The intracellular amino acid pool within lysosome is a signal that stimulates the nutrient-sensing mTORC1 signalling pathway. The signal transduction cascade has garnered much attention, but little is known about the sequestration of the signalling molecules within the lysosome. Using human HEK293 cells as a model, we found that suppression of the BHD syndrome gene FLCN reduced the leucine level in lysosome, which correlated with decreased mTORC1 activity. Both consequences could be reversed by supplementation with high levels of leucine, but not other tested amino acids. Conversely, overexpressed FLCN could sequester lysosomal leucine and stimulate mTORC1 in an amino acid limitation environment. These results identify a novel function of FLCN: it controls mTORC1 by modulating the leucine signal in lysosome. Furthermore, we provided evidence that FLCN exerted this role by inhibiting the accumulation of the amino acid transporter PAT1 on the lysosome surface, thereby maintaining the signal level within the organelle.

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Apple grain aphid, Rhopalosiphum padi (Linnaeus), is an important wheat pest. In China, it has been reported that R. padi has developed high resistance to carbamate and organophosphate insecticides. Previous work cloned from this aphid 2 different genes encoding acetylcholinesterase (AChE), which is the target enzyme for carbamate and organophosphate insecticides, and its insensitive alteration has been proven to be an important mechanism for insecticide resistance in other insects. In this study, both resistant and susceptible strains of R, padi were developed, and their AChEs were compared to determine whether resistance resulted from this mechanism and whether these 2 genes both play a role in resistance. Bioassays showed that the resistant strain used was highly or moderately resistant to pirimicarb, omethoate, and monocrotophos (resistance ratio, 263.8, 53.8, and 17.5, respectively), and showed little resistance to deltamethrin or thiodicarb (resistance ratio, 5.2 and 3.4, respectively). Correspondingly, biochemistry analysis found that AChE from resistant aphids was very insensitive to the first 3 insecticides (I50 increased 43.0-, 15.2-, and 8.8-fold, respectively), but not to thiodicarb (I50 increased 1.1-fold). Enzyme kinetics tests showed that resistant and susceptible strains had different AChEs. Sequence analysis of the 2 AChE genes cloned from resistant and susceptible aphids revealed that 2 mutations in Ace2 and 1 in Ace1 were consistently associated with resistance. Mutation F368(290)L in Ace2 localized at the same position as a previously proven resistance mutation site in other insects. The other 2 mutations, S329(228)P in Ace1 and V435(356)A in Ace2, were also found to affect the enzyme structure. These findings indicate that resistance in this aphid is mainly the result of insensistive AChE alteration, that the 3 mutations found might contribute to resistance, and that the AChEs encoded by both genes could serve as targets of insecticides.

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The ATP-binding cassette (ABC) transporters are important transmembrane proteins encoded by a supergene family. The majority of ABC proteins are primary active transporters that bind and hydrolyze ATP to mediate the efflux of a diverse range of substrates across lipid membranes. In this study, we cloned and characterized a putative multidrug resistance associated protein 1 (MRP1) from Rhopalosiphum padi encoded by ABCC1. Structural analysis showed that this protein has structural features typical of the ABC transporter family. Phylogenetic analysis indicated that the amino acid sequence was highly similar that of the corresponding protein from Acyrthosiphon pisum. Real-time quantitative polymerase chain reaction (PCR) analysis showed that ABCC1 was expressed throughout all R. padi developmental stages, with the highest level of expression in the fourth larval instar. We also examined ABCC1 expression in four different tissue types and found that it was most highly expressed in the midgut. Exposing R. padi to imidacloprid and chlorpyrifos increased ABCC1 expression. Furthermore, ABCC1 expression was higher in the imidacloprid-resistant (IR) and chlorpyrifos-resistant (CR) strains than in an insecticide-susceptible strain (SS) of R. padi. Exposing R. padi to verapamil in combination with insecticides significantly increased the toxicity of the insecticides. The respective synergy factor of CR and IR R. padi strain was 1.33 and 1.26, which was lower than that (2.72 and 1.64, respectively) of the SS. Our results clarify the biological function of ABCC1 in R. padi, particularly its role in insecticide resistance, and suggest novel strategies for pest management that use ABC transporter inhibitors to increase the effectiveness of insecticides.

Fruit-bagging is being used as a pest management tool to control fruit moths on several continents. However, nothing is known about how this physical control method affects population genetics of these moths. Focusing on the oriental fruit moth, Grapholita (= Cydia) molesta (Busck) (Lepidoptera: Tortricidae), we used five polymorphic microsatellite loci and two mitochondrial gene sequences to investigate the genetic diversity and genetic structure of populations from three fruit-bagged and two unbagged apple orchards in five regions of a major fruit-growing area of China. We sampled each orchard 39 during the season and found high genetic diversity in the samples of populations collected. Populations from fruit-bagged and unbagged orchards were genetically significantly differentiated, indicating a clear effect of the pest management method applied. Genetic structures of the populations under a given management regime changed in the course of the season in a characteristic manner, and within the same regime, patterns were always similar at a given sampling time. There was no significant isolation by geographical distance. Population genetics from both orchard types did not resemble one another at the beginning of the season, but they did rather at the end. Potential reasons for this unexpected direction of change in population genetic structure are discussed, and we hypothesize that diapause-related characteristics may account for the patterns observed. The current study documents that pest management methods applied can have an important impact on the genetic diversity and genetic structure of targeted pest insect populations.

Cydia pomonella is a serious invasive insect pest in China, and has caused severe damage to the production of apple and pear in its invaded areas. This species is distributing in the northwest and northeast of China, but no occurrence of it has been recorded in the large areas (about 3000-5000 km away) between the invaded northwestern and northeastern regions despite continuous monitoring. As yet the genetic diversity and invasion origin of the C. pomonella populations in Northwestern and Northeastern China is obscure. In this study, we investigate the genetic diversity of 14 populations of C. pomonella sampled throughout the main distribution regions in Northwestern (Xinjiang and Gansu Provinces) and Northeastern (Heilongjiang Province) China and compared them with nine populations from Europe and other continents using the mitochondrial COI, COII and Cytb genes. Both the populations from Northeastern and Northwestern China shared some haplotypes with populations from other countries. Haplotypes of the three mitochondrial genes had a different distribution in Northeastern and Northwestern China. The northeastern populations had more private haplotypes than the northwestern populations. A large number of the individuals from northwestern populations shared a few haplotypes of each of the three genes. The haplotype numbers and haplotype diversities of the northeastern populations were similar to those of field populations in other countries, but were higher than those of the northwestern populations. Populations from the Northwestern China showed similar haplotype number and haplotype diversity. We conclude that the population genetic background of C. pomonella populations in Northeastern and Northwestern China varies due to different invasion sources and that this should be considered before the application of new pest control tactics.

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