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ObjectiveTo characterize Torso-like (tsl) gene and investigate its expression characteristics in Anopheles dirus, so as to provide a theoretical basis for subsequent functional studies of the tsl gene. MethodsAccording to the coding sequences of Drosophila melanogaster and An. gambiae tsl genes, the complete genome of An. dirus was retrieved and the An. dirus tsl gene was characterized. Specific primers were designed and the target gene was amplified using PCR and reverse-transcription PCR assays. The physicochemical properties, signal peptide, transmembrane structure, secondary structure and tertiary structure of the encoded protein TSL were analyzed using bioinformatics tools, and a phylogenetic analysis was performed. In addition, the specific expression of the tls gene was detected in various tissues of An. dirus using a quantitative real-time PCR assay. Results The An. dirus tsl gene was 16 751 bp in length with a CDS region of 1 134 bp, encoding 377 amino acids, and the encoded TSL protein was a stably hydrophilic protein. The TSL protein was predicted to be a secretory protein that was located in extra-membrane regions containing signal peptides. The secondary structure of the TSL protein contained α-helix (51.72%), extended strand (12.20%), β-bridge (4.78%) and random coil (31.30%) in the secondary structure, and a 3D homology model was generated using 5cj9.1.A as a template. Phylogenetic analysis revealed a close genetic relationship in the TSL protein between An. dirus and An. farauti. In addition, quantitative real-time PCR assay detected the tsl gene expression in the head, chest, abdomen and foot of An. dirus, with the highest expression in the head and low expression in the foot. Conclusions The tsl gene is characterized in An. dirus at a genomic level, and the prediction of the TSL protein structure and the elucidation of the tissue-specific tsl gene expression in An. dirus provide a basis for the further studies on the gene functions.
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Objective: To investigate the effect of sublethal dose of Bacillus sphaericus (Bs) 2 362 strain on the malaria transmission ability of Anopheles (An.) dirus (Hainan strain) and its molecular mechanism. Methods: Firstly, the fourth instar larvae of An. dirus was treated with sublethal dose of Bacillus sphaericus. The surviving larvae were then collected and placed into mosquito cages, where they were hatched into adult mosquitoes. These mosquitoes were recorded as Bs group. Meanwhile, the control group was set without any treatment. Then, for infection with Plasmodium yoelii BY265 RFP, 3- to 5-day old female adults were kept at 24 °C and fed on Plasmodium yoelii BY265 RFP-infected Kunming mice with a gametocytemia above 0.5%. On day 9-11 post infection, mosquitoes were dissected, and the oocysts on the midguts were examined under a fluorescence microscope. Thirdly, total RNA was extracted from mosquitoes of Bs group and the control group at different time-points respectively, and the cDNA were synthesized later. Finally, SYBR quantitative PCR was conducted to investigate the expression of Imd pathway anti-malaria molecules at different time-points, including TEP1 and Rel2, in Bs and control group mosquitoes. Results: Bs treatment remarkably reduced the infection rate of Plasmodium from 23.71% (124/523) to 16.23% (87/536) (Chi-square test, P=0.002 0.05). Additionally, the intensities of melanized oocysts were compared between the two groups, and no significant difference was found, either (P=0.566>0.05). Interestingly, compared with the control group, the expression levels of TEP1 and Rel2 in Bs group were obviously up-regulated in larval, adult and infected mosquitoes. Especially in 3 dpi and 7 dpi, the expression level of TEP1 in Bs group was nearly 4 times higher than that of the control group, while Rel2 reached to approximately 7 times. Conclusions: We firstly found that the sublethal dose of Bs significantly suppressed the vector competence of An. dirus to malaria parasites, which revealed a new important role of Bs on the basis of killing mosquito larvae. Furthermore, the Imd signaling pathway might play an effective way in Bs impacting the vector competence of An. dirus through upregulating the expression of NF-kB transcription factor Rel2, enhancing the expression of TEP1, which killed the Plasmodium, but not through melanization.
