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.

[Analysis of the Influence of Temperature on the Retrieval of Ozone Vertical Profiles Using the Thermal Infrared CrIS Sounder].

Ozone is a particularly critical trace gas in the Earth's atmosphere, since this molecule plays a key role in the photochemical reactions and climate change. The TIR measurements can capture the variability of ozone and are weakly sensitive to the lowermost tropospheric ozone content but can provide accurate measurements of tropospheric ozone and higher vertical resolution ozone profiles, with the additional advantage that measurements are also possible during the night. Because of the influence of atmospheric temperature, the ozone profile retrieval accuracy is severely limited. This paper analyze and discuss the ozone absorption spectra and weighting function sensitivity of temperature and its influence on ozone profile retrieval in detail. First, we simulate the change of atmospheric transmittance and radiance by importing 1 K temperature uncertainty, using line-by-line radiative transfer mode under 6 different atmosphere modes. The results show that the transmittance change ratio for 1 K temperature variation was consistent with the transmittance change ratio for 5%-6% change of ozone density variation in all layers of the profile. Then, we calculate the change of weighting function by a temperature error of 1 K, using the Community Radiative Transfer Model (CRTM) for the Cross-track Infrared Sounder (CrIS) on the Suomi National Polar-orbiting Partnership (Suomi NPP) satellite and calculate the corresponding change of retrieval result. The results demonstrate that CrIS is sensitive to Ozone in the middle to upper stratosphere, with the peak vertical sensitivity between 10-100 hPa and the change of weighting function for 1 K temperature variation was consistent with 6% change in the ozone profile. Finally, the paper retrieves ozone profiles from the CrIS radiances with a nonlinear Newton iteration method and use the eigenvector regression algorithm to construct the a priori state. In order to resolve the problem of temperature uncertainty and get high accuracy ozone profile, atmospheric temperature profile and ozone profile are simultaneously retrieved. Comparison of the CrIS retrieved ozone profile with high-vertical-resolution ozonesonde profiles provided by the World Ozone and Ultraviolet Radiation Data Centre (WOUDC) and ERA-Interim ozone profiles indicated that the retrieved ozone profiles are in good agreement with the ozonesonde profiles, and a notable improvement in this algorithm than the retrieval without atmospheric temperature profile, are also better than the ECMWF model profiles. The relative differences are less than 20% for the stratosphere and 50% for the lower troposphere.

[Simulation of atmospheric temperature and moisture profiles retrieval from CrIS observations].

In order to get higher vertical resolution atmosphere profile information, the present paper retrieves atmospheric temperature and moisture profiles from the Cross-track Infrared Sounder (CrIS) on the newly-launched Suomi National Polar-orbiting Partnership (Suomi NPP) and future Joint Polar Satellite System (JPSS) with a nonlinear Newton iteration method by using the profiles retrieved via statical regression method as the first guess, and the issue of channel selection is discussed. The retrieved profiles are compared with radiosonde observations, and National Centers for Environmental Prediction (NCEP) Global Data Assimilation System (GDAS) analyses show that the physical retrievals of temperature and moisture are in good agreement with the distributions from GDAS analysis fields and radiosonde observations, and have a notable improvements of the atmospheric profile retrieval accuracy as compared with the eigenvector regression algorithm. For pressures between 200 and 700 hPa the accuracy is of the order of 1 K for the temperature profile, and 20% for the relative humidity profile is consistent with the jacobian peaks of the selected channels.

Sast1-mediated manifold effects inhibit Plutella xylostella fertility.

BACKGROUND: Plutella xylostella (Linnaeus) is a destructive pest of cruciferous crops due to its strong reproductive capacity and extensive resistance to pesticides. Seminal fluid proteins (SFPs) are the main effective factors that determine the reproductive physiology and behaviour of both sexes. Although an increasing number of SFPs have been identified, the effects of astacins in SFPs on agricultural pests have not yet been reported. Here, we elucidated the mechanisms by which Sast1 (seminal astacin 1) regulates the fertility of Plutella xylostella (L.). RESULTS: PxSast1 was specifically expressed in the testis and accesssory gland. CRISPR/Cas9-induced PxSast1 knockout successfully constructed two homozygous mutant strains. Sast1 impaired the fertility of P. xylostella by separately regulating the reproductive capacity of males and females. Loss of PxSast1, on the one hand, significantly decreased the ability of males to mate and fertilize, mainly manifested as shortened mating duration, reduced mating competitiveness and decreased eupyrene sperm production; on the other hand, it significantly inhibited the expression of chorion genes in females, resulting in oogenesis deficits. Simultaneously, for mated females, the differentially expressed genes in signalling pathways related to oogenesis and chorion formation were significantly enriched after PxSast1 knockout. CONCLUSION: These analyses of the functions of PxSast1 as the regulator of spermatogenesis and oogenesis establish its importance in the fertility process of P. xylostella, as well as its potential as a promising target for genetic regulation-based pest control. © 2024 Society of Chemical Industry.

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.

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.

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.

Transcriptome profiling of the Plutella xylostella (Lepidoptera: Plutellidae) ovary reveals genes involved in oogenesis.

BACKGROUND: As a specialized organ, the insect ovary performs valuable functions by ensuring fecundity and population survival. Oogenesis is the complex physiological process resulting in the production of mature eggs, which are involved in epigenetic programming, germ cell behavior, cell cycle regulation, etc. Identification of the genes involved in ovary development and oogenesis is critical to better understand the reproductive biology and screening for the potential molecular targets in Plutella xylostella, a worldwide destructive pest of economically major crops. RESULTS: Based on transcriptome sequencing, a total of 7.88Gb clean nucleotides was obtained, with 19,934 genes and 1861 new transcripts being identified. Expression profiling indicated that 61.7% of the genes were expressed (FPKM≥1) in the P. xylostella ovary. GO annotation showed that the pathways of multicellular organism reproduction and multicellular organism reproduction process, as well as gamete generation and chorion were significantly enriched. Processes that were most likely relevant to reproduction included the spliceosome, ubiquitin mediated proteolysis, endocytosis, PI3K-Akt signaling pathway, insulin signaling pathway, cAMP signaling pathway, and focal adhesion were identified in the top 20 'highly represented' KEGG pathways. Functional genes involved in oogenesis were further analyzed and validated by qRT-PCR to show their potential predominant roles in P. xylostella reproduction. CONCLUSIONS: Our newly developed P. xylostella ovary transcriptome provides an overview of the gene expression profiling in this specialized tissue and the functional gene network closely related to the ovary development and oogenesis. This is the first genome-wide transcriptome dataset of P. xylostella ovary that includes a subset of functionally activated genes. This global approach will be the basis for further studies on molecular mechanisms of P. xylostella reproduction aimed at screening potential molecular targets for integrated pest management.