Initial study and phylogenetic analysis of hard ticks (Acari: Ixodidae) in Nantong, China along the route of avian migration.

The growing concern about migratory birds potentially spreading ticks due to global warming has become a significant issue. The city of Nantong in this study is situated along the East Asia-Australasian Flyway (EAAF), with numerous wetlands serving as roosting sites for migratory birds. We conducted an investigation of hard ticks and determined the phylogenetic characteristics of tick species in this city. We utilized three different genes for our study: the mitochondrial cytochrome oxidase subunit 1 (COX1) gene, the second internal transcribed spacer (ITS2), and the mitochondrial small subunit rRNA (12 S rRNA) gene. The predominant tick species were Haemaphysalis flava (H. flava) and Haemaphysalis longicornis (H. longicornis). Additionally, specimens of Haemaphysalis campanulata (H. campanulata) and Rhipicephalus sanguineus (R. sanguineus) were collected. The H. flava specimens in this study showed a close genetic relationship with those from inland provinces of China, as well as South Korea and Japan. Furthermore, samples of H. longicornis exhibited a close genetic relationship with those from South Korea, Japan, Australia, and the USA, as well as specific provinces in China. Furthermore, R. sanguineus specimens captured in Nantong showed genetic similarities with specimens from Egypt, Nigeria, and Argentina.

Dynamics of cuticle-associated transcript profiles during moulting of the bed bug Cimexlectularius.

The bed bug Cimex lectularius is a worldwide human pest. The sequenced genome allows molecular analyses of all aspects of bed bug biology. The present work was conducted to contribute to bed bug cuticle biology. As in other insect species, the C. lectularius cuticle consists of the three horizontal layers procuticle, epicuticle and envelope. To analyse the genes needed for the establishment of the stratified cuticle, we studied the expression pattern of 42 key cuticle-related genes at the transition of the penultimate nymphal stage to adult animals when a new cuticle is formed. Based on gene expression dynamics, in simplified model, we distinguish two key events during cuticle renewal in C. lectularius. First, upon blood feeding, modulation of ecdysone signalling culminates in the transcriptional activation of the transcription factor Clec-Ftz-F1 that possibly controls the expression of 32 of the 42 genes tested. Second, timed expression of Clec-Ftz-F1 seems to depend also on the insulin signalling pathway as RNA interference against transcripts of the insulin receptor delays Clec-Ftz-F1 expression and stage transition. An important observation of our transcript survey is that genes needed for the construction of the three cuticle layers are largely expressed simultaneously. Based on these data, we hypothesise a considerable synchronous mechanism of layer formation rather than a strictly sequential one. Together, this work provides a basis for functional analyses of cuticle formation in C. lectularius.

Identification and RNAi-based function analysis of trehalase family genes in Frankliniella occidentalis (Pergande).

BACKGROUND: Insects utilize trehalases (TREs) to regulate energy metabolism and chitin biosynthesis, which are essential for their growth, development, and reproduction. TREs can therefore be used as potential targets for future insecticide development. However, the roles of TREs in Frankliniella occidentalis (Pergande), a serious widespread agricultural pest, remain unclear. RESULTS: Three TRE genes were identified in F. occidentalis and cloned, and their functions were then investigated via feeding RNA interference (RNAi) and virus-induced gene silencing (VIGS) assays. The results showed that silencing FoTRE1-1 or FoTRE1-2 significantly decreased expression levels of FoGFAT, FoPGM, FoUAP, and FoCHS, which are members of the chitin biosynthesis pathway. Silencing FoTRE1-1 or FoTRE2 significantly down-regulated FoPFK and FoPK, which are members of the energy metabolism pathway. These changes resulted in 2-fold decreases in glucose and glycogen content, 2-fold increases in trehalose content, and 1.5- to 2.0-fold decreases in chitinase activity. Furthermore, knocking down FoTRE1-1 or FoTRE1-2 resulted in deformed nymphs and pupae as a result of hindered molting. The VIGS assay for the three FoTREs revealed that FoTRE1-1 or FoTRE2 caused shortened ovarioles, and reduced egg-laying and hatching rates. CONCLUSION: The results suggest that FoTRE1-1 and FoTRE1-2 play important roles in the growth and development of F. occidentalis, while FoTRE1-1 and FoTRE2 are essential for its reproduction. These three genes could be candidate targets for RNAi-based management and control of this destructive agricultural pest. © 2024 Society of Chemical Industry.

