G1 Generation Research Articles

Growth, survival and fatty acids profile of sea urchins, Paracentrotus lividus juveniles fed with Ulva spp. and maize in aquaculture production. First results using G1 generation in Portugal

Growth, survival and fatty acids profile of sea urchins, Paracentrotus lividus juveniles fed with Ulva spp. and maize in aquaculture production. First results using G1 generation in Portugal

Review of two-phase emission detectors R&D (Dedicated to the memory of Prof. Boris A. Dolgoshein)

This review is dedicated to the 90th anniversary of the outstanding experimental physicist Boris Anatolyevich Dolgoshein, in whose laboratory exactly 50 years ago the first two-phase emission detector has been created. Today two-phase emission detectors found the best application in the most sensitive at the moment experiments searching for cold dark matter in the form of weakly interacting massive particles (WIMPs). Multi-ton active mass WIMP detectors of the upcoming G3 generation shall become sensitive to solar neutrinos interactions, to double-beta decay of isotopes containing in the working media with naturally occurring isotope abundances. The RED-100 detector constructed at NRNU MEPhI will be used for investigation of the reactor neutrino elastic coherent scattering off xenon nuclei at the Kalinin NPP in 2021. ProtoDUNE-DP prototype emission detector (6x6x6 m3 300 tons LAr) is currently being assembled at the CERN Neutrino Platform in support of the DUNE project focused on investigation of high energy neutrino oscillations. Thus the detector technology invented at MEPhI 50 years has demonstrated a great potential to be used in a variety of fundamental research programs.

Genetic transformation of mosquitoes by microparticle bombardment.

Mosquitoes constitute the major living beings causing human deaths in the world. They are vectors of malaria, yellow fever, dengue, zika, filariases, chikungunya, among other diseases. New strategies to control/eradicate mosquito populations are based on newly developed genetic manipulation techniques. However, genetic transformation of mosquitoes is a major technical bottleneck due to low efficiency, the need of sophisticated equipment, and highly trained personnel. The present report shows the transgenerational genetic transformation of Aedes aegypti, using the particle inflow gun (PIG), by integrating the ecfp gene in the AAEL000582 mosquito gene with the CRISPR-Cas9 technique, achieving a mean efficiency of 44.5% of bombarded individuals (G0) that showed ECFP expression in their tissues, and a mean of 28.5% transformation efficiency measured on G1 individuals. The same transformation technique was used to integrate the egfp/scorpine genes cloned in the Minos transposon pMinHygeGFP into the Anopheles albimanus genome, achieving a mean efficiency of 43.25% of bombarded individuals (G0) that showed EGFP expression in their tissues. Once the technique was standardized, transformation of Ae. aegypti neonate larvae and An. albimanus eggs was achieved when exposed to gold microparticle bombardment. Integration of genes and heterologous protein expression were confirmed by PCR, sequencing, fluorescent microscopy, mass spectrometry, Western blot and dot blot analyses. Transgenerational inheritance of the transgenes was observed only on Ae. aegypti, as all transformed An. albimanus individuals died at the pupal stage of the G0 generation.

Status of insecticide resistance and biochemical characterization of chlorpyrifos resistance in Sogatella furcifera (Hemiptera:Delphacidae) in Sichuan Province, China

