Culex Pipiens Pallens Research Articles

Adaptation and carry over effects of extreme sporadic heat stress in Culex mosquitoes

Mosquitoes, as temperature-sensitive ectothermic vectors, exhibit temperature-dependence. This study investigates Culex pipiens pallens (Cx. pallens) responses to abrupt temperature increases and their implications on mosquito physiology.First instar larvae (24hr post hatching) and newly enclosed adults (24hr post emergence) were separately exposed to heat shock regimes of 33 °C, 37 °C, and 42 °C for 3 days alongside a control temperature of 27 °C. Results showed that mortality was triggered at 42 °C within a day. Adult male mosquitoes were less tolerant to all temperatures than larvae and adult females (p < 0.05). Exposing larvae to constant temperatures for 3 days significantly decreased larvae's development time, growth rate and adult emergence (p < 0.05). Reproductive fitness was significantly reduced (p < 0.05) in males emerging from larvae exposed to 37 °C. Life table parameters showed significant increased mortality rate, kill power and decreased life expectancy at the embryonic stage (p < 0.05). Furthermore, heatwaves deactivated the Transient receptor protein ankyrin 1 at 37 °C (p < 0.05) in larvae but not adults. Calmodium, Heat shock protein 90, and small heat shock protein expression were significantly decreased in larvae at 37 °C (p < 0.05) as compared to larvae raised at 33 °C and 27 °C.In conclusion, we classified the heat waves into three categories: adaptable (33 °C), critical (37 °C), and fatal (42 °C). Prolonged exposure of Culex pallens larvae to extreme heat affects the male reproductive output. These findings may serve as an important reference for forecasting vector and pest dynamics and used to tailor mosquito prevention and control measures.

Wolbachia and mosquitoes: Exploring transmission modes and coevolutionary dynamics in Shandong Province, China.

Vector-borne diseases leave a large footprint on global health. Notable culprits include West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and Japanese encephalitis virus (JEV), all transmitted by Culex mosquitoes. Chemical insecticides have been widely used to reduce the spread of mosquito-borne diseases. Still, mosquitoes are becoming more and more resistant to most chemical insecticides which cause particular harm to the ecology. Wolbachia belongs to the family Ehrlichiaceae in the order Rickettsiales and is a matrilineally inherited endosymbiont present in 60% of insects in nature. Wolbachia is capable of inducing a wide range of reproductive abnormalities in its hosts, such as cytoplasmic incompatibility, and can alter mosquito resistance to pathogen infection. Wolbachia has been proposed as a biological alternative to chemical vector control, and specific research progress and effectiveness have been achieved. Despite the importance of Wolbachia, this strategy has not been tested in Culex pipiens pallens, the most prevalent mosquito species in Shandong Province, China. Little is known about how the mass release of Wolbachia-infected mosquitoes may impact the genetic structure of Culex pipiens pallens, and how the symbiotic bacterium Wolbachia interacts with mitochondria during host mosquito transmission. Based on the population genetic structure of Culex pipiens pallens in Shandong Province, this study investigated the infection rate and infection type of Wolbachia in Shandong Province and jointly analysed the evolutionary relationship between the host mosquito and the symbiotic bacterium Wolbachia. Our study showed that Wolbachia naturally infected by Culex pipiens pallens in Shandong Province was less homologous to Wolbachia infected by Aedes albopictus released from mosquito factory in Guangzhou. Our results also show that Culex pipiens pallens is undergoing demographic expansion in Shandong Province. The overall Wolbachia infection rate of Culex pipiens pallens was 92.8%, and a total of 15 WSP haplotypes were detected. We found that the genetic diversity of Wolbachia was low in Culex pipiens pallens from Shandong Province, and the mosquitoes were infected only with type B Wolbachia. Visualizing the relationship between Culex pipiens pallens and Wolbachia using a tanglegram revealed patterns of widespread associations. A specific coevolutionary relationship exists between the host mosquito and Wolbachia. Knowledge of this mosquito-Wolbachia relationship will provide essential scientific information required for Wolbachia-based vector control approaches in Shandong Province and will lead to a better understanding of the diversity and evolution of Wolbachia for its utility as a biocontrol agent.

