Characterization of an intestinal mucin from the peritrophic matrix of the diamondback moth,Plutella xylostella

The peritrophic matrix (PM) of Plutella xylostella larvae was found to contain twelve integral and eighteen loosely associated proteins. An antiserum against Mamestra configurata integral PM proteins cross-reacted with several P. xylostella PM proteins and was used to isolate a partial cDNA encoding an insect intestinal mucin (PxIIM). PxIIM was expressed primarily in the larval midgut. The deduced protein sequence of the partial cDNA contained three potentially glycosylated, mucin-like domains and six cysteine-rich chitin-binding domains (CBDs). An additional chitin-binding domain was proposed to reside at the amino terminus of the protein based on comparison with other IIM. The organization of mucin domains and CBDs exhibited features, including an internal triplet of regularly spaced CBDs and a carboxyl terminal CBD with two additional conserved cysteine residues, that were found to be common to other lepidopteran IIMs.     

Germline transformation of the diamondback moth, Plutella xylostella L., using the piggyBac transposable element

The diamondback moth, Plutella xylostella, is one of the most economically important agricultural pests. The larvae of this moth cause damage by feeding on the foliage of cruciferous vegetables such as cabbage, broccoli, cauliflower and rapeseed. Control generally comprises chemical treatment; however, the diamondback moth is renowned for rapid development of resistance to pesticides. Other methods, such as biological control, have not been able to provide adequate protection. Germline transformation of pest insects has become available in recent years as an enabling technology for new genetics‐based control methods, such as the Release of Insects carrying a Dominant Lethal (RIDL^®). In the present study, we report the first transformation of the diamondback moth, using the piggyBac transposable element, by embryo microinjection. In generating transgenic strains using four different constructs, the function of three regulatory sequences in this moth was demonstrated in driving expression of fluorescent proteins. The transformation rates achieved, 0.48–0.68%, are relatively low compared with those described in other Lepidoptera, but not prohibitive, and are likely to increase with experience. We anticipate that germline transformation of the diamondback moth will permit the development of RIDL strains for use against this pest and facilitate the wider use of this species as a model organism for basic studies.     

Revised annotation of Plutella xylostella microRNAs and their genome‐wide target identification

The diamondback moth, Plutella xylostella, is the most devastating pest of brassica crops worldwide. Although 128 mature microRNAs (miRNAs) have been annotated from this species in miRBase, there is a need to extend and correct the current P. xylostella miRNA repertoire as a result of its recently improved genome assembly and more available small RNA sequence data. We used our new ultra‐deep sequence data and bioinformatics to re‐annotate the P. xylostella genome for high confidence miRNAs with the correct 5p and 3p arm features. Furthermore, all the P. xylostella annotated genes were also screened to identify potential miRNA binding sites using three target‐predicting algorithms. In total, 203 mature miRNAs were annotated, including 33 novel miRNAs. We identified 7691 highly confident binding sites for 160 pxy‐miRNAs. The data provided here will facilitate future studies involving functional analyses of P. xylostella miRNAs as a platform to introduce novel approaches for sustainable management of this destructive pest.     

A new aminopeptidase from diamondback moth provides evidence for a gene duplication event in Lepidoptera

We screened a midgut cDNA library from diamondback moth, Plutella xylostella, with a probe generated using sequence information from an aminopeptidase N gene from Manduca sexta (MsAPN-1). The sequence recovered (PxAPN-A) encodes a protein of 988 resides with a 60% sequence identity to MsAPN-1. The two proteins share a signal peptide which directs processing by the endoplasmic reticulum, a C-terminal hydrophobic region satisfying the criterion for a GPI anchor and cleavage, and the possibility of an O-glycosylated rigid stalk attached to the GPI anchor. PxAPN-A is more closely related to MsAPN-1 than it is to another aminopeptidase recently reported from P. xylostella. Sequence comparisons with other species suggests that at least one aminopeptidase gene duplication occurred in an ancestral lepidopteran.     

