An aquaporin and an aquaglyceroporin have roles in low temperature adaptation of mosquitoes (Anopheles sinensis).

Mosquitoes (Anopheles sinensis), widely geographically distributed in Asia including China, are the primary vector of the malaria parasite Plasmodium vivax and other parasitic diseases such as Malayan filariasis. An. sinensis can survive through low winter temperatures. Aquaporin channels are found in all life forms, where they facilitate environmental adaptation by allowing rapid trans-cellular movement of water (classical aquaporins) or water and solutes such as glycerol (aquaglyceroporins). Here, we identified and characterized 2 aquaporin (AQP) homologs in An. sinensis: AsAQP2 (An. sinensis aquaglyceroporin) and AsAQP4 (An. sinensis aquaporin). When expressed in frog (Xenopus laevis) oocytes, AsAQP2 transported water, glycerol, and urea; AsAQP4 transported only water. Water permeation through AsAQP2 and AsAQP4 was inhibited by mercuric chloride. AsAQP2 expression was slightly higher in adult female mosquitoes than in males, and AsAQP4 expression was significantly higher in adult males. The 2 AsAQPs were highly expressed in Malpighian tubules and midgut. AsAQP2 and AsAQP4 expression was up-regulated by blood feeding compared with sugar feeding. At freezing point (0 °C), the AsAQP4 expression level increased and An. sinensis survival time reduced compared with those at normal temperature (26 °C). At low temperature (8 °C), the AsAQP2 and AsAQP4 expression levels decreased and survival time was significantly longer compared with those at 26 °C. These results suggest that AsAQP2 and AsAQP4 have roles in water homeostasis during blood digestion and in low temperature adaptation of A. sinensis. Together, our results show that the 2 AQPs are important for mosquito diuresis after blood feeding and when exposed to low temperatures.

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.

Genetic Diversity Analysis of Surface-Related Antigen (SRA) in Plasmodium falciparum Imported From Africa to China.

Plasmodium falciparum surface-related antigen (SRA) is located on the surfaces of gametocyte and merozoite and has the structural and functional characteristics of potential targets for multistage vaccine development. However, little information is available regarding the genetic polymorphism of pfsra. To determine the extent of genetic variation about P. falciparum by characterizing the sra sequence, 74 P. falciparum samples were collected from migrant workers who returned to China from 12 countries of Africa between 2015 and 2019. The full length of the sra gene was amplified and sequenced. The average pairwise nucleotide diversities (π) of P. falciparum sra gene was 0.00132, and the haplotype diversity (Hd) was 0.770. The average number of nucleotide differences (k) for pfsra was 3.049. The ratio of non-synonymous (dN) to synonymous (dS) substitutions across sites (dN/dS) was 1.365. Amino acid substitutions of P. falciparum SRA could be categorized into 35 unique amino acid variants. Neutrality tests showed that the polymorphism of PfSRA was maintained by positive diversifying selection, which indicated its role as a potential target of protective immune responses and a vaccine candidate. Overall, the ability of the N-terminal of PfSRA antibodies to evoke inhibition of merozoite invasion of erythrocytes and conserved amino acid at low genetic diversity suggest that the N-terminal of PfSRA could be evaluated as a vaccine candidate against P. falciparum infection.

Identification of a Novel Variant of ARHGAP29 in a Chinese Family with Nonsyndromic Cleft Lip and Palate.

BACKGROUND: Cleft lip with or without cleft palate (CL/P) is the most common facial birth defect, with a worldwide incidence of 1 in 700-1000 live births. CL/P can be divided into syndromic CL/P (SCL/P) and nonsyndromic CL/P (NSCL/P). Genetic factors are an important component to the etiology of NSCL/P. ARHGAP29, one of the NSCL/P disease-causing genes, mediates the cyclical regulation of small GTP binding proteins such as RhoA and plays an essential role in cellular shape, proliferation, and craniofacial development. METHODS: The present study investigated a Chinese family with NSCL/P and explored potential pathogenic variants using whole-exome sequencing (WES). Variants were screened and filtered through bioinformatic analysis and prediction of variant pathogenicity. Cosegregation was subsequently conducted. RESULTS: We identified a novel heterozygous missense variant of ARHGAP29 (c.2615C > T, p.A872V) in a Chinese pedigree with NSCL/P. CONCLUSION: We detected the disease-causing variant in this NSCL/P family. Our identification expands the genetic spectrum of ARHGAP29 and contributes to novel approaches to the genetic diagnosis and counseling of CL/P families.

[Effect of different temperatures on development of Aedes albopictus].

