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The extensive use of chemical insecticides for public health and agricultural purposes has increased the occurrence and development of insecticide resistance. This study used transcriptome sequencing to screen 10 upregulated metabolic detoxification enzyme genes from Aedes albopictus resistant strains. Of these, CYP6A14 and CYP6N6 were found to be substantially overexpressed in the deltamethrin-induced expression test, indicating their role in deltamethrin resistance in Ae. albopictus. Furthermore, the corresponding 60-kDa recombinant proteins, CYP6A14 and CYP6N6, were successfully expressed using the Escherichia coli expression system. Enzyme activity studies revealed that CYP6A14 (5.84 U/L) and CYP6N6 (6.3 U/L) have cytochrome P450 (CYP450) enzyme activity. In vitro, the metabolic analysis revealed that the recombinant proteins degraded deltamethrin into 1-oleoyl-sn-glycero-3-phosphoethanolamine and 2′,2′-dibromo-2′-deoxyguanosine. Subsequently, the CYP450 genes in larvae of Ae. albopictus were silenced by RNA interference technology to study deltamethrin resistance in vivo. The silencing of CYP6A14 and CYP6N6 increased the mortality rate of mosquitoes without affecting their survival time, spawning quantity, hatching rate, and other normal life activities. Altogether, CYP6A14 and CYP6N6 belong to the CYP6 family and mutually increase deltamethrin resistance in Ae. albopictus.

Malaria is one of the most important parasitic diseases that causes a serious public health problem. The genetic diversity of malaria parasites may affect malaria transmission and malaria control strategies. In China, imported malaria was significantly increased in recent years, among which numerous migrant workers were infected with Plasmodium falciparum from Africa. However, little was known about genetic diversity of these populations in China. In this study, we evaluated genetic polymorphism and allele frequencies of msp1, msp2, and glurp genes in P. falciparum among Chinese migrant workers returnee from Africa between 2013 and 2017. Of the 381 P. falciparum isolates, 89.0% for msp1 gene, 71.7% for msp2 gene, and 78.0% for glurp gene were successfully genotyped. In msp1, 29 different alleles were observed, among which the K1 allelic family (71.7%) was predominant. In msp2, 21 different alleles were detected, of which the 3D7 allelic family (91.2%) was more frequent than FC27 allelic family (72.5%). For glurp, 12 individual alleles were detected in the samples. Taken together, the findings showed a high genetic diversity of these isolates, which provided the baseline data for African P. falciparum population imported to China.

Background. Future distribution of dengue risk is usually predicted based on predicted climate changes using general circulation models (GCMs). However, it is hard to validate the GCM results and assess the uncertainty of the predictions. Observed changes in climate may be very different from GCM results. Methods.We collected Aedes albopictus surveillance data and observed climate records from about 90 meteorological stations for the period 1970–2021. We analyzed the trends of climate change in China and made predictions on future climate for the years 2050 and 2080 based on trend analyses. We analyzed the relationship between climatic variables and the prevalence of Ae. albopictusin different months/seasons. We built a series of machine learning classification tree models to predict the monthly/seasonal Ae. albopictus distribution based on the average climate from 1970 to 2000 and assessed the contributions of different climatic variables to the Ae. albopictus distribution. Using these models, we projected the future distributions of Ae. albopictus for the years of 2050 and 2080. Results. The study included Ae. albopictus surveillance from 259 sites in China and found that winter to early spring (November–February) temperatures were strongly correlated with Ae. albopictus prevalence – the higher the temperature the higher the prevalence, while precipitation in summer (June–September) was important predictors for Ae. albopictus prevalence. The machine learning tree models predicted the current prevalence of Ae. albopictus with high agreement (accuracy > 90% and Kappa agreement > 80% for all months). Overall, winter temperature contributed the most to Ae. albopictus distribution, followed by summer precipitation. Increase in temperature was observed in most places in China and rates of annual change varied substantially among sites, with the highest increase in temperature occurring from February to April (annual increase of 1.4 – 4.7ºC for monthly mean, 0.6 – 4.0ºC for monthly minimum, and 1.3 – 4.3ºC for monthly maximum temperature) and the lowest in November and December. Temperature increases were lower in the tropics/subtropics compared to the high-latitude areas. The projected temperatures in 2050 and 2080 by this study were about 1~1.5°C higher than projected by GCMs. The estimated current Ae. albopictus risk distribution had a northern boundary of north-central China and the southern edge of northeastern China, with a risk period of June–September. The projected future Ae. albopictus risks in 2050 and 2080 cover nearly all of China, with an expanded risk period of April–October. The current at-risk population was estimated to be 960 million and the future at-risk population was projected to be 1.2 billion. Conclusions.The magnitude of climate change in China is likely to surpass the GCM predictions. Future dengue risks will expand to cover nearly all of China if the current climate trends continue.

