Lijuan Liu

Development of insecticide resistance in malaria vector Anopheles sinensis populations from Shandong province in China

BackgroundAnopheles sinensis is a major vector of malaria and among the dominant species in Shandong province of China. Insecticide resistance is an important threat to vector-borne disease control. However, there are only few reports about insecticide resistance of An. sinensis populations from Shandong province.MethodsFrom 2003 to 2012, six districts in Shandong province were selected as the study areas. Insecticide susceptibility bioassay were tested on F1 progeny of An. sinensis to 4% DDT, 0.05% deltamethrin, 0.15% cyfluthrin, and 5% malathion, using the standard WHO resistance tube assay.ResultsThe resistance status of An. sinensis showed a significant decrease in the mortality rates in DDT, deltamethrin and cyfluthrin during the past ten years. Whereas obvious increase of mortality to malathion was observed throughout the assay, ranging from 47.37% to 86.62%.

Identification of TCT, a novel knockdown resistance allele mutation and analysis of resistance detection methods in the voltage-gated Na+ channel of Culex pipiens pallens from Shandong Province, China

The present study aimed to investigate deltamethrin resistance in Culex pipiens pallens (C. pipiens pallens) mosquitoes and its correlation with knockdown resistance (kdr) mutations. In addition, mosquito‑resistance testing methods were analyzed. Using specific primers in polymerase chain reaction (PCR) and allele-specific (AS)-PCR, kdr gene sequences isolated from wild C. pipiens pallens mosquitoes were sequenced. Linear regression analysis was used to determine the correlation between the mutations and deltamethrin resistance. A kdr allelic gene was cloned and sequenced. Analysis of the DNA sequences revealed the presence of two point mutations at the L1014 residue in the IIS6 transmembrane segment of the voltage‑gated sodium channel (VGSC): L1014F, TTA→TTT, replacing a leucine (L) with a phenylalanine (F); L1014S, TTA→TCA, replacing leucine (L) with serine (S). Two alternative kdr-like mutations, L1014F and L1014S, were identified to be positively correlated with the deltamethrin-resistant phenotype. In addition a novel mutation, TCT, was identified in the VGSC of C. pipiens pallens. PCR and AS-PCR yielded consistent results with respect to mosquito resistance. However, the detection rate of PCR was higher than that of AS-PCR. Further studies are required to determine the specific resistance mechanism. PCR and AS-PCR demonstrated suitability for mosquito resistance field tests, however, the former method may be superior to the latter.

Resistance Level of Mosquito Species (Diptera: Culicidae) from Shandong Province, China

This study describes the aquatic habitats, species composition, and the insecticide resistance level of the mosquito Culex pipiens pallens in Shandong Province, China. A cross-sectional survey of mosquito larval habitats was conducted from May to November 2014 to determine the species composition and larval abundance. Larvae were collected using the standard dipping technique, and a total of four habitat types were sampled. The fourth instar larvae of Cx. pipiens pallens collected in each habitat type were tested for resistance to five insecticides according to a WHO bioassay. A total of 7,281 mosquito larvae were collected, of which 399 (5.48%) were categorized as Anopheles mosquito larvae (An. sinensis), 6636 (91.14%) as culicine larvae (Cx. pipiens pallens, Cx. tritaeniorhynchus, Cx. halifaxii, and Cx. bitaeniorhynchus), 213 (2.93%) as Armigeres larvae, and 33 (0.45%) as Aedes larvae (Aedes albopictus). In addition, a total of 1,149 mosquito pupae were collected. Culex larvae were distributed in all habitats investigated. Tukeys HSD analysis showed that roadside drainages were the most productive habitat type for Culex larvae. Armigeres species were found only in drains, Aedes only in water tanks, and Anopheles in water that was comparatively clear and rich in emergent plants. Bioassay showed that the maximum resistance level of Cx. pipiens pallens was to deltamethrin, while it was lowest to plifenate. The productivity of various mosquitoes in different habitat types is very heterogeneous. It is particularly important to modify human activity and the environment to achieve effective mosquito vector control. For effective larval control, the type of habitat should be considered, and the most productive habitat type should be given priority in mosquito abatement programs.

