[Partial sequence of sporogony stage-specific 18S ribosomal DNA of Plasmodium yoelii and its application for detection of parasites].

OBJECTIVE: To determine sequence of sporogony stage-specific (S type) 18S ribosomal RNA gene of Plasmodium yoelii (P. y) By265 strain, and by using it to detect the malaria parasites within vector mosquito. METHODS: A pair of conserved DNA primers, universe primer (Pu) and reverse transcription one (Pr), was designed and synthesized according to sequence of the 18S rRNA gene of Plasmodium berghei (P. b). The segment of the S type 18S rDNA of P. y was amplified by reverse transcript-polymerase chain reaction (RT-PCR) from dissected midguts of Anopheles stephensi infected with P. y on the 7th day after infective blood-meal, and its sequence was then determined. One P. y sporogony stage-specific primer (Pys) was selected according to the sequence. Using this primer and Pr, the parasites within mosquitoes were semi-quantitatively detected through RT-PCR between 1-7 d post-infection. RESULTS: The length of the amplified segment was 920 bp. Alignment in match region of the 18S rDNA among S type of P. y (PyS), S type of P. b (PbS) and asexual blood stage-specific one of P. y (PyA) revealed that the similarity between the former and the latter two reached 95.3% and 94.0% respectively. The density of amplified band was significantly concordance with the intensity of oocyst in the midgut. Sensitivity of RT-PCR method was higher than that of the traditional dissection and oocyst observation also. The assay could detect the 18S rRNA molecule of the parasites on the third day post-infection while their oocysts were difficult to be recognized under an optical microscope at that time. CONCLUSION: This S type 18S rDNA sequence in P. y species was first reported (AF266261). As a molecular marker, it could be applied to monitoring the parasite development in its vector at an earlier stage semi-quantitatively with an adequate sensitivity and specificity.

[Enrichment and screening of up-regulated genes of the mosquito Anopheles stephensi in response to malaria parasite].

OBJECTIVE: To isolate and identify genes related to malaria parasite infection in vector mosquito, and to explore the mechanisms. METHODS: Anopheles stephensi infected with Plasmodium yoelii was used as tester (T) group, while uninfected but normal blood fed as driver (D) one. Engorged female mosquitoes of two groups were collected separately at 24 hours after biting. An enriched subtractive cDNA pool was generated through the course of suppression subtractive hybridization (SSH) and selective PCR amplification. The subtracted library was screened by hybridization using T and D cDNA mixture as probes, respectively. The positive clones, which produced stronger signal when probed with T than with D, were sequenced and their sequence homologues in GenBank database were searched with BLAST by internet. RESULTS: The analysis of subtraction efficiency showed that the differentially expressed genes in T comparing to in D were enriched significantly. In dot blot screening, 24 of 58 randomly selected clones (41.4%) were shown up-regulation in malaria infected mosquitoes. The BLAST search of 23 genes revealed that 12 were homologous to functionally known genes, 4 were homologous to functionally unknown entries, and 7 were novel without any relatives. Nine of the 23 genes (39.1%) also hit homologous sequences in the An. gambiae EST database generated from an immune competent cell line treated with lipopolysaccharide (LPS). CONCLUSION: An enriched cDNA pool of the mosquito genes which up-regulated responsively at the early stage of malaria parasite infection was obtained. Expression screening against the pool indicated that various biochemical processes and mechanisms might be involved in the response of mosquito to parasite infection, especially those related with the innate immune system and energy metabolism.

[On molecular identification and taxonomic status of Anopheles lesteri and Anopheles anthropophagus in China (Diptera: Culicidae)].

OBJECTIVE: To clarify the taxonomic status of Anopheles lesteri and An. anthropophagus in China. METHODS: Using molecular identification (PCR assay and rDNA-ITS2 sequencing) to examine the field anopheline mosquito specimens from Liaoning and Shandong. According to the ITS2 sequences, molecular phylogenetic tree was made. RESULTS: According to the molecular identification, An. lesteri and An. anthropophagus were distributed both in Liaoning Province and Shandong Province. The length and GC content of rDNA-ITS2 sequence were 451 bp, 46.2% in An. lesteri (n = 6), and 448 bp, 46.0% in An. anthropophagus (n = 10), respectively. The ITS2 sequences from presentation sites were same in An. lesteri, while the intraspecies difference in An. anthropophagus was 0.88%. The specific difference between An. lesteri and An. anthropophagus was 25.7%. By analyzing molecular phylogenetic tree, the relationship between An. lesteri and An. sinensis, An. anthropophagus and An. liangshanensis was found to be closer. CONCLUSION: According to the molecular identification, it was defined that An. lesteri and An. anthropophagus were sympatric independent species in China.

[Comparison of rDNA-ITS2 sequences and morphological characters of Anopheles kunmingensis and Anopheles langshanensis in China, with discussion on taxonomic status].

