Anopheles Anthropophagus Research Articles

The complete mitochondrial genome of major malaria vector Anopheles anthropophagus (Diptera: Culicidae) in China

Anopheles anthropophagus (Xu and Feng 1975) is the major vector of malaria in Eastern and Southern China. The species An. anthropophagus is considered a synonym of An. lesteri (Baisas & Hu, 1936), although they differ in several key biological characteristics. Here, we report the complete mitochondrial genome of An. anthropophagus for the first time. The mitogenome of An. anthropophagus is a typical circular, double-stranded molecule with a total length of 15,413 base pairs, and contains 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and an AT-rich control region. A phylogenetic analysis of the complete mitogenomes of 16 species of Anopheles (Culicidae) revealed that An. anthropophagus is closely related to An. sinensis (Wiedemann 1828), in the family Culicidae. The An. anthropophagus mitogenome provides new data for further taxonomic and phylogenetic studies of the genus Anopheles.

Epidemiological characteristics of malaria in Henan Province from 1950 to 2019

To analyze the epidemiological characteristics of malaria in Henan Province from 1950 to 2019, so as to provide the scientific evidence for consolidating malaria elimination achievements in the province. The epidemiological situation of malaria and demographic data in Henan Province from 1950 to 2019 were collected, and the statistical analyses were performed using a descriptive epidemiological method. The spatial temporal distribution of malaria cases was identified using the software ArcGIS version 10.3. During the period from 1950 through 2019, the progress of malaria elimination was divided into 4 stages in Henan Province, including the baseline-survey and key-control stage, morbidity-control and incidence-reduction stage, basic-eradication and achievement-consolidation stage and elimination stage. The spatial distribution of malaria cases shifted from south of the Huai River and the plain regions between the Yellow River and Taihang Mountain to the Huang-Huai-Hai Plain and Nanyang Basin, then was concentrated in eastern part of southern Huai River where Anopheles anthropophagus was distributed, and finally was gradually under control following malaria outbreak in Eastern Henan Plain. In addition, the species of Plasmodium changed from P. vivax, P. falciparum and P. malariae co-endemics to a single P. vivax infection, and the current co-endemics of 5 invasive malaria parasites, and the malaria vectors shifted from co-existence of Anopheles sinensis and An. anthropophagus to An. sinensis alone. There has been a large change in the epidemiological characteristics of malaria in Henan Province from 1950 to 2019. Although malaria has been eliminated in Henan Province, the consolidation of the malaria elimination achievements remain a great challenge due to overseas imported malaria.

Larvicidal activity of Zanthoxylum acanthopodium essential oil against the malaria mosquitoes, Anopheles anthropophagus and Anopheles sinensis

BackgroundZanthoxylum acanthopodium has insecticidal effect in Chinese traditional medicine. In this study, the essential oil from the dried Zanthoxylum plant was used as a larvicidal compound against the malaria mosquitoes, Anopheles anthropophagus and Anopheles sinensis.MethodsCompounds in the Zanthoxylum essential oil were investigated by gas chromatography and mass spectroscopy (GC–MS). The larvicidal bioassays of the whole oil, as well as the main compounds in the oil (estragole and eucalyptol) were performed using WHO method.ResultsIn total, 63 main compounds (99.32%) were found in the oils, including estragole (15.46%), eucalyptol (10.94%), β-caryophyllene (5.52%), cis-linalool oxide (3.76%), cis-limonene oxide (3.06%). A dose-dependent effect on mortality was recorded with increasing concentrations of essential oil and compounds increasing mortality of the larvae. Larvicidal bioassays revealed that 24 h LC50 of the whole essential oil was 36.00 mg/L and LC90 was 101.49 mg/L against An. anthropophagus, while LC50 was 49.02 mg/L and LC90 was 125.18 mg/L against An. sinensis. Additionally, 24 h LC50 of estragole were 38.56 and 41.67 mg/L against An. anthropophagus and An. sinensis, respectively, while the related LC90 were 95.90 and 107.89 mg/L. LC50 of eucalyptol were 42.41 and 45.49 mg/L against An. anthropophagus and An. sinensis, while the related LC90 were 114.45 and 124.95 mg/L.ConclusionThe essential oil of Z. acanthopodium and its several major compounds may have potential for use in the control of malaria mosquitoes.

