Vitelline Membrane Protein 26 Mutagenesis, Using CRISPR/Cas9, Results in Egg Collapse in Plutella xylostella.

Vitelline membrane proteins (VMPs) are the main proteins that form the inner shell (vitelline membrane layer) of insect eggs and are an integral part of egg formation and embryo development. Here, we characterized the molecular structure and expression patterns of the VMP26 gene and analyzed its reproductive functions in diamondback moth, Plutella xylostella (L.), a worldwide migratory pest of cruciferous plants. The PxVMP26 gene was shown to be a single exon gene that contained an open reading frame of 852 base pairs (bp) encoding 283 amino acids. Both qPCR and western blot analyses showed that PxVMP26 was specifically expressed in female adults and was significantly highly expressed in the ovary. Further anatomical analysis indicated that the expression level of PxVMP26 in the ovarian tube with an incomplete yolk was significantly higher than that in the ovarian tube with a complete yolk. CRISPR/Cas9-induced PxVMP26 knockout successfully created two homozygous strains with 8- and 46-bp frameshift mutations. The expression deficiency of the PxVMP26 protein was detected in the mutant strains using immunofluorescence and western blot. No significant difference was found in the number of eggs laid within three days between wild and mutant individuals, but there was a lower egg hatchability. The loss of the PxVMP26 gene changed the mean egg size, damaged the structure of the vitelline membrane, and increased the proportion of abnormal eggs due to water loss, resulting in egg collapse. This first analysis of the roles of the VMP gene in the oocyte formation and embryonic development of P. xylostella, using CRISPR/Cas9 technology, provides a basis for screening new genetic control targets of P. xylostella.

Functional Characterization of Two RNA Methyltransferase Genes METTL3 and METTL14 Uncovers the Roles of m6A in Mediating Adaptation of Plutella xylostella to Host Plants.

N6-methyladenosine (m6A) is one of the major epigenetic modifications in eukaryotes. Although increasing functions of m6A have been identified in insects, its role in Plutella xylostella L. for host plant adaptation remains unclear. In the current study, we show that the m6A content of P. xylostella was relatively low in different developmental stages and tissues, with no significant differences. Two RNA methyltransferase genes, PxMETTL3 (methyltransferase-like 3) and PxMETTL14 (methyltransferase-like 14), were identified and characterized. PxMETTL3 could be transcribed into two transcripts, and PxMETTL14 had only one transcript; both of these genes were highly expressed in egg and adult stages and reproductive tissues. The CRISPR/Cas9-mediated knockout of PxMETTL3 (ΔPxMETTL3-2) or PxMETTL14 (ΔPxMETTL14-14) confirmed their function in m6A installation into RNA. Furthermore, upon transfer from an artificial diet to the host plant, the mutant strains were affected in terms of larval and pupal weight or adult emergence rate, while the wildtype (WT) strain did not exhibit any difference. In addition, the fecundity and egg hatching rate of the WT strain decreased significantly, whereas only the ΔPxMETTL14-14 mutant strain displayed significantly decreased fecundity. There seemed to be a tradeoff between the stress adaptation and reproduction in P. xylostella mediated by m6A modification. During host transfer, the expression of PxMETTL14 was consistent with the change in m6A content, which implied that PxMETTL14 could respond to host plant defense effectively, and may regulate m6A content. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the differentially expressed transcripts with changes in m6A levels revealed that the potential functions of m6A-related genes may be involved in steroid biosynthesis for larval performance and metabolic pathways for adult reproduction. Overall, our work reveals an epigenetic regulation mechanism for the rapid adaptation of P. xylostella to variations in the host environment.

Mass spectrometry imaging: An emerging technology for the analysis of metabolites in insects.

Mass spectrometry imaging (MSI) can visualize the composition, abundance, and spatial distribution of molecules in tissues or cells, which has been widely used in the research of life science. Insects, especially the agricultural pests, have received a great deal of interests from the scientists in biodiversity and food security. This review introduces the major characteristics of MSI, summarizes its application to the investigation of insect endogenous metabolites, exogenous metabolites, and the spatiotemporal changes of metabolites between insects and plants, and discusses its shortfalls and perspectives. The significance of these concerns is beneficial for future insect research such as physiology and metabolism.

Differential Profiles of Gut Microbiota and Metabolites Associated with Host Shift of Plutella xylostella.

