Interactions between the gut micro-community and transcriptome of Culex pipiens pallens under low-temperature stress.

BACKGROUND: Culex pipiens pallens (Diptera: Culicidae) can survive at low temperature for long periods. Understanding the effects of low-temperature stress on the gut microflora and gene expression levels in Cx. pipiens pallens, as well as their correlation, will contribute to the study of the overwintering mechanism of Cx. pipiens pallens. METHODS: The gut bacteria were removed by antibiotic treatment, and the survival of Cx. pipiens pallens under low-temperature stress was observed and compared with the control group. Then, full-length 16S rRNA sequencing and the Illumina HiSeq X Ten sequencing platform were used to evaluate the gut microflora and gene expression levels in Cx. pipiens pallens under low-temperature stress. RESULTS: Under the low-temperature stress of 7 °C, the median survival time of Cx. pipiens pallens in the antibiotic treatment group was significantly shortened by approximately 70% compared to that in the control group. The species diversity index (Shannon, Simpson, Ace, Chao1) of Cx. pipiens pallens decreased under low-temperature stress (7 °C). Non-metric multidimensional scaling (NMDS) analysis divided all the gut samples into two groups: control group and treatment group. Pseudomonas was the dominant taxon identified in the control group, followed by Elizabethkingia and Dyadobacter; in the treatment group, Pseudomonas was the dominant taxon, followed by Aeromonas and Comamonas. Of the 2417 differentially expressed genes (DEGs), 1316 were upregulated, and 1101 were downregulated. Functional GO terms were enriched in 23 biological processes, 20 cellular components and 21 molecular functions. KEGG annotation results showed that most of these genes were related to energy metabolism-related pathways. The results of Pearson's correlation analysis showed a significant correlation between the gut microcommunity at the genus level and several DEGs. CONCLUSIONS: These results suggest that the mechanism of adaptation of Cx. pipiens pallens to low-temperature stress may be the result of interactions between the gut bacterial community and transcriptome.

Chigger Mite (Acari: Trombiculidae) Survey of Rodents in Shandong Province, Northern China.

Chigger mites are parasites of rodents and other vertebrates, invertebrates, and other arthropods, and are the only vectors of scrub typhus, in addition to other zoonoses. Therefore, investigating their distribution, diversity, and seasonal abundance is important for public health. Rodent surveillance was conducted at 6 districts in Shandong Province, northern China (114-112°E, 34-38°N), from January to December 2011. Overall, 225/286 (78.7%) rodents captured were infested with chigger mites. A total of 451 chigger mites were identified as belonging to 5 most commonly collected species and 3 genera in 1 family. Leptotrombidium scutellare and Leptotrombidium intermedia were the most commonly collected chigger mites. L. scutellare (66.2%, 36.7%, and 49.0%) was the most frequently collected chigger mite from Apodemus agrarius, Rattus norvegicus, and Microtus fortis, respectively, whereas L. intermedia (61.5% and 63.2%) was the most frequently collected chigger mite from Cricetulus triton and Mus musculus, respectively. This study demonstrated a relatively high prevalence of chigger mites that varied seasonally in Shandong Province, China.

Epidemiological analysis of 133 malaria cases in Shanxian county, Shandong Province, China.

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

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.

[Sequence analysis of ribosomal DNA internal transcribed spacer 2 of sympatric populations of Anopheles sinensis of different feeding preferences].