20E biosynthesis gene CYP306A1 confers resistance to imidacloprid in the nymph stage of Bemisia tabaci by detoxification metabolism.

BACKGROUND: Difference in physiology level between the immature and mature stages of insects likely contribute to different mechanisms of insecticide resistance. It is well acknowledged that insect 20-hydroxyecdysone (20E) plays an important role in many biological processes in the immature stage, whether 20E confers insecticide resistance at this specific stage is still poorly understood. By gene cloning, reverse transcription quantitative real-time PCR, RNA interference (RNAi) and in vitro metabolism experiments, this study aimed to investigate the potential role of 20E-related genes in conferring imidacloprid (IMD) resistance in the immature stage of the whitefly Bemisia tabaci Mediterranean. RESULTS: After identification of low to moderate IMD resistance in the whitefly, we found CYP306A1 of the six 20E-related genes was overexpressed in the nymph stage of the three resistant strains compared to a laboratory reference susceptible strain, but not in the adult stage. Further exposure to IMD resulted in an increase in CYP306A1 expression in the nymph stage. These results together imply that CYP306A1 may be implicated in IMD resistance in the nymph stage of the whitefly. RNAi knockdown of CYP306A1 increased the mortality of nymphs after treatment with IMD in bioassay, suggesting a pivotal role of CYP306A1 in conferring IMD resistance in the nymph stage. Additionally, our metabolism experiments in vivo showed that the content of IMD reduced by 20% along with cytochrome P450 reductase and heterologously expressed CYP306A1, which provides additional evidence for the important function of CYP306A1 in metabolizing IMD that leads to the resistance. CONCLUSION: This study uncovers a novel function of the 20E biosynthesis gene CYP306A1 in metabolizing imidacloprid, thus contributing to such resistance in the immature stage of the insect. These findings not only advance our understanding of 20E-mediated insecticide resistance, but also provide a new target for sustainable pest control of global insect pests such as whitefly. © 2023 Society of Chemical Industry.

The loss of Halloween gene function seriously affects the development and reproduction of Diaphorina citri (Hemiptera: Liviidae) and increases its susceptibility to pesticides.

The citrus industry has suffered severe losses as a result of Huanglongbing spread by Diaphorina citri. Controlling the population of D. citri is the key to preventing and controlling the spread of Huanglongbing. Ecdysteroids are key hormones that regulate insect development and reproduction. Therefore, the Halloween gene family involved in the ecdysone synthesis of D. citri is an ideal target for controlling the population growth of this insect. In this study, we successfully cloned four Halloween genes expressed during D. citri development. Silencing of one of the four genes resulted in a significant decrease in 20E titers in nymphs and significant decreases in the developmental, survival and emergence rates. Inhibiting Halloween gene expression in adults impeded the growth of the female ovary, diminished yolk formation, lowered vitellogenin transcription levels, and hence impaired female fecundity. This showed that Halloween genes were required for D. citri development and reproduction. DcCYP315A1 and DcCYP314A1 were highly expressed when D. citri was exposed to thiamethoxam and cypermethrin, and silencing these two genes made D. citri more sensitive to these two pesticides. Inhibition of DcCYP315A1 and DcCYP314A1 expression not only significantly delayed the development and reproduction of D. citri but also increased its susceptibility to pesticides. Therefore, these two genes are more suitable as potential target genes for controlling D. citri.

Topical delivery of dsRNA in two hemipteran species: Evaluation of RNAi specificity and non-target effects.