The white-backed planthopper, Sogatella furcifera (Horváth) (Hemiptera, Delphacidae), is an energetic rice insect pest in rice production or rice-growing areas. Due to excessive use of the chemical insecticide, S. furcifera has produced the high resistance to some frequently used insecticides. In this paper, the resistance levels of S. furcifera from the eight different areas of Sichuan Province against the five chemicals were monitored by using the rice seedling dipping during 2017–2018 to understand the resistance levels. The results showed that most of all populations have developed low or moderate level of resistance for chlorpyrifos (3.4 to 44.3-fold) and thiamethoxam (3.9- to 15.5-fold), the populations in the LS (1.7 to 5.4- fold)and WS (1.6 to 5.0- fold) regions were still sensitive or low resistance levels compared with other local populations. Almost all populations displayed the susceptible to imidacloprid (0.9- to 5.0-fold), buprofezin (0.9- to 4.3-fold) or low levels of resistance to pymetrozine (1.5- to 6.8-fold). The synergism experiment indicated that P450 enzymes may be important contributed to the metabolic detoxification of chlorpyrifos. The cross-resistance bioassay showed that there was no cross-resistance between chlorpyrifos and triflumezopyrim, but for sulfoxaflor, in the XY17 population. The relative expression level of twelve insecticide resistant-related P450 genes were analyzed by using qRT-PCR and found that CYP4C77, CYP418A1, CYP418A2, CYP408A3 and CYP6ER4 were significantly more expressed in the 3rd-instar nymph of the XY17 and XY18 field populations. To determine the main resistant-related P450 gene for chlorpyrifos, the relative expression level of five P450 genes were detected by using qRT-PCR from the G2 and G4 generation of XY17 under the pressure with LC50 of chlorpyrifos. The results showed that CYP6ER4 was significantly up-regulated expression in XY17 G2 and G4 generations population over 700-fold (P < 0.01). The full length and proteins tertiary structure were also cloned and predicted. Meanwhile, the function of CYP6ER4 was analyzed by RNA interference and the results indicated that the relative expression of CYP6ER4 in the XY17 (G4) population after injected dsRNA was lower than that in the dsGFP injected group. Moreover, the mortality rates of the S. furcifera treated with the LC50 concentration of chlorpyrifos after dsRNA microinjection was significantly higher than that of the dsGFP injected group 72 h after treatment (P < 0.01). Therefore, the overexpression of CYP6ER4 may be one of the primary factors in the development of chlorpyrifos resistance in S. furcifera.

Introduction and evaluation on the US West Coast of a new strain (Midori) of Pacific oyster (Crassostrea gigas) collected from the Ariake Sea, southern Japan

We describe the introduction, taxonomic identification and genetic parameters for farm traits of the Midori strain of the Pacific oyster Crassostrea gigas - a possible alternative to the widely farmed Miyagi strain on the US West Coast. Broodstock (G0) oysters collected from the mouth of the Midori river and near Iwajima Island, Ariake Bay, Japan were identified as C. gigas based on the nuclear internal transcribed spacer (ITS) region and 28S gene as well as a mitochondrial (COI) marker. G0 and G1 generations and G2 larvae and spat were maintained in quarantine conditions and subjected to numerous histological exams and PCR tests for detection of pathogens of concern. After the G1 generation and G2 early life stages were found to be free of detectable diseases, G2 spat were planted at farm test sites and adults were used to produce a G3 generation. This G3 generation was cultured under non-quarantine conditions and families were planted at sub-tidal and inter-tidal test sites, in Oregon and California, respectively. Field performance traits were measured to determine the potential for improvement through selection. Overall, Midori oysters showed significantly lower values for harvest yield, survival, individual average weight and dry meat weight compared with oysters from a selected stock of the Miyagi strain of Pacific oyster; however, shell shape index (a measure of shell depth) was significantly greater in Midori oysters. Heritability estimates for group traits at harvest (survival, yield and individual average harvest weight) were high, whereas heritability estimates for individual traits (individual weight, wet meat weight and dry meat weight) and for shell dimension measurements (shell length, shell width and shell depth) were moderate-to-high. Family-by-site (GxE) correlations were low-to-moderate for all measured traits. Genetic correlations were positive and moderate-to-high among group traits and shell dimensions. In contrast, correlations between shell shape and other traits had small-to-moderate negative values but these were not statistically significant. Estimated potential genetic gain for dry meat weight was high (46%) whereas estimated gains for yield, individual average weight, individual weight were high-to-moderate (approximately 10%). In contrast, shell shape showed very little potential for genetic gain through selection. Overall, the Midori strain of the Pacific oyster shows promise as a deep-cupped oyster for the half-shell trade. Statement of relevanceWe describe the introduction of a new strain of Pacific oyster (Midori) collected from southern Japan to the US West Coast. Rigorous quarantine protocols and disease tests were applied to ensure that the risks of introducing diseases were minimized. The Midori strain was found to have a deeper cup than the commonly farmed Miyagi strain but the potential estimated gain for improving this trait through selection was small.

