Genetic characteristics of P. falciparum parasites collected from 2012 to 2016 and anti-malaria resistance along the China-Myanmar border.

BACKGROUNDS: The therapeutic efficacy studies of DHA-PIP for uncomplicated Plasmodium falciparum patients were implemented from 2012 to 2016 along China (Yunnan province)-Myanmar border, which verified the high efficacy of DHA-PIP. With the samples collected in these studies, the genetic characteristics of P. falciparum parasites based on in vivo parasite clearance time (PCT) was investigated to explore if these parasites had developed resistance to DHA and PIP at molecular level. METHODS: The genetic characteristics were investigated based on K13 genotypes, copy numbers of genes pfpm2 and pfmdr1, and nine microsatellite loci (Short Tandem Repeats, STR) flanking the K13 gene on chromosome 13. The PCT 50s were compared based on different K13 genotypes, sites, periods and copy numbers. RESULTS: In the NW (North-West Yunnan province bordering with Myanmar) region, F446I was the main K13 genotype. No significant differences for PCT 50s presented among three K13 genotypes. In SW (South-West Yunnan province bordering with Myanmar) region, only wild K13 genotype was detected in all parasite isolates whose PCT 50s was significantly longer than those in NW region. For the copy numbers of genes, parasite isolates containing multiple copies of pfmdr1 gene were found in both regions, but only single copy of pfpm2 gene was detected. Though the prevalence of parasite isolates with multiple copies of pfmdr1 gene in SW region was higher than that in NW region, no difference in PCT 50s were presented between isolates with single and multiple copies of pfmdr1 gene. The median He values of F446I group and Others (Non-F446I K13 mutation) group were 0.08 and 0.41 respectively. The mean He values of ML group (Menglian County in SW) and W (wild K13 genotype in NW) group were 0 and 0.69 respectively. The mean Fst values between ML and W groups were significantly higher than the other two K13 groups. CONCLUSIONS: P. falciparum isolates in NW and SW regions had very different genetic characteristics. The F446I was hypothesized to have independently appeared and spread in NW region from 2012 and 2016. The high susceptibility of PIP had ensured the efficacy of DHA-PIP in vivo. Multiple copy numbers of pfmdr1 gene might be a potential cause of prolonged clearance time of ACTs drugs along China-Myanmar border. TRIAL REGISTRATION: Trial registration: ISRCTN, ISRCTN 11775446. Registered 17 April 2020-Retrospectively registered, the registered name was Investigating resistance to DHA-PIP for the treatment of Plasmodium falciparum malaria and chloroquine for the treatment of Plasmodium vivax malaria in Yunnan, China. http://www.isrctn.com/ISRCTN11775446.

The impact of enterprise resilience and HRM practices on performance: Findings from fsQCA.

Background: Both enterprise resilience and HRM practices can have a positive impact on enterprise performance. The impact of enterprise resilience or human resource management (HRM) practices on enterprise performance independently has been studied widely. But few studies have combined the above two aspects to discuss their impact on enterprise performance. Objective: In order to provide positive conclusions for improving enterprise performance, the theoretical model is established to expound the relationship between enterprise resilience, HRM practices including their internal influencing factors and enterprise performance. According to this model, a series of hypotheses about the influence of the combination from these internal factors on enterprise performance are presented. Method: Based on the statistical data of the questionnaire survey with managers and general employees at different levels in enterprises as respondents, the correctness of these hypotheses is proved by the fuzzy set qualitative comparative analysis (fsQCA) method. Results and discussion: The impact of the combination of enterprise resilience for high enterprise performance is shown in Table 3. The positive impact on the configuration of HRM practices for enterprise performance is shown in Table 4. The influences of the various combinations of internal factors about enterprise resilience and HRM practices on enterprise performance are shown in Table 5. From Table 4, it is discovered that performance appraisal and training have a significant positive effect on high enterprise performance. From Table 5, it is found that information sharing capabilities play a critical role, and enterprise resilience capabilities have a relatively positive impact on enterprise performance. Therefore, managers need to seek the development of enterprise resilience and HRM practices simultaneously and choose the most suitable combination configuration according to the actual situation of the enterprise itself. Moreover, a meeting system should be set up to ensure the transmission of internal information efficiently and accurately.

