Genotoxicity and cytotoxicity in male reproductive cells caused by sediment pollutants.

In recent years, mounting evidence has highlighted a global decline in male semen quality, paralleling an increase in male infertility problems. Such developments in the male reproductive system are likely due to a range of environmental factors, which could negatively affect the outcomes of pregnancy, reproductive health, and the well-being of fetuses. Different environmental contaminants ultimately accumulate in riverbed sediments due to gravity, so these sediments are frequently considered hotspots for pollutants. Therefore, understanding the detrimental effects of river sediment pollution on human reproductive health is crucial. This study indicates male germ cells' high vulnerability to environmental contaminants. There is a strong positive correlation between the concentration of complex accumulated pollutants from human activities and the reproductive toxicity observed in human testicular embryonic cell lines NCCIT and NTERA-2. This toxicity is characterized by increased levels of reactive oxygen species, disruption of critical cellular functions, genotoxic impacts, and the induction of cell apoptosis. This research marks a significant step in providing in vitro evidence of the damaging effects of environmental pollutants on the human male germline.

Chromosome-level genome assembly of the bethylid ectoparasitoid wasp Sclerodermus sp. 'alternatusi'.

The Bethylidae are the most diverse of Hymenoptera chrysidoid families. As external parasitoids, the bethylids have been widely adopted as biocontrol agents to control insect pests worldwide. Thus far, the genomic information of the family Bethylidae has not been reported yet. In this study, we crystallized into a high-quality chromosome-level genome of ant-like bethylid wasps Sclerodermus sp. 'alternatusi' (Hymenoptera: Bethylidae) using PacBio sequencing as well as Hi-C technology. The assembled S. alternatusi genome was 162.30 Mb in size with a contig N50 size of 3.83 Mb and scaffold N50 size of 11.10 Mb. Totally, 92.85% assembled sequences anchored to 15 pseudo-chromosomes. A total of 10,204 protein-coding genes were annotated, and 23.01 Mb repetitive sequences occupying 14.17% of genome were pinpointed. The BUSCO results showed that 97.9% of the complete core Insecta genes were identified in the genome, while 97.1% in the gene sets. The high-quality genome of S. alternatusi will not only provide valuable genomic information, but also show insights into parasitoid wasp evolution and bio-control application in future studies.

The genomic history and global migration of a windborne pest.

Many insect pests, including the brown planthopper (BPH), undergo windborne migration that is challenging to observe and track. It remains controversial about their migration patterns and largely unknown regarding the underlying genetic basis. By analyzing 360 whole genomes from around the globe, we clarify the genetic sources of worldwide BPHs and illuminate a landscape of BPH migration showing that East Asian populations perform closed-circuit journeys between Indochina and the Far East, while populations of Malay Archipelago and South Asia undergo one-way migration to Indochina. We further find round-trip migration accelerates population differentiation, with highly diverged regions enriching in a gene desert chromosome that is simultaneously the speciation hotspot between BPH and related species. This study not only shows the power of applying genomic approaches to demystify the migration in windborne migrants but also enhances our understanding of how seasonal movements affect speciation and evolution in insects.

Chromosome-level Genome Assembly and Sex-specific Differential Transcriptome of the White-backed Planthopper, Sogatella furcifera.

Background: The white-backed planthopper (WBPH), Sogatella furcifera, causes great damage to many crops (mainly rice) by direct feeding or transmitting plant viruses. The previous genome assembly was generated by second-generation sequencing technologies, with a contig N50 of only 51.5 kb, and contained a lot of heterozygous sequences. Methods: We utilized third-generation sequencing technologies and Hi-C data to generate a high-quality chromosome-level assembly. We also provide a large amount of transcriptome data for full-length transcriptome analysis and gender differential expression analysis. Results: The final assembly comprised 56.38 Mb, with a contig N50 of 2.20 Mb and a scaffold N50 of 45.25 Mb. Fourteen autosomes and one X chromosome were identified. More than 99.5% of the assembled bases located on the 15 chromosomes. 95.9% of the complete BUSCO Hemiptera genes were detected in the final assembly and 16,880 genes were annotated. 722 genes were relatively highly expressed in males, while 60 in the females. Conclusion: The integrated genome, definite sex chromosomes, comprehensive transcriptome profiles, high efficiency of RNA interference and short life cycle substantially made WBPH an efficient research object for functional genomics.

Horizontally Transferred Salivary Protein Promotes Insect Feeding by Suppressing Ferredoxin-Mediated Plant Defenses.