OBJECTIVE: To explore the effect of different temperatures on the different development stages of Aedes albopictus. METHODS: The changes at different development stages of mosquitoes (egg, larva, pupae) and gonotrophic cycle were observed at different temperature conditions of 10, 15, 20, 25, 30, 35 °C and 40 °C. The full developmental cycles were compared during different temperatures. RESULTS: All the stages of the mosquitoes could not develop at 10 °C. Under the different temperatures of 15, 20, 25, 30, 35 °C and 40 °C, the hatchabilities of the mosquitoes were 0, 32%, 82%, 83%, 82% and 59% respectively; the pupation rates of the mosquitoes were 38%, 53%, 84%, 88%, 72% and 42% respectively; and the emergence rates of the mosquitoes were 92%, 95%, 97%, 97%, 83% and 17% respectively. The mosquitoes could well develop at 20, 25, 30 °C and 35 °C, the development time was 37.73, 18.50, 16.92 and 13.66 days respectively. CONCLUSION: The development time of Aedes albopictus is shorter at the higher temperature. The optimum temperature for the mosquitoes to develop is between 25-30 °C, and higher or lower the temperatures will suppress the development of the mosquitoes.

[Epidemiological analysis of malaria prevalence in Jiangsu Province in 2012].

OBJECTIVE: To understand the malaria epidemic situation and characteristics in Jiangsu Province in 2012, so as to provide the evidence for formulating and adjusting effective malaria elimination strategies and measures. METHODS: The reported malaria cases from the Internet Reporting System and the epidemiological data of malaria in Jiangsu Province were collected and analyzed. RESULTS: A total of 198 malaria cases were reported in Jiangsu Province in 2012 with the incidence of 0.026/10 000, which decreased by 47.06% compared with that in 2011(374 cases). A total of 198 malaria cases were reported from 13 prefectures of Jiangsu and the cases were mainly distributed in Yangzhou (34 cases), Nantong (31 cases), Nanjing (22 cases), Taizhou (21 cases), Xuzhou (17 cases) and Huaian (17 cases), which accounted for 71.72% (142/198) among the total cases of the province. There were no local malaria cases reported from Jiangsu in 2012, and the imported malaria cases from other countries decreased by 45.15% compared with that in 2011. CONCLUSIONS: For the first time, there are no local malaria cases reported from Jiangsu in 2012. However, the imported case distribution is further expanded and the infected plasmodium parasites are more diverse. Imported malaria from other countries remains the key for malaria control in Jiangsu Province.

Development and application of an AllGlo probe-based qPCR assay for detecting knockdown resistance (kdr) mutations in Anopheles sinensis.

BACKGROUND: Anopheles sinensis is one of the most important malaria vectors in China and other Southeast Asian countries. High levels of resistance have been reported in this species due to the long-term use of insecticides, especially pyrethroids, for public health and agricultural purposes. Knockdown resistance (kdr) caused by a single base pair mutation in the gene encoding the sodium channel is strongly associated with pyrethroid insecticide resistance in many Anopheles mosquitoes. There are few methods currently available for detecting kdr mutations in An. sinensis. METHODS: A novel AllGlo probe-based qPCR (AllGlo-qPCR) method was developed to screen for the predominant kdr mutations in An. sinensis mosquitoes from the Jiangsu Province. The results from AllGlo-qPCR, allele-specific PCR (AS-PCR), and TaqMan-MGB probe-based qPCR (TaqMan-qPCR) were compared. A comparative analysis of the equipment required, ease of use and cost of the available methods was also performed. Finally, the AllGlo-qPCR method was used to detect the frequencies of kdr mutations from the other four provinces in central China. RESULTS: Six kdr genotypes were detected in An. sinensis from the Jiangsu Province by DNA sequencing. The AllGlo-qPCR method detected all of the kdr genotypes with a high level of accuracy (97% sensitivity and 98% specificity). AllGlo-qPCR correctly determined the kdr genotypes of 98.73% of 158 An. sinensis samples, whereas TaqMan-qPCR and AS-PCR correctly identified 96.84% and 88.61% of mutations, respectively. Furthermore, the AllGlo-qPCR method is simpler to perform, requires less equipment, and exhibits a moderate expense cost comparing with the other tested methods of kdr mutation detection. Samples collected from four of the other provinces in central China showed a high frequency of kdr mutation in An. sinensis, as detected by the established AllGlo-qPCR method. CONCLUSION: The novel AllGlo-qPCR method developed for kdr mutation detection in An. sinensis exhibits greater specificity and sensitivity than currently available methods and is more cost-effective; therefore, it represents a useful tool for entomological surveillance.