Identification and functional analysis of a Masculinizer orthologue in Trilocha varians (Lepidoptera: Bombycidae)

We recently showed that the Masculinizer gene (Masc) plays a primary role in sex determination in the lepidopteran model insect Bombyx mori. However, it remains unknown whether this Masc protein‐dependent sex determination system is conserved amongst lepidopteran insects or within the family Bombycidae. Here we cloned and characterized a Masc homologue (TvMasc) in Trilocha varians (Lepidoptera: Bombycidae), a species closely related to B. mori. To elucidate the role of TvMasc in the sex determination cascade of T. varians, TvMasc expression was knocked down in early embryos by the injection of small interfering RNAs (siRNAs) that targeted TvMasc mRNAs. Both female‐ and male‐type splice variants of Tvdsx, a doublesex (dsx) homologue in T. varians were observed in control siRNA‐injected embryos. By contrast, only female‐type splice variants were observed in TvMasc siRNA‐injected embryos. These results indicate that the TvMasc protein directly or indirectly regulates the splicing patterns of Tvdsx. Furthermore, we found that male‐type splice variants of B. mori dsx (Bmdsx) were produced in TvMasc‐overexpressing BmN4 cells. The mRNA level of B. mori Imp, a gene whose product induces male‐specific Bmdsx splicing also increased. These results suggest that Masc genes play similar roles in the sex‐determination cascade in Bombycidae .     

A viral histone H4 suppresses expression of a transferrin that plays a role in the immune response of the diamondback moth,Plutella xylostella

A transferrin (Tf) gene has been predicted from an expressed sequence tag of the diamondback moth, Plutella xylostella. It encodes 681 amino acid residues that share 80–90% sequence homologies with other lepidopteran Tfs. The gene was constitutively expressed in all developmental stages of P. xylostella. Double‐stranded RNA (dsRNA) specific to the Tf gene was prepared and microinjected into the larvae. We hypothesize that the dsRNA treatment suppressed the Tf gene expression level and it significantly inhibited haemocyte nodule formation in response to bacterial challenge. The larvae treated with dsRNA also showed a significantly enhanced susceptibility to an entomopathogenic bacterium, Bacillus thuringiensis. An endoparasitoid wasp, Cotesia plutellae, parasitized the larvae of P. xylostella, which showed significant reduction of Tf expression. The suppression of Tf expression was mimicked by transient expression of a viral gene CpBV‐H4, encoded in the symbiotic virus of C. plutellae. A truncated form of CpBV‐H4 prepared by deleting an extended N‐terminal 38 amino acid residue lost its inhibitory activity against the Tf gene expression. These results suggest that Tf of P. xylostella plays an immunological role in P. xylostella and that the suppression of its expression in the parasitized larvae is caused by a viral histone H4 in an epigenetic mode.     

Superfamily of genes encoding G protein‐coupled receptors in the diamondback moth Plutella xylostella (Lepidoptera: Plutellidae)

G protein‐coupled receptors (GPCRs) are the largest and most versatile superfamily of cell membrane proteins, which mediate various physiological processes including reproduction, development and behaviour. The diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), is one of the most notorious insect pests, preferentially feeding on cruciferous plants. P. xylostella is not only one of the world's most widespread lepidopteran insects, but has also developed resistance to nearly all classes of insecticides. Although the mechanisms of insecticide resistance have been studied extensively in many insect species, few investigations have been carried out on GPCRs in P. xylostella. In the present study, we identified 95 putative GPCRs in the P. xylostella genome. The identified GPCRs were compared with their homologues in Bombyx mori and Drosophila melanogaster. Our results suggest that GPCRs in different insect species may have evolved by a birth‐and‐death process. One of the differences among compared insects is the duplication of short neuropeptide F receptor and adipokinetic hormone receptors in P. xylostella and B. mori. Another divergence is the decrease in quantity and diversity of the stress‐tolerance gene, Mth, in P. xylostella. The evolution by the birth‐and‐death process is probably involved in adaptation to the feeding behaviour, reproduction and stress responses of P. xylostella. Some of the genes identified in the present study could be potential targets for the development of novel pesticides.     