Nanostructures of PAMAM Dendrimers in Drug Delivery System for 5-Fluorouracil

In this article, we studied five noncovalent structures for adsorption of 5 fluorouracil drug (5 FL) on poly(amidoamine) G0 generation dendrimer (PAMAMG0) carrier using M06-2X and B3LYPfunctionals. We investigate the quantum molecular descriptors and the binding and solvation energies in gas phase and aqueous solution. The energetic stability of non-bonded species (PAMAMG0/5-FL1-5) was shown through evaluation of binding free energies. The solvation free energies of PAMAMG0/5-FL1-5 are negative, indicating that the solvation process is spontaneous. We considered quantum molecular descriptors such as electrophilicity power and global hardness and found reduced toxicity of 5-FL drug near PAMAMG0 carrier as well as facilitated drug release. The AIM (Atoms In Molecule) analysis for all PAMAMG0/5-FL1-5 structures demonstrated that the pseudo-hydrogen and hydrogen bonds are essential in the functionalization of PAMAMG0 with 5-FL drug. We found thatthe structure in which 5-FL drug interacts with CO functional groups of PAMAMG0 is the most stable configuration

Bombyx mori kynurenine 3‐monooxygenase gene editing and insect molecular breeding using the clustered regularly interspaced short palindromic repeat/CRISPR associated protein 9 system

Genome editing by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated protein (Cas)9, a third-generation gene scissors, and molecular breeding at the genome level are attracting considerable attention as future breeding techniques. In the present study, genetic and phenotypic analyses were conducted to examine the molecular breeding of Bombyx mori through CRISPR/Cas9-mediated editing of the kynurenine 3-monooxygenase (KMO) gene. The synthesized guide RNAs (gRNAs) were analyzed using T7 endonuclease I after introduction into the BM-N silkworm cell line. To edit the silkworm gene, K1P gRNA, and Cas9 complexes were microinjected into silkworm embryos. After microinjection, the hatching rate and the incidence of mutation were determined as 18.1% and 60%, respectively. Gene mutation was verified in the heterozygous G0 generation, but no phenotypic change was observed; however, certain embryos and moths produced through sib-mating had significant differences compared to the wild-type. In successive generations, a distinct phenotypic change was also observed by continuous mating. Thus, although there are limitations in the phenotypic expression in breeding through the induction of deletion mutations, as in the present study, the process is believed to yield successful results within a shorter period compared to traditional breeding and is safer than transgenic technology.

Genome-Wide Association Study of Body Weights in Hu Sheep and Population Verification of Related Single-Nucleotide Polymorphisms.

Body weight (BW) is a critical economic trait for meat production in sheep. The current study aimed to perform a genome-wide association study (GWAS) to detect significant single-nucleotide polymorphisms (SNPs) that are associated with BW in Hu sheep. The comparison and analysis of the G1 and G2 generations of a nucleus meat Hu sheep breeding herd revealed four SNPs identified by GWAS. The subsequent verification of the significant SNP loci in the Hu sheep G3 generation nucleus herd also detected nine SNPs in significant SNP regions. Two SNPs were significantly associated with the BW of Hu sheep (p < 0.05). OARX_76354330.1 and s64890.1 could be identified as functional SNPs for the growth traits of Hu sheep. CAPN6, as a candidate gene, was significantly different in the biceps femoris and longissimus dorsi muscles of weaning (60-day) and 6-month sheep, which facilitated the discovery of causal variants for BW and contributed to the marker-assisted selection breeding of Hu sheep.