Comparative expression profile of microRNAs in Anopheles anthropophagus midgut after blood-feeding and Plasmodium infection

BackgroundAnopheles anthropophagus is one of the major vectors of malaria in Asia. MicroRNAs (miRNAs) play important roles in cell development and differentiation as well as in the cellular response to stress and infection. In a former study, we have investigated the global miRNA profiles in relation to sex in An. anthropophagus. However, the miRNAs contributing to the blood-feeding and infection with Plasmodium are still unknown.MethodsHigh-throughput sequencing was performed to identify miRNA profiles of An. anthropophagus midguts after blood-feeding and Plasmodium infection. The expression patterns of miRNA in different midgut libraries were compared based on transcripts per million reads (TPM), and further confirmed by Northern blots. Target prediction and pathway analysis were carried out to investigate the role of regulated miRNAs in blood-feeding and Plasmodium infection.ResultsWe identified 67 known and 21 novel miRNAs in all three libraries (sugar-feeding, blood-feeding and Plasmodium infection) in An. anthropophagus midguts. Comparing with the sugar-feeding, the experssion of nine (6 known and 3 novel) and ten (9 known and 1 novel) miRNAs were significantly upregulated and downregulated respectively after blood-feeding (P < 0.05, fold change ≥ 2 and TPM ≥ 10). Plasmodium infection induced the expression of thirteen (9 known and 4 novel) and eleven (9 known and 2 novel) miRNAs significantly upregulated and downregulated, respectively, compared with blood-feeding. The representative upregulated miR-92a in blood-feeding and downregulated miR-275 in Plasmodium infection were further confirmed by Northern Blot. Putative targets of these regulated miRNAs were further investigated and classified into their pathways.ConclusionsThis study suggests that miRNAs are involved in the blood-feeding and Plasmodium infection in An. anthropophagus midgut. Further studies of the function of these differential expressed miRNAs will facilitate in better understanding of mosquito biology and anti-parasite immunity.

Impact of Malaria Vector Control Interventions at the Beginning of a Malaria Elimination Stage in a Dominant Area of Anopheles anthropophagus, Hubei Province, China.

Three towns with similar socio-ecological characteristics, malaria morbidities, and populations were selected for this study to explore economic and effective malaria control measures.The sources of infection were controlled in each town. Impregnated mosquito nets with 2.5% deltamethrin (15 mg/m(2)) combined with residual spraying of 5% cypermethrin (25 mg/m(2)) was implemented in cattle and pig pens, as well as in crowded sites in Chenji, whereas the mosquito nets were treated with 2.5% deltamethrin only in Guanqiao Town. All the control measures implemented in Fengling (control town) were the same as those implemented in the towns of Chenji and Guanqiao, except for mosquito elimination control. Results were evaluated and compared based on pathogens and entomology. The densities of Anopheles anthropophagus mosquitoes in houses, outside houses (man bait), as well as in cattle pens and pig pens were reduced by 100%, 71.96%, 94.01%, and 67.42%, respectively at all 4 sites in Chenji Town, whereas the density increased at 1 site (the outside house [man bait]) by 12.38%, while the densities at the other 3 sites (in houses, cattle pens and pig pens) were reduced by 99.63%, 18.71% and 69.44% respectively in Guanqiao Town. The biting rates of An. anthropophagus in the 3 towns were 0.11, 0.22, and 1.1 respectively in Chenji, Guanqiao, and Fengling. The incidence of malaria in the 3 towns decreased by 73.12%, 57.71%, and 65.71% in terms of annual average. Both impregnated mosquito nets combined with residual spraying and impregnated mosquito nets only reduced the density of An. anthropophagus in houses in the 2 towns, but reduction was more rapid in Chenji Town.