Larvicidal activity prediction of Essential oils against Culex pipiens pallens using QSAR Modeling

The search for an eco-freindly larvicide suitable for vector control requires a budget and considerable time to carry out experiments. Fortunately, the use of QSAR modeling allows the prediction of larvicidal activity of structurally diverse chemicals against mosquitoes in a way quick and costless. This approach can be helpful to study for making biolarvicide with highest ability to destroy mosquito larvae. We propose a QSAR model using two different statistical methods, multiple linear regression (MLR) and Support vector machine (SVM) for predicting the larvicidal activity of 30 compounds of essential oil (EOs) isolated from the root of Asarum heterotropoides against Culex pipiens pallens. A model with four theoretical descriptors derived from Dragon software was developed applying the genetic algorithm (GA)-variable subset selection (VSS) procedure. The statistical parameters, R2 = 0.9716, Q2LOO = 0.9595, s = 0.1690 of the model developed by MLR showed a good predictive capability for log LC50 values. The comparison between the results of SVM and MLR models showed that the SVM model present a good alternative to construct a QSAR model for the prediction of the larvicidal activity.

Application of circular statistics in temporal distribution of adult mosquitoes in Qingdao, Shandong Province, China, 2021–2023

BackgroundAnalyses of the temporal distribution of mosquitoes are presented in statistical charts, but it is difficult to prove in statistics whether differences in peak periods exist among different years or habitats. This study aimed to investigate the application of circular statistics in determining the peak period and a comparison of differences.MethodsSurveillance of adult mosquitoes was conducted twice a month by light traps in five different habitats from March to November for 3 years (2021–2023) in Qingdao, Shandong Province, China. The Kruskal–Wallis test was performed to determine the differences in mosquito density among different years and habitats. Circular statistics and line charts were employed to determine the peak period and a comparison of differences.ResultsAmong a total of 14,834 adult mosquitoes comprising five mosquito species from four genera, Culex pipiens pallens was dominant and accounted for 89.6% of the specimens identified. Aedes albopictus, Armigeres subalbatus, and Anopheles sinensis made up 5.7%, 4.2%, and 0.5%, respectively. Culex tritaeniorhynchus accounted for less than 0.1%. The mean mosquito density (females/trap night) for the trapping period was 10.3 in 2021, 5.6 in 2022, and 3.6 in 2023. Among five habitats, the highest mosquito density was 8.9 in livestock sheds, followed by 6.8 in parks, 5.9 in rural dwellings, 5.5 in urban dwellings, and 5.4 in hospitals. No statistically significant differences were found among different years (H = 1.96, d.f. 2, P = 0.376) and habitats (H = 0.45, d.f. 4, P = 0.978). Overall, the peak period of mosquito activity fell in the months from June to September. The peak period among 3 years differed significantly (F(2,7022) = 119.17, P < 0.01), but there were no statistically significant differences in peak period among different habitats (F(4,7020) = −159.09, P > 0.05). ConclusionCircular statistics could be effectively combined with statistical charts to elucidate the peak period of mosquitoes and determine the differences in statistics among different years and habitats. These findings will provide valuable information for mosquito control and public health management.Graphical

Species Composition and Feeding Behaviors of Vector Mosquitoes of Avian Infectious Diseases at a Wild Bird Rehabilitation Facility in Japan.

Although wild bird rehabilitation facilities are important for the conservation of wild species, individuals may be kept within the facilities for long periods, consequently posing a risk for the bird to be infected with pathogens to which they are not naturally exposed. In turn, novel pathogens may be introduced through rescued migratory species. Avian malaria and West Nile fever are important avian diseases transmitted by mosquitoes. To understand the transmission dynamics of such diseases at rehabilitation facilities, the ecology of vector mosquitoes, including species composition, seasonality, and feeding behaviors, were explored. Mosquitoes were collected at a wild bird rehabilitation facility and wildlife sanctuary in Japan from 2019 to 2020 using mouth aspirators, sweep nets, and light traps. A total of 2,819 mosquitoes of 6 species were captured, all of which are potential vectors of avian diseases. Culex pipiens pallens and Cx. pipiens form molestus were the dominant species (82.9% of all collected mosquitoes). Density and seasonality differed between sampling locations, presumably because of differences in mosquito behaviors including feeding preferences and responses to climatic factors. Blood-fed Culex mosquitoes fed solely on birds, and many mosquito species are thought to have fed on birds within the facility. Particularly, Cx. pipiens group probably fed on both rescued and free-living birds. The rehabilitation facility may be an important site for the introduction and spread of pathogens because 1) numerous mosquitoes inhabit the hospital and its surroundings; 2) blood-fed mosquitoes are caught within the hospital; 3) there is direct contact between birds and mosquitoes; 4) both birds within the hospital and wild birds are fed upon. Furthermore, blood-fed Cx. pipiens form molestus were observed in the winter, suggesting that pathogens might be transmitted even during the winter when other mosquito species are inactive.

Lethal and sublethal effects of spinosad on dengue vector mosquito, Aedes albopictus and bancroftian filariasis vector mosquito, Culex pipiens pallens.