Evolutionary and ecological forces are important factors that shape gut microbial profiles in hosts, which can help insects adapt to different environments through modulating their metabolites. However, little is known about how gut microbes and metabolites are altered when lepidopteran pest species switch hosts. In the present study, using 16S-rDNA sequencing and mass spectrometry-based metabolomics, we analyzed the gut microbiota and metabolites of three populations of Plutella xylostella: one feeding on radish (PxR) and two feeding on peas (PxP; with PxP-1 and PxP-17 being the first and 17th generations after host shift from radish to peas, respectively). We found that the diversity of gut microbes in PxP-17 was significantly lower than those in PxR and PxP-1, which indicates a distinct change in gut microbiota after host shift. Kyoto Encyclopedia of Genes and Genomes analysis revealed that the functions of energy metabolism, signal transduction, and xenobiotics biodegradation and metabolism were increased in PxP-17, suggesting their potential roles in host adaptation. Metabolic profiling showed a significant difference in the abundance of gut metabolites between PxR and PxP-17, and significant correlations of gut bacteria with gut metabolites. These findings shed light on the interaction among plants, herbivores, and symbionts, and advance our understanding of host adaptation associated with gut bacteria and metabolic activities in P. xylostella.

Endophytic effects of Aspergillus oryzae on radish (Raphanus sativus) and its herbivore, Plutella xylostella.

MAIN CONCLUSION: We provide evidence that Aspergillus oryzae not only acts as an endophyte in Raphanus sativus, but also works as a plant growth promoter and provides some protection against its herbivore, Plutella xylostella affecting its feeding rate, mortality and fitness parameters, thereby contributing to the pest population suppression. Seed inoculation of radish seeds with the fungus Aspergillus oryzae allowed its establishment as an endophyte promoting plant growth and negatively affecting fitness parameters of its major herbivore, diamondback moth, Plutella xylostella. Endophytic fungi may contribute to the growth of their host plants and enhance resistance to herbivores and diseases. We evaluated the effect of A. oryzae (Ahlburg) E. Cohn as an endophyte in radish (Raphanus sativus L.) on growth and development of the plants themselves and their major herbivore, the diamondback moth P. xylostella (L). A. oryzae colonization rates in leaves were significantly higher than in roots and stems, with a rate of 80% in leaves, 40% in stems and 20% in roots 1 week after seed inoculation. Colonization gradually decreased in the various plant tissues, and disappeared completely in roots, stems and leaves within 2, 5 and 7 weeks, respectively. A. oryzae did not affect seed germination; however, it promoted radish growth with endophytic plants attaining average heights of 116 mm compared to 99.6 mm in the controls at the third week post-inoculation. The P. xylostella fitness parameters, consumption, larval and pupal weights, and feeding on the endophytic plants were significantly lower than the controls, while larval mortality was significantly higher. Larvae fed on endophytic plants consumed 0.46 mg less leaf matter in the first week post seed inoculation and weighed 0.83 mg less as mature 4th instars than controls. We have demonstrated that A. oryzae can establish as an endophyte in R. sativus through seed inoculation providing some plant growth promotion and protection against its herbivore by increasing its mortality and negatively affecting its fitness parameters, suggesting that adopting seed treatments with A. oryzae may be beneficial in the commercial cultivation of radish.

Identification of Empoasca onukii (Hemiptera: Cicadellidae) and Monitoring of its Populations in the Tea Plantations of South China.

Tea green leafhoppers (Empoasca spp.) are considered one of the major pests in tea plantations in Asia. They are, however, difficult to monitor due to their size and flying and jumping abilities. In this study, we clarified the identification of the leafhopper species encountered in our study plantations and examined the impacts of sampling methods in estimating population abundance and sex ratio. The natural sex ratio of eggs, nymphs, and adults of tea green leafhopper and the differences between male and female were tested. Despite previous reports that Empoasca vitis (Goethe) was the major leafhopper present in our study area, our results showed that only Empoasca onukii Matsuda was found. Variation in population size over time and bias in sex ratio depending on the sampling methods were found in our monitoring experiments. In general, adult males were more attracted to yellow sticky cards than females. We believe that because female leafhoppers should be the target in pest control, yellow sticky cards may not be the most suitable monitoring or effective control of tea green leafhopper. We demonstrate the importance of understanding the implications of sampling techniques for population estimation and sex ratio bias as well as how temporal variation may affect monitoring results. Precise monitoring should take into consideration the different life histories of male and female.