Culex pipiens molestus and Culex pipiens pallens are two distinct bioforms in the Culex pipiens complex that are important vectors of several pathogens and are widely distributed around the world. In the current study, we present a high-quality chromosome-level genome of Cx. pipiens f. molestus and describe the genetic characteristics of this genome. The assembly genome was 559.749 Mb with contig and scaffold N50 values of 200.952 Mb and 0.370 Mb, and more than 94.78% of the assembled bases were located on 3 chromosomes. A total of 19,399 protein-coding genes were predicted. Many gene families were expanded in the genome of Cx. pipiens f. molestus, particularly those of the chemosensory protein (CSP) and gustatory receptor (GR) gene families. In addition, utilizing Hi-C data, we improved the previously assembled draft genome of Cx. pipiens f. pallens, with scaffold N50 of 186.195 Mb and contig N50 of 0.749 Mb, and more than 97.02% of the assembled bases were located on three chromosomes. This reference genome provides a foundation for genome-based investigations of the unique ecological and evolutionary characteristics of Cx. pipiens f. molestus, and the findings in this study will help to elucidate the mechanisms involved in species divergence in the Culex pipiens complex.

Due to the development of insecticide resistance in mosquitoes, with worldwide mosquito-borne diseases resurgence in recent years, recent advances in proteome technology have facilitated a proteome-wide analysis of insecticide resistance-associated proteins in mosquitoes. Understanding the complexity of the molecular basis of insecticide resistance mechanisms employed by mosquitoes will help in designing the most effective and sustainable mosquito control methods.After 30 generations, insecticide-selected strains showed elevated resistance levels to the cypermethrin used for selection. Proteome data allowed the detection of 2892 proteins, of which 2885 differentially expressed proteins (DEPs) achieved quantitative significances in four stages (egg, larvae, pupae, adult) of Culex pipiens pallens cypermethrin-resistant strain as compared to the susceptible strain. Among them, a significant enrichment of proteins, including cuticular proteins, enzymes involved in the detoxification (cytochrome P450, glutathione S-transferases, esterase, ATP-binding cassette) and some biological pathways (oxidative phosphorylation, hippo signalling) that are potentially involved in cypermethrin resistance, was observed. Thirty-one representative DEPs (cytochrome P450, glutathione S-transferase, cuticle protein) during Cx. pipiens pallens developmental stages were confirmed by a parallel reaction monitoring strategy.The present study confirmed the power of isobaric tags for relative and absolute quantification for identifying concomitantly quantitative proteome changes associated with cypermethrin in Cx. pipiens pallens. Proteome analysis suggests that proteome modifications can be selected rapidly by cypermethrin, and multiple resistance mechanisms operate simultaneously in cypermethrin-resistance of Cx. pipiens pallens, Our results interpret that an up-regulated expression of proteins and enzymes like cytochrome P450, glutathione S-transferases, esterase etc. has an impact in insecticide resistance. Previously neglected penetration resistance (cuticular proteins) may play an important role in the adaptive response of Cx. pipiens pallens to insecticides. This information may serve as a basis for future work concerning the possible role of these proteins in cypermethrin resistance in mosquito Cx. pipiens pallens. © 2022 Society of Chemical Industry.

China used to be one of the most heavily endemic countries for lymphatic filariasis (LF) in the world. Bancroftian filariasis, which is caused by the filarioid nematode Wuchereria bancrofti, is the only filariasis in Shandong Province. A total of 864 endemic counties (cities) in 16 provinces/autonomous regions/municipalities with a total population of 330 million people were at risk of infection. Shandong Province was a highly LF-endemic area in the 1950s, the epidemiological investigation of LF conducted in 1957 indicated that the disease was endemic in 74 counties and the highest microfilaria rate was up to 26%. There were ∼5 million people in the province infected with LF; among which almost 2.5 million people had lymphedema, elephantiasis, or hydrocele. Through vigorous prevention and scientific research, Shandong Province was the first to propose to treat LF with fortified diethylcarbamazine salt, more than 25 million people in the province had taken the salt, and more than 3 million people took intermittent medication. After more than 50 years of unremitting efforts, Shandong Province basically eliminated LF in 1983 and became the first province in China that successfully eradicated LF in 2004, which has played an important role in accelerating the elimination of filariasis in the country in 2007 and has made tremendous contributions to social and economic development of China. Since 1980, Shandong Province has carried out extensive international cooperation as the WHO Collaborating Center for Lymphatic Filariasis. This article is intended to share the experience in eliminating LF to other parts of the world and improve public health capacity in regions such as Africa and Oceania where the disease is still endemic for interest.

