies (mostly diurnal) that occurred ~119 million years ago. In moths, it is the females that typically emit pheromones to attract male mates, but in butterflies males emit pheromones that are used by females for mate choice. The molecular bases of sex pheromone communication are well understood in moths, but they have remained relatively unexplored in butterflies. We used a combination of transcriptomics, real time qPCR, and phylogenetics to identify genes involved in the different steps (i.e., production, regulation, and reception) of sex pheromone communication of the butterfly Bicyclus anynana. Our results show that the biosynthesis and reception of sex pheromones relies both on moth-specific gene families (reductases) and on more ancestral insect gene families (desaturases, olfactory receptors, odorant binding proteins). Interestingly, B. anynana appears to use what was believed to be the moth-specific neuropeptide Pheromone Biosynthesis Activating Neuropeptide (PBAN) for regulating sex pheromone production. Altogether, our results suggest that a mosaic pattern best explains how sex pheromone communication evolved in butterflies, with some molecular components derived from moths, and others conserved from more ancient insect ancestors. This is the first large-scale investigation of the genetic pathways underlying sex pheromone communication in a butterfly.

A male-specific doublesex isoform reveals an evolutionary pathway of sexual development via distinct alternative splicing mechanisms

The doublesex/mab-3 related transcription factor (Dmrt) genes regulate sexual development in metazoans. Studies of the doublesex (dsx) gene in insects, in particular Drosophila melanogaster, reveal that alternative splicing of dsx generates sex-specific Dsx isoforms underlying sexual differentiation. Such a splicing-based mechanism underlying sex-specific Dmrt function is thought to be evolved from a transcription-based mechanism used in non-insect species, but how such transition occurs during evolution is not known. Here we identified a male-specific dsx transcript (dsxM2) through intron retention (IR), in addition to previously identified dsxM and dsxF transcripts through alternative polyadenylation (APA) with mutually exclusive exons. We found that DsxM2 had similarly masculinizing function as DsxM. We also found that the IR-based mechanism generating sex-specific dsx transcripts was conserved from flies to cockroaches. Further analysis of these dsx transcripts suggested an evolutionary pathway from sexually monomorphic to sex-specific dsx via the sequential use of IR-based and APA-based alternative splicing.

REDfly: An Integrated Knowledgebase for Insect Regulatory Genomics.

Simple SummaryUnderstanding how genes are regulated is a vital area of current biological research and a crucial adjunct to ongoing efforts to sequence entire genomes. Knowing the DNA sequences responsible for gene regulation—transcriptional cis-regulatory modules (CRMs, e.g., “enhancers”) and transcription factor binding sites (TFBSs)—is important for many areas of research including interpretation and validation of data developed by large-scale genomics projects, providing training data for machine-learning CRM-discovery methods, genome annotation, modeling gene-regulatory networks, studying the evolution of gene regulation, and numerous aspects of the basic biology of transcriptional regulation. Knowledge of insect CRMs is also an important step in developing biotechnology methods for control of insect disease vectors and for eliminating pathogen transmission. The REDfly (Regulatory Element Database for Fly) database integrates all of the available insect cis-regulatory information from multiple sources to provide a comprehensive collection of known regulatory elements. In this paper, we describe REDfly’s basic contents and data model, emphasizing recently added features, and provide illustrated walk-throughs of some common search scenarios.We provide here an updated description of the REDfly (Regulatory Element Database for Fly) database of transcriptional regulatory elements, a unique resource that provides regulatory annotation for the genome of Drosophila and other insects. The genomic sequences regulating insect gene expression—transcriptional cis-regulatory modules (CRMs, e.g., “enhancers”) and transcription factor binding sites (TFBSs)—are not currently curated by any other major database resources. However, knowledge of such sequences is important, as CRMs play critical roles with respect to disease as well as normal development, phenotypic variation, and evolution. Characterized CRMs also provide useful tools for both basic and applied research, including developing methods for insect control. REDfly, which is the most detailed existing platform for metazoan regulatory-element annotation, includes over 40,000 experimentally verified CRMs and TFBSs along with their DNA sequences, their associated genes, and the expression patterns they direct. Here, we briefly describe REDfly’s contents and data model, with an emphasis on the new features implemented since 2020. We then provide an illustrated walk-through of several common REDfly search use cases.