Oxygen stress on age-stage, two-sex life tables and transcriptomic response of diamondback moth (Plutella xylostella).

Elucidating the genetic basis of local adaption is one of the important tasks in evolutionary biology. The Qinghai-Tibet Plateau has the highest biodiversity for an extreme environment worldwide, and provides an ideal natural laboratory to study adaptive evolution. The diamondback moth (DBM), Plutella xylostella, is one of the most devastating pests of the global Brassica industry. A highly heterozygous genome of this pest has facilitated its adaptation to a variety of complex environments, and so provides an ideal model to study fast adaptation. We conducted a pilot study combining RNA-seq with an age-stage, two-sex life table to study the effects of oxygen deprivation on DBM. The developmental periods of all instars were significantly shorter in the hypoxic environment. We compared the transcriptomes of DBM from Fuzhou, Fujian (low-altitude) and Lhasa, Tibet (high-altitude) under hypoxia treatment in a hypoxic chamber. Some DEGs are enriched in pathways associated with DNA replication, such as DNA repair, nucleotide excision repair, base excision repair, mismatch repair and homologous recombination. The pathways with significant changes were associated with metabolism process and cell development. Thus, we assumed that insects could adapt to different environments by regulating their metabolism. Our findings indicated that although adaptive mechanisms to hypoxia in different DBM strains could be similar, DBM individuals from Tibet had superior tolerance to hypoxia compared with those of Fuzhou. Local adaptation of the Tibetan colony was assumed to be responsible for this difference. Our research suggests novel mechanisms of insect responses to hypoxia stress.

Identification and characterization of odorant binding proteins in the diamondback moth, Plutella xylostella.

Odorant binding proteins (OBPs) are a group of soluble proteins functioning as odorant carriers in insect antennae, mouth parts and other chemosensory organs. However, multiple insect OBPs have been detected in other tissues and various functions have been proposed. Therefore, a detailed expression profile including stages, tissues and sexes where OBPs are expressed will assist in building the links to their potential functions, enhancing the functional studies of insect OBPs. Here, we identified 39 putative OBP genes from its genome and transcriptome sequences of diamondback moth (DBM), Plutella xylostella. The expression patterns of identified PxylOBPs were further investigated from eggs, larvae, pupae, virgin adults, mated adults, larval midgut, larval heads, adult antennae, adult heads and adult tarsi. Moreover, P. xylostella larvae and adults with and without host plants for 5 h were utilized to study the interactions between OBP expression and host plants. The results showed that most PxylOBPs were highly expressed in male and female adult antennae. The expression levels of certain PxyOBPs could be regulated by mating activities and feeding host plants. This study advances our knowledge of P. xylostella OBPs, which may help develop new strategies for more environmentally sustainable management of P. xylostella.

Spatio-temporal distribution patterns of Plutella xylostella (Lepidoptera: Plutellidae) in a fine-scale agricultural landscape based on geostatistical analysis.

A detailed knowledge on the spatial distribution of pests is crucial for predicting population outbreaks or developing control strategies and sustainable management plans. The diamondback moth, Plutella xylostella, is one of the most destructive pests of cruciferous crops worldwide. Despite the abundant research on the species's ecology, little is known about the spatio-temporal pattern of P. xylostella in an agricultural landscape. Therefore, in this study, the spatial distribution of P. xylostella was characterized to assess the effect of landscape elements in a fine-scale agricultural landscape by geostatistical analysis. The P. xylostella adults captured by pheromone-baited traps showed a seasonal pattern of population fluctuation from October 2015 to September 2017, with a marked peak in spring, suggesting that mild temperatures, 15-25 °C, are favorable for P. xylostella. Geostatistics (GS) correlograms fitted with spherical and Gaussian models showed an aggregated distribution in 21 of the 47 cases interpolation contour maps. This result highlighted that spatial distribution of P. xylostella was not limited to the Brassica vegetable field, but presence was the highest there. Nevertheless, population aggregations also showed a seasonal variation associated with the growing stage of host plants. GS model analysis showed higher abundances in cruciferous fields than in any other patches of the landscape, indicating a strong host plant dependency. We demonstrate that Brassica vegetables distribution and growth stage, have dominant impacts on the spatial distribution of P. xylostella in a fine-scale landscape. This work clarified the spatio-temporal dynamic and distribution patterns of P. xylostella in an agricultural landscape, and the distribution model developed by geostatistical analysis can provide a scientific basis for precise targeting and localized control of P. xylostella.