Herbivorous insects such as whiteflies, planthoppers, and aphids secrete abundant orphan proteins to facilitate feeding. Yet, how these genes are recruited and evolve to mediate plant-insect interaction remains unknown. In this study, we report a horizontal gene transfer (HGT) event from fungi to an ancestor of Aleyrodidae insects approximately 42 to 190 million years ago. BtFTSP1 is a salivary protein that is secreted into host plants during Bemisia tabaci feeding. It targets a defensive ferredoxin 1 in Nicotiana tabacum (NtFD1) and disrupts the NtFD1-NtFD1 interaction in plant cytosol, leading to the degradation of NtFD1 in a ubiquitin-dependent manner. Silencing BtFTSP1 has negative effects on B. tabaci feeding while overexpressing BtFTSP1 in N. tabacum benefits insects and rescues the adverse effect caused by NtFD1 overexpression. The association between BtFTSP1 and NtFD1 is newly evolved after HGT, with the homologous FTSP in its fungal donor failing to interact and destabilize NtFD1. Our study illustrates the important roles of horizontally transferred genes in plant-insect interactions and suggests the potential origin of orphan salivary genes.

Chromosome-level Assembly, Dosage Compensation and Sex-biased Gene Expression in the Small Brown Planthopper, Laodelphax striatellus.

In insects, sex chromosome differentiation often results in unequal gene dosages between sexes. Dosage compensation mechanisms evolve to balance gene expression, but the degree and mechanism of regulation often vary by insect species. In hemipteran species, the small brown planthopper (SBPH), Laodelphax striatellus, is an injurious crop pest, with a sex chromosome type XX in females and XO in males. This species offers the opportunity to study dosage compensation and sex-biased gene expression. In this study, we generated a chromosome-level genome of SBPH using Oxford Nanopore Technologies and high-throughput chromatin conformation capture (Hi-C) technology. We also sequenced RNA-seq data from 16 tissue samples to annotate the genome and analyze gene dosage compensation. We finally obtained a 510.2 megabases (Mb) genome with 99.12% of the scaffolds anchored on 15 chromosomes (14 autosomes and 1 X chromosome) and annotated 16,160 protein-coding genes based on full-length cDNA sequencing data. Furthermore, we found complete dosage compensation in all L. striatellus somatic tissues, but lack of dosage compensation in gonad tissue testis. We also found that female-biased genes were significantly enriched on the X chromosome in all tissues, whereas male-biased genes in gonad tissues were enriched on autosomes. This study not only provides a high-quality genome assembly but also lays a foundation for a better understanding of the sexual regulatory network in hemipteran insects.

Lateral oviduct-secreted proteins in the brown planthopper.

Oviducts are the "traffic hubs" of the female reproductive system, serving as the crucial conduits for egg transportation. By performing LC-MS/MS proteomic detection together with transcriptomic analysis, 80 lateral oviduct-secreted proteins were identified, and 5 genes (NlOdsp, NlOdsp1, NlOdsp2, NlOdsp3 and NlOdsp4) specifically expressed in the oviducts of the brown planthopper Nilaparvata lugens, the most destructive rice pest, were authenticated. qRT-PCR analysis revealed that these genes and proteins were mainly/specifically expressed in the female reproductive system in adulthood. RNA interference (RNAi) against the 5 NlOdsp genes significantly affected the survival rates (3.4% - 68.7% of the control) and fecundities of female adults (3.9% - 57.6% of the control) at 8 d post injection (p.i.). In addition, the lack of NlOdsp1 caused decreases in the gel-like brown secretions inside the lateral oviducts, while increased secretions were found in the dsNlOdsp2-treated groups. In addition, NlOdsp3 is a pleiotropic gene involved in both oocyte development and egg movement through the lateral oviducts, similar to the role of NlOdsp in egg transportation. The results deepen our understanding of oviduct-secreted proteins in female insects and provide novel target genes for RNAi-based insect pest control. SIGNIFICANCE: Oviduct plays a vital role in animal reproductive processes and it serves as the crucial conduit for egg transportation. Though oviduct secretes have been well documented in high animals, the proteomic information of insect oviduct secretes remains poorly understood. The present study revealed 80 oviduct secreted proteins, including 19 unknown proteins, from the rice planthopper, the most destructive rice pest which lay eggs in plant tissues. Five of the 19 proteins were further functionally characterized. The results not only deepen our understanding of the oviduct secreted proteins in insect reproductive biology, but also provide basis for interaction between insects and host plants, and provide novel target genes for RNAi-based insect pest control.

