Molecular characterization and functional analysis of a novel candidate of cuticle carboxylesterase in Spodoptera exigua degradating sex pheromones and plant volatile esters.

Odorant-degrading enzymes (ODEs) are considered to play key roles in odorant inactivation to maintain the odorant receptor sensitivity of insects. Some members of carboxylesterase (CXE) is a major sub-family of ODEs. However, only a few CXEs have been functionally characterized so far. In the present study, we cloned the antennal esterase SexiCXE11 cDNA full-length sequences from the male antennae of a notorious crop pest, Spodoptera exigua, and its encoded 538 amino acids. It was similar to other insect esterases and had the characteristics of a carboxylesterase. We expressed recombinant enzyme in High-Five insect cells and obtained the high level purified recombinant protein by affinity column. Furthermore we test enzyme activity toward its two acetate sex pheromone components (Z9,E12-Tetradecadienyl acetate, Z9E12-14:Ac and Z9-Tetradecenyl acetate, Z9-14:Ac) and other 18 ester plant volatiles. Our results demonstrated that SexiCXE11 degraded acetate sex pheromone components with similar degradation activities (about 15.75% with Z9E12-14:Ac and 19.28% with Z9-14:Ac) and plant volatiles with a relatively high activity such as pentyl acetate and (Z)-3-hexenyl caproate. SexiCXE11 had high hydrolytic activity with these two ester odorants (>50% degradation), which is characterized that although a ubiquitous expression esterase SexiCXE11 may be partly involved with olfaction. This study may facilitate a better understanding of moth ODE differentiation and suggest strategies for the development of new pest behavior inhibitors.

Binding affinity characterization of an antennae-enriched chemosensory protein from the white-backed planthopper, Sogatella furcifera (Horváth), with host plant volatiles.

The white-backed planthopper (WBPH) Sogatella furcifera is a notorious rice pest in Asia. Olfaction is crucial for the WBPH to seek and locate rice plants. However, its mechanism is still not fully understood. Chemosensory proteins (CSPs) are some of the important olfactory-related proteins. In this study, we first used a bacterial system to successfully express the recombinant, antennae-enriched protein SfurCSP5. Further, competitive fluorescence binding assays with 86 candidate ligands, including some known rice plant volatiles, showed that SfurCSP5 has high affinities for 2-tridecanone, 2-pentadecanone, and ß-ionone, which are known to be present in volatile mixtures that can attract rice planthoppers, and produced Ki values of 4.89, 4.09, and 1.39 µmol/L, respectively. Additionally, homology modeling of the protein structure of SfurCSP5 showed that it possesses five α-helixes (α-1, α-2, α-3, α-4, and α-5), which is a non-typical feature of the insect CSPs. Finally, ligand docking results revealed that Leu-44, Ile-64, Phe-90, Trp-98, and Phe-101 are five hydrophobic residues that interact with all of the ligands, indicating their key involvement in the binding of SfurCSP5. Our study lays the foundation for an understanding of the olfaction mechanism of rice planthoppers.

Higher viral load and genetic diversity of HIV-1 in seminal compartments than in blood of seven Chinese men who have sex with men and have early HIV-1 infection.

To date, there have been no reports characterizing HIV-1 in the semen of Chinese men who have sex with men (MSM) with early infection. In this study, genetic diversity and viral load of HIV-1 in the seminal compartments and blood of Chinese MSM with early HIV-1 infection were examined. Viral load and genetic diversity of HIV-1 in paired samples of semen and blood were analyzed in seven MSM with early HIV-1 infection. HIV-1 RNA and DNA were quantitated by real-time PCR assays. Through sequencing the C2-V5 region of the HIV-1 env gene, the HIV-1 genotype and genetic diversity based on V3 loop amino acid sequences were determined by using Geno2pheno and PSSM programs co-receptor usage. It was found that there was more HIV-1 RNA in seminal plasma than in blood plasma and total, and more 2-LTR circular and integrated HIV-1 DNA in seminal cells than in peripheral blood mononuclear cells from all seven patients with early HIV-infection. There was also greater HIV-1 genetic diversity in seminal than in blood compartments. HIV-1 in plasma displayed higher genetic diversity than in cells from the blood and semen. In addition, V3 loop central motifs, which present some key neutralizing antibody epitopes, varied between blood and semen. Thus, virological characteristics in semen may be more representative when evaluating risk of transmission in persons with early HIV infection.

Characterization of Different Phosphorus Forms in Flooded and Upland Paddy Soils Incubated with Various Manures.

