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Objective To explore primarily the differentially expressed proteins in the hemolymph from adult female Anopheles stephensi ( An stephensi ) infected with Plasmodium yoelii ( P yoelii ) after being fed with sucrose solution containing nitroquine or not at different time points Methods Hemolymph of 2 groups of adult female An stephensi was collected with the expulsion method from the first day to the fifth day after the feeding Hemolymph samples were examined with SDS PAGE The protein gels were visualized by either Coomassie brilliant blue or silver staining, scanned and automatically analyzed by the BioRad1000 gel image analysis system for differential proteins bands Results On the second day of feeding with nitroquine, a few oocysts were partially melanized Furthermore, during the period from the fifth day to the ninth day, the number of mosquitoes with malanized oocysts and the number of melanized oocysts gradually increased The number of hemolymph protein binds in the treatment group was markedly more than that in the control Many different bands, mainly located at the molecular weight of (20～40)?10 3 and (60～80)?10 3, were visualized in the 2 groups The number of protein bands stained by the silver staining was more than that by the Coomassie brilliant blue staining Conclusion There are differentially expressed proteins in the hemolymph in An stephensi infected with P yoelii after being fed with sucrose solution containing nitroquine These differential proteins may be the melanization engaging proteins
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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 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 study the gene expression change of circumsporozoite protein (CSP) and erythrocyte binding protein (MAEBL) in the Plasmodium yoelii sporozoites from oocyst and salivary gland at different phases during Plasmodium yoelii development in mosquitos. Methods The total RNA of plasmodium was extracted from the abdomen on day 5, 7, 10 and the thoraces on day 12, 15, 18 after Anopheles stephensi was infected by Plasmodium yoelii, then the expression of CSP and MAEBL mRNA was analyzed by semiquantitative RT-PCR with rRNA 28S as control. Results The transcription of CSP and MAEBL of plasmodium sporozoite were obviously up-regulated during the mosquito stage (P
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Objective To study the expression levels of B7.1 and B7.2 in the macrophagus of rats infected with the Plasmodium yoelii sporozoites and investigate the roles of these costimulators in parasite infection immunity. Methods The mammal model was established by infecting rats with the sporozoites, then macrophagus was separated from the rat abdominal cavity 2, 12, 24, 48, 72 h after infection. The expressions were quantitative analyzed by immunofluorescence staining and laser scanning confocal microscopy. Result The expressions of B7.1 and B7.2 were upregulated after infected with the sporozoites. The expression of B7.1 was slowly induced and then descended at 72 h. B7.2 was rapidly induced and maximally expressed at 48 h and downregulation at 72 h. The expression of B7.2 was significantly higher than that of B7.1 at all time points assayed after stimulation (P
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Objective To explore the relationship between hemolymph phenol oxidase and the melanization of Plasmodium yoelii oocysts in Anopheles dirus. Methods An Anopheles dirus-Plasmodium yoelii system was used Anopheles dirus were divided into 3 groups, that is, non-blood-fedding (N), normal-blood-fedding (B) and infected-blood-fedding (I). The activities of MPO and o-DPO in hemolymph from 3 groups were determined with native polyacrylamide gel electrophoresis (PAGE) and density scanning at 5, 7, 11 and 15 d after blood feeding. Results Both MPO and o-DPO activity were significantly higher in group I than group N and B (P<0.05). But with the melanization of Plasmodium yoelii oocysts, both MPO and o-DPO activity in group I were decreased in comparison with group N, especially on the 15 th day after infected-blood feeding. MPO and o-DPO activity in group B were significantly stronger than those of group N. Conclusion Blood feeding and infection of Plasmodium yoelii both can activate the cascade. The heamolymph phenol oxidase may play an important role in the melanization of Plasmodium yoelii oocysts in Anopheles dirus.