Chromosome-level genome assembly for the horned-gall aphid provides insights into interactions between gall-making insect and its host plant.

The aphid Schlechtendalia chinensis is an economically important insect that can induce horned galls, which are valuable for the medicinal and chemical industries. Up to now, more than twenty aphid genomes have been reported. Most of the sequenced genomes are derived from free-living aphids. Here, we generated a high-quality genome assembly from a galling aphid. The final genome assembly is 271.52 Mb, representing one of the smallest sequenced genomes of aphids. The genome assembly is based on contig and scaffold N50 values of the genome sequence are 3.77 Mb and 20.41 Mb, respectively. Nine-seven percent of the assembled sequences was anchored onto 13 chromosomes. Based on BUSCO analysis, the assembly involved 96.9% of conserved arthropod and 98.5% of the conserved Hemiptera single-copy orthologous genes. A total of 14,089 protein-coding genes were predicted. Phylogenetic analysis revealed that S. chinensis diverged from the common ancestor of Eriosoma lanigerum approximately 57 million years ago (MYA). In addition, 35 genes encoding salivary gland proteins showed differentially when S. chinensis forms a gall, suggesting they have potential roles in gall formation and plant defense suppression. Taken together, this high-quality S. chinensis genome assembly and annotation provide a solid genetic foundation for future research to reveal the mechanism of gall formation and to explore the interaction between aphids and their host plants.

The Genetic Network of Forkhead Gene Family in Development of Brown Planthoppers.

We identified 18 distinct Fox genes in the genome of the brown planthopper, Nilaparvata lugens, and further found a novel insect-specific subfamily that we temporarily named FoxT. A total of 16 genes were highly expressed in the eggs, while NlFoxL2 and NlFoxT are female- and male-specific genes, respectively. Large scale RNAi and RNA-seq analyses were used to reveal the functions and potential targets of NlFoxs. In the eggs, NlFoxA, NlFoxN1 and NlFoxN2 are indispensable to early embryogenesis by regulating different target genes; NlFoxG and NlFoxQ co-regulate NlSix3 for brain development; and NlFoxC, NlFoxJ1 and NlFoxP have complementary effects on late embryogenesis. Moreover, NlFoxA, NlFoxNl and NlFoxQ have pleiotropism. NlFoxA and NlFoxQ regulate the expression of NlCHS1 and cuticular proteins, respectively, thereby participating in the formation of cuticles. NlFoxN1, which regulates the expression of NlKrt9 is involved in the formation of intermediate filament frameworks. Our previous studies have revealed that NlFoxL2 and NlFoxO play important roles in chorion formation and wing polyphenism. Altogether, N. lugens Fox genes exhibit functional diversity in embryonic development and organogenesis. This comprehensive study combines genomics, transcriptomics and phenomics, thereby constructing a complex genetic network that spans the entire life cycle of the brown planthopper.

Intersex Plays a Role in Microbial Homeostasis in the Brown Planthopper.

Insects harbor a wide variety of symbiotic microorganisms that are capable of regulating host health and promoting host adaptation to their environment and food sources. However, there is little knowledge concerning the mechanisms that maintain the microbial community homeostasis within insects. In this study, we found that the intersex (ix) gene played an essential role in maintaining microbial homeostasis in the brown planthopper (BPH), Nilaparvata lugens. Injection of the double-strand RNA targeting N. lugens ix (Nlix) into the newly emerged females resulted in abnormal expansion of the copulatory bursa of BPH after mating. Further observation by transmission electron microscopy (TEM) revealed that the abnormally enlarged copulatory bursa resulting from dsNlix treatment was full of microorganisms, while in contrast, the copulatory bursa of dsGFP-treated individuals stored a large number of sperm accompanied by a few bacteria. Moreover, RNA-seq analysis showed that the gene responses to bacteria were remarkably enriched in differentially expressed genes (DEGs). In addition, 16s rRNA sequencing indicated that, compared with control samples, changes in the composition of microbes presented in dsNlix-treated copulatory bursa. Together, our results revealed the immune functions of the Nlix gene in maintaining microbial homeostasis and combating infection in BPH.

Cuticular Hydrocarbon Plasticity in Three Rice Planthopper Species.