Aedes albopictu and Culex pipiens pallens are important vectors of many viruses and have had resistance to chemical pesticide. Spinosad is a selective biological insecticide to control urban mosquito. The aim of this study was to reveal the sublethal effects of spinosad on mosquito and provide reference basis for integrated mosquito management. The toxicity of spinosad against Ae. albopictus and Cx. pipiens pallens were determined under laboratory conditions by exposing early third-instar larvae to different concentrations. The LC50 values of spinosad to Ae. albopictus and Cx. Pipiens pallens larvaes were 4.44×10-3 mg∙L-1 and 1.93×10-3 mg∙L-1 respectively after 72 h exposure. Spinosad at sublethal concentrations has many negative effects on Ae. albopictus and Cx. Pipiens pallens larval, pupae, adult and offspring eggs, including significantly reduced their larvae pupation rate by 51.37% and 58.47%, significantly prolonged pupae length by 21.43% and 16.18%, reduced female wing-spans by 20.19% and 14.89%, reduced male wing-spans by 3.84% and 7.54%, reduced female weight by 29.04% and 31.52%, reduced male weight by 7.47% and 9.07%, reduced female and male ratio by 51.98% and 45.21%, reduced individual egg-laying amount by 15.73% and 35.51%, in addition, offspring egg hatchability were dramatically decreased by 25.71% and 34.04%, egg periods were significantly prolonged by 14.42% and 62.82% respectively. No significant effect on larval period, pupae emergence rate, female bite rates were observed. These results suggest that spinosad might affect pest population dynamics significantly and is fairly expected to be a candidate biological pesticide for mosquito control.

Isolation, characterization and functional analysis of a bacteriophage targeting Culex pipiens pallens resistance-associated Aeromonas hydrophila

BackgroundCulex pipiens pallens is a well-known mosquito vector for several diseases. Deltamethrin, a commonly used pyrethroid insecticide, has been frequently applied to manage adult Cx. pipiens pallens. However, mosquitoes can develop resistance to these insecticides as a result of insecticide misuse and, therefore, it is crucial to identify novel methods to control insecticide resistance. The relationship between commensal bacteria and vector resistance has been recently recognized. Bacteriophages (= phages) are effective tools by which to control insect commensal bacteria, but there have as yet been no studies using phages on adult mosquitoes. In this study, we isolated an Aeromonas phage vB AhM-LH that specifically targets resistance-associated symbiotic bacteria in mosquitoes. We investigated the impact of Aeromonas phage vB AhM-LH in an abundance of Aeromonas hydrophila in the gut of Cx. pipiens pallens and its effect on the status of deltamethrin resistance.MethodsPhages were isolated on double-layer agar plates and their biological properties analyzed. Phage morphology was observed by transmission electron microscopy (TEM) after negative staining. The phage was then introduced into the mosquito intestines via oral feeding. The inhibitory effect of Aeromonas phage vB AhM-LH on Aeromonas hydrophila in mosquito intestines was assessed through quantitative real-time PCR analysis. Deltamethrin resistance of mosquitoes was assessed using WHO bottle bioassays.ResultsAn Aeromonas phage vB AhM-LH was isolated from sewage and identified as belonging to the Myoviridae family in the order Caudovirales using TEM. Based on biological characteristics analysis and in vitro antibacterial experiments, Aeromonas phage vB AhM-LH was observed to exhibit excellent stability and effective bactericidal activity. Sequencing revealed that the Aeromonas phage vB AhM-LH genome comprises 43,663 bp (51.6% CG content) with 81 predicted open reading frames. No integrase-related gene was detected in the vB AH-LH genome, which marked it as a potential biological antibacterial. Finally, we found that Aeromonas phage vB AhM-LH could significantly reduce deltamethrin resistance in Cx. pipiens pallens, in both the laboratory and field settings, by decreasing the abundance of Aeromonas hydrophila in their midgut.ConclusionsOur findings demonstrate that Aeromonas phage vB AhM-LH could effectively modulate commensal bacteria Aeromonas hydrophila in adult mosquitoes, thus representing a promising strategy to mitigate mosquito vector resistance.Graphical

Evaluating the mosquito vector range for two orthobunyaviruses: Oya virus and Ebinur Lake virus