Objectives The aim of this study was to investigate the epidemic scope, epidemic pattern and degree of damage of clonorchiasis as well as to summarize the diagnosis and control effect of clonorchiasis in Shandong Province, China. Methods A retrospective study was conducted to summarize the research on the dynamic epidemiology and the prevention and treatment measures of clonorchiasis in Shandong Province, China from 1962 to 2015. Results After unremitting efforts and three nationwide surveys on the distribution and infection of human parasites, the infection rate of clonorchiasis decreased from 1.51% (1,500, 000) in the1960s to 0.16% in 1991, 0.04% in 2000 and 0.007% in 2015, respectively. The infection rate for villages with perennial water accumulation and rainy season water accumulation was higher than that of villages with rainy season water accumulation and non-water accumulation. The egg flotation in 70% potassium iodide solution method was applied to accurately test the specific proportion of C. sinensis eggs, which was 1.42. Using the modified Kato thick smear method, the detection rate of eggs was over 98%; hexachloroparaxylene yielded satisfactory therapeutic effects with slight adverse reactions, it is effective in the treatment of clonorchiasis. Conclusions After 50 years of active prevention and treatment, the disease has been successfully controlled in Shandong Province and had been used for reference in other endemic areas of clonorchiasis in China.

BackgroundDengue fever outbreaks tend to spread northward in China, and Jining is the northernmost region where local dengue fever cases have been detected. Therefore, it is important to investigate the density ofAedes albopictusand its resistance to deltamethrin.MethodsThe Breteau index (BI) and container index (CI) were calculated to assess the larval density ofAe. albopictusand human-baited double net trap (HDN) surveillance was performed in six subordinate counties (Rencheng, Yanzhou, Sishui, Liangshan, Zoucheng and Jiaxiang) of Jining City in 2017 and 2018. The resistance ofAe. albopictusadults to deltamethrin was evaluated using the World Health Organization (WHO) standard resistance bioassay. The mutations atVgsccodons 1532 and 1534 were also analysed to determine the association betweenkdrmutations and phenotypic resistance in adult mosquitoes.ResultsThe average BI, CI and biting rate at Jining were 45.30, 16.02 and 1.97 (female /man/hour) in 2017 and 15.95, 7.86 and 0.59 f/m/h in 2018, respectively. In August 26, 2017, when the first dengue fever case was diagnosed, the BI at Qianli village in Jiaxiang County was 107.27. The application of prevention and control measures by the government sharply decreased the BI to a value of 4.95 in September 3, 2017. The mortality of field-collectedAe. albopictusfemales from Jiaxiang was 41.98%. I1532T, F1534L and F1534S mutations were found in domain III of theVgscgene. This study provides the first demonstration that both I1532T and F1534S mutations are positively correlated with the deltamethrin-resistant phenotype.ConclusionsMosquito density surveillance, resistance monitoring and risk assessment should be strengthened in areas at risk for dengue to ensure the sustainable control ofAe. albopictusand thus the prevention and control of dengue transmission.

Background The increasing insecticide resistance of Aedes albopictus puts many countries in Asia and Africa, including China, at great risk of a mosquito-borne virus epidemic. To date, a growing number of researches have focused on the relationship between intestinal symbiotic bacteria and their hosts’ resistance to insecticides. This provides a novel aspect to the study of resistant mechanisms. Methods/Findings This study reveals significant composition and dynamic changes in the intestinal symbiotic bacteria of Ae. albopictus between the resistant and susceptible strains based on full-length sequencing technology. The relative abundance of Serratia oryzae was significantly higher in the resistance strain than in the susceptible strains; also, the relative abundance of S. oryzae was significantly higher in deltamethrin-induced Ae. albopictus than in their counterpart. These suggested that S. oryzae may be involved in the development of insecticide resistance in Ae. albopictus. To explore the insecticide resistance mechanism, adult mosquitoes were fed with GFP-tagged S. oryzae, which resulted in stable bacterial enrichment in the mosquito gut without affecting the normal physiology, longevity, oviposition, and hatching rates of the host. The resistance measurements were made based on bioassays as per the WHO guidelines. The results showed that the survival rate of S. oryzae-enriched Ae. albopictus was significantly higher than the untreated mosquitoes, indicating the enhanced resistance of S. oryzae-enriched Ae. albopictus. Also, the activities of three metabolic detoxification enzymes in S. oryzae-enriched mosquitoes were increased to varying degrees. Meanwhile, the activity of extracellular enzymes released by S. oryzae was measured, but only carboxylesterase activity was detected. HPLC and UHPLC were respectively used to measure deltamethrin residue concentration and metabolite qualitative analysis, showing that the deltamethrin degradation efficiency of S. oryzae was positively correlated with time and bacterial amount. Deltamethrin was broken down into 1-Oleoyl-2-hydroxy-sn-glycero-3-PE and 2’,2’-Dibromo-2’-deoxyguanosine. Transcriptome analysis revealed that 9 cytochrome P450s, 8 GSTs and 7 CarEs genes were significantly upregulated. Conclusions S. oryzae can be accumulated into adult Ae. albopictus by artificial feeding, which enhances deltamethrin resistance by inducing the metabolic detoxification genes and autocrine metabolic enzymes. S. oryzae is vertically transmitted in Ae. albopictus population. Importantly, S. oryzae can degrade deltamethrin in vitro, and use deltamethrin as the sole carbon source for their growths. Therefore, in the future, S. oryzae may also be commercially used to break down the residual insecticides in the farmland and lakes to protect the environment.