Insect cuticular hydrocarbons (CHCs) are organic compounds of the surface lipid layer, which function as a barrier against water loss and xenobiotic penetration, while also serving as chemical signals. Plasticity of CHC profiles can vary depending upon numerous biological and environmental factors. Here, we investigated potential sources of variation in CHC profiles of Nilaparvata lugens, Laodelphax striatellus and Sogatella furcifera, which are considered to be the most important rice pests in Asia. CHC profiles were quantified by GC/MS, and factors associated with variations were explored by conducting principal component analysis (PCA). Transcriptomes were further compared under different environmental conditions. The results demonstrated that CHC profiles differ among three species and change with different developmental stages, sexes, temperature, humidity and host plants. Genes involved in cuticular lipid biosynthesis pathways are modulated, which might explain why CHC profiles vary among species under different environments. Our study illustrates some biological and ecological variations in modifying CHC profiles, and the underlying molecular regulation mechanisms of the planthoppers in coping with changes of environmental conditions, which is of great importance for identifying potential vulnerabilities relating to pest ecology and developing novel pest management strategies.

Chromosome-level genome assembly of the bean bug Riptortus pedestris.

The bean bug (Riptortus pedestris) causes great economic losses of soybeans by piercing and sucking pods and seeds. Although R. pedestris has become the focus of numerous studies associated with insect-microbe interactions, plant-insect interactions, and pesticide resistance, a lack of genomic resources has limited deeper insights. Here, we report the first R. pedestris genome at the chromosomal level using PacBio, Illumina, and Hi-C technologies. The assembled genome was 1.080 Gb in size with a contig N50 of 2.882 Mb. More than 96.3% of the total genome bases were successfully anchored to six unique chromosomes. Genome resequencing of male and female individuals and chromosomic staining demonstrated that the sex chromosome system of R. pedestris is XO, and the shortest chromosome is the X chromosome. In total, 19,026 protein-coding genes were predicted, 18,745 of which were validated as being expressed. Temporospatial expression of R. pedestris genes in six tissues and 37 development stages revealed 4,657 and 7,793 genes mainly expressed in gonads and egg periods, respectively. Evolutionary analysis demonstrated that R. pedestris and Oncopeltus fasciatus formed a sister group and split ∼80 million years ago (Mya). Additionally, a 5.04 Mb complete genome of symbiotic Serratia marcescens Rip1 was assembled, and the virulence factors that account for successful colonization in the host midgut were identified. The high-quality R. pedestris genome provides a valuable resource for further research, as well as for the pest management of bug pests.

Chromosome-level assembly of the brown planthopper genome with a characterized Y chromosome.

Hundreds of insect genome sequences have been reported; however, most sequencing projects have not determined the sex chromosomes, and no Y chromosomes from a heterometabolous insect have been identified and characterized to date. The brown planthopper (Nilaparvata lugens Stål) is the most economically damaging pest to rice and is also an ideal research subject for paddy ecology and functional genomics. We previously assembled a draft female genome mainly using second-generation sequencing technologies, with a contig N50 of only 24 kb, due to the large size and excessive repetitive regions in the N. lugens genome. Here, we utilize third-generation sequencing technologies and Hi-C data to generate a high-quality male N. lugens assembly with a contig N50 of 1.01 Mb, a scaffold N50 of 69.96 Mb and more than 95.6% of the assembled bases located on 16 chromosomes. Fourteen autosomes and two sex chromosomes (X + Y) were identified, filling in the gap related to the Y chromosome in heterometabolous insects. A total of 18,021 protein-coding genes and 6423 long-noncoding RNAs were predicted with full-length cDNA sequencing data. All 315 of the Y chromosome genes (Y-genes) were derived from autosomal and X-chromosome duplications. Large-scale RNA interference (RNAi) experiments were conducted against the N. lugens Y-genes, demonstrating that 7 Y-genes were essential for normal BPH development or male organ development, suggesting the importance of Y-genes. The first identified Y chromosome in heterometabolous insects will help gain more insight into sex determination, fertility and chromosome evolution.

Pleiotropic Functions of FoxN1: Regulating Different Target Genes during Embryogenesis and Nymph Molting in the Brown Planthopper.