BackgroundMosquito-borne viruses cause various infectious diseases in humans and animals. Oya virus (OYAV) and Ebinur Lake virus (EBIV), belonging to the genus Orthobunyavirus within the family Peribunyaviridae, are recognized as neglected viruses with the potential to pose threats to animal or public health. The evaluation of vector competence is essential for predicting the arbovirus transmission risk.MethodsTo investigate the range of mosquito vectors for OYAV (strain SZC50) and EBIV (strain Cu20-XJ), the susceptibility of four mosquito species (Culex pipiens pallens, Cx. quinquefasciatus, Aedes albopictus, and Ae. aegypti) was measured through artificial oral infection. Then, mosquito species with a high infection rate (IR) were chosen to further evaluate the dissemination rate (DR), transmission rate (TR), and transmission efficiency. The viral RNA in each mosquito sample was determined by RT-qPCR.ResultsThe results revealed that for OYAV, Cx. pipiens pallens had the highest IR (up to 40.0%) among the four species, but the DR and TR were 4.8% and 0.0%, respectively. For EBIV, Cx. pipiens pallens and Cx. quinquefasciatus had higher IR compared to Ae. albopictus (1.7%). However, the EBIV RNA and infectious virus were detected in Cx. pipiens pallens, with a TR of up to 15.4% and a transmission efficiency of 3.3%.ConclusionsThe findings indicate that Cx. pipiens pallens was susceptible to OYAV but had an extremely low risk of transmitting the virus. Culex pipiens pallens and Cx. quinquefasciatus were susceptible to EBIV, and Cx. pipiens pallens had a higher transmission risk to EBIV than Cx. quinquefasciatus.Graphical

Comparison of the microbiota diversity between autogenous and anautogenous Culex pipiens pallens

To investigate the microbiota composition and diversity between autogenous and anautogenous Culex pipiens pallens, so as to provide insights into unraveling the pathogenesis of autogeny in Cx. pipiens pallens. Autogenous and anautogenous adult Cx. pipiens pallens samples were collected at 25 ℃, and the hypervariable regions of the microbial 16S ribosomal RNA (16S rRNA) gene was sequenced on the Illumina NovaSeq 6000 sequencing platform. The microbiota abundance and diversity were evaluated using the alpha diversity index, and the difference in the microbiota structure was examined using the beta diversity index. The microbiota with significant differences in the abundance between autogenous and anautogenous adult Cx. pipiens pallens samples was identified using the linear discriminant analysis effect size (LEfSe). The microbiota in autogenous and anautogenous Cx. pipiens pallens samples belonged to 18 phyla, 28 classes, 70 orders, 113 families, and 170 genera, and the dominant phyla included Proteobacteria, Bacteroidetes, and so on. At the genus level, Wolbachia was a common dominant genus, and the relative abundance was (77.6 ± 11.3)% in autogenous Cx. pipiens pallens samples and (47.5 ± 8.5)% in anautogenous mosquito samples, while Faecalibaculum (0.4% ± 0.1%), Dubosiella (0.5% ± 0.0%) and Massilia (0.5% ± 0.1%) were specific species in autogenous Cx. pipiens pallens samples. Alpha diversity analysis showed that higher Chao1 index and ACE index in autogenous Cx. pipiens pallens samples than in anautogenous samples (both P values > 0.05), and lower Shannon index (P > 0.05) and Simpson index (P < 0.05) in autogenous Cx. pipiens pallens samples than in anautogenous samples. LEfSe analysis showed a total of 48 significantly different taxa between autogenous and anautogenous Cx. pipiens pallens samples (all P values < 0.05). There is a significant difference in the microbiota diversity between autogenous and anautogenous Cx. pipiens pallens.

Evaluation of vector susceptibility in Aedes aegypti and Culex pipiens pallens to Tibet orbivirus.

Mosquito-borne viruses cause various infectious diseases in humans and animals. Tibet orbivirus (TIBOV), a newly identified arbovirus, efficiently replicates in different types of vertebrate and mosquito cells, with its neutralizing antibodies detected in cattle and goats. However, despite being isolated from Culicoides midges, Anopheles, and Culex mosquitoes, there has been a notable absence of systematic studies on its vector competence. Thus, in this study, Aedes aegypti and Culex pipiens pallens were reared in the laboratory to measure vector susceptibility through blood-feeding infection. Furthermore, RNA sequencing was used to examine the overall alterations in the Ae. aegypti transcriptome following TIBOV infection. The results revealed that Ae. aegypti exhibited a high susceptibility to TIBOV compared to Cx. p. pallens. Effective replication of the virus in Ae. aegypti midguts occurred when the blood-feeding titer of TIBOV exceeded 105 plaque-forming units mL-1. Nevertheless, only a few TIBOV RNA-positive samples were detected in the saliva of Ae. aegypti and Cx. p. pallens, suggesting that these mosquito species may not be the primary vectors for TIBOV. Moreover, at 2 dpi of TIBOV, numerous antimicrobial peptides downstream of the Toll and Imd signaling pathways were significantly downregulated in Ae. aegypti, indicating that TIBOV suppressed mosquitos' defense to survive in the vector at an early stage. Subsequently, the stress-activated protein kinase JNK, a crucial component of the MAPK signaling pathway, exhibited significant upregulation. Certain genes were also enriched in the MAPK signaling pathway in TIBOV-infected Ae. aegypti at 7 dpi.IMPORTANCETibet orbivirus (TIBOV) is an understudied arbovirus of the genus Orbivirus. Our study is the first-ever attempt to assess the vector susceptibility of this virus in two important mosquito vectors, Aedes aegypti and Culex pipiens pallens. Additionally, we present transcriptome data detailing the interaction between TIBOV and the immune system of Ae. aegypti, which expands the knowledge about orbivirus infection and its interaction with mosquitoes.