Fitness comparison of Plutella xylostella on original and marginal hosts using age-stage, two-sex life tables.

The diamondback moth, Plutella xylostella, is an important agricultural pest that severely damages cruciferous vegetables. Although previously considered a threat only to Brassica species, P. xylostella has been observed to feed on noncruciferous vegetables. Here, we established a population of P. xylostella on the pea Pisum sativum (PxP population). We compared this PxP population's performance on the pea host plant to a population (PxR) reared on the original host plant radish (Raphanus sativus) for several generations using an age-stage, two-sex life table and analyzed the correlations between different fitness parameters. In the 1st generation of the PxP population, survival rate of immature stage was 17%, while the survival rate of PxR was 68%; the duration of the 4th larval instar (5.30 d) and mortality (25%) of this generation were significantly longer (2.8 d) and higher (1%) than that of PxR, respectively (both p < .001). Upon long-term acclimation, the PxP fitness improved significantly, especially that the survival rate of immature stages increased to approximately 60% in the 15th, 30th, and 45th generations. However, PxP feeding on pea exhibited poorer fitness with longer larval developmental time, shorter total life span, lighter pupa, and lower fecundity in different generations compared with PxP feeding on radish. PxP feeding on pea also showed a significantly lower intrinsic rate of increase (r), net reproduction rate (R 0), finite increase rate (λ), and longer mean generation time (T) than PxP feeding on radish in all generations tested. Significant positive correlations were observed between pupal weight and female fecundity in pea-fed populations, and between female longevity and female fecundity in pea-fed and radish-fed populations. Our findings suggest that P. xylostella adaptation to pea does not improve overall fitness compared with the original host radish, making pea a marginal host for P. xylostella.

Predation Pressure in Tea (Camellia sinensis) Plantations in Southeastern China Measured by the Sentinel Prey Method.

Tea (Camellia sinensis) is an important food product with thousands of years of human use. Being a non-washable food, no pesticide residues are allowed, which increases the importance of natural means of plant protection. Predation, a component of natural pest control, is an important contributor to this, but its level and sustainability are not known in most of the areas of tea production. We quantified predation intensity using the artificial sentinel prey method in a tea-growing landscape containing remnants of the original forest vegetation in Fujian Province, China. The most common predators were chewing arthropods (49.8% of predation events) and birds (48.1%). Overall, predation rates at the edges of forest fragments (18.9% d-1) were lower than either in fragment interiors (25.4%d-1) or in the surrounding tea plantations (19.2-24.1%d-1). Arthropod predation was higher inside, and at the edge of, forest fragments than within plantations, and generally decreased with increasing distance from a fragment edge, indicating limited spillover of arthropod predators from the native habitat remnants to the cultivated matrix at the local scale. Bird predation, though, showed a different trend: it was lower on the inside of forest fragments than in the tea planation, and bird attack rates increased at increasing distances (up to 40 m) from the forest fragment edge. We also found a reciprocal relationship between attack rates by birds and arthropods, suggesting intra-guild predation. Measures protecting arthropod natural enemies could increase the combined pest suppression effect, contributing to pesticide-free tea production in China.

Mechanism and consequences for avoidance of superparasitism in the solitary parasitoid Cotesia vestalis.

A parasitoid's decision to reject or accept a potential host is fundamental to its fitness. Superparasitism, in which more than one egg of a given parasitoid species can deposit in a single host, is usually considered sub-optimal in systems where the host is able to support the development of only a single parasitoid. It follows that selection pressure may drive the capacity for parasitoids to recognize parasitized hosts, especially if there is a fitness cost of superparasitism. Here, we used microsatellite studies of two distinct populations of Cotesia vestalis to demonstrate that an egg laid into a diamondback moth (Plutella xylostella) larva that was parasitized by a conspecific parasitoid 10 min, 2 or 6 h previously was as likely to develop and emerge successfully as was the first-laid egg. Consistent with this, a naive parasitoid encountering its first host was equally likely to accept a healthy larva as one parasitized 10 min prior, though handling time of parasitized hosts was extended. For second and third host encounters, parasitized hosts were less readily accepted than healthy larvae. If 12 h elapsed between parasitism events, the second-laid egg was much less likely to develop. Discrimination between parasitized and healthy hosts was evident when females were allowed physical contact with hosts, and healthy hosts were rendered less acceptable by manual injection of parasitoid venom into their hemolymph. Collectively, these results show a limited capacity to discriminate parasitized from healthy larvae despite a viability cost associated with failing to avoid superparasitism.