FoxN1 gene belongs to the forkhead box gene family that comprises a diverse group of "winged helix" transcription factors that have been implicated in a variety of biochemical and cellular processes. In the brown planthopper (BPH), FoxN1 is highly expressed in the ovaries and newly laid eggs, where it acted as an indispensable gene through its molecular targets to regulate early embryonic development. Moreover, the results of the RNAi experiments indicated that Nilaparvata lugens FoxN1 (NlFoxN1) exhibited pleiotropism: they not only affected the embryogenesis, but also played an important role in molting. RNA-seq and RNAi were further used to reveal potential target genes of NlFoxN1 in different stages. In the eggs, ten downregulated genes were defined as potential target genes of NlFoxN1 because of the similar expression patterns and functions with NlFoxN1. Knockdown of NlFoxN1 or any of these genes prevented the development of the eggs, resulting in a zero hatchability. In the nymphs, NlFoxN1 regulated the expression of a keratin gene, type I cytoskeletal keratin 9 (NlKrt9), to participate in the formation of an intermediate filament framework. Depletion of NlFoxN1 or NlKrt9 in nymphs, BPHs failed to shed their old cuticle during nymph-to-nymph or nymph-to-adult molting and the mortality was almost 100%. Altogether, the pleiotropic roles of NlFoxN1 during embryogenesis and nymph molting were supported by the ability to coordinate the temporal and spatial gene expression of their target genes.

Salivary DNase II from Laodelphax striatellus acts as an effector that suppresses plant defence.

Extracellular DNA, released by damaged plant cells, acts as a damage-associated molecular pattern (DAMP). We demonstrated previously that the small brown planthopper (Laodelphax striatellus, SBPH) secreted DNase II when feeding on artificial diets. However, the function of DNase II in insect feeding remained elusive. The influences of DNase II on SBPHs and rice plants were investigated by suppressing expression of DNase II or by application of heterogeneously expressed DNase II. We demonstrated that DNase II is mainly expressed in the salivary gland and is responsible for DNA-degrading activity of saliva. Knocking down the expression of DNase II resulted in decreased performance of SBPH reared on rice plants. The dsDNase II-treated SBPH did not influenced jasmonic acid (JA), salicylic acid (SA), ethylene (ET) pathways, but elicited a higher level of H2 O2 and callose accumulation. Application of heterogeneously expressed DNase II in DNase II-deficient saliva slightly reduced the wound-induced defence response. We propose a DNase II-based invading model for SBPH feeding on host plants, and provide a potential target for pest management.

A comprehensive omics analysis and functional survey of cuticular proteins in the brown planthopper.

Cuticle, mainly composed of chitin and cuticular proteins (CPs), is a multifunctional structure of arthropods. CPs usually account for >1% of the total insect proteins. Why does an insect encode so many different CP genes in the genome? In this study, we use comprehensive large-scale technologies to study the full complement of CPs (i.e., the CP-ome) of the brown planthopper (BPH), Nilaparvata lugens, a major rice plant pest. Eight CP families (CPR, CPF, TWDL, CPLCP, CPG, CPAP1, CPAP3, and CPAPn) including 140 proteins in BPH, in which CPAPn is a CP family that we discovered. The CPG family that was considered to be restricted to the Lepidoptera has also been identified in BPH. As reported here, CPLCP family members are characterized by three conserved sequence motifs. In addition, we identified a testis protein family with a peritrophin A domain that we named TPAP. We authenticated the real existence of 106 proteins among the 140 CPs. RNA interference (RNAi) experiments were conducted against 135 CP genes in early- and late-instar nymphs and newly emerged female adults, demonstrating that 32 CPs were essential for BPH normal development or egg production. Combined RNAi experiments suggested redundant and complementary functions of the large number of CPs. Transcriptomic data revealed that the CP genes were expressed in a tissue-specific manner, and there were four clusters of developmental expression patterns. This study gives a comprehensive understanding of the roles of CPs in an insect cuticle.

Insights into Bacterial Cellulose Biosynthesis from Different Carbon Sources and the Associated Biochemical Transformation Pathways in Komagataeibacter sp. W1.

Cellulose is the most abundant and widely used biopolymer on earth and can be produced by both plants and micro-organisms. Among bacterial cellulose (BC)-producing bacteria, the strains in genus Komagataeibacter have attracted wide attention due to their particular ability in furthering BC production. Our previous study reported a new strain of genus Komagataeibacter from a vinegar factory. To evaluate its capacity for BC production from different carbon sources, the present study subjected the strain to media spiked with 2% acetate, ethanol, fructose, glucose, lactose, mannitol or sucrose. Then the BC productivity, BC characteristics and biochemical transformation pathways of various carbon sources were fully investigated. After 14 days of incubation, strain W1 produced 0.040⁻1.529 g L-1 BC, the highest yield being observed in fructose. Unlike BC yields, the morphology and microfibrils of BCs from different carbon sources were similar, with an average diameter of 35⁻50 nm. X-ray diffraction analysis showed that all membranes produced from various carbon sources had 1⁻3 typical diffraction peaks, and the highest crystallinity (i.e., 90%) was found for BC produced from mannitol. Similarly, several typical spectra bands obtained by Fourier transform infrared spectroscopy were similar for the BCs produced from different carbon sources, as was the Iα fraction. The genome annotation and Kyoto Encyclopedia of Genes and Genomes analysis revealed that the biochemical transformation pathways associated with the utilization of and BC production from fructose, glucose, glycerol, and mannitol were found in strain W1, but this was not the case for other carbon sources. Our data provides suggestions for further investigations of strain W1 to produce BC by using low molecular weight sugars and gives clues to understand how this strain produces BC based on metabolic pathway analysis.