CRISPR/Cas9-based functional characterization of the pigmentation gene ebony in Plutella xylostella

Body pigmentation is an important character of insects in adapting to biotic and abiotic environmental challenges. Additionally, based on the relative ease of screening, several genes involved in insect melanization have been used in classic genetic studies or as visual markers in constructing transgenic insects. Here, a homologue of the Bombyx mori melanization-inhibiting gene ebony, associated with the conversion of dopamine to N-β-alanyl dopamine, was identified in a global pest, Plutella xylostella. The CRISPR/Cas9 system was applied to generate multiple Pxebony knockout alleles which were crossed to produce a Pxebony knockout strain, showing darker pigmentation in larvae, pupae and adults, compared with wildtype. Interestingly, we observed that Pxebony heterozygotes displayed an intermediate darkened phenotype, indicating partial dominance between the knockout and wildtype alleles. The fitness costs of Pxebony deficiency were also assessed in the mutant strain, indicating that embryo hatchability and larval survival were significantly reduced, while the eclosion rate was not obviously affected. Our work provides a potential target for exploring CRISPR-based genetics-control systems in this economically important pest lepidopteran.     

The Bactrocera tryoni homologue of the Drosophila melanogaster sex-determination gene doublesex

A homologue of the bifunctional sex-determining gene, doublesex (dsx), has been identified in the tephritid fruit fly, Bactrocera tryoni, and has been found to be expressed in a sex-specific manner in adult flies. The male- and female-specific cDNAs are identical at their 5′ ends but differ at their 3′ ends and appear to be the products of alternate splicing. The level of identity of the sex-specific DSX proteins of B. tryoni with the D. melanogaster DSX proteins, across the region corresponding to the DNA binding domain and the oligomerization domains, is greater than 85%. Four sequence motifs which are ten to thirteen bases identical to the TRA/TRA-2 binding sites (thirteen-nucleotide repeat sequences) are present in the female-specific exon of the B. tryoni dsx gene.     

Identification and characterization of doublesex in Bemisia tabaci

Bemisia tabaci (Gennadius) is an important agricultural pest with a worldwide distribution. Although B. tabaci is known to have a unique haplodiploid reproductive strategy, its sex determination mechanism is largely unknown. In this study, we cloned the full‐length sequence of B. tabaci doublesex (Btdsx) and found that Btdsx has 28 splicing isoforms. We found two new splicing isoforms of transformer 2 (Bttra2), which encode two proteins. We also confirmed that both genes lack sex‐specific splicing isoforms. Real‐time quantitative PCR analysis showed that the expression of Btdsx and Bttra2 is higher in males than in females. RNA interference of Bttra2 affected the expression of Btdsx and vice versa. Furthermore, silencing of Bttra2 or Btdsx caused malformation of the male genitalia (anal style). It did not affect the female phenotype, but reduced the expression of vitellogenin gene in females. These results indicate that Btdsx is associated with sex determination in B. tabaci and that Btdsx and Bttra2 affect each other and are important for male genitalia formation. In addition to increasing our understanding of the roles of dsx and tra2 in the sex determination of B. tabaci, the results will be useful for studies of sex determination in other haplodiploid species.     

Cloning and functional identification of moricins from the diamondback moth,Plutella xylostella (L.)

Antimicrobial peptides (AMPs) are small‐molecule peptides that play crucial roles in insect innate immune responses. To better understand the function of AMPs in Plutella xylostella, one of the main pests of cruciferous vegetables, three full‐length cDNAs encoding moricins were cloned from Pl. xylostella. Two variants of the moricin named PxMor2 and PxMor3 were heterologously expressed and purified. A secondary structure analysis using circular dichroism demonstrated that the two peptides adopted an α‐helical structure in the membrane‐like environment, but in aqueous solution, they were present in random coiled conformation. Antimicrobial activity assays demonstrated that PxMor2 exhibited high activity against Gram‐positive Staphylococcus aureus and Gram‐negative Escherichia coli; however, PxMor3 only demonstrated high activity against E. coli. Scanning electron microscopy and confocal laser‐scanning microscopy analyses suggest that PxMors can lead to the disruption of bacterial membrane, which might be the mechanism by which PxMors inhibit bacterial growth. This study contributes to the understanding of Pl. xylostella AMPs and immune responses, and also enriches the knowledge of insect moricin.     