Establishing and applying an adaptive strategy and approach to eliminating malaria: practice and lessons learnt from China from 2011 to 2020.

ABSTRACTOn 30 June 2021, China was certified malaria-free by the World Health Organization. In this study, the evolution, performance, outcomes, and impact of China's adaptive strategy and approach for malaria elimination from 2011 to 2020 were analysed using 10-year data. The strategy and approach focused on timely detection and rapid responses to individual cases and foci. Indigenous cases declined from 1,308 in 2011 to 36 in 2015, and the last one was reported from Yunnan Province in April 2016, although thousands of imported cases still occur annually. The "1-3-7" approach was implemented successfully between 2013 and 2020, with 100% of cases reported within 24 h, 94.5% of cases investigated within three days of case reporting, and 93.4% of foci responses performed within seven days. Additionally, 81.6% of patients attended the first healthcare visit within 1-3 days of onset and 58.4% were diagnosed as malaria within three days of onset, in 2017-2020. The adaptive strategy and approach, along with their universal implementation, are most critical in malaria elimination. In addition to strengthening surveillance on drug resistance and vectors and border malaria collaboration, a further adapted three-step strategy and the corresponding "3-3-7" model are recommended to address the risks of re-transmission and death by imported cases after elimination. China's successful practice and lessons learnt through long-term efforts provide a reference for countries moving towards elimination.

No evidence of amplified Plasmodium falciparum plasmepsin II gene copy number in an area with artemisinin-resistant malaria along the China-Myanmar border.

BACKGROUND: The emergence and spread of artemisinin resistance in Plasmodium falciparum poses a threat to malaria eradication, including China's plan to eliminate malaria by 2020. Piperaquine (PPQ) resistance has emerged in Cambodia, compromising an important partner drug that is widely used in China in the form of dihydroartemisinin (DHA)-PPQ. Several mutations in a P. falciparum gene encoding a kelch protein on chromosome 13 (k13) are associated with artemisinin resistance and have arisen spread in the Great Mekong subregion, including the China-Myanmar border. Multiple copies of the plasmepsin II/III (pm2/3) genes, located on chromosome 14, have been shown to be associated with PPQ resistance. METHODS: The therapeutic efficacy of DHA-PPQ for the treatment of uncomplicated P. falciparum was evaluated along the China-Myanmar border from 2010 to 2014. The dry blood spots samples collected in the efficacy study prior DHA-PPQ treatment and from the local hospital by passive detection were used to amplify k13 and pm2. Polymorphisms within k13 were genotyped by capillary sequencing and pm2 copy number was quantified by relative-quantitative real-time polymerase chain reaction. Treatment outcome was evaluated with the World Health Organization protocol. A linear regression model was used to estimate the association between the day 3 positive rate and k13 mutation and the relationship of the pm2 copy number variants and k13 mutations. RESULTS: DHA-PPQ was effective for uncomplicated P. falciparum infection in Yunnan Province with cure rates > 95%. Twelve non synonymous mutations in the k13 domain were observed among the 268 samples with the prevalence of 44.0% and the predominant mutation was F446I with a prevalence of 32.8%. Only one sample was observed with multi-copies of pm2, including parasites with and without k13 mutations. The therapeutic efficacy of DHA-PPQ was > 95% along the China-Myanmar border, consistent with the lack of amplification of pm2. CONCLUSION: DHA-PPQ for uncomplicated P. falciparum infection still showed efficacy in an area with artemisinin-resistant malaria along the China-Myanmar border. There was no evidence to show PPQ resistance by clinical study and molecular markers survey. Continued monitoring of the parasite population using molecular markers will be important to track emergence and spread of resistance in this region.

The contributions and achievements on malaria control and forthcoming elimination in China over the past 70 years by NIPD-CTDR.