Forkhead box transcription factor L2 activates Fcp3C to regulate insect chorion formation.

Most animals are oviparous. However, the genes regulating egg shell formation remain not very clear. In this study, we found that Nilaparvata lugens Forkhead box transcription factor L2 (NlFoxL2) directly activated follicle cell protein 3C (NlFcp3C) to regulate chorion formation. NlFoxL2 and NlFcp3C had a similar expression pattern, both highly expressed in the follicular cells of female adults. Knockdown of NlFoxL2 or NlFcp3C also resulted in the same phenotypes: obesity and female infertility. RNA interference (RNAi) results suggested that NlFcp3C is a downstream gene of NlFoxL2 Furthermore, transient expression showed that NlFoxL2 could directly activate the NlFcp3C promoter. These results suggest that NlFcp3C is a direct target gene of NlFoxL2. Depletion of NlFoxL2 or NlFcp3C prevented normal chorion formation. Our results first revealed the functions of Fcp3C and FoxL2 in regulation of oocyte maturation in an oviparous animal.

Characteristics of the draft genome of "Candidatus Arsenophonus nilaparvatae", a facultative endosymbiont of Nilaparvata lugens.

There exists a kind of symbiotic bacterium named "Candidatus Arsenophonus nilaparvatae" in the brown planthopper (BPH), Nilaparvata lugens. After being filtered and assembled from the BPH genome sequencing project, the genome sequence of this bacterial symbiont was obtained. After initial analysis based on the genome, we have found its potential role to synthesize B vitamins for the host. In order to better understand the lifestyle and the genomic changes of this symbiotic bacterium after the symbiotic relationship was established, we further report the characteristics of this draft genome. Compared with several other related bacteria, "Candidatus Arsenophonus nilaparvatae" has proven to be a facultative endosymbiont at the genomic level. Concurrently, the presence of fimbriae and flagella formation related genes indicates this maternally transmitted endosymbiont is most likely to retain the capacity to invade new hosts. Through further analysis of annotated gene sets, we also find evidence of genome reduction in its secretion system and metabolic pathways. These findings reflect its evolutionary trend to be an obligate one and enable a deeper study of microbe-insect interactions.

The multicopper oxidase gene family in the brown planthopper, Nilaparvata lugens.

The multicopper oxidase (MCO) family of enzymes includes laccases, ascorbate oxidases, bilirubin oxidases and a subgroup of metal oxidases. On the basis of a bioinformatics investigation, we identified 7 genes encoding putative multicopper oxidase proteins in the genome of the brown planthopper (BPH), Nilaparvata lugens (Hemiptera: Delphacidae). MCO1 and MCO2 are conserved, while others diverse in insects. Analysis of developmental and tissue-specific expression patterns revealed the following: NlMCO2 was mainly expressed in the integument, and its expression peaked periodically during molting; NlMCO3 was an ovary-specific MCO gene with a high expression level only at the adult stage; NlMCO4 was a salivary gland-specific MCO gene that was expressed at all developmental stages; NlMCO5 only had short-term expression in the middle of the fourth instar stage and was expressed mainly in the gut; NlMCO6 had a developmental expression pattern similar to that of NlMCO2 and was expressed in most N. lugens tissues; and NlMCO1 was expressed in most N. lugens tissues except for the testis, whereas NlMCO7 was mainly expressed in the gut and the Malpighian tube. BPHs injected with double-stranded RNA (dsRNA) targeting NlMCO2 failed to pigment and sclerotize, were colorless and soft-bodied and subsequently died in a short time. Lethal phenotypes were also observed in insects challenged by dsRNA targeting NlMCO6. However, no observable morphological or internal structural abnormality was obtained in the insects treated with dsRNA for NlMCO1, NlMCO3, NlMCO4, NlMCO5 or NlMCO7.