Experimental demonstration of the transmission of Spiroplasma between different arthropod taxa.

Spiroplasma (Mycoplasmatales: Spiroplasmataceae) is one of the most widely distributed symbionts of arthropods. Spiroplasma species can infect their hosts via vertical or horizontal transmission. However, the mode of transmission of Spiroplasma between different arthropod taxa has not been elucidated. In this study, we investigated the potential for the transmission of Spiroplasma to non-native arthropod species, using 2 Spiroplasma spp. isolated from ticks, namely Spiroplasma ixodetis and Spiroplasma mirum, and 3 species of mosquito laboratory colonies, namely Aedes albopictus, Aedes aegypti, and Culex pipiens pallens (Diptera: Culicidae). After feeding the adult mosquitoes with Spiroplasma-containing artificial meals, they were kept at 25 °C for 10 days. Homogenates prepared from Spiroplasma-fed mosquitoes were used to re-isolate Spiroplasma using the in vitro culture method. Nine weeks after culture initiation, the presence of Spiroplasma was tested using the polymerase chain reaction (PCR). The results revealed that only S. ixodetis was detected from all 3 species of mosquitoes and re-isolated from 2 of them. The differences in the infection ability of different Spirolasma species could be attributed to several factors, including environmental effects. Nevertheless, this is the first experimental demonstration of Spiroplasma transmission among different arthropod taxa. Further studies are needed to elucidate the evolutionary mechanism that supports the survival of Spiroplasma in nature.

MicroRNA-989 targets 5-hydroxytryptamine receptor1 to regulate ovarian development and eggs production in Culex pipiens pallens

BackgroundFemale mosquitoes need a blood meal after mating for their eggs to develop, and this behavior leads to the spread of pathogens. Therefore, understanding the molecular regulation of reproduction in female mosquitoes is essential to control mosquito vector populations. In this study, we reported that microRNA-989 (miR-989), which targets 5-HTR1 (encoding secreted 5-hydroxytryptamine receptor1), is essential for mosquito reproduction.MethodsThe spatiotemporal expression profile of miR-989 was detected using quantitative real-time reverse transcription PCR (RT-qPCR). miR-989 antagomirs and antagomir-negative control (NC) were designed and synthesized to knock down the expression of endogenous miR-989 in female mosquitoes. RNA sequencing was used to analyze the ovarian response to miR-989 deletion. The targets of miR-989 were predicted and confirmed using RNAhybrid and dual-luciferase assays.ResultsmiR-989 is exclusively expressed in female mosquito ovaries and responds to blood feeding. Injection of the miR-989 antagomir resulted in smaller ovaries and reduced egg production. 5-HTR1 was demonstrated as a target of miR-989. The deletion of miR-989 contributed to the upregulation of 5-HTR1 expression. Knockdown of 5-HTR1 rescued the adverse egg production caused by miR-989 silencing. Thus, miR-989 might play an essential role in female reproduction by targeting 5-HTR1.ConclusionsWe found that miR-989 targets 5-HTR1 and participates in the regulation of reproduction in female mosquitoes. These findings expand our understanding of reproduction-related miRNAs and promote new control strategies for mosquitoes.Graphical

Adverse effects of knocking down chitin synthase A on female reproduction in Culex pipiens pallens (Diptera: Culicidae).