Positive Correlation of the Gene Rearrangements and Evolutionary Rates in the Mitochondrial Genomes of Thrips (Insecta: Thysanoptera).

Simple SummaryAeolothrips, commonly known as banded thrips, is the largest genus of the family Aeolothripidae (predatory thrips). In the current study, we sequenced the mitochondrial genome (mitogenome) of the banded thrip species Aeolothrips xinjiangensis. We found a novel gene arrangement in this mitogenome that has not been reported in Thysanoptera. By comparing the gene order and rearrangement patterns, we found seven identical gene blocks and three identical rearrangement events in two mitogenomes of banded thrips. There was marked variation in the mitochondrial gene order across thrip species, with only two conserved gene blocks shared by all 14 thrips. In addition, we found a positive correlation between the degree of gene rearrangement and evolutionary rate. Our results suggested that the mitogenomes of thrips have tended to be stable since their massive rearrangement.Extensive gene rearrangement is characteristic in the mitogenomes of thrips (Thysanoptera), but the historical process giving rise to the contemporary gene rearrangement pattern remains unclear. To better understand the evolutionary processes of gene rearrangement in the mitogenomes of thrips, we sequenced the mitogenome of the banded thrip species Aeolothrips xinjiangensis. First, we found a novel mitochondrial gene order in this species. This mitogenome is 16,947 bp in length and encodes the typical 37 coding genes (13 protein-coding genes, 22 tRNA genes, and two rRNA genes) of insects. The gene arrangement was dramatically different from the putative ancestral mitogenome, with 26 genes being translocated, eight of which were inverted. Moreover, we found a novel, conserved gene block, trnC-trnY, which has not been previously reported in the mitogenomes of thrips. With this newly assembled mitogenome, we compared mitogenome sequences across Thysanoptera to assess the evolutionary processes giving rise to the current gene rearrangement pattern in thrips. Seven identical gene blocks were shared by two sequenced banded thrip mitogenomes, while the reversal of ND2 combined with TDRL events resulted in the different gene orders of these two species. In phylogenetic analysis, the monophyly of the suborders and families of Thysanoptera was well supported. Across the gene orders of 14 thrips, only two conserved gene blocks, ATP8-ATP6 and ND4-ND4L, could be found. Correlation analysis showed that the degree of gene rearrangement was positively correlated with the non-synonymous substitution rate in thrips. Our study suggests that the mitogenomes of thrips remain stable over long evolutionary timescales after massive rearrangement during early diversification.

Silencing of the Prophenoloxidase Gene BtPPO1 Increased the Ability of Acquisition and Retention of Tomato chlorosis virus by Bemisia tabaci.

Tomato chlorosis virus (ToCV) has seriously impacted tomato production around the world. ToCV is semi-persistently transmitted by the whitefly, Bemisia tabaci, which is a serious agricultural pest in the world. However, the interaction mechanism between ToCV and its whitefly vector is still poorly understood. Our previous transcriptome analysis demonstrated that the expression level of an immune-related gene, prophenoloxidase (PPO), in B. tabaci increased after ToCV acquisition, which indicates that the PPO may be involved in the interaction mechanism between the ToCV and its vector. To determine the role of the PPO in the acquisition and retention of ToCV by B. tabaci, we cloned the complete Open Reading Frames (ORF) of the BtPPOs (BtPPO1 and BtPPO2), and then structure and phylogenetic analyses were performed. BtPPOs were closely related to the PPO genes of Hemiptera insects. Spatial-temporal expression detection was qualified by using reverse transcription quantitative PCR (RT-qPCR), and this revealed that BtPPOs were expressed in all tissues and developmental stages. We found that only BtPPO1 was significantly upregulated after B. tabaci acquired ToCV for 12 and 24 h. According to the paraffin-fluorescence probe-fluorescence in situ hybridization (FISH) experiment, we verified that ToCV and BtPPO1 were co-located in the thorax of B. tabaci, which further revealed the location of their interaction. Finally, the effects of the BtPPOs on ToCV acquisition and retention by B. tabaci were determined using RNA interference (RNAi). The results showed that the RNAi of the responsive gene (BtPPO1) significantly increased the titer of ToCV in B. tabaci. These results demonstrate that BtPPO1 participates in ToCV acquisition and retention by B. tabaci.

Reference-Guided De Novo Genome Assembly of the Flour Beetle Tribolium freemani.