Although the past decades have seen a remarkable decrease in malaria-caused mortality and morbidity, the infection remains a significant challenge to global health. In the battle against malaria, China has gained notable feat and achievement since the 1940s through the efforts of several generations. Notably, China has not recorded a single indigenous malaria case since August 2016. The National Institute of Parasitic Diseases of the Chinese Center for Disease Control and Prevention (NIPD), as the only specialized institution for parasitic disease at the national level, has played a significant role in the malaria control, prevention, and elimination in China in the different historical periods. In order to transfer Chinese experiences on malaria control and elimination to other Low and Middle Income Countries (LMICs) and to improve global health collaboration, we have summarized and reviewed the contributions and achievements by the NIPD over the past 70 years, covering the epidemic situation; field investigation and laboratory experimental research on both parasite and vector; research and development on diagnostics, drugs, and insecticides; surveillance and response; technical and international. Support and cooperation. In addition, we also focus in particular on malaria retransmission risk, strategies on management of imported malaria cases and mobile populations, surveillance and response capacity to be maintained in post-elimination stage, challenges on diagnosis, drug resistance, and insecticide resistance as future concerns.

Genomic Epidemiology of Antimalarial Drug Resistance in Plasmodium falciparum in Southern China.

Emerging artemisinin resistance in Southeast Asia poses a significant risk to malaria control and eradication goals, including China's plan to eliminate malaria nationwide by 2020. Plasmodium falciparum was endemic in China, especially in Southern China. Parasites from this region have shown decreased susceptibility to artemisinin and delayed parasite clearance after artemisinin treatment. Understanding the genetic basis of artemisinin resistance and identifying specific genetic loci associated with this phenotype is crucial for surveillance and containment of resistance. In this study, parasites were collected from clinical patients from Yunnan province and Hainan island. The parasites were genotyped using a P. falciparum-specific single nucleotide polymorphism (SNP) microarray. The SNP profiles examined included a total of 27 validated and candidate molecular markers of drug resistance. The structure of the parasite population was evaluated by principal component analysis by using the EIGENSOFT program, and ADMIXTURE was used to calculate maximum likelihood estimates for the substructure analysis. Parasites showed a high prevalence of resistance haplotypes of pfdhfr and pfdhps and moderate prevalence of pfcrt. There was no mutation identified on pfmdr1. Candidate SNPs on chromosomes 10, 13, and 14 that were associated with delayed parasite clearance showed a low prevalence of mutants. Parasites from Southern China were clustered and separated from those from Southeast Asia. Parasites from Yunnan province were substructured from parasites from Hainan island. This study provides evidence for a genomic population with drug resistance in Southern China and also illustrates the utility of SNP microarrays for large-scale parasite molecular epidemiology.

Artemisinin resistance-associated markers in Plasmodium falciparum parasites from the China-Myanmar border: predicted structural stability of K13 propeller variants detected in a low-prevalence area.

BACKGROUND: Malaria reduction and future elimination in China is made more difficult by the importation of cases from neighboring endemic countries, particularly Myanmar, Laos, and Vietnam, and increased travel to Africa by Chinese nationals. The increasing prevalence of artemisinin resistant parasites across Southeast Asia highlights the importance of monitoring the parasite importation into China. Artemisinin resistance in the Mekong region is associated with variants of genes encoding the K13 kelch domain protein (pf13k), found in specific genetic backgrounds, including certain alleles of genes encoding the chloroquine resistance transporter (pfcrt) and multidrug resistance transporter PgH1 (pfmdr1). METHODS: In this study we investigated the prevalence of drug resistance markers in 72 P. falciparum samples from uncomplicated malaria infections in Tengchong and Yingjiang, counties on the Yunnan-Myanmar border. Variants of pf13k, pfcrt and pfmdr1 are described. RESULTS: Almost all parasites harboured chloroquine-resistant alleles of pfcrt, whereas pfmdr1 was more diverse. Major mutations in the K13 propeller domain associated with artemisinin resistance in the Mekong region (C580Y, R539T and Y493H) were absent, but F446I and two previously undescribed mutations (V603E and V454I) were identified. Protein structural modelling was carried out in silico on each of these K13 variants, based on recently published crystal structures for the K13 propeller domain. Whereas F446I was predicted to elicit a moderate destabilisation of the propeller structure, the V603E substitution is likely to lead to relatively high protein instability. We plotted these stability estimates, and those for all previously described variants, against published values for in vivo parasitaemia half-life, and found that quadratic regression generates a useful predictive algorithm. CONCLUSION: This study provides a baseline of P. falciparum resistance-associated mutations prevalent at the China-Myanmar border. We also show that protein modelling can be used to generate testable predictions as to the impact of pfk13 mutations on in vivo (and potentially in vitro) artemisinin susceptibility.