Gene Knockout in a Cephalopod

Research using squid as a model led to seminal breakthroughs in diverse areas of biology and biophysics. Despite their biological novelties, such as possessing by far the largest brains among invertebrates, highly plastic systems for camouflage, and an advanced system for recoding genetic information as it passes through mRNA, squid and other cephalopods are not widely used in research today. This is mostly because at present they are genetically intractable. In this work we demonstrate the first gene knockout in a cephalopod. The pigments in squid chromatophores and retinas are ommochromes. In many organisms, the first step in ommochrome biosynthesis is catalyzed by Tryptophan 2,3 Dioxygenase (TDO). We show that in the squid Doryteuthis pealeii TDO message is highly expressed in chromatophores and eyes, and that pigmentation can be blocked in developing embryos using TDO-specific antagonists. Likewise, TDO knockouts in developing embryos using the CRISPR-Cas9 system efficiently eliminated pigmentation. By fertilizing oocytes in vitro and precisely timing CRISPR-Cas9 delivery, the degree of pigmentation could be finely controlled. Genotyping of hatchlings through deep sequencing revealed highly efficient gene knockouts (routinely greater than 90%) in the G0 generation. These results open a pathway for exploring gene function in cephalopods and the associated biological innovations.

Subjective employment perspective among older workers with and without migrant background in Germany—Results of the lidA cohort study

ObjectivesThe aim of this study was to investigate the subjective employment perspective in higher working age for different employee groups with migrant background (EMB) and without (non‐EMB), meaning willing, being able, and planning to work until the individual state pension age (iSPA).MethodsA representative sample of socially insured employees born in 1959 or 1965 was surveyed in 2011, 2014, and 2018 with computer‐assisted personal interviews. The current cross‐sectional analysis is based on data from the third study wave (n = 3286) of the lidA cohort study. EMB were differentiated via generation (first generation, G1, vs second generation, G2) or nationality (German vs foreign). Applying bivariate statistics with the tests of independence and block‐wise logistic regressions, group differences were investigated. Sex, age, educational level, net household income, health, and work factors were considered as covariates.ResultsWhen comparing subgroups of EMB, significant differences appeared in bivariate analyses for willing and planning to work. G1 were to a higher degree planning to work longer than G2 and those with foreign nationality were more willing and planning than those with German nationality. Multivariate analyses revealed significant differences of G1 and non‐EMB for planning, being significant in the fully adjusted model, but not for willing.ConclusionThe findings underline the need for differentiation of migrant groups in social research and policy. When it comes to extended working lives, the first‐generation migrant group, as well as foreigners may constitute risk groups and require increased attention from a work, health, and economic point of view.

Functionalization of PAMAM dendrimers with curcumin: Synthesis, characterization, fluorescent improvement and application on PET polymer

Environmentally friendly and healthier demands of people lead to grow the application of the natural dyes, although some difficulties limit their usages. This means that overcoming these issues will suggest the exchange of synthetic dyes having many environmental problems with natural dyes. In the current study, to develop the natural dyes, we have enhanced the fluorescence intensity of yellow-emitting curcumin dye, by its covalently bonding to PAMAM dendrimer from G0, G1, G2 generations. The chemical structures of modified materials have been confirmed by FTIR, 1HNMR, 13CNMR, UV–Vis, DSC and Elemental Analysis techniques. The synthesized compounds have been used for polyethylene terephthalate (PET) fabrics dyeing at high temperature to study their color characteristics. On the other hand, the spectral characteristics of dendrimers have been evaluated in organic solvents with different polarity. The influence of dendrimer generation on the fluorescence intensity and quantum yield has also been described. The results showed that the new dendrimers can be used for PET fabrics coloration.

Accelerated Synthesis of Surface Functionalized Mannosylated Dendrimers Built on Cyclotriphosphazene Core

ABSTRACTThe syntheses of five propargylated dendrimer scaffolds ranging from 2, 3, 4, 6, and 12 surface groups are described together with a 2-azidoethyl α-D-mannopyranoside. The former is appended to the core structure using highly efficient copper-catalysed azide-alkyne cycloaddition (CuAAC) (“click reaction”) to provide glycodendrimers in an accelerated approach. Two of the core structures are based on cyclotrisphosphazene, thus expanding the scope of the “onion-peel” strategy to build dendritic architectures with a large number of surface groups at the G1 generation only.

Identification and functional characterization of doublesex gene in the testis of Spodoptera litura.