Phosphorus (P) is an essential nutrient for crop production, and animal manures are rich in P. When using animal manures as alternatives to synthetic fertilizers, it is important to know the kinetics of P release from different animal manures and the forms, amounts, and dynamics of P in manure-treated soils. We chose four types of manure, viz., pig manure (PM), chicken manure (CM), dairy manure (DM), and commercial organic compost (OM), and evaluated the P release rate and availability in water solution and flooded/upland paddy soils. The WEP/total P (TP) and the water-extractable P (WEP) concentrations are highest for OM with the order: OM > PM > CM > DM. An increase in soil Olsen-P concentration was observed for the addition of manure with a varying application rate of P from low to moderate to high. The release capacity of Olsen-P in flooded conditions was higher than that in upland conditions. Under the flooded soil, PM and OM have faster release rates than CM and OM in the upland soil. Moreover, PM significantly increased available P by 29% in the flooded paddy soil while moderately inorganic P increased by 17% in the upland paddy soil. Olsen-P has a significant linear relationship with available P (Resin-P + NaHCO3-Pi; R 2 = 0.104; P < 0.01) and moderately inorganic P (NaOH-Pi + HCl-P; R 2 = 0.286; P < 0.01). The structural equation model showed that the organic input was beneficial to the conversion of moderately inorganic P to available P. Our results indicate that PM amendment promotes the release of available P in paddy soil.

Silencing the odorant coreceptor (Orco) disrupts sex pheromonal communication and feeding responses in Blattella germanica: toward an alternative target for controlling insect-transmitted human diseases.

BACKGROUND: The German cockroach, Blattella germanica, is one of the most severe pests of urban and rural areas. High-throughput genetic screening approaches indicate that the olfactory system of this pest is extremely powerful because it has an extensive array of olfactory receptor genes compared with many other insect species. Several of these genes have been identified previously, but their functions have not yet been characterized. RESULTS: This study describes the sequence of five transcriptomes of B. germanica adult male antennae, female antennae, maxillary palps, legs, and fifth-instar nymph antennae to investigate expression patterns of odorant receptors (ORs). Approximately 90% of ORs were found to be the most highly expressed genes in adult or nymph antennae. Additionally, every OR requires an odorant co-receptor (Orco) to become fully functional, and this was selected and successfully inhibited by injection of the corresponding double-stranded (ds)RNA targeting the Orco. A strong RNA interference (RNAi) effect was observed in which > 75% of Orco messenger RNA (mRNA) was clearly suppressed after 72 h of treatment. Olfactory behavioral assays showed that Orco-impaired B. germanica respond more slowly and show less attraction to one volatile sex pheromone and food resources compared with a control group. CONCLUSION: The results show that Orco plays a pivotal role in both sex pheromone and food-seeking olfactory processes, and provide an alternative genetic technique for controlling this urban pest species by olfactory disruption. © 2020 Society of Chemical Industry.

Expression, Affinity, and Functional Characterization of the Specific Binding of Two Putative Pheromone-Binding Proteins in the Omnivorous German Cockroach Blattella germanica.

The German cockroach Blattella germanica (L.) is an important pest in medical, veterinary, and public health. Studies on the olfaction mechanism of hemimetabolous insects have rarely been reported, especially in cockroaches. Pheromone-binding proteins (PBPs) play a vital role in insect sex pheromone recognition, which solubilize and carry the hydrophobic pheromonal compounds through the antennal lymph to receptors. In this study, two potential PBPs (BgerOBP26 and BgerOBP40) were identified on the basis of their biased expression in male antennae using tissue transcriptome data and verified by the quantitative real-time polymerase chain reaction approach. We then expressed and purified the two identified odorant-binding proteins (OBPs) using the Escherichia coli expression system and affinity purification. In vitro binding studies showed that the two OBPs display stronger binding affinities to the female volatile sex pheromone blattellaquinone than to its analogues and contact sex pheromone components. Finally, three-dimensional modeling of the two OBPs and dock conformation with sex pheromone molecules showed BgerOBP26 has a larger odorant cavity and more conservative active amino acid residues than BgerOBP40. These results illuminated the binding characteristics of potential PBPs of B. germanica, which could lay the groundwork for improved understanding of many aspects of the chemical ecology of B. germanica. Moreover, this information complements the understanding of the olfactory molecular mechanism in cockroaches and provides potential gene targets for B. germanica control.

Evolution and functional analysis of odorant-binding proteins in three rice planthoppers: Nilaparvata lugens, Sogatella furcifera, and Laodelphax striatellus.