[Establishment and Application of Multiplex PCR System for Detecting Four Human Plasmodium Species].

OBJECTIVE: To establish a multiplex PCR detection system for identifying 4 human Plasmodium species and evaluate its applicability. METHODS: The sequences of 18S rDNA gene of the 4 human Plasmodium species were compared using DNAman software, and 4 downstream primers were designed using Oligo 6.0 software, which targeted the region of variability between conserved regions 5 and 6 of the sequences. Using these primers, the specificity and sensitivity of the multiplex PCR system were evaluated, with plasmids containing the 18S rDNA gene sequence as a template. Further, a new nest PCR system (M-Nest) was established by combining the multiplex PCR system with the first-cycle genus-specific primer of the NP-1993 system. The sensitivities of the multiplex PCR system and the M-nest system were evaluated in serial dilutions of blood DNA samples from patients infected by P. falciparum and P. vivax. In addition, the NP-1993 and M-Nest systems were applied to screen the Plasmodium species in 307 blood samples from people returning to Guangxi from Ghana, a malaria epidemic area. And the NP-2002 and M-Nest systems were applied to re-check Plasmodium species in 66 blood samples collected in Guangxi from 2014 January to May, which were identified by microscopy to be infected mainly by P. ovale. RESULTS: The sizes of multiplex PCR products for P. falciparum, P. vivax, P. ovale, and P. malariae were 268 bp, 323 bp, 394 bp, and 446 bp, respectively, located in-between 50-bp DNA ladders. However, their melting curves had similar Tm values, thus could not be used to identify the 4 species. The minimum detection limits of P. falciparum, P. malariae, P. ovale, and P. vivax 18S rDNA gene by the multiplex PCR system were 5.58x102, 1.56x103, 1.66x103, and 1.80 x 10(2) copies/[A. The minimum detection limit of blood DNA from falciparum malaria patients by the multiplex PCR system was 1.43 x 10(2)-8.84 x 10(3) copies/p.1 or 5.10 x 10-4.92 x 10(2) parasites/µl, higher than that of P. vivax (17.4-69.1 copies/L or 13.5-83.2 parasites/p). Compared with this multiples PCR system, The M-Nest system further reduced the minimum detection limit of Plasmodium by 10-100 folds. Further, the M-Nest and NP-1993 systems reached inconsistent detection results in 307 blood samples from people returned to from Ghana; the former detected 2 cases of P. ovale infection while the latter failed. In addition, the NP-2002 and M-Nest systems came to the same results in re-checking Plasmodium species in the 66 blood samples. CONCLUSION: The established multiplex PCR system can identify 4 human Plasmodium species simultaneously and has good applicability in practice.

A Single Mutation in K13 Predominates in Southern China and Is Associated With Delayed Clearance of Plasmodium falciparum Following Artemisinin Treatment.

BACKGROUND: Artemisinin resistance in Plasmodium falciparum has emerged in Southeast Asia and poses a threat to malaria control and elimination. Mutations in a P. falciparum gene encoding a kelch protein on chromosome 13 have been associated with delayed parasite clearance following artemisinin treatment elsewhere in the region, but not yet in China. METHODS: Therapeutic efficacy studies of artesunate and dihydroartemisinin-piperaquine were conducted from 2009 to 2012 in the Yunnan Province of China near the border with Myanmar. K13 mutations were genotyped by capillary sequencing of DNA extracted from dried blood spots collected in these clinical trials and in routine surveillance. Associations between K13 mutations and delayed parasite clearance were tested using regression models. RESULTS: Parasite clearance half-lives were prolonged after artemisinin treatment, with 44% of infections having half-lives >5 hours (n = 109). Fourteen mutations in K13 were observed, with an overall prevalence of 47.7% (n = 329). A single mutation, F446I, predominated, with a prevalence of 36.5%. Infections with F446I were significantly associated with parasitemia on day 3 following artemisinin treatment and with longer clearance half-lives. CONCLUSIONS: Plasmodium falciparum infections in southern China displayed markedly delayed clearance following artemisinin treatment. F446I was the predominant K13 mutation and was associated with delayed parasite clearance.