Current mosquito-borne disease vector control strategies, largely based on chemical insecticides, are seriously threatened by increasing resistance worldwide. There is also growing concerned about the adverse effects of insecticides on nontarget organisms and the environment, therefore effective and ecologically friendly alternative approaches are urgently needed. Targeting critical steps of reproduction is considered a potential way to control mosquito populations. Herein, we focused on the roles of chitin synthase A (encoded by chsa) in the reproduction of female mosquitoes. The injection of small interfering RNA targeting Cpchsa in female Culex pipiens pallens (Diptera: Culicidae) had antireproductive effects, including decreased follicle numbers, egg-laying, and hatching rate. Scanning electron microscopy observations showed that Cpchsa silencing caused a defective egg envelope, including absence of the vitelline membrane and cracked chorion layers, which resulted in abnormal permeability. Widely distributed nurse cell apoptosis and follicular epithelial cell autophagy were observed in Cpchsa-silenced ovaries during the vitellogenesis phase. Consistent with the detective egg envelope formation during oogenesis, the exochorionic eggshell structures were also affected in eggs deposited by Cpchsa-silenced mosquitoes. This study provided fundamental evidence for the role of chitin synthase A in the female reproductive process of mosquitoes and might result in a novel alternative strategy for mosquito control. © 2023 Society of Chemical Industry.

Identification and functional analysis of ABC transporter genes related to deltamethrin resistance in Culex pipiens pallens.

Pathogens that reproduce or develop in mosquitoes can transmit several diseases, endanger human health, and overwhelm health systems. Synthetic pyrethroids are the most widely used insecticides against adult mosquitoes, but their widespread use has led to resistance. The adenosine triphosphate (ATP)-binding cassette (ABC) transporters are involved in the resistance monitoring of insects, but their role and underlying mechanisms in insecticide resistance have not been fully elucidated. In the present study, we identified ABC transporter genes in Culex pipiens and investigated their role in the development of insecticide resistance. We identified 63 ABC transporter genes in Cx. pipiens and classified them as per the ABC transporter subfamilies. We also performed phylogenetic analysis. The knockdown rate of the mosquitoes orally fed with the ABC transporter inhibitor verapamil increased after deltamethrin treatment compared with that of the control group. Several genes from the ABCB, ABCC, and ABCG subfamilies were highly expressed in resistant mosquitoes. Immunofluorescence analysis revealed that ABCG6032427 was expressed in the head, chest, abdomen, wings, and legs, and the expression was the highest in the legs. Subsequently, knockdown of ABCG6032427 using RNA interference (RNAi) increased the sensitivity of the mosquitoes to deltamethrin, and scanning and transmission electron microscopy revealed that ABCG6032427 knockdown reduced cuticle thickness and the cuticle became loose and irregular. ABCG6032427 may modulate cuticle thickness and structure, thus play an important role in the development of deltamethrin resistance in mosquitoes. Thus, it could be a potential target for deltamethrin resistance management in Cx. pipiens. © 2023 Society of Chemical Industry.

Design, Synthesis and Various Bioactivity of Acylhydrazone-Containing Matrine Analogues.

Compounds with acylhydrazone fragments contain amide and imine groups that can act as electron donors and acceptors, so they are easier to bind to biological targets and thus generally exhibit significant biological activity. In this work, acylhydrazone fragments were introduced to the C-14 or C-11 position of matrine, a natural alkaloid, aiming to enhance their biological activities. The result of this bioassay showed that many synthesized compounds exhibited excellent anti-virus activity against the tobacco mosaic virus (TMV). Seventeen out of 25 14-acylhydrazone matrine derivatives and 17 out of 20 11-butanehydrazone matrine derivatives had a higher inhibitory activity against TMV than the commercial antiviral agent Ribavirin (the in vitro activity, in vivo inactivation, curative and protection activities at 500 µg/mL were 40.9, 36.5 ± 0.9, 38.0 ± 1.6 and 35.1 ± 2.2%, respectively), and four 11-butanehydrazone matrine derivatives even had similar to or higher activity than the most efficient antiviral agent Ningnanmycin (55.4, 57.8 ± 1.4, 55.3 ± 0.5 and 60.3 ± 1.2% at 500 µg/mL for the above four test modes). Among them, the N-benzyl-11-butanehydrazone of matrine formed with 4-bromoindole-3-carboxaldehyde exhibited the best anti-TMV activity (65.8, 71.8 ± 2.8, 66.8 ± 1.3 and 69.5 ± 3.1% at 500 µg/mL; 29, 33.5 ± 0.7, 24.1 ± 0.2 and 30.3 ± 0.6% at 100 µg/mL for the above four test modes), deserving further investigation as an antiviral agent. Other than these, the two series of acylhydrazone-containing matrine derivatives were evaluated for their insecticidal and fungicidal activities. Several compounds were found to have good insecticidal activities against diamondback moth (Plutella xylostella) and mosquito larvae (Culex pipiens pallens), showing broad biological activities.

Vector competence evaluation of mosquitoes for Tahyna virus PJ01 strain, a new Orthobunyavirus in China.