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Objective: To assess the repellency to female Aedes aegypti (Ae. aegypti), Anopheles dirus (An. dirus) and Culex quinquefasciatus (Cx. quinquefasciatus) of seven essential oils using two treatment methods. Methods: Topical applications of three dose concentrations (0.02, 0.10 and 0.21 mg/cm2) were made on the forearms of volunteers. Dose-response study and protection time study were employed in the experiment. Results: In the dose-response test, Cymbopogon citratus (C. citratus), Cymbopogon nardus (C. nardus), Syzygium aromaticum (S. aromaticum) and Ocimum basilicum (O. basilicum) exhibited a high repellency against Ae. aegypti with ED50 at < 0.045 mg/cm2, whereas C. citratus, C. nardus and S. aromaticum showed repellency against An. dirus with ED50 at <0.068 mg/cm2. Furthermore, the essential oils of C. citratus, C. nardus, S. aromaticum, O.basilicum and Cananga odorata gave strong effective dose (ED 50) values at <0.003 mg/cm2 when tested against Cx. quinquefasciatus. For testing by arm in cage method, at 0.21 mg/cm2, protection time of C. citratus gave the longest lasting period against three mosquito species, 72 min for Ae. aegypti, 132 min for An. dirus and 84 min for Cx. quinquefasciatus. In addition, the two essential oils exhibited moderate repellency against Ae. aegypti, An. dirus and Cx. quinquefasciatus, at 60, 90 and 78 min with C. nardus, and 54, 96 and 72 min with S. aromaticum, respectively. Conclusions: The percentage repellency increased when the concentration of essential oils increased. In contrast, biting rates decreased when the concentration of essential oils increased.C. citratus exhibited high efficiency for the protection time and the percentage of biting deterrent against all of 3 mosquito species.
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Objective: To investigate the repellent activity of herbal essential oils from garlic (Allium sativum), clove (Syzygium aromaticum), lemon grass (Cybopogon citratus), citronella grass (Cymbopogon nardus), peppermint (Mentha piperita), eucalyptus (Eucalyptus globulus), orange (Citrus sinensis) and sweet basil (Ocimum basilicum) and their combinations against Aedes aegypti (Ae. aegypti) (L.) and Anopheles dirus (An. dirus) Peyton & Harrion under laboratory conditions.Methods:In laboratory condition, 0.1 mL of each essential oil was applied to 3-10 cm of exposed area on a volunteer’s forearm. The test was carried out every 30 min until fewer than two mosquitoes bit or land during the 3 min study period and then the repellency test was stopped.Results:Essential oil from lemon grass exhibited protection against biting from two mosquito species, for Ae. aegypti [(98.66±11.56) min protection time and 0.97% biting rate] and for An. dirus [(98.00±15.28) min protection time and 0.80% biting rate]. The combinations from eucalyptus oil and sweet basil oil were effective as repellents and feeding deterrents against Ae. aegypti [(98.87±10.28) min protection time and 0.90% biting rate] and An. dirus [(210±10.70) min protection time and 0.93% biting rate]. All herbal repellents exhibited the period of protection time against Ae. aegypti which was lower than 120 min. Conlussions: It can be concluded that oils of lemon grass and combination from eucalyptus-sweet basil are the most effective in repellent activity.
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The eggs of Anopheles dirus originated from China and currently colonized at the National Institute of Malariology, Parasitology and Entomology kept at 24 - 26°C on wet cotton for 20 - 25 days gave hatching rate approximately of 40%. The suitable food for Anopheles dirus larvae consisted of 6g bread powder, 2g green bean powder and 0.5mg vitamin B 1. The appropriate density for rearing An. dirus larvae is 0.4 larva per square centimetre of water surface
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Malaria , Anopheles , LaboratoriesABSTRACT
Anopheles dirus mosquitoes collected in the field of Khanh Phu commune, Khanh Vinh district, Khanh Hoa province were reared in the insectarium. The typical karyotype (2n=6) have 2 pairs of chromosomes and one pair of sex chromosomes. The chromosome III are the longest and submetacentric and the II are shorter and metacentric. The sex chromosome is heteromorphic. Three types of X (Xl; X2; X3) chromosomes and 2 types of Y (Yl; Y2) chromosomes were identified. The study on biological characteristics shows that An. dirus are anthropophilous has interest in human blood but they can feed on mice. Mosquitoes fed by human blood were found to lay more eggs (45 - 258 eggs) that those fed on white mice (15 - 148 eggs). The appropriate water level for the advanced development of larval stage should be 10mm to 20mm. Mass death of larvae was observed while protozoa intensively developed in the rearing aquarium.