OBJECTIVE: To determine the taxonomic status of Anopheles kunmingensis (AK) and An. liangshanensis(AL) from China. METHODS: Sequence differences of rDNA-ITS2 and main morphological characters variability between different sources of AK and AL were compared. RESULTS: The level of rDNA-ITS2 sequence differences among eight samples was under 2.9%. The total occurrence rates of main morphological characters examined in the female mosquitoes with pale fringe spot on V5.2, white basal band (or spot) on hind tarsomere IV, and larvae with bifurcated head hair 2-C were 43%(9/21), 89%(17/19), 40(4/10) in AK, and 79%(34/43), 44%(17/39), 20%(4/20) in AL, respectively. These rates calculated from separate colonies fluctuated within a wide range and overlapped, suggesting that there was no definite, stable morphological difference between AK and AL. CONCLUSION: The morphological and molecular variations between AK and AL were small and belong to intraspecific level. The AK and AL may be considered as the same species, and that the An. kunmingensis may be recognized as the synonym of An. liangshanensis.

Ribosomal DNA difference between species A and D of the Anopheles dirus complex of mosquitoes from China.

Species A and D of the Anopheles dirus complex were found in China. Ribosomal DNA second internal transcribed spacers (ITS2) of both species A and D were sequenced and found to be 716 and 710 base-pairs in length, respectively, with 69% GC content. No evidence of intraspecific variation was detected in the ITS2 sequence of species A, whereas the sequence of species D showed variation at one position in the ITS2. A large number of simple repeat motifs were dispersed throughout the ITS2 sequences. The level of interspecific difference was 5.4% of the nucleotide sequences. Some of the interspecific differences were located in regions with subrepeat structure.

Detection of 54-kDa protein overexpressed by chloroquine-resistant Plasmodium berghei ANKA strain in pyronaridine-resistant P berghei ANKA strain.

AIM: A 54-kDa protein overexpressed by chloroquine-resistant (CR) Plasmodium berghei ANKA strain was first reported by us. This study is conducted to detect the protein in pyronaridine-resistant (PR) P berghei ANKA strain. METHODS: Immunoblotting analysis and immunoelectron microscopy were used. RESULTS: PR parasites, like CR parasites, mainly overexpressed 2 major bands of 37 (36-38) kDa and 16 (15-17) kDa which were considered to be 2 subunits of 54 (52-62) kDa protein. Three of 7 times of experiments showed a 54-kDa and a 96 (95-100) kDa bands. The proteins were localized to be mainly scattered in cytoplasm of trophozoites, schizonts, and merozoites of erythrocytic stage of P berghei. Some of them were distributed in cytoplasm of erythrocytes infected with parasites. CONCLUSION: Both PR and CR parasites overexpressed the same proteins.

A 54-kDa protein overexpressed by chloroquine-resistant Plasmodium berghei ANKA strain.

Using an insoluble chloroquine-adsorbent, a 54-kDa protein (with a range of 50-60 kDa) was extracted from serum of mice infected with chloroquine-resistant (CR) Plasmodium berghei ANKA strain. Immunoblotting assay with antiserum against the 54-kDa protein showed that the content of the protein was higher in serum of mice infected with the CR parasites than that of mice infected with chloroquine-sensitive (CS) P berghei ANKA strain, and that instead of the 54-kDa protein, a set of 15-, 16-, and 23-kDa proteins was found to be highly overexpressed in lysate of purified CR parasites in comparison with that of purified CS parasites, suggesting the 54-kDa protein probably to be composed of 3 subunits. These findings may bear great importance in probing mechanism of chloroquine resistance in malaria parasites.

[Immunoelectron-microscopic localization of a 54-kDa protein overexpressed by chloroquine-resistant Plasmodium berghei ANKA strain].

A 54-kDa protein overexpressed by chloroquine-resistant Plasmodium berghei ANKA strain was first reported by us. In this paper, the localization of this protein by immunoelectron microscopy is presented. The results showed that the protein was mainly scattered inside the cytoplasm of the early, late trophozoites and schizonts of erythrocytic stage of P. berghei ANKA strain, and some of it was also found in cytoplasm of erythrocytes infected with parasites. The protein content was much higher in chloroquine-resistant P. berghei ANKA strain than in chloroquine-sensitive P. berghei ANKA strain, suggesting the importance of this protein in understanding mechanism of chloroquine resistance in malaria parasites.

[Experimental infection index of Anopheles sinensis and melanization of periodic Brugia malayi larvae].

This paper deals with the infection index of Anopheles sinensis infected with the blood meals of 9 different microfilarial densities (5-300 mf/10 microliters) of periodic Brugia malayi. The results indicated that the mean number of microfilariae (mf) ingested by mosquitoes increased with the mf-density of the blood meal. The infection rates of vectors were 30, 65, 93 and 100%, when mf-densities were 5, 10, 20 and 50 mf/10 microliters respectively. Although the infection rate was 100% when mf-densities were larger than 50 (100, 150, 200, 250, 300) mf/10 microliters, the infection intensities were gradually increased from 17.2 to 51.4 and the host efficiencies were decreased from 0.4135 to 0.2328. The infection intensities and host efficiencies of low mf-densities (5, 10, 20, 50 mf/10 microliters) were 1.22-8.40 and 0.5669-0.6356 respectively. Under the condition of 27.5 +/- 0.5 degrees C, RH 75 +/- 5%, the developmental period from mf to third stage larva was 8 days and numerous infective larvae could be harvested when mf-density was 200 mf/10 microliters. The relationship between mf-density and host efficiency, number of melanized larvae and susceptibility of vector, experimental infection indices of Kartman and of Wharton were discussed.