Expression patterns and colocalization of two sensory neurone membrane proteins in Ectropis obliqua Prout,a geometrid moth pest that uses Type‐II sex pheromones

Sensory neurone membrane proteins (SNMPs) function as essential cofactors for insect sex pheromone detection. In this study, we report two SNMPs in Ectropis obliqua Prout, a serious geometrid pest that produces typical Type‐II sex pheromones. Sequence alignments and phylogenetic analyses showed that EoblSNMP1 and EoblSNMP2 belong to two distinct SNMP subfamilies. Quantitative real‐time PCR suggested that EoblSNMP1 was male antennae‐biased, whereas EoblSNMP2 was highly expressed on male antennae but was also expressed on female antennae and other chemosensory tissues. Additionally, EoblSNMP1 and EoblSNMP2 differed in their developmental expression profiles. In situ hybridization revealed that EoblSNMP1 was sensilla trichodea I specific, whereas EoblSNMP2 was expressed in sensilla trichodea I and the sensilla basiconica; furthermore, EoblSNMP1 and EoblSNMP2 were co‐expressed in sensilla trichodea I but in different cells. This study suggests that EoblSNMP1 is functionally distinct from EoblSNMP2 in E. obliqua; EoblSNMP1 may specifically contribute to the recognition of sex pheromones, whereas EoblSNMP2 exhibits multiple olfactory roles. Our findings comprehensively reveal the expression patterns of SNMPs in a lepidopteran species that uses Type‐II sex pheromones, providing new insights into the functional evolution of SNMPs from lepidopteran moths with Type‐I sex pheromones to those with Type‐II sex pheromones.     

Identification and characterization of troponin genes in Locusta migratoria

Troponin complex comprises three subunits, namely troponin C (TpnC), troponin I (TpnI) and troponin T (TpnT), and regulates the contraction of striated muscle. We found that the locust Locusta migratoria genome has one TpnT gene (LmTpnT), one TpnI gene (LmTpnI) and three TpnC genes (LmTpnC1, LmTpnC2 and LmTpnC3). Through alternative splicing, LmTpnT and LmTpnI potentially encode two and eight isoforms, respectively. The flight muscle and the jump muscle of L. migratoria express an identical LmTpnT isoform, but different LmTpnC isoforms and LmTpnI isoforms. LmTpnC2 and LmTpnC3 both contain highly conserved residues essential for calcium binding in the EF‐hand II and IV, thus belonging two‐site isoform. LmTpnC1 contains non‐conserved substitutions in the EF‐hand II and all highly conserved residues for calcium binding in the EF‐hand IV. Mutagenesis and tyrosine fluorescence spectroscopic analysis show that both the EF‐hand II and IV of LmTpnC1 can serve as calcium‐binding site. Therefore, all three LmTpnC isoforms belong to two‐site isoform. This is in contrast to the situation in the insect with asynchronous flight muscle, which expresses both one‐site isoform and two‐site isoform of TpnC. Those results suggest that the origination of insect asynchronous flight muscle is associated with the emergence of one‐site isoform of TpnC.     

Identification and characterization of a putative sevenless homologue in the malaria vector Anopheles gambiae

Tyrosine kinase sequences were identified and characterized in Anopheles gambiae, the major vector of malaria in subsaharan Africa. One of these sequences has the characteristics expected for a homologue of the Drosophila sevenless gene, which is necessary for R7 photoreceptor cell fate determination in the developing compound eye. The putative Anopheles seven-less gene homologue is located in a telomeric region of the X chromosome and is expressed in the head of late larval and pupal stage mosquitoes. Identification of the Anopheles homologue of the sevenless gene is a first step towards the development of a dominant phenotypic marker that could be used for detecting transformed Anopheles mosquitoes in a wide variety of genetic backgrounds and, as such, could be used in the development of transgenic mosquitoes for the control of parasite transmission. Preliminary evidence for sevenless sequences were also found in DNA from blackfly, Mediterranean fruit fly and the honeybee.