OBJECTIVE: To investigate the existence of genetic divergence of sympatric populations of Anopheles sinensis of different feeding preferences based on the rDNA-ITS2 sequence differences. METHODS: A large number of wild anopheles populations were trapped all night by man-baited net and calf-baited net that had been set up between high-density natural villages of An. sinensis populations and vector-breeding sites, from which two groups of An. sinensis were separated by morphological identification and brought back to the lab for conventional breeding. A large closed greenhouse which temperature and humidity was appropriate was selected as research settings of mark-release-recapture methods by female mosquitoes, in the center of which above An. sinensis populations baited by man and calf and respectively correspondingly marked by red and yellow phosphors were released in together, in each side of which An. sinensis were recaptured simultaneously by man-baited net and calf-baited net. An. sinensis populations trapped by man twice were brought back to the lab and bred with man-blood, correspondingly ones trapped by calf with calf-blood. Man-preferring and calf-preferring strains were screened respectively from An. sinensis which had been baited by man and calf by the mark-release-recapture methods after parent and F1 mosquitoes, and sequencing and aligning of both rDNA-ITS2 were conducted via PCR amplification. RESULTS: The recapture ratios of wild parental mosquitoes An. sinensis of man-preferring group by man-baited net and calf-baited net were 54.07% (339/627) and 45.93% (288/627) respectively, and ones of calf-preferring group by man-baited net and calf-baited net were 58.01% (409/705) and 41.99% (296/ 705) respectively. Two groups of parental mosquitoes trended towards selecting the original blood hosts in host-seeking preference (Χ2 = 19.42, P < 0.01). The recapture ratios of F1 mosquitoes An. sinensis of man-preferring group by man-baited net and calf-baited net were 63.43% (765/1 206) and 36.57% (441/1 206), and ones of calf-preferring group by man-baited net and calf-baited net were 68.22% (1 039/1 523) and 31.78% (484/1 523). Two groups of F1 mosquitoes had more significant characteristics of selecting the original blood hosts in host-seeking preference (Χ2 = 271.69, P < 0.01) and showed the genetic differentiation phenomenon, but the results of sequencing and aligning of the rDNA-ITS2 via PCR amplification showed no difference in base sequence between the two strains and both were 469 bp. CONCLUSIONS: The genetic divergence based on the rDNA-ITS2 sequence does not happen in An. sinensis sympatric populations of different feeding preferences.

[Experimental observation on host preference of Anopheles sinensis].

OBJECTIVE: To observe the host preference of Anopheles sinensis captured by outdoor human or cattle baits. METHODS: A large number of non-blood-fed An. sinensis females were collected by overnight trapping outdoor with human and cattle in the rice paddy field in Shan County of Shandong Province, and took back to the lab, and individually labeled as human baits group and cattle baits group, fed with mouse blood. The host preference of parent, F1 and F25 generations of the two groups were observed by mark-release-recapture methods in a large greenhouse. RESULTS: The recapture rates of parent, F1 and F25 generations were 39.02% (1332/3414), 37.97% (2583/6803), and 30.55% (1523/4986), respectively. In parent generation, the proportion of mosquitoes from human baits group and cattle baits group collected by human-bait and cattle-bait was 54.07% (339/627) and 58.01% (409/705), respectively (χ2=19.42, P<0.01); in F1 generation, that of the two groups was 51.03% (669/1311) and 55.11% (701/1272), respectively (χ2= 9.75, P<0.01); in F25 generation, that of the two groups was 51.98% (342/658) and 52.37 (453/865), respectively (χ2=2.82, P>0.05). CONCLUSION: After culture for 25 generations in an experimental condition, the host preference for human or cattle of An. sinensis maybe change.

[Study on biological characteristics of cypermethrin-resistant and -susceptible strains of Aedes albopictus at different temperatures].

OBJECTIVE: To study the biological characteristics of cypermethrin-resistance strain and -susceptible strain of Aedes albopictus under different controlled temperatures in the laboratory. METHODS: The two strains were raised at three different temperatures, 20, 25 degrees C and 28 degrees C respectively, and the biological characteristics of the two mosquito strains, such as reproduction, development and life expectancy, were observed and recorded in the laboratory. RESULTS: The life expectancy of both strains became shorter as the temperature raised, and the resistant strain(69.37% ± 0.01%, 77.04% ± 0.07%) lived shorter than the susceptible strain(85.24% ± 0.03%, 88.23% ± 0.05%)in average. Under 25 degrees C, the hatching rate of resistant strain decreased by 25.88%, and the pupation rate decreased by 11.18%. In the three temperatures, all the life expectancy expanded as the temperature went up, the periods for the susceptible strain were 19.75 ± 0.10, 23.65 ± 0.07 d and 25.08 ± 0.08 d under 28, 25 degrees C and 20 degrees C. While life expectancy for the resistant strain decreased to 17.21 ± 0.09, 20.95 ± 0.09, 22.58 ± 0.10 d. Under the same temperature, the development timing of the resistance strain was longer than that of the susceptible strain, and the period was the longest under 28 degrees C (156.2 h, 137.1 h). In the three temperatures, all the development periods expanded as the temperature went up, the susceptible and resistant larvae developed 137.1 d and 163.3 d, 247.7 d and 156.2 d, 182.3 d and 263.2 d under 28, 25 degrees C and 20 degrees C. The differences show statistic significance (P < 0.05). CONCLUSION: The resistance of A. albopictus to cypermethrin results in the decrease of adaptability to the environment change and the disadvantage of reproduction at different temperatures.