Double-stranded (ds) RNA-based technologies could provide novel and potential tool for pest management with efficiency and specificity of action. However, before applying this technique in the field, it is necessary to identify effective delivery methods and evaluate the non-target effects that may occur. In this article, we evaluated the effectiveness of dsRNA by topical delivery on a species of great agricultural interest, Halyomorpha halys. The specificity of action of the dsRNA was also investigated in Rhodnius prolixus, an insect phylogenetically close to H. halys. Of the three investigated genes (putative ATPase N2B, ATPase, serine/threonine-protein phosphatase PP1-ß catalytic subunit, PP1, and IAP repeat-containing protein 7-B-like, IAP), IAP and ATPase were able to induce higher mortality in H. halys nymphs compared to the control, with specific concentrations for each gene targeted. However, when the same RNAs were topically delivered to both R. prolixus 2nd and 3rd instar nymphs, no gene silencing and mortality were observed. For this reason, to assess dsRNA application-mediated non-target effects, we injected both H. halys and R. prolixus specific dsRNA in R. prolixus 5th instar nymphs. When the dsRNA targeting H. halys IAP was microinjected into R. prolixus 5th instar nymphs, no mortality was observed, suggesting a strong RNAi specificity. Together, these data suggest that the topical delivery could be suitable for the dsRNA to control H. halys population. Furthermore, its specificity of action would allow treatments towards single harmful species with limited non-target effects.

Description of two new species in the Ixodes ricinus complex from the New World (Acari: Ixodidae), and redescription of Ixodes affinis Neumann, 1899.

Ixodes chacoensis n. sp. is described based on males, females, nymphs and larvae collected from vegetation, ungulates and passerine birds in northeastern Argentina. Ixodes affinis Neumann, 1899 is redescribed based on the original type specimens (females) from Leopardus pardalis, and from recently collected specimens from Costa Rica. Ixodes keiransi n. sp., previously treated as North American populations of Ixodes affinis, is described based on males and females from carnivores and ungulates from the southeastern United States. Concatenated total evidence phylogenetics based on combined DNA sequence analyses from mitochondrial genes (12SrDNA, 16SrDNA and COI) and a nuclear gene (ITS2) corroborate the recognition of these species.

SPARC plays an important role in the oviposition and nymphal development in Nilaparvata lugens Stål.

BACKGROUND: The brown planthopper (Nilaparvata lugens Stål)is a notorious rice pest in many areas of Asia. Study on the molecular mechanisms underlying its development and reproduction will provide scientific basis for effective control. SPARC (Secreted Protein, Acidic and Rich in Cysteine) is one of structural component of the extracellular matrix, which influences a diverse array of biological functions. In this study, the gene for SPARC was identified and functionally analysed from N.lugens. RESULTS: The result showed that the NlSPARC mRNA was highly expressed in fat body, hemolymph and early embryo. The mortality increased significantly when NlSPARC was downregulated after RNA interference (RNAi) in 3 ~ 4th instar nymphs. Downregulation of NlSPARC in adults significantly reduced the number of eggs and offspring, as well as the transcription level of NlSPARC in newly hatched nymphs and survival rate in progeny. The observation with microanatomy on individuals after NlSPARC RNAi showed smaller and less abundant fat body than that in control. No obvious morphological abnormalities in the nymphal development and no differences in development of internal reproductive organ were observed when compared with control. CONCLUSION: NlSPARC is required for oviposition and nymphal development mainly through regulating the tissue of fat body in N.lugens. NlSPARC could be a new candidate target for controlling the rapid propagation of N.lugens population. Our results also demonstrated that the effect of NlSPARC RNAi can transfer to the next generation in N.lugens.

The microRNAs in the antennae of Apolygus lucorum (Hemiptera: Miridae): Expression properties and targets prediction.