Fusion of the testis occurs in most Lepidoptera insects, including Spodoptera litura, an important polyphagous pest. Testicular fusion in S. litura is advantageous for male reproduction, and the molecular mechanism of fusion remains unknown. Doublesex influences the formation of genitalia, the behavior of courtship, and sexually dimorphic traits in fruit-fly and silkworm, and is essential for sexual differentiation. However, its purpose in the testis of S. litura remains unknown. The doublesex gene of S. litura (Sldsx) has male-specific SldsxM and female-specific SldsxF isoforms, and exhibits a higher expression level in the male testis. At the testicular fusion stage (L6D6), Sldsx attained the highest expression compared to the pre-fusion and post-fusion periods. Moreover, Sldsx had a higher expression in the peritoneal sheaths of testis than that of germ cells in the follicle. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9) was applied to S. litura to determine the role of Sldsx. A mixture of single guide RNA messenger RNA and Cas9 protein (300 ng/μL each) was injected into eggs within 2 h following oviposition. CRISPR/Cas9 successfully induced genomic mutagenesis of Sldsx at G0 generation. The mutant males had smaller testis surrounded by less tracheae. Moreover, the mutant males had abnormal external genitalia and could not finish mating with wild-type females. Additionally, testes were fused for almost all mutant males. The results showed that Sldsx was not related to testicular fusion, and is required for both testis development and the formation and function of external genitalia in S. litura. The main roles of doublesex on the male are similar to other insects.

Estimation of genetic parameters for upper thermal tolerance and growth-related traits in turbot Scophthalmus maximus using the Bayesian method based on Gibbs sampling

In order to carry out the genetic improvement of turbot upper thermal tolerance, it is necessary to estimate the genetic parameters of UTT (upper thermal tolerance) and growth-related traits. The objective of this study was to estimate genetic parameters for BW (body weight) and UTT in a two-generational turbot (Scophthalmus maximus L.) pedigree derived from four imported turbot stocks (England, France, Denmark and Norway). A total of 42 families including 20 families from G1 generation and 22 families from G2 generation were used to test upper thermal tolerance (40–50 animals per family) in this study and the body weight of individuals were measured. The heritability of BW and UTT and the correlation between these two traits were estimated based on an individual animal model using Bayesian method based on two types of animal models with and without maternal effects. These results showed that the heritabilities for BW and UTT and phenotypic and genetic correlations between the two traits estimated from model without maternal effects were 0.239±0.141, 0.111±0.080, 0.075±0.026 and–0.019±0.011, respectively. The corresponding values from model with maternal effects were 0.203±0.115, 0.055±0.026, 0.047±0.034 and–0.024±0.028, respectively. The maternal effects of BW and UTT were 0.050±0.017 and 0.013±0.004, respectively. The maternal effects had a certain influence on the genetic evaluation of the two traits. The findings of this paper provided the necessary background to determine the best selection strategy to be adopted in the genetic improvement program.

Dendrimer ligands-capped CH3NH3PbBr3 perovskite nanocrystals with delayed halide exchange and record stability against both moisture and water

CH3NH3PbBr3 perovskite nanocrystals (NCs) suffer from poor stability because of their high sensitivity to environmental moisture and water. To solve this problem, previous works mainly focus on embedding perovskite NCs into water-resistant matrix to form large composites (size of microns or larger). As an alternative solution without serious changing of NC size, enhancing the stability of perovskite NCs themselves by ligand engineering is rarely reported. In this work, we used hyperbranched polyamidoamine (PAMAM) dendrimers with two different generations (G0 and G4) to synthesize CH3NH3PbBr3 perovskite NCs with high photoluminescence (PL) quantum yields (QY) above 70% and a new record stability. A novel dendrimers generation-dependent stability of perovskite NCs was observed. The water-resistance time is 18 h (27 h) for perovskite NCs capped by G0 (G4) generation of PAMAM, which is 7 times (11 times) longer than that of traditional oleic acid-capped NCs. Similar PAMAM generation-related stability is also observed in moisture-resistance tests. The stability time against moisture is 500 h (800 h) for G0 (G4) generation of PAMAM-capped perovskite NCs, which is a new record stability time against moisture for CH3NH3PbBr3 perovskite NCs. In addition, our results also indicate that PAMAM ligands outside perovskite NCs can dramatically slow down the speed of halide exchange. Even for the mixture of perovskite NCs with two different halide composition, the original luminescence properties of PAMAM-capped perovskite NCs can retain after mixing. In view of slow halide exchange speed, excellent water and moisture stability, PAMAM dendrimers-capped perovskite NCs and their mixture are available as color conversion single layer in fabrication of light-emitting diodes (LED).