Japanese encephalitis (JE), which is caused by the Japanese encephalitis virus (JEV), is a zoonotic, vector-borne neurotropic disease that remains a major cause of viral encephalitis in Asia. JEV is spread to humans through mosquitoes, and its primary transmission vector is Culex tritaeniorhynchus. Mosquitoes were sampled from three collection sites: Hanzhuang town in Weishan County, Taibai Lake in Jining city, and Dongping Lake in Shandong Province, China. Pyrethroid insecticide resistance bioassays were conducted using adult mosquitoes. Cx. tritaeniorhynchus and C. pipiens pallens populations in Hanzhuang town and Dongping Lake showed resistance to pyrethroid insecticides, and populations in Taibai Lake showed incipient resistance. Coquillettidia ochracea populations in Hanzhuang town presented resistance as well, while in Taibai Lake, resistance was incipient. A total of 16,711 mosquitoes were collected, identified, and divided into 346 pools for JEV testing. Cx. tritaeniorhynchus had the advantage of being a local mosquito species. Overall, 31 (22.96) of the 135 pools of Cx. Tritaeniorhynchus were positive for JEV. The overall maximum likelihood estimates of Cx. tritaeniorhynchus, C. pipiens pallens, and Cq. ochracea indicated pooled infection rates of 5.29/1000 mosquitoes (95% confidence interval [CI] = 3.67–7.42), 1.60/1000 mosquitoes (95% CI = 0.82–2.85), and 6.39/1000 mosquitoes (95% CI = 0.39–32.23), respectively. There were no significant differences in the pooled infection rates between the districts. The resistance to pyrethroids has increased the difficulty in controlling the mosquito vectors, especially JEV-positive mosquitoes. Given the changes in the JEV transmission vectors, the spatial and temporal diversity and the dynamic variety of mosquito species, insecticide resistance and global warming have the potential to facilitate the transmission of JE to humans.

The intestinal symbiotic bacteria of Aedes albopictus play a potential role in host resistance to insecticides. In this study, we sequenced the full‐length of 16S rRNA and analyzed the differences in the intestinal microbiota between deltamethrin‐resistant and ‐sensitive Ae. albopictus. Symbiotic bacteria were cultured and analyzed using six types of culture media in aerobic and anaerobic environments. We found significant differences in the diversity and abundance of the intestinal microbiota of the two strains of Ae. albopictus. The symbiotic bacteria cultured in vitro were found to be mainly facultative anaerobes. The cultured bacteria such as Serratia oryzae and Acinetobacter junii may function to promote the development of insecticide resistance. This work indicates that intestinal bacteria may contribute to the enhancement of insecticide resistance of Ae. albopictus It also highlights the analytical advantage of full‐length 16S rRNA sequencing to study the intestinal microbiota of mosquitoes., In this study, we used third‐generation sequencing technology to determine the full‐length of 16S rRNA and to analyze the differences in the intestinal symbionts between deltamethrin‐resistant and sensitive Aedes albopictus. Significant differences in diversity and abundance have been found. Symbiotic bacteria were cultured and analyzed using 6 different types of culture media under aerobic and anaerobic environments. The cultured bacteria, such as Serratia oryzae and Acinetobacter junii, may contribute to the enhancement of insecticide resistance in Ae. albopictus.

Mosquito control based on chemical insecticides is considered as an important element of the current global strategies for the control of mosquito-borne diseases. Unfortunately, the development of insecticide resistance of important vector mosquito species jeopardizes the effectiveness of insecticide-based mosquito control. In contrast to target site resistance, other mechanisms are far from being fully understood. Global protein profiles among cypermethrin-resistant, propoxur-resistant, dimethyl-dichloro-vinyl-phosphate-resistant and susceptible strain of Culex pipiens pallens were obtained and proteomic differences were evaluated by using isobaric tags for relative and absolute quantification labeling coupled with liquid chromatography/tandem mass spectrometric analysis. A susceptible strain of Culex pipiens pallens showed elevated resistance levels after 25 generations of insecticide selection, through iTRAQ data analysis detected 2,502 proteins, of which 1,513 were differentially expressed in insecticide-selected strains compared to the susceptible strain. Finally, midgut differential protein expression profiles were analyzed, and 62 proteins were selected for verification of differential expression using iTRAQ and parallel reaction monitoring strategy, respectively. iTRAQ profiles of adaptation selection to three insecticide strains combined with midgut profiles revealed that multiple insecticide resistance mechanisms operate simultaneously in resistant insects of Culex pipiens pallens. Significant molecular resources were developed for Culex pipiens pallens, potential candidates were involved in metabolic resistance and reducing penetration or sequestering insecticide. Future research that is targeted towards RNA interference of the identified metabolic targets, such as cuticular proteins, cytochrome P450s, glutathione S-transferases and ribosomal proteins proteins and biological pathways (drug metabolism—cytochrome P450, metabolism of xenobiotics by cytochrome P450, oxidative phosphorylation, ribosome) could lay the foundation for a better understanding of the genetic basis of insecticide resistance in Culex pipiens pallens., Author summary Global protein profiles were compared among a susceptible strain of Cx. pipiens pallens and strains that were cypermethrin-resistant, propoxur-resistant, and dimethyl-dichloro-vinyl-phosphate-resistant after 25 generations of selection by distinct chemical insecticide families, multiple mechanisms were found to operate simultaneously in resistant mosquitoes of Cx. pipiens pallens, including mechanisms to lower penetration of or sequester the insecticide or to increase biodegradation of the insecticide via subtle alterations in either the cuticular protein levels or the activities of detoxification enzymes (P450s and glutathione S-transferases).