Identification and characterization of the expression profile of microRNAs in Anopheles anthropophagus

BackgroundAnopheles anthropophagus, one of the most important mosquito-borne disease vectors in Asia, mainly takes blood meals from humans and transmits both malaria and filariae. MicroRNAs (miRNAs) are small non-coding RNAs, and play a critical role in many cellular processes, including development, differentiation, apoptosis and innate immunity.MethodsWe investigated the global miRNA expression profile of male and female adults of A. anthropophagus using illumina Hiseq2000 sequencing combined with Northern blot.ResultsBy using the miRNAs of the closely-related species Anopheles gambiae and Aedes aegypti as reference, we obtained 102 miRNAs candidates out of 12.43 million raw sequencing reads for male and 16.51 million reads for female, with 81 of them found as known miRNAs in An. gambiae and/or Ae. aegypti, and the remaining 21 miRNAs were considered as novel. By analyzing the revised read count of miRNAs in male and female, 29 known miRNAs show sexual difference expression: >2-fold in the read count of the same miRNAs in male and female. Especially for miR-989, which is highly expressed in the female mosquitoes, but shows almost no detected expression in male mosquitoes, indicating that miR-989 may be involved in the physiological activity of female mosquito adults. The expression of four miRNAs in different growth stages of mosquito were further identified by Northern blot. Several miRNAs show the stage-specific expression, of which miR-2943 only expressed in the egg stage, suggesting that miR-2943 may be associated with the development of mosquito eggs.ConclusionsThe present study represents the first global characterization of An. anthropophagus miRNAs in sexual differences and stage-specific functions. A better understanding of the functions of these miRNAs will offer new insights in mosquito biology and has implications for the effective control of mosquito-borne infectious diseases.

Surveillance and response strategy in the malaria post-elimination stage: case study of Fujian province.

Malaria used to be a serious health problem in Fujian province in the past, but no local malaria transmission has been found since 2000. In order to eliminate the potential residual cases and prevent re-introduction of malaria so as to achieve the final goal of malaria elimination in Fujian province, various strategy and intervention approaches were tailored to the local settings. For instance, the monitoring of febrile patients by blood smear examinations and vector surveillance and control were strengthened in addition to the routine intervention in the mountainous area of Fujian province, where malaria was highly endemic and the mosquito Anopheles anthropophagus distributed with a high vectorial capacity. There were two local cases who got infected due to imported cases found in the building site of an expressway in 2004 and 2005, respective. All other imported malaria cases were detected during post-elimination stage through surveillance system. Based on results from post-transmission surveillance, malaria transmission has been interrupted in Fujian province for 13years. Therefore, post-transmission surveillance and response is an important intervention to maintain the malaria elimination achievements in Fujian province.

Susceptibility of Anopheles sinensis to Plasmodium vivax in malarial outbreak areas of central China

BackgroundAnopheles sinensis, Anopheles anthropophagus, Anopheles minimus and Anopheles dirus are the major vectors of malaria transmission in China. Anopheles sinensis is considered a secondary vector due to its relatively low malaria-transmission ability. However, in 2005, an outbreak of over 40,000 Plasmodium vivax malaria cases was reported in areas where Anopheles sinensis was the only major vector. Therefore, it is necessary to reassess the malaria transmission ability of this vector species in China.MethodsLaboratory colonies of An. sinensis and An. anthropophagus, and first-generation progeny (F1) of An. sinensis that had been collected in central China, were infected by direct membrane feeding assay with mono-vivax gametocyte-containing blood collected from vivax-infected patients. The mosquitoes were kept for 7 to 14 days post-blood feeding to allow parasites to develop into oocysts and sporozoites. Infectivity was measured by dissecting midguts and salivary glands. The presence of oocysts and sporozoites was determined by microscopy at 7 and 14 days post-blood feeding, and the numbers of gametocytes and asexual parasites, as well as mosquito parasite infections, were determined.ResultsThe positive oocyst and sporozoite feed rates of the 142 pairs of lab-colony An. sinensis and An. anthropophagus were not significantly different, and the same results were found with the 10 pairs of laboratory and F1 An. sinensis. An. sinensis had more oocysts/midgut at 7 days post-feeding than An. anthropophagus, but the gametocytemia, asexual parasitemia, and ratio of macrogametocytes to microgametocytes, did not correlate with either oocyst or sporozoite infection. However, in the oocyst-positive mosquitoes, there was a correlation between gametocytemia and the average oocyst number/midgut.ConclusionsThe susceptibility of An. sinensis (both laboratory and F1) to P. vivax-infected blood is similar to Anopheles anthropophagus, when evaluated by membrane feeding assay under laboratory conditions. In recent years, in central China, the vivax malaria transmission ability of An. sinensis has probably been underestimated. Further studies of this species in other regions are needed. An. sinensis could also be a good candidate vector for evaluating candidate malaria transmission-blocking vaccines (TBV).