MicroRNAs (miRNAs) regulate gene expression and contribute to numerous physiological processes. However, little is known about the functions of miRNAs in insect chemosensation. In this study, nine small RNA libraries were constructed and sequenced from the antennae of nymphs, adult males, and adult females of Apolygus lucorum. In total, 399 (275 known and 124 novel) miRNAs were identified. miR-7-5p_1 was the most abundant miRNA. Altogether, 69,708 target genes related to biogenesis, membrane, and binding activities were predicted. In particular, 15 miRNAs targeted 16 olfactory genes. Comparing the antennae of nymphs and adult males and females, 94 miRNAs were differentially expressed. Alternatively, a subset of differentially expressed miRNAs was verified by qPCR, supporting the reliability of the sequencing results. This study provides a global miRNA transcriptome for the antennae of A. lucorum and valuable information for further investigations of the functions of miRNAs in the regulation of chemosensation.

Molecular Characterization of UDP-N-Acetylglucosamine Pyrophosphorylase and Its Role in the Growth and Development of the White-Backed Planthopper Sogatella furcifera (Hemiptera: Delphacidae).

UDP-N-acetylglucosamine pyrophosphorylase (UAP) is a key enzyme in the chitin biosynthesis pathway of insects. Here, we described the gene SfUAP in the white-backed planthopper Sogatella furcifera (Horváth) with an open reading frame of 1470 bp. Quantitative real-time polymerase chain reaction (qPCR) suggested that SfUAP exhibits a different developmental expression pattern and a higher expression after molting. The highest expression of SfUAP was observed in the integument tissues of adults, whereas head tissues showed negligible expression. RNAi-based gene silencing decreased the mRNA transcript levels in S. furcifera nymphs injected with double-stranded RNA of SfUAP. Finally, SfUAP silencing led to 84% mortality and malformed phenotypes in nymphs. Thus, our results can help better understand the role of SfUAP in S. furcifera.

Identification and profiling of Sogatella furcifera microRNAs and their potential roles in regulating the developmental transitions of nymph-adult.

Sogatella furcifera is one of the most serious insect pests that affect rice in Asia. One class of small RNAs (sRNAs; ~22 nt long) is miRNAs, which participate in various biological processes by regulating the expression of target genes in a spatiotemporal manner. However, the role of miRNAs in nymph-to-adult transition in S. furcifera remains unknown. In this study, we sequenced sRNA libraries of S. furcifera prepared from individuals at three different developmental stages (pre-moult, moulting and early adult). A total of 253 miRNAs (134 known and 119 novel) were identified, of which 12 were differentially expressed during the nymph-to-adult developmental transition. Moreover, Real time quantitative PCR (RT-qPCR) analysis revealed that all 12 miRNAs were differentially expressed among five different nymph tissues and 14 different developmental stages (first to fifth instar nymphs and 1-day-old adults). Injection of miR-2a-2 mimic/antagomir and miR-305-5p-1 mimic/antagomir into 1-day-old fifth instar nymphs significantly increased the mortality rate. In addition, a defective moulting phenotype was observed in nymphs injected with miR-2a-2 and miR-305-5p-1, suggesting that these miRNAs are involved in S. furcifera nymph-adult transition. In conclusion, these results reveal the function of critical miRNAs in S. furcifera nymph-adult transition, and also provide novel potential targets of insecticides for the long-term sustainable management of S. furcifera.

Resistance of True Citrus species to Diaphorina citri.

BACKGROUND: Host genetic resistance is a promising strategy for the management of Diaphorina citri Kuwayama (Hemiptera: Psyllidae), and consequently Huanglongbing (HLB). To date, no study has investigated the resistance to D. citri in the clonal and vegetatively propagated plants of the Microcitrus, Eremocitrus, and Atalantia genera. This study assesses Near and True Citrus genotype antixenosis and antibiosis against D. citri, with trichome density and volatile emission as possible mechanisms of resistance. RESULTS: All genotypes were oviposited by D. citri, however, 8 of 14 genotypes were less oviposited than Citrus × sinensis 'Valencia' (susceptible control). Diaphorina citri nymphs had lower nymphal viability in E. glauca (31%) and M. warburgiana (58%) than that in Citrus × sinensis (77%). The behavioral assay showed that 30% of D. citri nymphs in the last instars evaded E. glauca shoots, whereas no nymphs evaded Citrus × sinensis shoots. A higher trichome density was observed in E. glauca shoots compared to the other genotypes. Chemical analysis revealed differences in the volatile profiles of E. glauca and Citrus × sinensis. CONCLUSION: Eremocitrus glauca and M. warburgiana genotypes were more resistant to D. citri than Citrus × sinensis. Higher trichome density in the shoots may negatively influence the development of D. citri nymphs. Eremocitrus glauca volatiles may also be involved in their resistance to D. citri. © 2022 Society of Chemical Industry.