The flour beetle Tribolium freemani is a sibling species of the model organism and important pest Tribolium castaneum. The two species are so closely related that they can produce hybrid progeny, but the genetic basis of their differences has not been revealed. In this work, we sequenced the T. freemani genome by applying PacBio HiFi technology. Using the well-assembled T. castaneum genome as a reference, we assembled 262 Mb of the T. freemani genomic sequence and anchored it in 10 linkage groups corresponding to nine autosomes and sex chromosome X. The assembly showed 99.8% completeness of conserved insect genes, indicating a high-quality reference genome. Comparison with the T. castaneum assembly revealed that the main differences in genomic sequence between the two sibling species come from repetitive DNA, including interspersed and tandem repeats. In this work, we also provided the complete assembled mitochondrial genome of T. freemani. Although the genome assembly needs to be ameliorated in tandemly repeated regions, the first version of the T. freemani reference genome and the complete mitogenome presented here represent useful resources for comparative evolutionary studies of related species and for further basic and applied research on different biological aspects of economically important pests.

DIPA-CRISPR is a simple and accessible method for insect gene editing

SummaryCurrent approaches for insect gene editing require microinjection of materials into early embryos. This severely limits the application of gene editing to a great number of insect species, especially to those whose reproduction systems preclude access to early embryos for injection. To overcome these limitations, we report a simple and accessible method for insect gene editing, termed “direct parental” CRISPR (DIPA-CRISPR). We show that injection of Cas9 ribonucleoproteins (RNPs) into the haemocoel of adult females efficiently introduces heritable mutations in developing oocytes. Importantly, commercially available standard Cas9 protein can be directly used for DIPA-CRISPR, which makes this approach highly practical and feasible. DIPA-CRISPR enables highly efficient gene editing in the cockroaches, on which conventional approaches cannot be applied, and in the model beetle Tribolium castaneum. Due to its simplicity and accessibility, DIPA-CRISPR will greatly extend the application of gene editing technology to a wide variety of insects.

Why did only one genus of insects, Halobates, take to the high seas?

Oceans cover more than 70% of the Earth’s surface and house a dizzying array of organisms. Mammals, birds, and all manner of fish can be commonly sighted at sea, but insects, the world’s most common animals, seem to be completely absent. Appearances can deceive, however, as 5 species of the ocean skater Halobates live exclusively at the ocean surface. Discovered 200 years ago, these peppercorn-sized insects remain rather mysterious. How do they cope with life at the ocean surface, and why are they the only genus of insects to have taken to the high seas?

Resistance to RNA interference by plant‐derived double‐stranded RNAs but not plant‐derived short interfering RNAs in Helicoverpa armigera

Plant-mediated RNA interference (RNAi) has emerged as a promising technology for pest control through expression of double-stranded RNAs (dsRNAs) targeted against essential insect genes. However, little is known about the underlying molecular mechanisms and whether long dsRNA or short interfering RNAs (siRNAs) are the effective triggers of the RNAi response. Here we generated transplastomic and nuclear transgenic tobacco plants expressing dsRNA against the Helicoverpa armigera ATPaseH gene. We showed that expression of long dsRNA of HaATPaseH was at least three orders of magnitude higher in transplastomic plants than in transgenic plants. HaATPaseH-derived siRNAs are absent from transplastomic plants, while they are abundant in transgenic plants. Feeding transgenic plants to H. armigera larvae reduced gene expression of HaATPaseH and delayed growth. Surprisingly, no effect of transplastomic plants on insect growth was observed, despite efficient dsRNA expression in plastids. Furthermore, we found that dsRNA ingested by H. armigera feeding on transplastomic plants was rapidly degraded in the intestinal fluid. In contrast, siRNAs are relatively stable in the digestive system. These results suggest that plant-derived siRNAs may be more effective triggers of RNAi in Lepidoptera than dsRNAs, which will aid the optimization of the strategies for plant-mediated RNAi to pest control.