In vivo monitoring of dihydroartemisinin-piperaquine sensitivity in Plasmodium falciparum along the China-Myanmar border of Yunnan Province, China from 2007 to 2013.

BACKGROUND: Artemisinin-based combination therapy (ACT) is the recommended first-line treatment of falciparum malaria in all endemic countries. Artemisinin resistance in Plasmodium falciparum has been confirmed in the Greater Mekong subregion (GMS). Dihydroartemisinin-piperaquine (DAPQ) is the most commonly used ACT in China. To understand the DAPQ sensitivity of P. falciparum, DAPQ resistance was monitored in vivo along the China-Myanmar border from 2007 to 2013. METHODS: Eligible patients with mono-infections of P. falciparum were recruited to this study after obtaining full informed consent. DAPQ tablets for different categories of kg body weight ranges were given once a day for three days. Patients were followed up for 42 days. Polymerase chain reaction (PCR) was conducted to distinguish between re-infection and recrudescence, to confirm the Plasmodium species. The data were entered and analysed by the Kaplan-Meier method. Treatment outcome was assessed according to the WHO recommended standards. RESULTS: 243 patients were completed valid follow-up. The fever clearance time (FCT) and asexual parasite clearance times (APCT) were, respectively, 36.5 ± 10.9 and 43.5 ± 11.8 hours, and there was an increasing trend of both FCT (F = 268.41, P < 0.0001) and APCT (F = 88.6, P < 0.0001) from 2007 to 2013. Eight (3.3%, 95% confidence interval, 1.4-6.4%) patients present parasitaemia on day three after medication; however they were spontaneous cure on day four. 241 (99.2%; 95% CI, 97.1-99.9%) of the patients were adequate clinical and parasitological response (ACPR) and the proportions of ACPR had not changed significantly from 2007 to 2013 (X(2) = 2.81, P = 0.7288). CONCLUSION: In terms of efficacy, DAPQ is still an effective treatment for falciparum malaria. DAPQ sensitivity in P. falciparum had not significantly changed along the China-Myanmar border of Yunnan Province, China. However more attentions should be given to becoming slower fever and parasite clearance.

[Opportunities, Challenges and Strategies for Malaria Elimination in the Tibet Autonomous Region of China].

This review summarizes the endemic characteristics and influencing factors of malaria in the Tibet Autonomous Region of China, as well as the opportunities and challenges for malaria elimination in this region. Recommendations on strategies for malaria control are also made.

Lessons from malaria control to elimination: case study in Hainan and Yunnan provinces.

Reduction patterns of Plasmodium falciparum and P. vivax malaria transmission and the role of an integrated strategy of case management and vector control are compared between different ecological zones. The epidemiology of malaria in Hainan and Yunnan provinces was disparate, even though distinct malaria control strategies have been adapted to different situations based on risk group, vector behaviours, local health infrastructure, and environmental conditions. The island Hainan appears to be victorious in eliminating malaria. However, there is still a long way to go to prevent the reintroduction of malaria in Hainan province and eliminating malaria in the border areas of Yunnan province. This review of the experiences and challenges from malaria control to elimination in Hainan and Yunnan provinces of southern China will provide a basis for the future elimination of malaria in the whole country.

Transition from control to elimination: impact of the 10-year global fund project on malaria control and elimination in China.