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Objective] To analyse the soluble antigens of different developmental stages of Pagumogonimus skrjabini and deve lop a specific and sensitive serodiagnostic method for pagumogonimiasis. [Methods] The soluble antigens of P.skrjabini of various stages were separated by SDS PAGE. The specific antigen of the adult fluke was recognized immunologically by immunoblot assay. The protein bands between 10～30 kDa purified by SDS PAGE and electrophoretic elution were used in dot ELISA. [Results] Using dot ELISA, the soluble antigens of adult were recognized by sera infected with P skrjabini . More reactive bands appeared at 10～30 kDa, but major protein bands were at 22、24 and 26 kDa. However, using sera from patients infected with other trematodes including schistosome and Clonorchis , cross reaction bands appeared within 60 to 90 kDa. When compared with ELISA of crude adult antigens for detecting 28 suspected patients, there was no significant difference between the two methods. The sera of 38 patients with other diseases were also detected by the two tests. No cross reaction occurred with the purified adult antigen dot ELISA while 13 2%(5/38) of the sera cross reacted in ELISA of crude adult antigens. [Conclusion] Dot ELISA using 10～30 kDa antigen might be a specific and sensitive serodiagnostic method for diagnoing pagumogonimiasis.
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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 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 analyze the relationship between the TEP1 gene of Anopheles stephensi and melanotic encapsulation of Plasmodium yoelii induced by anti-malaria drug nitroquine. Methods Haemolymph samples from three groups of An. stephensi fed with sucrose solution, Plasmodium-infected blood and nitroquine, respectively, were collected at the 1st, 2nd, 3rd and 4th day after drug adminstration. Degenerate primers were designed according to the conserved amino acid sequence within TEPs of the mosquitoes. Fluorescent quantitation PCR was used to detect the variation of TEP1 gene transcript induced by nitroquine. The melanization of oocysts was observed by light microcopy. Results TEP1 gene was cloned, the predicted amino acid residues harbored a highly conserved canonical thioester motif GCGEQ. The fluorescent quantitation PCR revealed that nitroquine induced an up-regulation of TEP1 activity. The transcription of TEP1 gene in nitroquine treated group (2.423) was significantly higher than that of the infected blood-fed group(1.036) at the 3rd day after nitroquine treatment (P
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Objective To study the change of intracellular free Ca2+ in the oocyst when it melanized and to find out the relationship between the melanized oocyst and its intracellular level of free Ca2+ in a Plasmodium-refractory strain of Anopheles dirus. Methods The distribution and experimental condition of the intracellular free Ca2+ in oocyst of Plasmodium yoelii was measured with Ca2+ sensitive dye Fluo-3/AM and Plutonic F-127 under confocal laser scanning microscope (CLSM) at different time. Results The best load condition was that the oocysts were incubated in 3 ?mol/ml Fluo-3/AM adding 1 ?l/ml 25% Pluronic F-127 for 60 min at 37 ℃ . Fluorescent imaging of oocysts was affected by an increase or decrease of the concentration of Fluo-3/Am and incubation time. The distribution of intracellular free Ca2+ was heterogeneous in the oocysts. The mean value of Ca2 + in the mature oocysts was (137.15 ?7.02) nmol/L(X?S) but was (18.44? 1.75) nmol/L in melanized oocysts with Ca2+ sedimentation in the wall of oocyst. Conclusion The results suggest that the level of the intracellular free Ca2+ in oocyst decreased and excreted during its melanization in a Plasmodium-reiractory anopheline mosquito species.
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Two hours after the rats were infected with 125I - labeled sporozoites of Plasmo-dium yoelii,the sporzoites captured by the liver,the spleen and the lungs accounted for 1.67%,0.046% and 0.49% respectively.During the 2nd to the 18h,the sporozoites decreased in number slowly in the liver,remained unchanged in the spleen and decreased in number quickly in the lungs as time went on.When the liver and the lungs were perfused with quick M199 stream containing sporozoites,the capture rate of sporozoites was not affected in the liver but that of the lungs was markedly decreased.The numbr of sporozoites in the liver and the spleen was larger in those rats with their phagocytosis activated than in those with the phagocytosis inhibited.while in the lungs,the number of sporozoites was not influenced by the activity of macrophages.These findings suggest that the mechanism of capturing sporozoites in the lung is mainly mechanical and the alveolar macrophages play little role in that,but that in the liver and the spleen is mainly a biological process in which macrophages play an important role.