New Identification Tools for the Nymphs of Three Amblyomma spp. Koch (Ixodida: Ixodidae) in South Texas.

Taxonomic uncertainties and morphologic and molecular similarities may lead to incorrect or incomplete species identifications of immature ticks. This problem can be compounded when the diagnostic morphological features used are unreliable, unstable, or difficult to view. Our study attempts to sort out the similar morphologies of three Amblyomma spp. nymphs with partially overlapping geographic distributions in South Texas. Amblyomma americanum (L.), Amblyomma mixtum Koch, and Amblyomma tenellum Koch are three closely related species with similar nymphs and larvae. Previous attempts to morphologically distinguish among nymphs of these species have relied on difficult-to-observe and unreliable characters. Over the course of decades of routine diagnostic work with these ticks, we have observed and discovered consistent and useful morphologic features sufficient to practically distinguish among these nymphs. We confirmed our morphological studies with molecular genetic evidence (i.e., 12S mitochondrial genes), and we propose that practical, rapid morphological species-level identification of nymphs of the three named species is now possible with confidence.

Broad complex and wing development in cockroaches.

In hemimetabolan insects, the transcription factor Broad complex (Br-C) promotes wing growth and development during the nymphal period. We wondered whether Br-C could trigger the initiation of wing development, using the cockroach Blattella germanica as a model. We show that first instar nymphs have their unique identity of these three thoracic segments specified. During embryogenesis, the expression of Br-C and some wing-related genes show two matching waves. The first takes place before the formation of the germ band, which might be involved in the establishment of various developmental fields including a potential "wing field", and the second wave around organogenesis, possibly involved in the initiation of wing development. However, the expression of Br-C in early embryogenesis concentrates in the developing central nervous system, thus not co-localizing with the expression of the typical wing-related gene vestigial, which is expressed at the edge of the thoracic and abdominal segments. This suggests that Br-C is not specifically involved in the establishment of a potential "wing field" in early embryogenesis. Moreover, maternal RNAi for Br-C depletes the first wave of Br-C expression but does not affect the early expression of wing-related genes. As maternal Br-C RNAi did not deplete the second expression wave of Br-C, we could not evaluate if Br-C is involved in the initiation of wing development. Alternatively, using nymphal RNAi of Br-C and Sex combs reduced (Scr), we show that Br-C contributes to the formation of ectopic wing structures that develop in the prothorax when Scr is depleted. The gene most clearly influenced by Br-C RNAi is nubbin (nub), which, in nymphs is crucial for wing growth. Together, these results suggest that Br-C does not specifically contribute to the establishment of the "wing field", but it does seem important later, in the initiation of wing development, enhancing the expression of wing-related genes, especially nub. This supports the hypothesis previously proposed by the authors, whereby Br-C might have facilitated the evolution of holometaboly. However, there is no doubt that other factors have also contributed to this evolution.

The Function of Nilaparvata lugens (Hemiptera: Delphacidae) E74 and Its Interaction With ßFtz-F1.

Drosophila E74 is an early gene located in the polytene chromosome 74EF puff position. E74 controls the production of late genes, indicating that it plays a crucial role in this cascade model. Nilaparvata lugens E74 is closely related to Diaphorina citri, Bemisia tabaci, and Laodelphax striatellus. After downregulating E74, molting, and nymphal mortality were increased, and ovarian development was delayed. Moreover, the expression of Vg was reduced at the transcriptional level, as measured by qRT-PCR, and the content of Vg protein was reduced, as detected by Western blotting. After downregulating E74, the expression of hormone-related genes, including Tai, ßFtz-F1, Met, Kr-h1, UspA, UspB, E93, and Br, was changed. The expression of E74 was significantly decreased after downregulating hormone-related genes. When the expression of E74 and ßFtz-F1 was downregulated together, nymph mortality and molting mortality were higher than those when E74 or ßFtz-F1 was downregulated alone. Thus, E74 probably interacts with ßFtz-F1 at the genetic level. In summary, this study showed that E74 plays a crucial role in the development, metamorphosis and reproduction of N. lugens, possibly via the interaction with ßFtz-F1 at the genetic level. This study provides a basis for the development of new target-based pesticides and new methods for the effective control of N. lugens.