Excision of Nucleopolyhedrovirus Form Transgenic Silkworm Using the CRISPR/Cas9 System.

The CRISPR/Cas9-mediated genome engineering has been shown to efficiently suppress infection by disrupting genes of the pathogen. We recently constructed transgenic lines expressing CRISPR/Cas9 and the double sgRNA target Bombyx mori nucleopolyhedrovirus (BmNPV) immediate early-1 (ie-1) gene in the silkworm, respectively, and obtained four transgenic hybrid lines by G1 generation hybridization: Cas9(-)/sgRNA(-), Cas9(+)/sgRNA(-), Cas9(-)/sgRNA(+), and Cas9(+)/sgRNA(+). We demonstrated that the Cas9(+)/sgRNA(+) transgenic lines effectively edited the target site of the BmNPV genome, and large fragment deletion was observed after BmNPV infection. Further antiviral analysis of the Cas9(+)/sgRNA(+) transgenic lines shows that the median lethal dose (LD50) is 1,000-fold higher than the normal lines after inoculation with occlusion bodies. The analysis of economic characters and off-target efficiency of Cas9(+)/sgRNA(+) transgenic hybrid line showed no significant difference compared with the normal lines. Our findings indicate that CRISPR/Cas9-mediated genome engineering more effectively targets the BmNPV genomes and could be utilized as an insect antiviral treatment.

Immune response of Exopalaemon carinicauda infected with an AHPND-causing strain of Vibrio parahaemolyticus

To investigate the immune response of Exopalaemon carinicauda infected with an AHPND-causing strain of Vibrio parahaemolyticus (VPAHPND), three-generation breeding of shrimp selected for their survival to VPAHPND infection was applied to explore the relationship between immune parameters and AHPND-resistant capacity of E. carinicauda. In this study, the LD50 dose of 48 h and survival rates at 144 h of shrimp to VPAHPND increased from 106.0 to 106.6 cfu ml−1 and from 26.67% to 36.67% by three successive generations selection, respectively, while there was no significant difference between the first and second generation (p > .05). Then the immune parameters including vibrio density, total hemocyte counts (THCs), hemocyanin (HEM) concentration, antibacterial activity, activities of four immune enzymes, and expressions of eight immune-related genes were determined in the shrimp of the first (G1) and the third selective generation (G3). The results showed that the shrimp in G1 and G3 generation cleared most of VPAHPND infecting hepatopancreas during 24 h and 6 h post injection, respectively. The levels of THCs, HEM concentration, antibacterial activity, immune enzymes including lysozyme (LZM) activity, alkaline phosphatase (AKP) activity in cell-free hemolymph, and the expression levels of Tollip, ALF, cathepsin B in hemocytes and hepatopancreas, crustin, LZM, SR in hepatopancreas and LGBP in hemocytes were higher in G3 generation than in G1 generation after infection with VPAHPND, suggesting that these parameters may serve as potential disease-resistant indicators for evaluating the physiological status and disease-resistant capability of shrimp when infected with VPAHPND. To further test the role of above genes in the shrimp immune response, RNAi was used to suppress their expressions and a significant decrease in survival was observed in knockdown shrimp infected with VPAHPND as compared to controls.