Chemical composition and larvicidal activity of essential oil of Artemisia gilvescens against Anopheles anthropophagus

Artemisia gilvescens, a traditional Chinese medicinal plant, is chiefly distributed in the middle and lower reaches of the Yangtze River, China. Chemical composition of hydrodistilled essential oil from A. gilvescens was investigated by gas chromatography and mass spectroscopy (GC-MS), and larvicidal activity of the oil and its six main compounds against Anopheles anthropophagus was carried out by WHO method. In total, 56 compounds corresponding to 98.20% of the total oil were identified and the major compounds identified were camphor (13.49%), eucalyptol (12.13%), terpine-4-ol (9.65%), germacrene D (8.62%), caryophyllene oxide (4.65%), and caryophyllene (4.29%). Essential oil induced 8, 46, 80, 85, 94, and 100% larval mortality at the concentrations of 25, 50, 75, 100, 125, and 150mg/l, and the LC(50) and LC(90) values were 49.95 and 97.36mg/l, respectively. Among the six compounds, the most potent larvicidal compound was caryophyllene oxide and germacrene D, with LC(50) values of 49.46 and 49.81mg/l and LC(90) values of 115.38 and 106.19mg/l, respectively. Terpine-4-ol had LC(50) and LC(90) values of 76.70 and 139.42mg/l followed by camphor which showed LC(50) and LC(90) values of 129.17 and 192.42mg/l, respectively. The least potent among the six compounds were eucalyptol and caryophyllene, with and LC(50) value exceeding 200mg/l. In general, it also shows a dose-dependent effect on mortality, with increasing concentrations of essential oil and compounds increasing mortality of the larvae. The essential oil of A. gilvescens and its several major compounds may have potential for use in control of A. anthropophagus.

Chemical composition and larvicidal activity of Blumea densiflora essential oils against Anopheles anthropophagus: a malarial vector mosquito

Blumea densiflora, an edible and medicinal plant, is chiefly distributed in Southeast Asia and South Asia. Essential oils extracted by steam distillation from B. densiflora were investigated for their chemical composition and larvicidal activity against Anopheles anthropophagus, the primary vector of malaria in China and other East Asian countries. Totally, 46 compounds were identified by gas chromatography and mass spectroscopy. The major chemical compounds identified were borneol (11.43%), germacrene D (8.66%), β-caryophyllene (6.68%), γ-terpinene (4.35%), sabinene (4.34%), and β-bisabolene (4.24%). A series of concentrations of essential oil (that ranged from 6.25 to 150 ppm) were tested against A. anthropophagus fourth-instar larvae according to WHO recommendation. In general, larval mortality increased as concentration and exposure time increased, indicating a dose-dependent effect, and high insecticidal activity showed that 100% mortality occurred within 6 h at 150 ppm, 10 h at 100 ppm, 30 h at 50 ppm, and 30 h at 25 ppm essential oil concentration. The LC(50) values were 22.32 (after 12 h) and 10.55 ppm (after 24 h), and the LC(90) values were 54.04 (after 12 h) and 33.56 ppm (after 24 h). Pylarvex, the reference standard, had better larvicidal activity, causing 100% mortality within 2 h at 150 ppm and within 6 h at 6.25 ppm. The results clearly reveal that the essential oil of B. densiflora served as a potential, eco-friendly mosquito larvicide against the malarial vector mosquito A. anthropophagus.