Description of the female, nymph and larva and mitochondrial genome, and redescription of the male of Ixodes barkeri Barker, 2019 (Acari: Ixodidae), from the short-beaked echidna, Tachyglossus aculeatus, with a consideration of the most suitable subgenus for this tick.

BACKGROUND: Ixodes barkeri, a tick with a distinctive ventrolateral horn-like projection on palpal segment 1, was described in 2019 from two male ticks from the Wet Tropics of Far North Queensland, Australia. However, females lie at the core of the taxonomy and subgenus classification of Ixodes; hence, we sought specimens of female ticks, successfully recovering females, plus nymphs and larvae. Mitochondrial genomes are also desirable additions to the descriptions of species of ticks particularly regarding subgenus systematics. So, we sequenced the mt genomes of I. barkeri Barker, 2019, and the possible relatives of I. barkeri that were available to us (I. australiensis Neumann, 1904, I. fecialis Warburton & Nuttall, 1909, and I. woyliei Ash et al. 2017) with a view to discovering which if any of the subgenera of Ixodes would be most suitable for I. barkeri Barker, 2019. RESULTS: The female, nymph, larva and mitochondrial genome of Ixodes barkeri Barker, 2019, are described for the first time and the male of I. barkeri is redescribed in greater detail than previously. So far, I. barkeri is known only from a monotreme, the short-beaked echidna, Tachyglossus aculeatus (Shaw, 1792), from the highland rainforests of the Wet Tropics of Far North Queensland, Australia. CONCLUSIONS: Our phylogeny from entire mitochondrial genomes indicated that I. barkeri and indeed I. woyliei Ash et al., 2017, another tick that was described recently, are best placed in the subgenus Endopalpiger Schulze, 1935.

Morphological and Molecular Identification of Cimex hemipterus Fabricius, 1803 (Hemiptera: Cimicidae) and First Report of C. lectularius Linnaeus, 1758, in Madagascar.

Bed bugs are hematophagous insects with a long history of presence in human communities. Over the last three decades, infestations by bed bugs in human dwellings have drastically increased, leading to a rise in bed bug concerns. This study was conducted in May 2019 in the Miarinarivo district of Madagascar. Eight locations with suspected infestations were inspected. Of these, five locations were found with active bed bug infestations. Overall, 23 (19 adults and four nymphs) specimens with numbers per apartment ranging from 2 to 6 were collected and analyzed using morphological and molecular approaches. Of 19 adults collected, 18 were morphologically identified as Cimex hemipterus (Hemiptera: Cimicidae), with the final adult identified as C. lectularius. Morphological identification of adults was confirmed by sequencing 450 bp fragment of COI gene. All four nymphs were also identified molecularly as C. hemipterus. Maximum likelihood phylogenetic tree of COI gene and sequence alignment of C. hemipterus specimens revealed the presence of two groups (first group clustered with previously reported haplotype from Asian and African countries and the second group encompassed a lesser known haplotype exclusively from Africa (Kenya and Senegal). This study presents low genetic diversity among C. hemipterus specimens and, for the first time, reports the occurrence of C. lectularius, known as temperate species, in Madagascar. These results can be helpful in control management strategies against these ectoparasites in this country.

Abdominal-B contributes to abdominal identity in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae).