The Global Fund to Fight AIDS, Tuberculosis and Malaria (GFATM) supported a project on the control and elimination of malaria in People's Republic of China which was one of the biggest-scale international cooperation programmes to control malaria in the country during the past 10 years. The project promoted the effective implementation of the Chinese national malaria control programme. On the basis of epidemiologic data, an overview of the project activities and key performance indicators, the overall impact of the GFATM project was evaluated. We also reviewed relevant programme features including technological and management approaches, with a focus on best practice, innovations in implementation and the introduction of international standards. Last, we summarised the multi-stakeholder cooperation mechanism and comments on its sustainability in the post-GFATM period. Recommendations for the future management of the Chinese national malaria elimination programme are put forward after considering the challenges, shortcomings and lessons learnt during the implementation of the GFATM project in China to sustain past achievements and foster the attainment of the ultimate goal of malaria elimination for the country.

Monitoring Plasmodium vivax chloroquine sensitivity along China-Myanmar border of Yunnan Province, China during 2008-2013.

BACKGROUND: Plasmodium vivax is the most widespread of the malaria parasites infecting human hosts. In malaria-eliminating settings, both imported and local malaria predominantly occurs in border areas, and most of them are P. vivax. Chloroquine (CQ) is the first-line drug for P. vivax treatment in China. To understand CQ sensitivity in P. vivax, in vivo monitoring of CQ resistance was conducted along the China-Myanmar border from 2008 to 2013. METHODS: Eligible patients with mono-infections of P. vivax were recruited to this study after obtaining full informed consent. CQ tablets for different categories of kg body weight ranges were given once a day for three days. Patients were followed up for 28 days. PCR was conducted to distinguish between re-infection and recrudescence, to confirm the Plasmodium species. The data were entered and analysed by the Kaplan-Meier method. Treatment outcome and sensitivity were classified according to the WHO recommended standards. RESULTS: 603 patients were completed valid follow-up. The fever clearance time and asexual parasite clearance times were, respectively, 22.2 ± 10.2 and 38.1 ± 12.6 hours. 594 (98.5%) patients were adequate clinical and parasitological response (ACPR), and nine (1.5%) patients, who were late clinical failure (LCF) or resistant response level I (RI), were imported from the neighbouring districts of Myanmar. CONCLUSION: In terms of efficacy, CQ is still effective for vivax malaria treatment. Plasmodium vivax CQ sensitivity had not significantly changed along the China-Myanmar border of Yunnan Province, China.

[Overviews on Plasmodium ovale wallikeri and its genetic detection system].

In recent years, some microscopy-positive but nest-PCR negative (based on small subunit ribosomal RNA gene) ovale malaria cases were reported. The detection failure was due to the genetic variations between Plasomdoium ovale wallikeri and P. ovale curtisi. This paper reviews the discovery and validation processes of P. ovale wallikeri and the detection systems for identification of P. ovale.

Anopheles sinensis mosquito insecticide resistance: comparison of three mosquito sample collection and preparation methods and mosquito age in resistance measurements.

BACKGROUND: Insecticide resistance monitoring in malaria mosquitoes is essential for guiding the rational use of insecticides in vector control programs. Resistance bioassay is the first step for insecticide monitoring and it lays an important foundation for molecular examination of resistance mechanisms. In the literature, various mosquito sample collection and preparation methods have been used, but how mosquito sample collection and preparation methods affect insecticide susceptibility bioassay results is largely unknown. The objectives of this study were to determine whether mosquito sample collection and preparation methods affected bioassay results, which may cause incorrect classification of mosquito resistance status. METHODS: The study was conducted in Anopheles sinensis mosquitoes in two study sites in central China. Three mosquito sample collection and preparation methods were compared for insecticide susceptibility, kdr frequencies and metabolic enzyme activities: 1) adult mosquitoes collected from the field; 2) F1 adults from field collected, blood-fed mosquitoes; and 3) adult mosquitoes reared from field collected larvae. RESULTS: Mosquito sample collection and preparation methods significantly affected mortality rates in the standard WHO tube resistance bioassay. Mortality rate of field-collected female adults was 10-15% higher than in mosquitoes reared from field-collected larvae and F1 adults from field collected blood-fed females. This pattern was consistent in mosquitoes from the two study sites. High kdr mutation frequency (85-95%) with L1014F allele as the predominant mutation was found in our study populations. Field-collected female adults consistently exhibited the highest monooxygenase and GST activities. The higher mortality rate observed in the field-collected female mosquitoes may have been caused by a mixture of mosquitoes of different ages, as older mosquitoes were more susceptible to deltamethrin than younger mosquitoes. CONCLUSIONS: Female adults reared from field-collected larvae in resistance bioassays are recommended to minimize the effect of confounding factors such as mosquito age and blood feeding status so that more reliable and reproducible mortality may be obtained.