Tahyna virus (TAHV), an arbovirus of the genus Orthobunyavirus, is a cause of human diseases and less studied worldwide. In this study, a new strain of TAHV was isolated from Aedes sp. mosquitoes collected in Panjin city, Liaoning province. However, the competent vector of TAHV in China is still unknown. The genome of newly isolated TAHV was sequenced and phylogenetic analysis is performed. Aedes albopictus and Culex pipiens pallens were orally infected with artificial virus blood meals (1:1 of virus suspension and mouse blood), the virus was detected in the midgut, ovary, salivary gland and saliva of the mosquitoes. Then, the transmission and dissemination rates, vertical transmission and horizontal transmission of the virus by the mosquitoes were assessed. Phylogenetic analysis revealed that the virus shared high similarity with TAHV and was named the TAHV PJ01 strain. After oral infection with virus blood meals, Ae. albopictus showed positive for the virus in all tested tissues with an extrinsic incubation period of 2 days and a fluctuating increasement of transmission and dissemination rates. Whereas no virus was detected in the saliva of Cx. pipiens pallens. Suckling mice bitten by infectious Ae. albopictus developed obvious neurological symptoms, including inactivity, hind-leg paralysis and difficulty turning over, when the virus titer reached 1.70×105 PFU/mL in the brain. Moreover, TAHV was detected in the eggs, larvae and adults of F1 offspring of Ae. albopictus. Ae. albopictus is an efficient vector to transmit TAHV but Cx. pipiens pallens is not. Ae. albopictus is also a reservoir host that transmits the virus vertically, which further increases the risk of outbreaks. This study has important epidemiological implications for the surveillance of pathogenic viruses in China and guiding comprehensive vector control strategies to counteract potential outbreaks in future.

New native Bacillus thuringiensis strains induce high insecticidal action against Culex pipiens pallens larvae and adults

Mosquitoes of many species are key disease vectors, killing millions of people each year. Bacillus thuringiensis-based insecticide formulations are largely recognized as among the most effective, ecologically safe, and long-lasting methods of managing insect pests. New B. thuringiensis strains with high mosquito control effectiveness were isolated, identified, genetically defined, and physiologically characterized. Eight B. thuringiensis strains were identified and shown to carry endotoxin-producing genes. Using a scanning electron microscope, results revealed typical crystal forms of various shapes in B. thuringiensis strains. Fourteen cry and cyt genes were found in the strains examined. Although the genome of the B. thuringiensis A4 strain had twelve cry and cyt genes, not all of them were expressed, and only a few protein profiles were observed. The larvicidal activity of the eight B. thuringiensis strains was found to be positive (LC50: 1.4–28.5 g/ml and LC95: 15.3-130.3 g/ml). Bioassays in a laboratory environment demonstrated that preparations containing B. thuringiensis spores and crystals were particularly active to mosquito larvae and adults. These new findings show that the novel preparation containing B. thuringiensis A4 spores and crystals mixture might be used to control larval and adult mosquitoes in a sustainable and ecologically friendly manner.

Evaluation of the evolutionary genetics and population structure of Culex pipiens pallens in Shandong province, China based on knockdown resistance (kdr) mutations and the mtDNA-COI gene

BackgroundMosquitoes are important vectors for a range of diseases, contributing to high rates of morbidity and mortality in the human population. Culex pipiens pallens is dominant species of Culex mosquito in northern China and a major vector for both West Nile virus and Bancroftian filariasis. Insecticide application were largely applied to control the mosquito-mediated spread of these diseases, contributing to increasing rates of resistance in the mosquito population. The voltage-gated sodium channel (Vgsc) gene is the target site of pyrethroids, and mutations in this gene cause knockdown resistance (kdr). While these kdr mutations are known to be critical to pyrethroid resistance, their evolutionary origins remain poorly understood. Clarifying the origins of these mutations is potential to guide further vector control and disease prevention efforts. Accordingly, the present study was designed to study the evolutionary genetics of kdr mutations and their association with the population structure of Cx. p. pallens in Shandong province, China.MethodsAdult Culex females were collected from Shandong province and subjected to morphological identification under a dissection microscope. Genomic DNA were extracted from the collected mosquitoes, the Vgsc gene were amplified via PCR and sequenced to assess kdr allele frequencies, intron polymorphisms, and kdr codon evolution. In addition, population genetic diversity and related population characteristics were assessed by amplifying and sequencing the mitochondrial cytochrome C oxidase I (COI) gene.ResultsTotally, 263 Cx. p. pallens specimens were used for DNA barcoding and sequencing analyses to assess kdr allele frequencies in nine Culex populations. The kdr codon L1014 in the Vgsc gene identified two non-synonymous mutations (L1014F and L1014S) in the analyzed population. These mutations were present in the eastern hilly area and west plain region of Shandong Province. However, only L1014F mutation was detected in the southern mountainous area and Dongying city of Shandong Province, where the mutation frequency was low. Compared to other cities, population in Qingdao revealed significant genetic differentiation. Spatial kdr mutation patterns are likely attributable to some combination of prolonged insecticide-mediated selection coupled with the genetic isolation of these mosquito populations.ConclusionsThese data suggest that multiple kdr alleles associated with insecticide resistance are present within the Cx. p. pallens populations of Shandong Province, China. The geographical distributions of kdr mutations in this province are likely that the result of prolonged and extensive insecticide application in agricultural contexts together with frequent mosquito population migrations. In contrast, the low-frequency kdr mutation detected in central Shandong Province populations may originate from the limited selection pressure in this area and the relative genetic isolation. Overall, the study compares the genetic patterns revealed by a functional gene with a neutral marker and demonstrates the combined impact of demographic and selection factors on population structure.