Molecular cloning and preliminary function study of iron responsive element binding protein 1 gene from cypermethrin-resistant Culex pipiens pallens

BackgroundInsecticide resistance jeopardizes the control of mosquito populations and mosquito-borne disease control, which creates a major public health concern. Two-dimensional electrophoresis identified one protein segment with high sequence homology to part of Aedes aegypti iron-responsive element binding protein (IRE-BP).MethodRT-PCR and RACE (rapid amplification of cDNA end) were used to clone a cDNA encoding full length IRE-BP 1. Real-time quantitative RT-PCR was used to evaluate the transcriptional level changes in the Cr-IRE strain Aedes aegypti compared to the susceptible strain of Cx. pipiens pallens. The expression profile of the gene was established in the mosquito life cycle. Methyl tritiated thymidine (3H-TdR) was used to observe the cypermethrin resistance changes in C6/36 cells containing the stably transfected IRE-BP 1 gene of Cx. pipiens pallens.ResultsThe complete sequence of iron responsive element binding protein 1 (IRE-BP 1) has been cloned from the cypermethrin-resistant strain of Culex pipiens pallens (Cr-IRE strain). Quantitative RT-PCR analysis indicated that the IRE-BP 1 transcription level was 6.7 times higher in the Cr-IRE strain than in the susceptible strain of 4th instar larvae. The IRE-BP 1 expression was also found to be consistently higher throughout the life cycle of the Cr-IRE strain. A protein of predicted size 109.4 kDa has been detected by Western blotting in IRE-BP 1-transfected mosquito C6/36 cells. These IRE-BP 1-transfected cells also showed enhanced cypermethrin resistance compared to null-transfected or plasmid vector-transfected cells as determined by 3H-TdR incorporation.ConclusionIRE-BP 1 is expressed at higher levels in the Cr-IRE strain, and may confer some insecticide resistance in Cx. pipiens pallens.

Effects of ribosomal protein l39-L on the drug resistance mechanisms of lung cancer A549 cells.

BACKGROUND Cancer is a major threat to the public health whether in developed or in developing countries. As the most common primary malignant tumor, the morbidity and mortality rate of lung cancer continues to rise in recent ten years worldwide. Chemotherapy is one of the main methods in the treatment of lung cancer, but this is hampered by chemotherapy drug resistance, especially MDR. As a component of the 60S large ribosomal subunit, ribosomal protein L39-L gene was reported to be expressed specifically in the human testis and human cancer samples of various tissue origins. MATERIALS AND METHODS Total RNA of cultured drug-resistant and susceptible A549 cells was isolated, and real time quantitative RT-PCR were used to indicate the transcribe difference between amycin resistant and susceptible strain of A549 cells. Viability assay were used to show the amycin resistance difference in RPL39-L transfected A549 cell line than control vector and null-transfected A549 cell line. RESULTS The ribosomal protein L39-L transcription level was 8.2 times higher in drug-resistant human lung cancer A549 cell line than in susceptible A549 cell line by quantitative RT-PCR analysis. The ribosomal protein L39-L transfected cells showed enhanced drug resistance compared to plasmid vector-transfected or null-transfected cells as determined by methyl tritiated thymidine (3H-TdR) incorporation. CONCLUSIONS AND IMPLICATIONS FOR PRACTICE The ribosomal protein L39-L gene may have effects on the drug resistance mechanism of lung cancer A549 cells.

Mosquito Species (Diptera: Culicidae) Reported from Shandong Province, China

Field surveys were conducted in 2007 - 2009 to determine the diversity of mosquito species occurring in Shandong Province, China. As a result, 31 species of mosquitoes were identified. Culex pipiens pallens Coquillett was the most common species collected. Aedes albopictus Skuse, Anopheles sinensis Wiedemann, and Cx. tritaeniorhynchus Giles also were commonly collected. These 4 species have been documented as the vectors of malaria, dengue, and epidemic encephalitis in China. At least 10 species identified are disease vectors in Shandong Province, and other species could be considered potential vectors of other diseases. Mosquito larvae were routinely collected from natural and artificial structures containing water. The diversity of these water-filled containers might explain the cooccurrence of various culicid species in some localities surveyed. The ecology and breeding behavior of these mosquito species should be further studied to optimize surveillance and management programs and to prevent the emergence of mosquito-transmitted diseases in human and other animal populations in the area.