Efficient control of western flower thrips by plastid-mediated RNA interference

Plastid-mediated RNA interference (PM-RNAi) has emerged as a promising strategy for pest control. Expression from the plastid genome of stable double-stranded RNAs (dsRNAs) targeted against essential insect genes can effectively control some herbivorous beetles, but little is known about the efficacy of the transplastomic approach in other groups of pest insects, especially nonchewing insects that do not consume large amounts of leaf material. Here we have investigated the susceptibility of the western flower thrip (WFT, Frankliniella occidentalis), a notorious pest in greenhouses and open fields, to PM-RNAi. We show that WFTs ingest chloroplasts and take up plastid-expressed dsRNAs. We generated a series of transplastomic tobacco plants expressing dsRNAs and hairpin RNAs (hpRNAs) targeted against four essential WFT genes. Unexpectedly, we discovered plastid genome instability in transplastomic plants expressing hpRNAs, suggesting that dsRNA cassettes are preferable over hpRNA cassettes when designing PM-RNAi strategies. Feeding studies revealed that, unlike nuclear transgenic plants, transplastomic plants induced a potent RNAi response in WFTs, causing efficient suppression of the targeted genes and high insect mortality. Our study extends the application range of PM-RNAi technology to an important group of nonchewing insects, reveals design principles for the construction of dsRNA-expressing transplastomic plants, and provides an efficient approach to control one of the toughest insect pests in agriculture and horticulture.

Plastid Transformation of Micro-Tom Tomato with a Hemipteran Double-Stranded RNA Results in RNA Interference in Multiple Insect Species.

Plant-mediated RNA interference (RNAi) holds great promise for insect pest control, as plants can be transformed to produce double-stranded RNA (dsRNA) to selectively down-regulate insect genes essential for survival. For optimum potency, dsRNA can be produced in plant plastids, enabling the accumulation of unprocessed dsRNAs. However, the relative effectiveness of this strategy in inducing an RNAi response in insects using different feeding mechanisms is understudied. To investigate this, we first tested an in vitro-synthesized 189 bp dsRNA matching a highly conserved region of the v-ATPaseA gene from cotton mealybug (Phenacoccus solenopsis) on three insect species from two different orders that use leaf-chewing, lacerate-and-flush, or sap-sucking mechanisms to feed, and showed that the dsRNA significantly down-regulated the target gene. We then developed transplastomic Micro-tom tomato plants to produce the dsRNA in plant plastids and showed that the dsRNA is produced in leaf, flower, green fruit, red fruit, and roots, with the highest dsRNA levels found in the leaf. The plastid-produced dsRNA induced a significant gene down-regulation in insects using leaf-chewing and lacerate-and-flush feeding mechanisms, while sap-sucking insects were unaffected. Our results suggest that plastid-produced dsRNA can be used to control leaf-chewing and lacerate-and-flush feeding insects, but may not be useful for sap-sucking insects.

Taxonomic revision of Paraphasma Redtenbacher, 1906 (Phasmatodea, Pseudophasmatidae) based on phallic and external morphology.

Paraphasma Redtenbacher, 1906 is a genus of fully-winged stick insects occurring in central and northern South America. We carried out a morphology-based taxonomic revision of this genus with emphasis on the phallic organ, a structure that has been poorly explored for taxonomic purposes in Phasmatodea. Additionally, pairwise genetic distances between mitochondrial COI gene sequences were calculated for ten Paraphasma specimens representing six species. We recognize nine valid species in the genus plus one nomen dubium, Paraphasma fasciatum Gray, 1835. We redescribe Paraphasma and the species previously assigned to it, describe Paraphasma indistinctum Chiquetto-Machado sp. nov., Paraphasma sooretama Chiquetto-Machado sp. nov. and Paraphasma spinicauda Chiquetto-Machado sp. nov., and provide a key to the species in the genus. The male of Paraphasma minus Redtenbacher, 1906 is described for the first time, as well as the eggs of six species. We transfer Paraphasma amabile Redtenbacher, 1906 to Pseudophasma Kirby, 1896 (comb. nov.) and synonymize Pseudophasma xanthotaenidium Gnther, 1930 under this species (syn. nov.). In addition, Phasma perspicillaris Stoll, 1813 is removed from the synonymy of Paraphasma laterale (Fabricius, 1775) and synonymized under Parastratocles xanthomela (Olivier, 1792) (syn. nov.). The examination of the phallic organ was essential for species delimitation, as most species of Paraphasma are very similar in the external morphology of both sexes. The analysis of the COI sequences supported the species delimitation, resulting in remarkably lower pairwise distances between conspecific individuals (p-distance 2.0%) than between different species (p-distance 6.917.5%). We hope that this paper will stimulate further studies exploring the taxonomic and phylogenetic potential of the internal male genitalia of stick insects.