Chemical composition and larvicidal effects of essential oil of Blumea martiniana against Anopheles anthropophagus

Objective To determine chemical composition of the hydrodistilled essential oil of Blumea martiniana, and to evaluate its larvicidal activity against Anopheles anthropophagus, the natural vector of malaria. Methods Chemical composition of essential oils extracted by steam distillation was investigated by gas chromatography and mass spectroscopy (GC-MS). Larvicidal activity of essential oil and its four main compounds was carried out by WHO method. Twenty larvae of early fourth-instar stage were used in the larvicidal assay and five replicates were maintained for each concentration. The 24 h LC 50 and LC 90 values were determined following probit analysis. Results Totally 68 compounds corresponding to 98.55% of the total oil were identified and the major constituents of essential oil were: linalool(10.36%), germacrene D(9.09%), borneol(6.24%), γ-terpinene(5.38%). The oil and linalool, germacrene D, borneol, γ-terpinene exerted significant larvicidal activity with LC 50 values of 46.86, 35.87, 44.61, 35.89, and 29.21 mg/L, respectively. It also showed a dose dependent effect on mortality. Conclusions The essential oil of Blumea martiniana and its four major compounds may have potential for use in control of Anopheles anthropophagus larvae and could be useful in search of newer, safer and more effective natural compounds as larvicides.

PP-198 Differentially expressed genes between female and male adult Anopheles anthropophagus

PP-198 Differentially expressed genes between female and male adult Anopheles anthropophagus

Differentially expressed genes between female and male adult Anopheles anthropophagus

The aim of the present study was to identify sex-specific genes in adult Anopheles anthropophagus. As the major malaria vector and Brugia malayi vector in the Asian continent, female Anopheles mosquitoes take blood meals and transmit pathogens through this pathway, while males are nectar feeders. This complex behavior is controlled at several levels, but is probably initiated by the genetic background difference between these two groups. In our study, a subtractive cDNA library for female A. anthropophagus was constructed using the suppression subtractive hybridization (SSH) technique and then 3,074 clones from the female SSH library were analyzed using a microarray-based survey. Genes that were expressed differentially according to sex in A. anthropophagus were screened using real-time polymerase chain reaction and reverse transcription polymerase chain reaction. In our results, we report a series of genes which may be involved in female-specific mosquito behavior, including an inorganic phosphate transporter, a serine protease, the salivary protein GP35-2, and the D7 cluster salivary protein. These findings will provide clues to the nature of insect vectors and open up unprecedented opportunities to develop novel strategies for the control of mosquito-borne diseases.

MALARIA CONTROL IN HENAN PROVINCE, PEOPLE’S REPUBLIC OF CHINA

We analyzed malaria prevalence and evaluated the effect of malaria control measures in Henan Province, People's Republic of China between 1993 and 2004. Data relating to malaria epidemics, malaria control measures and their effects, and vector surveillance between 1993 and 2004 were collected and analyzed. Mean malaria incidence during this period was 2.96/100,000. After integrated vector control measures and appropriate treatment of malaria cases were carried out, the number of malaria cases in Henan Province decreased from 4,815 in 2000 to 2,112 in 2004. The parasite-positive rate and the density of Anopheles anthropophagus were also reduced. Malaria control measures were effective and malaria incidence decreased in Henan. However, there are still more cases in this province than in 1992. Local malaria outbreaks and epidemics have occurred in areas where Anopheles anthropophagus and An. sinensis are present. Thus, malaria control measures should be strengthened.

Molecular identification of the malaria vectors Anopheles anthropophagus and Anopheles sinensis (Diptera: Culicidae) in central China using polymerase chain reaction and appraisal of their position within the Hyrcanus group.

In central China, Anopheles anthropophagus is considered the primary malaria vector and Anopheles sinensis is a secondary vector. Identification of these two cryptic species would facilitate studies on malaria transmission and the application of control measures. At present, the only reliable morphological markers occur in the egg stage, making this approach impractical for any large scale field studies. In this study, we report on the development of a polymerase chain reaction (PCR)-restriction fragment length polymorphism procedure involving the ribosomal DNA ITS2 region for discrimination of these species. The PCR-amplified product size of the ITS2 was 574 bp for An. anthropophagus and 594 bp for An. sinensis. Diagnostic restriction fragment length polymorphisms appeared with the restriction enzymes RsaI or HinfI. This diagnostic PCR was tested on mosquitoes collected from different locations throughout China. Specimens identified morphologically as An. anthropophagus in the adult and egg stage from one location in Quangdong Province were found to be An. sinensis, while specimens from Liaoning Province, which were variable in their egg morphology, were found to be An. anthropophagus. The presence of An. anthropophagus in Liaoning Province extends the range of this species north to 42 degrees N. The ITS2 spacer sequence was used in a maximum parsimony phylogenetic reconstruction of six members of the Hyrcanus group, two members of the Lesteri subgroup, and one member of the Nigerrimus subgroup, with the resulting molecular groupings at odds with the current morphological groupings.