Background: We aimed to conduct a retrospective analysis of the epidemiological changes and prevention effects for malaria in Weifang, Shandong Peninsula, China from 1957 to 2017. Methods: The malaria data from a web-based reporting system were analyzed to explore malaria epidemiological characteristics and prevention effects in Weifang. Results: Overall, 1, 704, 890 malaria cases were reported in Weifang from 1957 to 2017, of which two major malaria epidemics occurred in 1961 (827.28/10, 000) and 1971 (366.14/10, 000). Prior to 1997, all malaria patients (1, 704, 829) were infected with Plasmodium vivax (P. vivax). After 2007, the cases of Plasmodium falciparum (P. falciparum) showed an upward trend (76.8%). The reported cases after the 21st century were mainly imported cases, and the last indigenous case was a patient that infected with P. vivax in 2006. Overall, 36 imported cases were reported from 2010 to 2017, of which 88.9% were acquired in Africa. Except for one 32-year-old woman, the rest were male (97.2%), in which laborers and farmers represented the vast majority (66.6%). From 1987 to 2017, there were 1, 224, 474 cases of fever with blood tests, and the average blood test rate was 4.9%. From 1957 to 2017, a total of 1, 704, 890 malaria patients were treated, 96 cases were treated during resting phase from 1987 to 2017. Conclusion: Weifang should continue to strengthen the management of the migrant population, making blood tests for fever patients and patient treatment as important means of malaria control and monitoring.

Background: Mosquito control based on chemical insecticides is considered as an important element in the current global strategies for the control of mosquito-borne diseases. Unfortunately, the development of insecticide resistance of important vector mosquito species jeopardizes the effectiveness of insecticide-based mosquito control. As opposed to target site resistance, other mechanisms are far from being fully understood.Results: Susceptible strain of Cx. pipiens pallen showed elevated resistance levels to after 25 generations insecticide-selected, through bioinformatics analysis allowed detecting 2,502 proteins, of which 1513 were differentially expression in insecticide-selected strains as compared to the susceptible strain. Finally, midgut differential expression protein profiles and 62 proteins were selected for verification of differential expression using parallel reaction monitoring strategy.Conclusions Significant molecular resources were developed for Cx. pipiens pallen potential candidates involved in metabolic resistance as well as those participating in lower penetration or sequestration of insecticide. Global protein profiles of change to three insecticide strains combined with midgut profiles revealed multiple insecticide resistance mechanisms operate simultaneously in resistant insects of Cx. pipiens pallens. Future research that is targeted towards RNA interference on the identified metabolic targets such as cuticular, cytochrome P450s and glutathione S-transferase proteins could lay the foundation for a better understanding of the genetic basis of insecticide resistance in Cx. pipiens pallen.

Background A long-lasting overdependence on insecticides has led to the rapid spread of pyrethroid resistance in mosquito vectors, which poses a great risk to the general public. Although there are many studies on metabolic resistance and target resistance, few have investigated cuticle resistance and behaviour resistance. The cuticle of mosquitoes has been hypothesized to play a role in insecticide resistance by reducing penetration or sequestering insecticides.Methods We used RNA sequencing (RNA-seq) to analyse the transcriptome of cypermethrin-resistant and cypermethrin-susceptible strains of Culex pipiens pallens. We sequenced 6 samples using an Illumina HiSeq platform and generated approximately 6.66 Gb bases from each sample on average. Mapping the sequenced reads to a reference genome and reconstructing the transcripts via gene expression analysis, we detected differentially expressed genes (DEGs) among the samples. Followed Gene Ontology (GO) classification and functional enrichment. Finally, we screened the genes of cuticle proteins associated with drug resistance throughout the genome, selected the significant DEGs with a log2 fold change > 3.0 and Padj < 0.05, and applied real-time fluorescence quantitative polymerase chain reaction (PCR) to verify the DEGs. Results We obtained 13,517 novel transcripts, of which 8,653 were previously unknown splicing events for known genes, 665 were novel coding transcripts without any known features, and 4,199 were long non-coding RNA (lncRNA). A total of 1035, 944, and 657 genes were upregulated in comparisons between the samples, and 2680, 1215, and 975 genes were downregulated in comparisons between the samples. Finally, among all samples, 167 genes were upregulated and 145 genes were downregulated. The GO classification and functional enrichment of DEGs were as follows: molecular function, 224 genes; cellular component, 149 genes; and biological process, 272 genes. The expression of XM_001863852 and XM_001845881 in resistant strains of Culex pipiens pallens was lower than that in the laboratory sensitive strain, with 0.177 and 0.548-fold change in expression, respectively; the expression of the XM_001845883.1 in the resistant strain was higher than that in the susceptible strain, with a 2.281-fold change in expression.Conclusions Our results provide a reference for resistance mechanisms via the mosquito cuticle as well as a new perspective for disease vector control.