CRISPR/Cas9-mediated knockout of two eye pigmentation genes in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae)

The brown planthopper Nilaparvata lugens is one of the most destructive insect pests in Asia, demonstrating high fertility and causing huge crop losses by sucking sap of rice as well as transmitting viruses. However, functional genomic studies on N. lugens are seriously constrained by lack of genetic tools. Here, we employed two eye pigmentation genes to generate germ-line mutations in N. lugens using the CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated) system. We showed that injection of single guide RNA of the cinnabar gene of N. lugens (Nl-cn) into pre-blastoderm eggs induced insertion and deletion (indels) in the founder generation (G0), which were heritably transmitted to the following G1 generation, leading to bright red compound eyes and ocelli. Mutations of N. lugens white (Nl-w) generated a high mutant rate of up to 27.3%, resulting in mosaic eyes consisting of white and lightly pigmented ommatidia in both G0 and G1 individuals. The specificity of CRISPR/Cas9-mediated mutagenesis was further bolstered by PCR and RNA interference-based knockdown analysis. These results show that CRISPR/Cas9-mediated gene editing is achievable in a hemipteran insect, offering a valuable tool for the study of functional genomics and pest management in this planthopper species.

CRISPR/Cas9-mediated PBP1 and PBP3 mutagenesis induced significant reduction in electrophysiological response to sex pheromones in male Chilo suppressalis.

Pheromone‐binding proteins (PBPs) are thought to bind and transport sex pheromones onto the olfactory receptors on the dendrite membrane of olfactory neurons, and thus play a vital role in sex pheromone perception. However, the function of PBPs has rarely been demonstrated in vivo. In this study, two PBPs (PBP1 and PBP3) of Chilo suppressalis, one of the most notorious pyralid pests, were in vivo functionally characterized using insects with the PBP gene knocked out by the CRISPR/Cas9 system. First, through direct injection of PBP‐single guide RNA (sgRNA)/Cas9 messenger RNA into newly laid eggs, a high rate of target‐gene editing (checked with polled eggs) was induced at 24 h after injection, 21.3% for PBP1‐sgRNA injected eggs and 19.5% for PBP3‐sgRNA injected eggs. Second, by an in‐crossing strategy, insects with mutant PBP1 or PBP3 (both with a premature stop codon) were screened, and homozygous mutants were obtained in the G3 generation. Third, the mutant insects were measured for electroantennogram (EAG) response to female sex pheromones. As a result, both PBP mutant males displayed significant reduction in EAG response, and this reduction in PBP1 mutants was higher than that in PBP3 mutants, indicating a more important role of PBP1. Finally, the relative importance of two PBPs and the possible off target effect induced by sgRNA‐injection are discussed. Taken together, our study provides a deeper insight into the function of and interaction between different PBP genes in sex pheromone perception of C. suppressalis, as well as a valuable reference in methodology for gene functional study in other genes and other moth species.

Sublethal effects of sulfoxaflor on biological characteristics and vitellogenin gene (AlVg) expression in the mirid bug, Apolygus lucorum (Meyer-Dür)

The mirid bug, Apolygus lucorum (Meyer-Dür) has evolved the resistance towards some traditional insecticides, especially pyrethroids and organophosphates. Sulfoxaflor, as a novel insecticide, is used for control of sap-feeding insects, like A. lucorum. Therefore, it is necessary to determine the acute toxicity and the potential sublethal effects of sulfoxaflor in A. lucorum. Here, the LD50 value of sulfoxaflor against A. lucorum was assayed as 3.347ng/adult at 48h via topical application. Besides, the effects of a sublethal dose (LD15) of sulfoxaflor on biological characteristics of A. lucorum were estimated by comparison of the life table parameters. The longevities and fecundity of parent generation did not exhibited significant difference between both control and treatment groups after exposure to LD15 dose of sulfoxaflor (0.568ng/adult) for 48-h. However, the parameters reflecting their progeny G1 generation population dynamics, including the intrinsic rate of increase (ri), the finite rate of increase (λ), the mean generation time (T), the net reproductive rate (R0) and gross reproduction rate (GRR) significantly reduced in the treatment group compared to the control. Furthermore, the expression level of AlVg mRNA significantly decreased by 43.8% in the progeny whose parents were treated with LD15 dose of sulfoxaflor in comparison with the control transgenerational female adults. These results suggested that sublethal dose of sulfoxaflor adversely affect the development and reproduction of transgenerational A. lucorum. The downregulation of AlVg might have negative impacts on the fecundity of A. lucorum.