[Ecological behavior comparison between Anopheles pseudowillmori and A. willmori in villages with malaria outbreaks in Motuo County, Tibet Autonomous Region].

OBJECTIVE: To understand the ecological behaviors of Anopheles pseudowillmori and A. willmori in medium or high altitude areas of Motuo County, Tibet Autonomous Region, and their transmission potential for malaria. METHODS: The methods of human net traps, cow baited trap, house baited trap, pig baited trap and CDC light traps were adopted for investigating the mosquito density, biting activity at night and in or out door preference of biting. All mosquitoes morphologically identified as A. maculatus group were labeled in accordance with the capture time, place and method, and PCR were used to identify the species. The resting habits were investigated with the morning capture and daytime collecting methods, and the larvae collected in different wa- ter bodies were reared to adults to study the breeding place. RESULTS: A total of 1,053 A. maculatus group were collected, and of which, 331 (31.43%) were identified as A. pseudowillmori, and 722 (68.57%) as A. willmori. The number of A. willmori were higher than A. pseudowillmori in both outer and inner doors (P < 0.01, P < 0.05) , while no statistically significant difference was found of the constituent ratio of the anopheles between outer and inner doors. The multiparous ratios of A. pseudowillmori and A. willmori were 65.90% and 69.86%, respectively, (P > 0.05). The A. pseudowillmori preferred to biting outer doors (P < 0.05), while A. willmori preferred both outer and inner doors (P > 0.05). Both mosquitoes had one biting activity peak in the night, and the biting activity peak of A. willmori was from 21 to 22 o'clock, while the peak of A. pseudowillmori was from 24 to next morning 1 o' clock. CONCLUSION: In the Anopheles, the constituent ratio and density of A. willmori are higher than those of A. pseudowillmori in semi-high altitudes area of Motuo County, Tibet, and there are obvious differences of ecological behaviors between A. willmori and A. pseudowillmori, and A. willmori has the more capacity of transmitting malaria than A. pseudowillmori.

[Changes of pyrethroid resistance and P450 monooxygenase activity with age in Anopheles sinensis in Huainan City, Anhui Province, China].

OBJECTIVE: To study the effect of mosquito age on the pyrethroid resistance and P450 monooxygenase activity in Anopheles sinensis in Huainan City, Anhui Province, China, as well as the molecular mechanism of pyrethroid resistance. METHODS: Anopheles mosquito larvae were collected in fields, and were emerged to adults. Then the pyrethroid resistance and P450 monooxygenase activity of female mosquitoes were detected 1, 3, 6, 9, 12, 15 d and 20 d after emergence. Meanwhile, the resistance level and P450 monooxygenase activity of the sensitive mosquitoes in lab were detected as the control group. RESULTS: curves of pyrethroid resistance and P450 monooxygenase activity changes with the mosquito age presented unsymmetrically reversed "U" type. At 1 d after emergence, the resistance level and P450 monooxygenase activity were lower, and then they increased quickly. When 3-9 d after emergence, the resistance level and P450 monooxygenase activity rose to the highest level, and entered into plateau, then declined gradually. Compared to the sensitive mosquitoes in lab, the P450 monooxygenase activity of those in the field was higher at 3-12 d after emergence, but lower at 1 d after emergence, and at 15 and 20 d after emergence, they were almost equal to each other. CONCLUSION: The mosquito age is a confounding factor in the detection of resistance level and enzyme activity. The promotion of activity of P450 monooxygenase may be one of the reasons of the resistance. Using female adults with different ages in the field as study subjects may underestimate the real resistance level of mosquitoes, and mislead the use of insecticides.