Objective To conduct an analysis of the epidemiological changes in malaria that have occurred in Shanxian county from 2002 to 2016. Methods A retrospective study was conducted and data were collected from web-based reporting system to explore the epidemiological characteristics in Shanxian county from 2002 to 2016. All individual case information was obtained from village malaria servicers organized by the local Shandong Institute of Parasitic Diseases. Results A total of 133 cases were identified as malaria in Shanxian county during this period, including 124 indigenous cases (93.2%) and 9 imported cases (6.8%). The 124 indigenous malaria cases were infected with Plasmodium vivax (P. vivax) , whereas 7 of the 9 confirmed imported cases were infected with Plasmodium falciparum (P. falciparum) , 1 was infected with Plasmodium ovale ( P. ovale ) and 1 patient was infected with P. falciparum mixed with P. vivax . The total number of malaria cases included 86 males (64.7%) and 47 females (35.3%). Age of the patients ranged from 1 to 83 years, although most (64.7%) infections occurred in the 21-to 60-year-old age group. Remarkably, 117 of the total malaria cases (98.0%) were reported from 2006 to 2011. The epidemic season was from June to October, with the peak occurring yearly from July to September. The most common occupation of the infected patients was farmer. In total, 58.1% of the cases occurred in 3 townships, namely, Fugang, Huanggang and Caozhuang. Conclusions In Shanxian county, the local malaria incidence experienced an emerge-peak-control-eliminate status. However, due to the numbers of migrant labourers returning from Africa, imported cases were continuous and presented an increasing annual trend, which became a non-negligible and a significant impediment for malaria elimination. Therefore, the need to eliminate instances of malaria reintroduction to receptive malaria-free areas should drive strategies to align with the epidemiological changes.

Background Long-lasting overdependence on insecticides has led to the rapid spread of pyrethroid resistance in mosquito vectors, which is of great concern to the general public. There are many studies on metabolic resistance and target resistance, but fewer studies have been conducted on cuticle resistance and behaviour resistance. The cuticle of mosquitoes has been hypothesized to play a role in insecticide resistance by reducing penetration or sequestering insecticides. Methods We used RNA sequencing (RNA-seq) to analyse the transcriptome of cypermethrin-resistant and cypermethrin-susceptible strains of Culex pipiens pallens . Sequenced 6 samples using an Illumina HiSeq platform, and generated approximately 6.66 Gb bases from each sample on average. Mapping the sequenced reads to a reference genome and reconstructing the transcripts, through gene expression analysis, we detected differentially expressed genes (DEGs) among the samples. Followed Gene Ontology (GO) classification and functional enrichment. Finally, we screened the genes of cuticle proteins associated with drug resistance throughout the genome, selected the significant DEGs with a log2 fold change>3.0 and Padj

Malaria, one of the most striking, re-emerging infectious diseases caused by the genus Plasmodium, places a huge burden on global healthcare systems. A major challenge in the control and eradication of malaria is the continuous emergence of increasingly widespread drug-resistant malaria, creating an urgent need to develop novel antimalarial agents. Chalcone derivatives are ubiquitous in nature and have become indispensable units in medicinal chemistry applications due to their diverse biological profiles. Many chalcone derivatives demonstrate potential in vitro and in vivo antimalarial activity, so chalcone could be a useful template for the development of novel antimalarial agents. This review covers the recent development of chalcone hybrids as antimalarial agents. The critical aspects of the design and structure-activity relationship of these compounds are also discussed.

Objectives: To study the epidemiological characteristics of malaria and the effects of comprehensive malaria control implementation in Yantai, as well as to provide a scientific basis for future malaria elimination. Materials and Methods: A retrospective analysis was conducted to explore the epidemiological changes and prevention effects in Yantai from 1951 to 2017. The malaria data before 2004 were collected from paper-based annual reports, and data after 2004 came from the Infectious Diseases Information Reporting Management System. Results: A total of 81,286 malaria cases were identified in Yantai from 1957 to 2017, with two peaks occurring in 1966 (120.93/100,000) and 1970–1972 (366.12/100,000). From 1951 to 2003, the case classification was mainly Plasmodium vivax (99.80%); the first case attributed to Plasmodium falciparum occurred in 2004, and P. falciparum became the main cause of malaria after 2011. All cases reported since 2010 have been imported cases, and the last indigenous case was caused by P. vivax in 2008. A total of 129 imported cases were reported from 2010 to 2017, most of which originated in Africa (93.80%), mainly in men aged 30–49 years (68.28%). From 1951 to 1973, 73,868 cases of malaria were diagnosed with clinical symptoms, and from 1974 to 2017, 818,943 cases in fever patients were diagnosed with blood tests. Conclusions: To achieve the goal of eliminating malaria by 2020, Yantai should continue to strengthen the management of migrant population, including improving malaria surveillance for returnees and immigrants from overseas endemic areas, and continue to increase the training of medical personnel to improve their diagnostic ability.