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Anopheles , Epidemiology , DiagnosisABSTRACT
Objective To construct cDNA library from adults of Anopheles dirus for cloning the immune genes or related genes for malaria parasites development. Methods The mRNA of adult Anopheles dirus was isolated. The library was constructed by using the Zap Express vector(Stratagene) and the quality was evaluated. Results The efficiency of the library was 2.1?10 6 pfu/ml with 98% clones positive. The average length of the insert fragment was over 1 kb. Conclusion cDNA library of adult Anopheles dirus with high efficiency can be constructed by using the Zap Express library construction Kit (Stratagene).
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Objective To observe the effect of blood feeding and plasmodium yoelii infection on the transcript abundance of ribosomal protein S7 from Anopheles dirus hemocyte Methods Anopheles dirus of 3～5 days old were divided into normal group (N), blood feeding group(B) and Plasmodium yoelii infection group(I) Hemocytes of 50 Anopheles dirus from each group were collected by centrifuge method on days 1, 2, 3, 4, 7 and 11 after blood feeding, respectively Then, all hemocyte samples were used for total RNA isolation and analyzed by RT PCR Finally, the same volume(10 ?l) of all the PCR products from each group was used for agarose gel electrophoresis and the data obtained were analyzed statistically Results There was no significant difference in ribosomal protein S7 signal between the three groups Conclusion Similar to Anopheles gambiae and human rpS7, Anopheles dirus rpS7 might be also used as an internal control for the studies of the role of Anopheles dirus related immune factors in attacking Plasmodium infection
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Objective To investigate the role of ribosomal protein S7(rpS7) in the defense of Anopheles dirus against infection. Methods rpS7 was amplified from Anopheles dirus hemocytes with degenerated primers designed according to the conservative region of S7, rpS7 was then cloned using T/A cloning kit and the inserted fragment was sequenced. The difference of the transcript abundance of rpS7 from Anopheles dirus hemocyte among non-blood-fed (N), normal-blood-fed (B) and Plasmodium yoelii infected groups (I) was also analyzed by RT-PCR and gel scanning system at d1, d2, d3, d4, d7 and d11 after blood feeding. Results There is no significant difference of rpS7 signal between the three groups. Conclusion Anopheles dirus S7 can be used as an internal control to study the role of Anopheles dirus related immune factors in Plasmodium infection.
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Objective To interpret genetic variation and population structure of Anopheles dirus A and D from China by molecular marker. Methods Samples included An. dirus A of Hainan laboratory colony (n=13), and field specimen from Mengla (n=17) and Jiangcheng (n=17) in Yunnan Province. The specimens were identified by PCR assay before study. mtDNA-COⅠ region was amplified and sequenced. Genetic variation and population structure was estimated according to sequence data. Results The mtDNA-COⅠ gene with a length of 959 bp was analyzed. There were three haplotypes in An. dirus A and six haplotypes in An. dirus D. The above haplotypes distributed in three populations unif-ormly. The average number of pairwise differences within Mengla population (7.441 2) was greater than that of Jiangcheng (1.279 4) and Hainan (1.051 3) populations, which suggested that the level of genetic divergence was the highest within Mengla population. The result of hierarchical AMOVA estimation showed a limited geneflow (Fst=0.799 9), therefore the variation level in a population (20.01%) was smaller than among the populations (79.99%). Conclusion The inter-specific genetic variation between An. dirus A and D in China was small and the level of divergence among individuals was high.
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Objective To establish a fast and sensitive method to detect Plasmodium sporozoites in mosquitoes. Methods Anopheles anthropophagus (An.a) artificially infected with Plasmodium vivax (P.v), Anopheles dirus (An.d) infected with Plasmodium falciparum (P.f) and both of P.v and P.f, field mosquitoes of Anopheles sinensis (An.s) captured in epidemic seasons were detected by nested-PCR. Results The results of 28 batches of An.a infected with P.v, 2 batches of An.d with P.f and 1 batch of An.d mixed with P.v and P.f by nested-PCR were accorded with the microscopical examination absolutely. Two positives of 589 An.s field mosquitoes were discovered with a positive rate of 0.34%. Conclusion The nested-PCR is fast and sensitive for detecting different species of Plasmodium in mosquitoes.