CRISPR/Cas9-induced vitellogenin knockout lead to incomplete embryonic development in Plutella xylostella.

Vitellogenin (Vg) is important for insect egg maturation and embryo development. In the present study, we characterized the molecular structure and expression profile of Vg gene, and analyzed its reproductive functions in diamondback moth, Plutella xylostella (L.), a destructive pest of cruciferous crops, using CRISPR/Cas9 system. The P. xylostella Vg (PxVg) included all conserved domains and motifs that were commonly found in most insect Vgs except for the polyserine tract. PxVg gene was highly expressed in female pupae and adults. PxVg protein was detected in eggs and female adults. PxVg was mainly expressed in the fat body and its protein was detected in most tissues, except in the midgut. CRISPR/Cas9-induced PxVg knockout successfully constructed a homozygous mutant strain with a 5-base pair nucleotide deletion. No PxVg protein was found in the mutant individuals and in their ovaries. There were no significant differences between wild (WT) and mutant (Mut-5) types of P. xylostella in terms of ovariole length and the number of fully developed oocytes in newly emerged females. No significant difference was observed in the number of eggs laid within two days, but there was a lower egg hatchability (84% for WT vs. 47% for Mut-5). This is the first study presenting the functions of Vg in ovary development, egg maturation, oviposition and embryonic development of P. xylostella. Our results suggest that the reproductive functions of Vg may be species-specific in insects. It is possible that Vg may not be the major egg yolk protein precursor in P. xylostella. Other "functional Vgs" closely involved in the yolk formation and oogenesis would need to be further explored in P. xylostella.

Cell lines from diamondback moth exhibiting differential susceptibility to baculovirus infection and expressing midgut genes.

Six new cell lines were established from embryonic tissues of the diamondback moth, Plutella xylostella (L.). The cell lines showed differential characteristics, including growth in attachment or in suspension, susceptibility to a baculovirus infection and expression of genes involved in the glucosinolate detoxification pathway in P. xylostella larvae. Five of the cell lines grew attached to the culture flask and one cell line grew unattached as a suspension cell line. The cell lines had population doubling times ranging from 18 to 23 h. Among five of the P. xylostella cell lines examined for infection of a nucleopolyhedrovirus from Autographa californica, AcMNPV, four cell lines were highly susceptible to AcMNPV infection, but one was only semi-permissive to AcMNPV infection. The production of two recombinant proteins, a ß-galactosidase of bacterial origin and a secreted alkaline phosphatase of eukaryotic origin, in the P. xylostella cell lines was examined in comparison with that in the cell line Sf9 which is commonly used for recombinant protein production. In the P. xylostella cell lines, expression of three important midgut genes involved in the glucosinolate detoxification pathway, including the glucosinolate sulfatase genes GSS1 and GSS2 and the sulfatase modifying factor gene SUMF1, was detected. The P. xylostella cell lines developed in this study could be useful in in vitro research systems for studying insec-virus interactions and complex molecular mechanisms in glucosinolate detoxification and insect-plant interactions.

The impact of cover crops on the predatory mite Anystis baccarum (Acari, Anystidae) and the leafhopper pest Empoasca onukii (Hemiptera, Cicadellidae) in a tea plantation.