Neurocysticercosis (NCC) has a wide spectrum of neurologic and psychiatric manifestations, including epileptic seizures, high intracranial pressure, cognitive dysfunction, and meningoencephalitis. This study presents the clinical diagnosis of 2539 NCC patients in Shandong Province, Eastern China, from 1997 to 2015. The diagnosis was based primarily on clinical features, neuroimaging, immunology, and electroencephalogramy studies. In all age groups, seizures were found to be the most common manifestation, followed by headaches, memory loss, and limb numbness, and disability. After antiparasitic treatment, computed tomography and magnetic resonance imaging showed that most of the lesions had been completely absorbed in 2106 (82.95%) patients, most of the lesions had been absorbed and a small proportion was converted into calcifications in 433 (17.05%) cases. Indirect hemagglutination (IHA), enzyme-linked immunosorbent assay (ELISA), and circulating antigen (CAg) serum tests initially gave 76.45%, 86.37%, and 80.66% positive results, respectively. After antiparasitic therapy, the positive rates of the IHA, ELISA, and CAg tests were 74.77%, 84.70%, and 3.75%, respectively, showing no significant difference in antibody levels (p > 0.05), but a significant difference in CAg levels (p

Background To investigate mosquito larval habitats and resistance to common insecticides in areas with high incidence rates of mosquito-borne diseases in Jining, Shandong Province, and to provide a scientific basis for the future prevention and control of mosquito-borne diseases and the rational use of insecticides. Methods and results From June to September 2018, mosquito habitat characteristics and species compositions in Jintun town were studied through a cross-sectional survey. Larvae and pupae were collected in different habitats using the standard dipping technique. A total of 7,815 mosquitoes, comprising 7 species from 4 genera, were collected. Among them, Culex pipiens pallens (n = 5,336, 68.28%) was the local dominant species and found in all four habitats (rice paddies, irrigation channels, water containers, drainage ditches). There were 1,708 Cx. tritaeniorhynchus (21.85%), 399 Anopheles sinensis (5.11%), 213 Armigeres subalbatus (2.72%), 124 Aedes albopictus (1.59%), and 35 other (Cx. bitaeniorhynchus and Cx. halifaxii) (0.45%) mosquito samples collected. Spearman correlation analysis was employed to evaluate the relationship between larval density and the physicochemical characteristics of the breeding habitat. It was found that the larval density of Cx. tritaeniorhynchus correlated positively with water depth (r = 0.927 p = 0.003), the larval density of An. sinensis correlated positively with dissolved oxygen (DO) (r = 0.775 p = 0.041) and the larval density of Cx. p. pallens correlated positively with ammonia nitrogen (r = 0.527 p = 0.002). Resistance bioassays were carried out on the dominant populations of Cx. p. pallens: mosquitoes presented very high resistance to cypermethrin and deltamethrin, moderate resistance to dichlorvos (DDVP), and low resistance to Bacillus thuringiensis israelensis (Bti), with decreased susceptibility to propoxur. Conclusion We showed that mosquito species vary across habitat type and that the mosquito larval density correlated positively with certain physicochemical characteristics in different habitats. In addition, Cx. p. pallens developed different levels of resistance to five insecticides. Vector monitoring should be strengthened after an epidemic, and further research should be conducted to scientifically prevent and kill mosquitoes.

Background Culex pipiens pallens is the most abundant Culex mosquito species in northern China and is an important vector of bancroftian filariasis and, potentially, West Nile virus. Insecticides, particularly pyrethroids, are widely used for adult mosquito control. Insecticide resistance has become common in several mosquito species, and vector control is the main method currently available to prevent disease transmission. The voltage-gated sodium channel (Vgsc) gene is the target site of pyrethroids, and mutations in this gene cause knockdown resistance (kdr). Methods Culex pipiens pallens larvae were collected from May to November over two decades, from 1992 to 2018, in four cities in Shandong Province, China. The World Health Organization (WHO) standard resistance bioassay was applied to test the resistance levels of Cx. p. pallens larvae to five different insecticides and to test deltamethrin resistance in adults, using the F1 generation. Mutations at Vgsc codon 1014 were also screened in 471 adult samples collected in 2014 to determine the association between kdr mutations and phenotypic resistance. Results Larval resistance against deltamethrin showed an increasing trend from the 1990s until 2018, which was statistically significant in all populations; resistance to cypermethrin increased significantly in mosquitoes from the Zaozhuang population. However, larval resistance to other insecticides remained relatively stable. Larval resistance against deltamethrin was consistent with adult bioassays in 2014, in which all tested populations were highly resistant, with mortality rates ranging from 39.4 to 55.23%. The L1014S and L1014F mutations were both observed in five Cx. p. pallens populations, with L1014F significantly associated with deltamethrin resistance. Conclusions The long-term dataset from Shandong demonstrates major increases in pyrethroid resistance over a 20-year period. The L1014F kdr mutation may be considered a viable molecular marker for monitoring pyrethroid resistance in Cx. p. pallens. Electronic supplementary material The online version of this article (10.1186/s13071-019-3416-9) contains supplementary material, which is available to authorized users.