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Objective To clone the partial cDNA sequence of prophenoloxidase (PPO) gene of Anopheles dirus . Methods Degenerative primers were designed according to the conserved sequence blocks within the prophenoloxidase of insects. RNA sequence of the larva of Anopheles dirus was amplified by RT PCR to get the prophenoloxidase cDNA which was then cloned into T vector and sequenced. The partial cDNA sequence of prophenoloxidase gene was analysed and compared with other prophenoloxidase gene of insects. Results The partial cDNA sequence of AdPPO4 was 597 bp, and its deduced amino acid sequence was 199aa. The cDNA sequence homology and amino acid sequence homology was 84% and 90%, respectively, as compared with the PPO4 gene of Anopheles gambiae . Conclusion The AdPPO4, with high sequence homology with the PPO4 gene of Anopheles gambiae , is successfully cloned from the larva of Anopheles dirus .
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Objective To investigate the expression patterns of serine proteases Adsp1 and Adsp3 in the main tissue of Anopheles dirus and the effects of blood feeding and Plasmodium infection on the transcript abundance of Adsp1 and Adsp3 from Anopheles dirus haemocytes. Methods Haemocytes were collected by centrifugation. The midguts and salivary glands were dissected from 50 adult female Anopheles dirus of 3-5 d old for the extraction of total RNA for amplification by RT PCR. Anopheles dirus of 3-5 d old were divided into normal group (N), blood feeding group (B), and Plasmodium yoelii infection group (I). Haemocytes of 50 adult female Anopheles dirus from each group after blood feeding were also collected, and the total RNA was isolated at 1, 2, 3, 4, 7, and 11 d, respectively. Then, the same volume (10 ?l) of total RNA was analyzed by semi quantitative RT PCR with Ads7 as the internal control. Agarose gel analysis was performed on PCR products from each group. Results Expression of both Adsp1 and Adsp3 mRNA was found in the haemocytes and salivary glands, but not in the midguts. Semi quantitative RT PCR indicated that transcript abundance of AdsP1 and AdsP3 from Anopheles dirus haemocytes was enhanced after blood feeding and Plasmodium yoelii infection, especially during melanotic encapsulation of Plasmodium yoelii . Conclusion AdsP1 and AdsP3 may be involved in the melanotic encapsulation response of Plasmodium yoelii by Anopheles dirus , and may be the prophenoloxidase activating enzyme of Anopheles dirus .
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Objective To investigate the relationship between the immune defence reaction against Plasmodium infection and the prophenoloxidase (PPO) of the midgut by comparative analysis of the distributions and the changes of PPO in the midgut of Anopheles stephensi and Anopheles dirus before and after infection with Plasmodium yoelii . Methods Midguts were dissected out from both Anopheles stephensi and Anopheles dirus at 3, 5, 7, 11 and 15 d before and after infection with Plasmodium yoelii . Immunohistochemistry and Western blotting were performed respectively on the collected midguts using Manduca Sexta PPO IgG polyantibody. Results PPO in the midguts from both Anopheles stephensi and Anopheles dirus was mainly located in the circulation conduit of midgut before infection with Plasmodium yoelii , but aggregated and distributed at the interspace of midguts as pieced or/and stripped forms after infection. Furthermore, PPO in the midgut of Anopheles dirus was more concentrated than that of Anopheles stephensi . Western blotting revealed that the PPO band with about molecular weight of 67?10 3 was detected in the midguts of both Anopheles stephensi and Anopheles dirus before and after Plasmodium yoelii infection. There was significant difference before and after infection, and the PPO band was obviously enhanced after infection. PPO bands in the midgut of Anopheles dirus were more prominent than those of Anopheles stephensi . Conclusion PPO in the midgut of Anopheles mosquitoes may come from the hemolymph by the circulation conduit before Plasmodium yoelii infection. However, the different distributions and changes of PPO in the midguts resulted from the Anopheles mosquitoes infected with Plasmodia may be closely correlated with Plasmodia infection, which may be of important physiological significance and may be involved in the immune defensive reaction against Plasmodium .