[Experimental observation of toxic effect of Bacillus thuringiensis var. israelensis against Aedes, Culex and Anopheles larvae].

OBJECTIVE: To evaluate the toxic effect of Bacillus thuringiensis var. israelensis (Bti) wettable powder against Aedes, Culex and Anopheles larvae. METHODS: The biological assay was applied to test the lethal concentration of 50% (LC50) of Bti wettable powder against Aedes, Culex and Anopheles larvae. RESULTS: The LC50(s) of Bti wettable powder against Aedes albopictus, Culex pipiens pallens and Anopheles sinensis larvae were 0.104, 0.160 microg/ml and 0.324 microg/ml, respectively; its biological potencies against them were 0.125, 0.192 IU/ml and 0.389 IU/ml, respectively. The LC50(s) of continuous contact of Bti wettable powder with An. sinensis stage III larvae for 1, 2 d and 3 d were 0.324, 0.092 microg/ml and 0.032 microg/ml, respectively, and its biological potencies were 0.389, 0.110 IU/ml and 0.038 IU/ml, respectively. The LC50(s) of the bacteria against An. sinensis stage I , II, III, IV were 0.024, 0.137, 0.324 microg/ml and 0.450 microg/ml, respectively, and the biological potencies were 0.029, 0.164, 0.389 IU/ml and 0.540 IU/ml, respectively. CONCLUSION: Bti wettable powder has a good toxicity to Aedes, Culex and Anopheles larvae, especially for the latter two. It is better to apply the bacteria at the early stage of mosquito larvae.

[Epidemiological analysis of malaria prevalence in Jiangsu Province in 2013].

OBJECTIVE: To understand the malaria epidemic situation and characteristics in Jiangsu Province in 2013, so as to provide the evidence for formulating and adjusting effective malaria elimination strategies and measures. METHODS: The reported malaria cases from the Internet Reporting System and the epidemiological data of malaria in Jiangsu Province were collected and analyzed. RESULTS: A total of 341 malaria cases were reported in Jiangsu Province in 2013 with the incidence of 0.050/10 000, which increased by 72.22% compared with that in 2012 (198 cases). All the cases were imported from other countries including one infected by blood transfusion resulted from imported infection. The cases were mainly distributed in Lianyungang City (15.84%, 54 cases), Nantong City (14.08%, 48 cases), Yangzhou City (14.08%, 48 cases), Huaian City (11.44%, 39 cases) and Yancheng City (8.50%, 29 cases). All the cases were confirmed in Jiangsu Provincial Reference Laboratory and there were 286 cases of Plasmodium falciparum, 8 cases of P. vivax, 9 cases of P. malariae, 30 cases of P. ovale and 8 cases of mixed infections. CONCLUSIONS: There were no local malaria cases reported from Jiangsu Province in the last two years which reflected effective achievements of malaria elimination. However, the situation of imported malaria is more serious and the species of infected plasmodium are more diverse. Imported malaria from other countries remains the key of malaria control in Jiangsu Province.

[Study on TaqMan-MGB real-time fluorescence quantitative PCR to detect gene mutation of kdr from Anopheles sinensis].

OBJECTIVE: To establish a Real-time Fluorescence Quantitative PCR to detect the kdr gene mutation in Anopheles sinensis. METHODS: One pair of primers and three TaqMan-MGB probes were designed based on kdr gene and its L1014 locus mutations of A. sinensis. After optimization, the Real-time Fluorescence Quantitative PCR was verified by using 6 types of A. sinensis samples with different kdr gene types. Additionally, 50 laboratory samples and 113 field samples were tested by this method. RESULTS: The established Real-time Fluorescence Quantitative PCR could identify 6 different kdr gene types in A. sinensis. The mutation could be detected by single-tube Fluorescence Quantitative PCR, and the detail mutation type could be further identified by double-tube Fluorescence Quantitative PCR. By using this method, 50 laboratory samples were confirmed as wild type homozygotes. Among 113 field samples, 12 were wild type homozygotes, others were L1014F or L1014C mutations, and the total mutation frequency was 87.61%. CONCLUSION: The new established TaqMan-MGB Real-time Fluorescence Quantitative PCR can be used to detect the kdr gene L1014 mutations of A. sinensis.

[Molecular identification of Anopheles hyrcanus complex by using single enzyme digestion PCR-RFLP method].