Background Anopheles sinensis is an important vector for the spread of malaria in China. Olfactory-related behaviours, particularly oviposition site seeking, offer opportunities for disrupting the disease-transmission process. Results This is the first report of the identification and characterization of AsinOrco and AsinOR10 in An. sinensis. AsinOrco and AsinOR10 share 97.49% and 90.37% amino acid sequence identity, respectively, with related sequences in Anopheles gambiae. A functional analysis demonstrated that AsinOrco- and AsinOR10-coexpressing HEK293 cells were highly sensitive to 3-methylindole, but showed no significant differences in response to other test odorants when compared to DMSO. Conclusions AsinOrco was characterized as a new member of the Orco ortholog subfamily. AsinOR10, which appears to be a member of the OR2-10 subfamily, is directly involved in identification of oviposition sites. This finding will help to elucidate the molecular mechanisms underlying olfactory signaling in An. sinensis and provide many more molecular targets for eco-friendly pest control. Electronic supplementary material The online version of this article (10.1186/s12936-018-2501-4) contains supplementary material, which is available to authorized users.

To reveal overwintering dormancy (diapause) mechanisms of Culex pipiens pallens (L.), global protein expression differences at three separate time points represent nondiapause, diapause preparation and overwintering diapause phases of Cx. pipiens pallens were compared using iTRAQ. Cx. pipiens pallens females accumulate more lipid droplets during diapause preparation and overwintering diapause maintenance than during the nondiapause phase. A total of 1030 proteins were identified, among which 1020 were quantified and compared. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG), Domain and Clusters of Orthologous Groups (COG) analyses revealed key groups of proteins, pathways and domains differentially regulated during diapause preparation and overwintering diapause maintenance phases in this mosquito, including major shifts in energy production and conversion, fatty acid metabolism, the citrate (TCA) cycle, and the cytoskeletal reorganization pathway. Our results provide novel insight into the molecular bases of diapause in mosquitoes and corroborate previously reported diapause-associated features in invertebrates. More interestingly, the phototransduction pathway exists in Cx. pipiens pallens, in particular, actin, rather than other proteins, appears to have substantial role in diapause regulation. In addition, the differential changes in calmodulin protein expression in each stage implicate its important regulatory role of the Cx. pipiens pallens biological clock. Finally, 24 proteins were selected for verification of differential expression using a parallel reaction monitoring strategy. The findings of this study provide a unique opportunity to explore the molecular modifications underlying diapause in mosquitoes and might therefore enable the future design and development of novel genetic tools for improving management strategies in mosquitoes.

Anopheles sinensis Wiedemann is a major vector of malaria and is among the dominant species in Shandong province of China. Knowledge of the blood-feeding patterns of mosquitoes is crucial for elimination of malaria vectors. However, little information is available on the blood-feeding behaviour of An. sinensis mosquitoes in Shandong province. This study was carried out to compare the blood-feeding behaviour of An. sinensis in malaria-endemic areas of Shandong province China.Adult Anopheles mosquitoes were collected from three malaria-endemic areas (Jimo, Yinan and Shanxian), during the peak months of mosquito population (August and September) from 2014 to 2015. Indoor-resting mosquitoes and outdoor-resting blood-fed females were sampled in the morning hours (0600 to 0900 hrs) from 10 randomly selected houses using pyrethrum spray catch method, and sweeping with an insect net. ELISA was used for the identification of blood meal. The blood meal of each mosquito was tested against antisera specific to human, pig, dog, cow, goat, horse (mule) and fowl.At all indoor study locations of Jimo, Yinan and Shanxian, 59.4, 68.1 and 98.8% blood-engorged female An. sinensis collected from cattle sheds fed almost exclusively on bovines, respectively. For outdoor locations, at Jimo site, 27.27 and 49.55% An. sinensis fed on cattle and pigs; at Yinan, 30.42% fed on cattle and 36.88% fed both on cattle and goats, while no pig antibodies were detected. At Shanxian, percent of An. sinensis that fed on cattle, pigs and cattle-goat was 20.72, 27.62 and 21.78%, respectively.The analysis of An. sinensis blood meals in all the three studied areas from human houses, cattle sheds, pig sheds and mixed dwellings revealed that An. sinensis prefers cattle hosts, and can feed on other available animal hosts if the cattle hosts are absent, and the mosquitoes readily feed on humans when domestic animals (cattle and pigs) are not nearby for feeding. The analysis of blood meal revealed that An. sinensis follow opportunistic feeding in Shandong province, China.