Facile synthesis, crystal structure, quantum calculation, and biological evaluations of novel selenenyl sulfide compounds as potential agrochemicals.

In order to design compounds with fresh molecular skeleton to break through the limitation of available agrochemicals, a series of 36 novel selenenyl sulfide compounds were chemically synthesized, and their biological activities were fully evaluated against tobacco mosaic virus (TMV), 14 plant pathogenic fungi, three insect species and plant acetohydroxyacid synthase (AHAS). All the target compounds were characterized by proton nuclear magnetic resonance (1 H-NMR), carbon-13 (13 C)-NMR, selenium-77 (77 Se)-NMR, and high-resolution mass spectrometry (HRMS). The crystal structure of 10j indicated that the Se-S bond was successfully constructed. Compounds 10d, 10h, 10s, 10u, 10aa, 10ac, 10ae, 10ag, and 10ai exhibited 40%, 43%, 39%, 41%, 47%, 46%, 47%, 42%, and 39% anti-TMV activities at 500 mg L-1 , better than that of ribavirin. The median effective concentration (EC50 ) against Sclerotinia sclerotiorum of 10ac was 6.69 mg L-1 and EC50 values against Physalospora piricola and Pyricularia grisea of 10z were 12.25 mg L-1 and 15.27 mg L-1 , respectively, superior to the corresponding values of chlorothalonil. Compounds 10c and 10v demonstrated 100% larvicidal activity against Culex pipiens pallens at 5mg L-1 , while 10a displayed 100% insecticidal activity against Mythimna separata at 200 mg L-1 . Compounds 10c, 10j, and 10o showed > 60% inhibitions against plant AHAS at 10μmol L-1 . From the quantum calculation, highest occupied molecular orbital (HOMO) was considered as a factor that affects the anti-TMV activity. The preliminary results suggested that more efforts should be devoted to exploring the selenenyl sulfides for the discovery of new leads of antiviral agent, fungicide, insecticide or AHAS inhibitors as potential agrochemicals for crop protection. © 2023 Society of Chemical Industry.

Design, Synthesis, and Insecticidal Evaluation of New Benzoylureas Containing Carbamate Groups Based on the Bipartite Model of Sulfonylurea Receptor Binding Site

Background: Benzoylureas are the most commonly used chitin synthesis inhibitors. But, the exact target site of benzoylurea insecticides has not been identified. Objective: To study whether benzoylphenylureas analogues with a bipartite model could be designed and synthesized as novel insecticides with improved activities. Methods: Based on the bipartite model of the sulfonylurea receptor binding site and commercial diflubenzuron, a series of novel benzoylureas containing carbamate groups were designed and synthesized via the key intermediate 2,6-difluoro-N-((4-hydroxyphenyl)carbamoyl)benzamide (2). The structures of the target compounds were confirmed by the 1H NMR and high-resolution mass spectrum (HRMS). Results: The results of bioassays indicated that these target compounds possessed good larvicidal activities against a broad spectrum of insects such as oriental armyworm (Mythimna Separata Walker), diamondback moth (Plutella xylostella), and mosquito (Culex pipiens pallens). A compound containing N, N-diisopropyl (3g) exhibited the highest insecticidal activity against oriental armyworm (40% at 10 mg kg-1), which was comparable with that of diflubenzuron. These compounds also had good larvicidal activities against diamondback moth and mosquito; most of these target compounds exhibited comparable larvicidal activities of diamondback moth with diflubenzuron and higher larvicidal activities of mosquito than diflubenzuron. Conclusion: The experimental data above preliminarily proved the rationality of our speculation and design ideology, and BPUs analogues with a bipartite model could improve the interaction with the target.