Genome Editing of Wnt-1, a Gene Associated with Segmentation, via CRISPR/Cas9 in the Pine Caterpillar Moth, Dendrolimus punctatus.
ABSTRACT: The pine caterpillar moth, Dendrolimus punctatus, is a devastating forest pest. Genetic manipulation of this insect pest is limited due to the lack of genomic and functional genomic toolsets. Recently, CRISPR/Cas9 technology has been demonstrated to be a promising approach to modify the genome. To investigate gene functions during the embryogenesis, we introduced CRISPR/Cas9 system in D. punctatus to precisely and effectively manipulate gene expressions inmutant embryos. Compared to controls, knocking out of DpWnt-1, a gene well known for its role in the early body planning, led to high embryonic mortality. Among these mutants, 32.9% of the embryos and larvae showed an abnormal development. DpWnt-1 mutants predominantly exhibited abnormal posterior segments. In addition, multiple phenotypes were observed, including the loss of limbs and the head deformation, suggesting that DpWnt-1 signaling pathway is necessary for anterior segmentation and appendage development. Overall, our results demonstrate that CRISPR/Cas9 system is feasible and efficient in inducing mutations at a specific locus in D. punctatus. This study not only lays the foundation for characterizing gene functions in a non-model species, but also facilitates the future development of pest control alternatives for a major defoliator.
Project description:Dendrolimus punctatus Walker is a serious pest affecting conifers in southern China. As extensive pesticide spraying is currently required to control D. punctatus, new control strategies are urgently needed. Chemosensory genes represent potential molecular targets for development of alternative pest control strategies, and the expression characteristics of these genes provide an indication of their function. To date, little information is available regarding chemosensory genes in D. punctatus or their expression profiles at different development stages and in various tissues. Here, we assembled and analyzed the transcriptomes of D. punctatus collected at different developmental stages and in a range of organs, using next-generation sequencing. A total of 171 putative chemosensory genes were identified, encoding 53 odorant binding proteins, 26 chemosensory proteins, 60 odorant receptors (OR), 12 gustatory receptors (GR), 18 ionotropic receptors (IR), and 2 sensory neuron membrane proteins (SNMPs). Expression analysis indicated that the antennae possess the largest number of highly expressed olfactory genes and that olfactory gene expression patterns in the eggs, larvae, and head were similar to one another, with each having moderate numbers of highly expressed olfactory genes. Fat body, ovary, midgut, and testis tissues also had similar olfactory gene expression patterns, including few highly expressed olfactory genes. Of particular note, we identified only two pheromone binding proteins and no pheromone receptors in D. punctatus, similar to our previous findings in Dendrolimus houi and Dendrolimus kikuchii, suggesting that insects of the Dendrolimus genus have different pheromone recognition characteristics to other Lepidopteran insects. Overall, this extensive expression profile analysis provides a clear map of D. punctatus chemosensory genes, and will facilitate functional studies and the development of new pest control methods in the future.
Project description:The insect chemosensory system is pivotal for interactions with their environments, and moths have especially sensitive olfaction. Exploration of the connection between the plasticity of olfactory-guided and molecular level pathways in insects is important for understanding the olfactory recognition mechanisms of insects. The pine caterpillar moth, Dendrolimus punctatus Walker, is a dominant conifer defoliator in China, and mating is the priority for adults of this species, during which sex pheromone recognition and oviposition site location are the main activities; these activities are all closely related to chemosensory genes. Thus, we aimed to identify chemosensory related genes and monitor the spectrum of their dynamic expression during the entire mating process in D. punctatus. In this study, we generated transcriptome data from male and female adult D. punctatus specimens at four mating stages: eclosion, calling, copulation, and post-coitum. These data were analyzed using bioinformatics tools to identify the major olfactory-related gene families and determine their expression patterns during mating. Levels of odorant binding proteins (OBPs), chemosensory proteins (CSPs), and odorant receptors (ORs) were closely correlated with mating behavior. Comparison with ORs from other Dendrolimus and Lepidoptera species led to the discovery of a group of ORs specific to Dendrolimus. Furthermore, we identified several genes encoding OBPs and ORs that were upregulated after mating in females; these genes may mediate the location of host plants for oviposition via plant-emitted volatiles. This work will facilitate functional research into D. punctatus chemosensory genes, provide information about the relationship between chemosensory genes and important physiological activities, and promote research into the mechanisms underlying insect olfactory recognition.
Project description:Dendrolimus punctatus walker (Lepidoptera: Lasiocampidae) is the most serious coniferous forest defoliator in China. This species has long life history, and shows different activity rhythms and light response behaviors at larval and adult stages. Insect vision system play important roles for survival and reproduction, and disturbance of photoreception may help us to control this pest. However, we know little about the visual system of D. punctatus. As opsins are the most important genes determining photoreceptor sensitivity of insects, we identified opsins of D. punctatus and analyzed their expression patterns at different development stages in this study. Four opsin genes were identified based on our transcriptome data. Phylogenetic analysis showed that there are three classical ultraviolet (UV), blue, and long-wavelength (LW) light sensitive opsin genes, and another UV-like opsin as homolog of a circadian photoreceptor, Rh7, in Drosophila melanogaster and other insects. Expression analysis indicated that the UV and UV-like opsins expression levels only fluctuated slightly during whole life stages of D. punctatus, while Blue and LW opsins were up-regulated many times at adult stage. Interestingly, the ratio of UV-opsin was much higher in eggs and larvae stages, and lower in pupa and adult stages; reversely, LW-opsin showed extremely high relative ratio in pupa and adult stages. High expression level of LW opsin in the adult stage may correlate to the nocturnal lifestyles of this species at adult stage, and different ratios of UV and LW opsins in larval and adult stages may help to explain the different visual ecologies of these two development stages of D. punctatus. This work is the foundation for further research of opsin functions and vision mechanisms of D. punctatus.