OBJECTIVE: To establish a novel molecular identification method for discrimination of members within Anopheles hyrcanus complex. METHODS: The sequences of the ribosomal DNA second internal transcribed spacer (rDNA ITS2) region of An. hyrcanus complex, including An. anthropophagus, An. lesteri, An. sinesis and An. yatsushiroensisi were analyzed by using molecular biology software Vector NTI 9.0, and a specificity restriction enzyme was selected based on the restriction fragment length polymorphism. Thus the single enzyme digestion PCR-RFLP method was established for genetic identification of An. hyrcanus complex, and 452 anopheline mosquitoes captured in the field were tested, comparing with the results of the previously established double enzyme digestion PCR-RFLP method and traditional morphological classification. RESULTS: The molecular software analysis revealed that the restriction enzyme Dde I could digest rDNA ITS2 region of An. hyrcanus complex into different fragments, thus it could be used for single enzyme PCR-RFLP for An. hyrcanus complex identification, and the result was further confirmed by laboratory experiment. Furthermore, a total of 452 anopheline mosquitoes captured from 4 malaria endemic areas were tested by this single enzyme digestion PCR-RFLP method, and 20 of them were identified as An. anthropophagus, 6 as An. lesteri, 391 as An. sinesis, and 35 as An. yatsushiroensisi. The results were 100% accordant to the double enzyme digestion PCR-RFLP method, and 93.4% accordant to the traditional morphological classification. CONCLUSIONS: The newly established single enzyme digestion PCR-RFLP method can be used for An. hyrcanus complex identification, and is more simple and reliable than the traditional morphological classification, and it is a suitable tool for field entomology surveillance.

[Cloning and sequence analysis of cDNA encoding aquaporin (AQP) gene from Anopheles sinensis].

OBJECTIVE: To clone and analyze the full-length sequence of aquaporin gene of Anopheles sinensis (AsAQP), so as to provide an insight into its biology functions. METHODS: The degenerate primers were used to amplify conserved region of AQP from An. sinensis cDNA. After then, the full-length cDNA of AsAQP was obtained by rapid amplification of cDNA ends (RACE). Concurrently, the bioinformatics methods were applied to analyze the obtained sequence. RESULTS: The obtained full-length cDNA of AsAQP consisted of 762 bp and 253 deduced amino acids with a predicted molecular mass of 63.2 kD. Bioinformatics analysis demonstrated that AsAQP had a typical structure with six membrane-spanning domains and an internal symmetry showing two highly conserved Asn-Pro-Ala (NPA) motif and possessing the consensus sequence of major intrinsic protein (MIP) superfamily. The AsAQP shared the identities of 76% and 78% with those of Culex quinquefasciatus and Aedes aegypti AQPs, respectively. Phylogenetic analysis indicated that AsAQP was clustered with Aedes and Culex AQPs. CONCLUSIONS: The full-length AsAQP is cloned by degenerate primers and RACE from An. sinensis. The AsAQP gene is a member of MIP protein family, and has the typical function region. The study lays the foundation for further research on the function of AsAQP.

[Establishment of early warning system of malaria epidemic situation in Jiangsu Province I impact of temperature on development of Plasmodium vivax in Anopheles sinensis in Jiangsu Province].

OBJECTIVE: To observe the developmental threshold temperature and the effective accumulated temperature of Plasmodium vivax sporozoites in Anopheles sinensis in Jiangsu Province, and to analyze the impact of temperature on the development of Plasmodium vivax. METHODS: The Anopheles sinensis mosquitoes were maintained under different temperatures of 16 +/- 0.5, 19 +/- 0.5, 22 +/- 0.5, 25 +/- 0.5, 28 +/- 0.5 degrees C and 31 +/- 0.5 degrees C in incubators after membrane feeding with Plasmodium vivax infected blood at laboratory. The salivary glands of Anopheles sinensis were dissected to confirm the development of sporozoite under different temperatures, and the developmental threshold temperature and the effective accumulated temperature of Plasmodium vivax sporozoites in Anopheles sinensis were calculated. The theoretical number of generations of Plasmodium vivax in Anopheles sinensis per year was calculated based on the average monthly temperatures of 13 municipalities in Jiangsu Province. RESULTS: The average developmental threshold temperature of Plasmodium vivax in Anopheles sinensis in Jiangsu Province was 15.31 degrees C, the average effective accumulated temperature was 109.81 day-degrees, and the optimal temperature for the proliferation of Plasmodium vivax was 25-28 degrees C. The theoretical generation number of Plasmodium vivax in sinensis in south Jiangsu was 1-3 generations being more than that in the north of the province. CONCLUSIONS: Temperature is one of the important influencing factors for the development of Plasmodium. The chance for Anopheles sinensis to transmit malaria increases under the temperature of 25-28 degrees C. However, other factors should be considered in the establishment of early warning system of malaria.