BACKGROUND: Empoasca onukii, the tea green leafhopper, is a key pest of tea whose control often requires the extensive use of insecticides. As a predator of the tea green leafhopper, the mite Anystis baccarum is a potential biological control agent worldwide, though little is known about how intercropping cover crops can impact its suppressing effect on E. onukii. Therefore, we conducted a field experiment to investigate how the relationship of the abundance of the predatory mite and its leafhopper prey is influenced by two different cover crops and a manually weeded inter-row treatment as a contrast to naturally growing vegetation in a tea plantation in China. RESULTS: The abundance of A. baccarum was significantly higher in tea canopies of intercropped treatments than in canopies over natural ground cover. Litter samples showed higher abundances of A. baccarum when tea was intercropped with Paspalum notatum than with natural ground cover in the first year of treatment. The abundance of E. onukii in tea canopies was higher over the bare ground treatment in the first year but the opposite was observed in the second year. CONCLUSIONS: Results suggest that the abundance of A. baccarum in a tea plantation is influenced by intercropping and it can affect its leafhopper prey, albeit with varying levels of suppression. For informing biological control and suppression of pests, long-term experiments are needed to investigate the interactions of both pest and predator with cover crop treatments. © 2019 Society of Chemical Industry.

Population characteristics of Macrocheles glaber (Acari: Macrochelidae) and Stratiolaelaps scimitus (Acari: Laelapidae) reared on a mushroom fly Coboldia fuscipes (Diptera: Scatopsidae).

Subterranean predatory mites are important biological control agents of pests in soil. In order to understand the population characteristics of two predatory mites, Macrocheles glaber Müller and Stratiolaelaps scimitus Womersley, we studied their development, survival and fecundity data under laboratory conditions using Coboldia fuscipes Meigen as a food source and analyzed them with the age-stage, two-sex life table. Macrocheles glaber had a significantly shorter developmental time, oviposition period, longevity and lower fecundity than those of S. scimitus. The intrinsic rate of increase (r), finite rate of increase (λ), net reproductive rate (R0 ), net predation rate (C0 ), and finite predation rate (ω) of M. glaber were significantly lower than those of S. scimitus. Both population parameters and computer simulation implied that S. scimitus is a potential powerful biocontrol agent compared to M. glaber.

Identification of Halloween Genes and RNA Interference-Mediated Functional Characterization of a Halloween Gene shadow in Plutella xylostella.

Ecdysteroids play an essential role in controlling insect development and reproduction. Their pathway is regulated by a group of enzymes called Halloween gene proteins. The relationship between the Halloween genes and ecdysteroid synthesis has yet to be clearly understood in diamondback moth, Plutella xylostella (L.), a worldwide Lepidoptera pest attacking cruciferous crops and wild plants. In this study, complete sequences for six Halloween genes, neverland (nvd), shroud (sro), spook (spo), phantom (phm), disembodied (dib), shadow (sad), and shade (shd), were identified. Phylogenetic analysis revealed a strong conservation in insects, including Halloween genes of P. xylostella that was clustered with all other Lepidoptera species. Three Halloween genes, dib, sad, and shd were highly expressed in the adult stage, while nvd and spo were highly expressed in the egg and pupal stages, respectively. Five Halloween genes were highly expressed specifically in the prothorax, which is the major site of ecdysone production. However, shd was expressed predominantly in the fat body to convert ecdysone into 20-hydroxyecdysone. RNAi-based knockdown of sad, which is involved in the last step of ecdysone biosynthesis, significantly reduced the 20E titer and resulted in a longer developmental duration and lower pupation of fourth-instar larvae, as well as caused shorter ovarioles and fewer fully developed eggs of P. xylostella. Furthermore, after the knockdown of sad, the expression levels of Vg and VgR genes were significantly decreased by 77.1 and 53.0%. Meanwhile, the number of eggs laid after 3 days was significantly reduced in sad knockdown females. These results suggest that Halloween genes may play a critical role in the biosynthesis of ecdysteroids and be involved in the development and reproduction of P. xylostella. Our work provides a solid basis for understanding the functional importance of these genes, which will help to screening potential genes for pest management of P. xylostella.

Host Plant-Derived miRNAs Potentially Modulate the Development of a Cosmopolitan Insect Pest, Plutella xylostella.