Host preferences and feeding patterns of Anopheles sinensis Wiedemann in three sites of Shandong province, China

Background & objectives: 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. Methods: 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. Results: 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. Interpretation & conclusion: 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.

Overwintering of Culex pipiens pallens (Diptera: Culicidae) in Shandong, China

Abstract  Culex pipiens pallens Coquillet can spread various mosquito-borne infectious diseases. This study identifies the overwintering sites and physiological condition of Cx. pipiens during hibernation in Shandong Province of P.R. China. Results show that female adult Cx. pipiens overwinter in habitats characterized by warm temperature, high humidity, and calm winds. Cellars yielded the highest densities, followed by air-raid shelters, caliducts, sewers, mountain caves, wells, stone caverns, etc. The adult density among overwintering sites differed significantly (F = 45.88, P < 0.01). Larvae and pupae were not captured after late December. In early winter, multiparous and bloodsucking mosquitoes accounted for a large proportion of the observed populations. However, their numbers gradually decreased as winter progressed, mainly due to their relatively high death rate in winter. Most mosquitoes overwinter in a state of diapause, and females have usually mated. Collections in midwinter showed that of those collected, 99.8% were in diapause and 100% had mated. Overwintering mosquitoes suffered few deaths under natural conditions and, upon emerging from overwintering diapause, could take blood meals and oviposit. The time taken for oviposition by mosquitoes emerging from diapause was longer than that observed with females continuously reared in the laboratory; however, there was no significant difference in the numbers of eggs (F = 0.571, P = 0.467) oviposited.

Measure Post-Bloodmeal Dispersal of Mosquitoes and Duration of Radioactivity by Using the Isotope 32P

Abstract The radioactive isotope 32 P-labeled disodium phosphate (Na 2 H 32 PO 4 ) was injected via the jugular vein into a cow kept in a shed in Maozhuang Village, Cao Township of Shanxian County, China. Over the following 5 d, mosquitoes feeding on the cow were captured at distances up to 400 m to determine dispersal distance. The duration of radioactivity in the cow and marked mosquitoes was 10 d. The results showed that after blood feeding, Anopheles sinensis and Culex tritaeniorhynchus temporarily rested in the cattle shed and then flew outdoors. In contrast, Culex pipiens pallens remained in the cattle shed after feeding. These findings confirmed that local An. sinensis and Cx. tritaeniorhynchus were partially endophilic and tended to rest out of doors, whereas Cx. pipiens pallens was endophilic. For marked An. sinensis and Cx. tritaeniorhynchus , there was a significant tendency for dispersal to be in a northeast and east direction, probably because of the presence of heavy shading by an agricultural field, a small river for mosquito oviposition sites, and locations downwind from the blood source. The furthest flight distances for An. sinensis and Cx. tritaeniorhynchus were 210 and 240 m; therefore, control of these mosquitoes should include resting places indoors and outdoors within a radius of 250 m from confirmed cases.

Feeding preference and resting habits of Anopheles sinensis in southwest of Shandong Province

In this study, feeding preference and resting habits of Anopheles sinensis Wiedemann ( An. sinensis ) were conducted by human, cattle-bait collections indoors, outdoors, and experiment hut in southwest of Shandong Province. The results showed that the number of An. sinensis caught indoors was about three times greater than outdoors by human landing collection. While, by cattle landing collection, the number of An. sinensis captured indoors and outdoors was similar. The number of An. sinensis caught was 12 times greater within 3 h in the evening than in the whole day. In cattle shed, about 50% An. sinensis rested within 1–10 min, 80% rested less than 30 min, only 10% rested more than 1 h. Overall, An. sinensis demonstrated preference to feed indoors compared with outdoors and fed preferentially on cows compared to human as offered.