[Surveillance and forecast system of schistosomiasis in Jiangsu Province. IV. Establishment of Schistosoma japonicum cercaria-killing method by spraying niclosamide suspension on water surface].

OBJECTIVE: To investigate the effect of suspension concentrate of niclosamide (SCN) on killing cercariae of Schistosoma japonicum on water surface, optimization and impact on fish, so as to establish an emergency-treatment intervention for rapidly killing cercariae and eliminating water infectivity. METHODS: SCN was formulated into different concentrations of solutions, and then the solutions were sprayed on the surface of water containing S. japonicum cercariae. The water infectivity was determined by using mice at 0, 10, 30 min after spraying SCN. SCN was formulated into a solution of 100 mg/L and then sprayed on the surface of the water by using the spraying values of 0.01, 0.02, 0.03 g/m2 and 0.04 g/m2. At 30 min and 60 min after spraying, the water infectivity was determined by using mice. Zebra fish were transferred into the static water, then 100 mg/L SCN (s), using spraying values of 0.01, 0.02, 0.03 g/m2 and 0.04 g/m2, were sprayed on water surface. At 0, 10, 30, 60 min after spraying, the samples were collected at water depths of 0, 10, 20, 30, 40 cm, and niclosamide was determined by using high-performance liquid chromatography. The death of zebra fish was continually observed within 96 h after spraying SCN. RESULTS: At 0, 10, 30 min after spraying 1 000, 100, 10, 1, 0.1 mg/L SCN on water surface, the infectivity of water significantly decreased. At 30 min after spraying 1 000 mg/L and 100 mg/L SCN, no schistosome infectivity was detected in the water. At 30 min after spraying 100 mg/L SCN, with spraying values of 0.01, 0.02, 0.03, 0.04 g/m2, the water infectivity significantly reduced, and no infectivity was found 60 min after spraying SCN. After the surface of static water was sprayed with 100 mg/L SCN, the peak concentration was found at 0 min, and the solution diffused to site with a water depth of 10 cm after 10 min, and 30 min later, SCN diffused to the whole water body, and distributed evenly. After spraying 100 mg/L SCN on the surface of water with a volume of (3.14 x 20(2) x 50) cm3, by using the spraying value of 0.02 g/m2, 96 h later, no death of zebra fish was found. CONCLUSIONS: From 30 to 60 min after spraying 100 mg/L SCN, with the value of 0.02 g/m2, on the surface of S. japonicum-infested water, the water infectivity can be eliminated, and there is no evident toxicity to fish. This cercaria-killing method, as an emergency-treatment intervention for infested water, can be applied in those surveillance and forecast sites.

[Enhancing protective immunity effects of TPI DNA vaccine against Schistosoma japonicum through codon optimization].

OBJECTIVE: To study the protective effect of codon optimized TPI DNA vaccine against Schistosoma japonicum infection. METHODS: Sixty female BALB/c mice were randomly divided into 5 groups. The mice were injected through musculus quadriceps femoris with 100 microg pcDNA 3.1 control (Group A), pcDNA3.1-TPI (Group B), pcDNA 3.1-TPI-mHSP70 (Group C), pcDNA3.1-TPI.opt (Group D), and pcDNA3.1-TPI.opt-mHSP70 (Group E) respectively. All mice were immunized for three times with an interval of two weeks. The mice were challenged with (40+/-1) cercariae of S. japonicum per mouse by abdominal skin penetration 4 weeks after the last immunization, and sacrificed at 42 days post-challenge, the number of worms or hepatic eggs was counted. Blood was taken for the detection of IgG, IgG1, and IgG2a 2 days before immunization and before challenge, respectively. Spleen cells of 2 mice from each group were cultured and stimulated with ConA and rSjCTPI peptide, and the supernatant was collected for detection of IL-2, IL-4, IL-5, IFN-gamma, and TNF by flow cytometry. RESULTS: ELISA showed that the mice in groups B, C, D, and E produced specific IgG and IgG1, IgG2a antibody isotypes, and the ratio of IgG2a/IgG1 was 1.73, 2.06, 2.44, and 3.09, respectively. The levels of IL-2, IFN-gamma and TNF in groups D and E were higher than that of groups B and C. The worm reduction rate and hepatic egg reduction rate in groups D (36.03%, 41.7%) and E (39.03%, 46.85%) were higher than those of groups B (26.28%, 28.35%) and C (28.38%, 31.39%) (P<0.01) . CONCLUSIONS: The codon optimized TPI DNA vaccine induces higher level of protective effect and Th1-biased cellular immune response than those of non-optimized TPI DNA vaccine.