The homeotic complex gene Abdominal-B (Abd-B) is involved in regulating the development of posterior abdomens and has been extensively studied in holometabolous insects. However, the function of Abd-B in hemimetabolous insects is not fully understood. Here, we functionally characterize an Abd-B homologue in the brown planthopper (BPH), Nilaparvata lugens. The full-length cDNA of the N. lugens Abd-B homologue (NlAbd-B) is 2334 nt, with an open reading frame of 1113 bp. NlAbd-B has the highest expression level at the egg stage relative to the nymphal and adult stages and is mainly expressed in the fourth to the ninth abdominal segment of embryos. RNA interference (RNAi)-mediated knockdown of NlAbd-B in nymphs disrupted the development of genitalia both in females and males and caused a genitalia-to-leg transformation. Parental RNAi of NlAbd-B in both female and male adults caused an extra abdominal segment in offspring nymphs, while parental RNAi of the N. lugens abdominal-A homologue in both female and males adults led to embryos with leg-like appendages on the second to the eighth abdominal segment. These findings suggest that NlAbd-B plays a pivotal role in genital development and posterior abdominal patterning and thus highlight the conservational role of Abd-B in holometabolous and hemimetabolous insects.

Cuticle Protein LmACP19 Is Required for the Stability of Epidermal Cells in Wing Development and Morphogenesis of Locusta migratoria.

Insect wing consists of a double layer of epidermal cells that produce and secrete the dorsal and ventral cuticular components. It is important for the stability of epidermal cells during wing development and morphogenesis, but its specific gene expression and physiological function during this process remain unclear. In our previous work, a wing cuticle protein gene LmACP19 was identified in Locusta migratoria based on transcriptomic data. Here, we report on its roles in wing development and morphogenesis. LmACP19 encodes a chitin-binding protein belonging to RR-2 subfamily of CPR family, which is highly homologous to CP19-like proteins in other insect species. RT-qPCR analysis revealed that LmACP19 is highly expressed in wing pads of fifth-instar nymphs, and its encoded protein is located in two layers of epidermal cells but not in the cuticle. Suppression of LmACP19 by RNA interference led to abnormal wing pad and wing morphogenesis with curved, unclosed, and wrinkled phenotypes during nymph-to-nymph and nymph-to-adult transition, respectively. Furthermore, deficiency of LmACP19 affected arrangement of epidermal cells, resulting in apoptosis. Our results indicate that LmACP19 is indispensable for wing development and normal morphological structure by maintaining the stability of epidermal cells during L. migratoria molting.

Silencing of Decapentaplegic (Dpp) gene inhibited the wing expansion in the white-backed planthopper, Sogatella furcifera (Horváth) (Hemiptera: Delphacidae).

The Decapentaplegic gene controls wing patterning and spreading by regulating downstream genes in many insect species. However, the molecular characteristics, expression, and biological function of Dpp in Sogatella furcifera remain poorly understood. In this study, we cloned the Dpp gene from S. furcifera and examined its expression levels in different development stages, wing typed adults, and tissues. Then, the function of SfDpp gene was analyzed using an RNA interference (RNAi)-based approach. The results showed that the full-length complementary DNA  of the SfDpp gene consists of 1034 bp and contains a 954-bp open reading frame encoding 317 amino acids. SfDpp has a transforming growth factor-ß (TGF-ß) propeptide superfamily domain and a TGF-ß superfamily domain, typical of members of the TGF-ß superfamily. Quantitative real-time polymerase chain reaction showed that the expression of SfDpp reached its highest expression level 40 min after eclosion. RNAi-based gene silencing inhibited transcript levels of the corresponding messenger RNA in S. furcifera nymphs injected with double-stranded RNA of SfDpp and resulted in death of 29.17% and 26.67% of 4th and 5th instar nymphs, respectively. The wing deformity rate of the adults was 74.12% and 3.41% after SfDpp gene silencing in 4th and 5th instar nymphs, respectively. Examining wing development-associated genes showed that two target genes of Dpp (Vestigial and Spalt) were both dramatically downregulated after SfDpp was silenced. Our results demonstrate that downregulated SfDpp in early development causes wing expansion failure in S. furcifera. Thus, Dpp may be a target gene for restricting the migration of rice-damaging planthoppers.