BACKGROUND: The white-backed planthopper (WBPH) Sogatella furcifera, the brown planthopper (BPH) Nilaparvata lugens, and the small brown planthopper (SBPH) Laodelphax striatellus are three notorious rice pests that cause annual losses in rice yield through sap-sucking and virus transmission. Odorant-binding proteins (OBPs) are crucial olfactory genes involved in host-seeking behavior. RESULTS: We discovered the presence of 12, 12, and 16 OBPs in WBPH, BPH, and SBPH, respectively, including two novel OBPs in BPH and seven novel OBPs in SBPH. Phylogenetic analysis indicated that most of these OBPs have homologous genes, and one group (SfurOBP11, NlugOBP8, and LstrOBP2) show a slower evolution rate and are more conserved. Further, in vitro binding studies demonstrated that the three OBPs have similar binding affinities for some rice plant volatiles. Finally, RNA interference (RNAi) successfully inhibited the mRNA expression of the three OBPs, and in vivo behavioral tests showed that the OBP-deficient rice planthoppers were partly anosmic and lost some of their ability to locate rice plants. CONCLUSION: These results demonstrate the crucial role of the rice planthopper OBP genes in seeking rice plants. This information complements the current genetic resources for the development of RNAi-based transgenic rice and other pest management technologies. © 2018 Society of Chemical Industry.

[Expression of alpha-smooth muscle actin in renal tubulointerstitium in patients with kidney collateral stasis].

OBJECTIVE: To investigate the expression of alpha-smooth muscle action (alpha-SMA) in the renal tubulointerstitium in patients with kidney collateral stasis. METHODS: The expression of alpha-SMA in the renal biopsy specimens from 54 patients with kidney collateral stasis was examined by immunohistochemical method. RESULTS: The degree of kidney collateral stasis was increased with the increasing of the degree of renal interstitial fibrosis (P<0.05), and there was significant positive correlation between kidney collateral stasis and alpha-SMA expression (P<0.05). CONCLUSION: Kidney collateral stasis is one of the main reasons of renal fibrosis. With the increasing of kidney collateral stasis, MFBs in the renal interstitium proliferate obviously, becoming one of the most important causes of renal interstitial fibrosis.

Molecular characterization and evolution of a chemosensory receptor gene family in three notorious rice planthoppers, Nilaparvata lugens, Sogatella furcifera and Laodelphax striatellus, based on genome and transcriptome analyses.

BACKGROUND: The white-backed planthopper (WBPH) Sogatella furcifera, the brown planthopper (BPH) Nilaparvata lugens, and the small brown planthopper (SBPH) Laodelphax striatellus (Hemiptera: Delphacidae) are rice pests that damage rice plants by sap-sucking and by transmitting viruses. Host-seeking behavior involves chemosensory receptor genes that include odorant receptors (ORs), ionotropic receptors (IRs) and gustatory receptors (GRs). RESULTS: We used genome and transcriptome data to identify 141 ORs, 28 GRs and 25 IRs in BPH; 135 ORs, 18 GRs and 16 IRs in WBPH; and 37 ORs, 14 GRs and 6 IRs in SBPH. A phylogenetic analysis identified several specific OR clades of rice planthoppers, the results indicating that these OR members might be used to respond to specific host volatiles. OR co-receptor (Orco) is the most conserved and essential OR gene among these species and RNA interference (RNAi) can decrease their mRNA expression level to <50%. RNAi knockdown rice planthoppers were anosmia and were unable to seek or locate rice plants in behavioral tests. CONCLUSION: The results demonstrate the importance of the planthopper Orco genes in locating rice plants. This information may aid in the development of RNAi-based transgenic rice and other pest management technologies. © 2018 Society of Chemical Industry.

Immune responses of piglets immunized by a recombinant plasmid containing porcine circovirus type 2 and porcine interleukin-18 genes.

In this study, two recombinant plasmids containing the ORF2 gene of porcine circovirus type 2 (PCV2) with or without porcine interleukin-18 (IL-18) were constructed and evaluated for their ability to protect piglets against PCV2 challenge. Transient expression of the plasmids in PK-15 cells could be detected using Western blot. Piglets were given two intramuscular immunizations 3 weeks apart and were challenged with a virulent Wuzhi strain of PCV2 at 42 days after the initial immunization. All animals vaccinated with pBudCE4.1-ORF2 or with pBudCE4.1-ORF2/IL18 developed PCV2-specific antibody and T-lymphocyte proliferative responses. The levels of T-lymphocyte proliferation in piglets immunized with pBudCE4.1-ORF2/IL18 were significantly higher than in those immunized with pBudCE4.1-ORF2, and pBudCE4.1-ORF2/IL18 stimulated a significantly increased production of IFN-γ and IL-2. Furthermore, PCV2 challenge experiments showed that the DNA vaccine-immunized groups can partially prevent PCV2 viremia and significantly reduce the amount of PCV2 virus in the lymphoid tissues, and the piglets immunized by pBudCE4.1-ORF2/IL18 exhibit a marked inhibition of PCV2 replication compared to the pBudCE4.1-ORF2 group. These data demonstrate that the plasmid pBudCE4.1-ORF2/IL18 may be an effective approach for increasing PCV2 DNA vaccine immunogenicity.