Plant microRNAs (miRNAs) have recently been reported to be involved in the cross-kingdom regulation of specific cellular and physiological processes in animals. However, little of this phenomenon is known for the communication between host plant and insect herbivore. In this study, the plant-derived miRNAs in the hemolymph of a cruciferous specialist Plutella xylostella were identified by small RNAs sequencing. A total of 39 miRNAs with typical characteristics of plant miRNAs were detected, of which 24 had read counts ≥ 2 in each library. Three plant-derived miRNAs with the highest read counts were validated, and all of them were predicted to target the hemocyanin domains-containing genes of P. xylostella. The luciferase assays in the Drosophila S2 cell demonstrated that miR159a and novel-7703-5p could target BJHSP1 and PPO2 respectively, possibly in an incomplete complementary pairing mode. We further found that treatment with agomir-7703-5p significantly influenced the pupal development and egg-hatching rate when reared on the artificial diet. The developments of both pupae and adults were severely affected upon their transfer to Arabidopsis thaliana, but this might be independent of the cross-kingdom regulation of the three plant-derived miRNAs on their target genes in P. xylostella, based on expression analysis. Taken together, our work reveals that the plant-derived miRNAs could break the barrier of the insect mid-gut to enter the circulatory system, and potentially regulate the development of P. xylostella. Our findings provide new insights into the co-evolution of insect herbivore and host plant, and novel direction for pest control using plant-derived miRNAs.

CRISPR/Cas9-Mediated Vitellogenin Receptor Knockout Leads to Functional Deficiency in the Reproductive Development of Plutella xylostella.

The vitellogenin receptor (VgR) belongs to the low-density lipoprotein receptor (LDLR) gene superfamily and plays an indispensable role in Vg transport, yolk deposition, and oocyte development. For this reason, it has become a promising target for pest control. The involvement of VgR in Vg transport and reproductive functions remains unclear in diamondback moths, Plutella xylostella (L.), a destructive pest of cruciferous crops. Here, we cloned and identified the complete cDNA sequence of P. xylostella VgR, which encoded 1805 amino acid residues and contained four conserved domains of LDLR superfamily. PxVgR was mainly expressed in female adults, more specifically in the ovary. PxVgR protein also showed the similar expression profile with the PxVgR transcript. CRISPR/Cas9-mediated PxVgR knockout created a homozygous mutant of P. xylostella with 5-bp-nucleotide deletion in the PxVgR. The expression deficiency of PxVgR protein was detected in the ovaries and eggs of mutant individuals. Vg protein was still detected in the eggs of the mutant individuals, but with a decreased expression level. However, PxVg transcripts were not significantly affected by the PxVgR knockout. Knockout of PxVgR resulted in shorter ovarioles of newly emerged females. No significant difference was detected between wild and mutant individuals in terms of the number of eggs laid in the first 3 days after mating. The loss of PxVgR gene resulted in smaller and whiter eggs and lower egg hatching rate. This study represents the first report on the functions of VgR in Vg transport, ovary development, oviposition, and embryonic development of P. xylostella using CRISPR/Cas9 technology. This study lays the foundation for understanding molecular mechanisms of P. xylostella reproduction, and for making use of VgR as a potential genetic-based molecular target for better control of the P. xylostella.

Structure and expression of sulfatase and sulfatase modifying factor genes in the diamondback moth, Plutella xylostella.

The diamondback moth, Plutella xylostella (L.), uses sulfatases (SULF) to counteract the glucosinolate-myrosinase defensive system that cruciferous plants have evolved to deter insect feeding. Sulfatase activity is regulated by post-translational modification of a cysteine residue by sulfatase modifying factor 1 (SUMF1). We identified 12 SULF genes (PxylSulfs) and two SUMF1 genes (PxylSumf1s) in the P. xylostella genome. Phylogenetic analysis of SULFs and SUMFs from P. xylostella, Bombyx mori, Manduca sexta, Heliconius melpomene, Danaus plexippus, Drosophila melanogaster, Tetranychus urticae and Homo sapiens showed that the SULFs were clustered into five groups, and the SUMFs could be divided into two groups. Profiling of the expression of PxylSulfs and PxylSumfs by RNA-seq and by quantitative real-time polymerase chain reaction showed that two glucosinolate sulfatase genes (GSS), PxylSulf2 and PxylSulf3, were primarily expressed in the midgut of 3rd- and 4th-instar larvae. Moreover, expression of sulfatases PxylSulf2, PxylSulf3 and PxylSulf4 were correlated with expression of the sulfatases modifying factor PxylSumf1a. The findings from this study provide new insights into the structure and expression of SUMF1 and PxylSulf genes that are considered to be key factors for the evolutionary success of P. xylostella as a specialist herbivore of cruciferous plants.