Project description:The pine moth Dendrolimus punctatus (Walker) is a common insect pest that confers serious damage to conifer forests in south of China. Extensive physiology and ecology studies on D. punctatus have been carried out, but the lack of genetic information has limited our understanding of the molecular mechanisms behind its development and resistance. Using RNA-seq approach, we characterized the transcriptome of this pine moth and investigated its developmental expression profiles during egg, larval, pupal, and adult stages. A total of 107.6 million raw reads were generated that were assembled into 70,664 unigenes. More than 30% unigenes were annotated by searching for homology in protein databases. To better understand the process of metamorphosis, we pairwise compared four developmental phases and obtained 17,624 differential expression genes. Functional enrichment analysis of differentially expressed genes showed positive correlation with specific physiological activities of each stage, and these results were confirmed by qRT-PCR experiments. This study provides a valuable genomic resource of D. punctatus covering all its developmental stages, and will promote future studies on biological processes at the molecular level.
Project description:In southern China, the masson pine caterpillar, Dendrolimus punctatus, has caused serious damage to the Pinus massoniana (Lamb.) pine forests. Here, the whole mitochondrial DNA (mtDNA) was employed to analyze the population evolution of D. punctatus and to understand the process underlying its current phylogenetic pattern. D. punctatus populations within its distribution range in China were categorized into five subgroups: central and eastern China (CEC), southwestern China (SWC), Yibin in Sichuan (SC), Baise in Guangxi (GX), and Luoding in Guangdong (GD), with a high level of haplotype diversity and nucleotide diversity among them. The genetic distances between subgroups are relatively large; however, the genetic distances between populations within the CEC subgroup were relatively small, suggesting that many populations were closely related in this subgroup. The mantel test showed that geographic distance had an important impact on the genetic distance of different geographic populations (r = 0.3633, P < 0.001). The neutrality tests, Bayesian skyline plot, and haplotype network showed that D. punctatus experienced a population expansion around 100,000 years ago. The divergence times of GX/SC, SWC, GD, and CEC were 0.347, 0.236, 0.200, and 0.110 million years ago, respectively. The SWC, CEC, and GD subgroups might have evolved from GX or SC subgroups. The population genetic structure of D. punctatus was closely related to its host tree species, geographic distance among populations, the weak flight capacity, and many eco-environment conditions.
Project description:Background:Pine moths, Dendrolimus spp. (Lasiocampidae), are serious economic pests of conifer forests. Six closely related species (Dendrolimus punctatus, D. tabulaeformis, D. spectabilis, D. superans, D. houi, and D. kikuchii) occur in China and cause serious damage to coniferophyte. The complete mito genomes of Dendrolimus genus are significant to resolve the phylogenetic relationship and provide theoretical support in pest control. Methods:The complete mitogenomes of three species (D. superans, D. houi, and D. kikuchii) were sequenced based on PCR-amplified with universal primers, which were used to amplify initial fragments. Phylogenetic analyses were carried out with 78 complete mitogenomes of lepidopteran species from 10 superfamilies. Results:The complete mitochondrial genomes of these three species were 15,417, 15,381, and 15,377 bp in length, separately. The phylogenetic analyses produced consistent results for six Dendrolimus species based on complete mitogenomes, two major clades were formed, one containing D. spectabilis clustered with D. punctatus + D. tabulaeformis, and D. superans as the sister group to this three-taxon clade, the other containing D. kikuchii and D. houi. Comparative analyses of the congeneric mitochondrial genomes were performed, which showed that non-coding regions were more variable than the A+T rich region. The mitochondrial nucleotide diversity was more variable when compared within than among genus, and the concatenated tRNA region was the most conserved and the nd6 genes was the most variable.
Project description:A novel cypovirus (designated DpCPV-MC) was isolated from the pine moth Dendrolimus punctatus using serial in vivo cloning procedures. DpCPV-MC occurs in typical polyhedral occlusion bodies, containing a number of spherical virions. Laboratory bioassays indicated that the infectivity of DpCPV-MC against second-instar Spodoptera exigua larvae does not differ significantly from that of Dendrolimus punctatus cypovirus 1. Full-length amplification of the DpCPV-MC cDNAs identified 16 dsRNA genome segments. Each segment encodes one open reading frame with unique conserved terminal sequences at the 5' and 3' ends, which differ from those of all previously reported cypoviruses. On a phylogenetic tree based on the amino acid sequences of the polyhedrin of 19 cypovirus species, DpCPV-MC was closest to the type-4 cypoviruses. Homology searches showed that ten segments of DpCPV-MC (S1, S2, S3, S4, S5, S7, S8, S9, S12, and S13) encode putative CPV structural and nonstructural proteins, three segments (S6, S10 and S14) encode putative insect proteins or other viral proteins, and the other three segments (S11, S15, and S16) encode proteins that have no obvious sequence similarity to any known protein. Based on RNA secondary structures analysis, two segments of them (S11 and S16) were predicted to possibly transcript less efficiently than the other segments. We speculate that DpCPV-MC is composed of several genotypes. The ten CPV-related segments constantly exist in all genotypes, and one or two of the six CPV-unrelated segments co-exist with the ten CPV-related segments in one DpCPV-MC genotype, thus each virion contains no more than 12 segments. Based on our results and the literature, DpCPV-MC is a new cypovirus (Cypovirus 22, strain DpCPV-22).