[Progress in application of genetic control of mosquitoes].

With the specific, environmental-friendly, fast and efficient characteristics, the genetic control of mosquitoes has been preliminary shown a good effect. However, the prejudices and misunderstandings to the genetic control limit its popularization and application. Therefore, in order to re-recognize and take full advantage of this method, the recent progresses towards applying the genetic control of mosquitoes are reviewed.

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, mosquito­resistance 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 voltage­gated 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.

[Research progress on antimicrobial peptides against malaria parasite].

Antimicrobial peptides have broad-spectrum antibacterial activity and high thermal stability. Researches prove that they can inhibit the development of Plasmodium or kill them. The paper focuses on research advances in their biological characteristics, natural or synthetic peptides as potential anti-Plasmodium agents in malaria research.

Cloning and overexpression of CYP6F1, a cytochrome P450 gene, from deltamethrin-resistant Culex pipiens pallens.

CYP6F1 (GenBank/EMBL accession No. AY662654), a novel gene with a complete encoding sequence in the cytochrome P450 family 6, was cloned and sequenced from deltamethrin-resistant 4th instar larvae of Culex pipiens pallens. The cDNA sequence of CYP6F1 has an open reading frame of 1527 bp, which encodes a putative protein of 508 amino acid residues. The deduced amino acid sequence of CYP6F1 indicated that the encoded P450 has conserved domains of a putative membrane-anchoring signal, putative reductase-binding sites, a typical heme-binding site, an ETLR motif and substrate recognition sites. Semi-quantitative RT-PCR analysis indicated that the CYP6F1 gene was expressed to a greater extent in the deltamethrin-resistant strain than in the susceptible strain of Cx. pipiens pallens. The expression levels of the CYP6F1 gene in the deltamethrin-resistant 1st, 2nd, 3rd, 4th instar larvae and adult female mosquitoes differed, with highest expression levels in the 4th instar larvae. In addition, the CYP6F1 gene was stably expressed in mosquito C6/36 cells, and the expected 61.2 kDa band was identified by Western blotting. The cells transfected with CYP6F1 had an increased resistance to deltamethrin as compared with control cells. These results indicate that CYP6F1 is expressed at higher levels in the deltamethrin-resistant strain, and may confer some insecticide resistance in Cx. pipiens pallens.

[The correlation between DDVP resistance of Culex pipiens pallens and esterase activity].

OBJECTIVE: To detect the resistance index and esterase activity of each generation of DDVP-resistant Culex mosquitoes and analyze the relationship between insecticide resistance and esterase. METHODS: WHO bioassay and micro-plate measurement were used for the detection. RESULTS: The resistance index increased to 12.17 after 43 generations' insecticide selection compared to 1.00 as sensitive isolate. The nonspecific esterase(NSE) activity of the mosquitoes became strengthened with the extension of the generations, and the individual frequency of those with OD values no less than 0.9 increased gradually, consistent basically to the bioassay. The AChE average inhibition rate decreased with the extended generation and increased resistance, and the individual frequency of those with inhibition rate less than 30% became strengthened with the extension of generations, showing a positive correlation. CONCLUSION: The activity of NSE and AChE shows a correlation with DDVP resistance.