Comparison of the effect of insecticide-treated bed nets and DDT residual spraying on the prevalence of malaria transmitted by Anopheles anthropophagus in China

In order to improve the control of malaria in a problem part of Hubei Province, China, where Anopheles anthropophagus is the vector of Plasmodium vivax, insecticide treatment of bed nets was introduced. The people were given the choice of DDT residual spraying, which had been used for many years, or deltamethrin treatment of their bed nets. Two counties, in which these 2 different methods had been introduced, and an untreated area were evaluated. DDT house spraying and insecticide treated bed nets were equally effective, but deltamethrin treatment was cheaper and so was considered the method of choice.

A successful control programme for falciparum malaria in Xinyang, China

This paper describes a large scale control programme for falciparum malaria in Xinyang prefecture, China, from the early 1980s to 1992. Falciparum malaria, transmitted mainly by Anopheles anthropophagus, was epidemic in Gusi, Huangchuan, Shangcheng and Huaibin counties in the prefecture in the early 1980s, 2922 cases being reported in 1984. From 1985 to 1992, DDT residual spraying and the use of bed nets impregnated with pyrethroid insecticide were introduced for 2 consecutive years in areas at higher risk of P. falciparum infection, and then gradually extended to cover all the endemic areas over a period of 8 years. Malaria control was also carried out through the primary health care system in 4 counties, as had been done since the 1970s. As a result, no falciparum malaria case has been found since 1988 in spite of extensive surveillance, and vivax malaria has also decreased greatly. Repeated cross-sectional surveys showed that A. anthropophagus could not be found in most sites after 2 consecutive years of the vector control programme. Vector control was a major factor in the successful elimination of falciparum malaria and decreasing the incidence of P. vivax in the 4 counties.

Alphamethrin-impregnated bed nets for malaria and mosquito control in China

A community-based intervention trial was carried out to evaluate the effectiveness of alphamethrin-impregnated bed nets for control of Plasmodium vivax malaria and its vector in an area of moderate endemicity in southern Henan province, central China in 1990. Malaria incidence was significantly lower in the intervention group than in the comparison group (2.03 vs. 3.57 per 100 person-years at risk). The protective efficacy for malaria incidence was 43%. The prevalence of malaria parasitaemia among children under 10 years old in the intervention group was about one-quarter of that in the comparison group (0·93% vs. 3·25% and 0·71% vs. 1·96% after one and 4 months use of impregnated nets, respectively). Alphamethrin-impregnated bed nets had a mass killing effect on vector mosquitoes. The outdoor person-biting density of Anopheles anthropophagus and A. sinensis decreased by 70·3% and 29·3% respectively. The density of these 2 mosquito species found resting inside treated nets was close to zero. No side effect was found among users of impregnated bed nets. Impregnation with alphamethrin was more effective on polyester than on cotton netting and residual effects lasted at least one year. Use of alphamethrin is less expensive than permethrin and deltamethrin.

Trial of Deltamethrin Impregnated Bed Nets for the Control of Malaria Transmitted by Anopheles sinensis and Anopheles anthropophagus

In 1985-1987, a field trial of malaria control using deltamethrin impregnated mosquito nets was carried out with 4,450 people in the Buji district, Bao'an County, Guangdong Province, China. The vectors were exophilic Anopheles sinensis (80%) and the endophilic and anthropophilic An. anthropophagus (20%). The first impregnation of the nets was in June 1985 and the second in April 1986. About 87% of the population slept under treated nets. The indoor vector density decreased by 93% and remained at the same level during the second year. During the second year the average monthly malaria incidence was 1.3% for January-June, a decrease of 74.6% as compared with pretreatment data, and 0.85% from July to December, a decrease of 92.7%. In April 1986 and April 1987, the treatment was extended to the whole Buji District, which has a total population of approximately 40,000. The average monthly malaria incidence (April-December 1986 and 1987) was 0.54% and 0.17%, a decrease of 64.7% and 89% respectively as compared with the same period for the previous year.