Young S. Hong

Modulation of Anopheles gambiae gene expression in response to o’nyong‐nyong virus infection

To determine if gene expression of An. gambiae is modulated in response to o’nyong‐nyong virus (ONNV) infection, we utilized cDNA microarrays including about 20 000 cDNAs. Gene expression levels of ONNV‐infected female mosquitoes were compared to that of the uninfected control females harvested at 14 days postinfection. In response to ONNV infection, expression levels of 18 genes were significantly modulated, being at least two‐fold up‐ or down‐regulated. Quantitative real‐time PCR analysis (qRT‐PCR) further substantiated the differential expression of six of these genes in response to ONNV infection. These genes have similarity to a putative heat shock protein 70, DAN4, agglutinin attachment subunit, elongation factor 1 alpha and ribosomal protein L35. One gene, with sequence similarity to mitochondrial ribosomal protein L7, was down‐regulated in infected mosquitoes. The expression levels and annotation of the differentially expressed genes are discussed in the context of host/virus interaction including host translation/replication factors, and intracellular transport pathways.

Comparative genomic analysis in the region of a major Plasmodium-refractoriness locus of Anopheles gambiae

We have sequenced six overlapping clones from a library of bacterial artificial chromosome (BAC) clones derived from a laboratory strain of the mosquito, Anopheles gambiae, the major vector of human malaria in Africa. The resulting uninterrupted 528-kb sequence is from the 8C region of the mosquito 2R chromosome, at or very near the major refractoriness locus associated with melanotic encapsulation of parasites. This sequence represents the first extensive view of the mosquito genome structure encompassing 48 genes. Genomic comparison reveals that the majority of the orthologues are found in six microsyntenic clusters in Drosophila melanogaster. A BAC clone that is wholly contained within this region demonstrates the existence of a remarkable degree of local polymorphism in this species, which may prove important for its population structure and vectorial capacity.

RNA Interference in Infectious Tropical Diseases

Introduction of double-stranded RNA (dsRNA) into some cells or organisms results in degradation of its homologous mRNA, a process called RNA interference (RNAi). The dsRNAs are processed into short interfering RNAs (siRNAs) that subsequently bind to the RNA-induced silencing complex (RISC), causing degradation of target mRNAs. Because of this sequence-specific ability to silence target genes, RNAi has been extensively used to study gene functions and has the potential to control disease pathogens or vectors. With this promise of RNAi to control pathogens and vectors, this paper reviews the current status of RNAi in protozoans, animal parasitic helminths and disease-transmitting vectors, such as insects. Many pathogens and vectors cause severe parasitic diseases in tropical regions and it is difficult to control once the host has been invaded. Intracellularly, RNAi can be highly effective in impeding parasitic development and proliferation within the host. To fully realize its potential as a means to control tropical diseases, appropriate delivery methods for RNAi should be developed, and possible off-target effects should be minimized for specific gene suppression. RNAi can also be utilized to reduce vector competence to interfere with disease transmission, as genes critical for pathogenesis of tropical diseases are knockdowned via RNAi.

Transcription Profiling Associated With Life Cycle of Anopheles gambiae

ABSTRACT Complex biological events occur during the developmental process of the mosquito Anopheles gambiae (Giles). Using cDNA expression microarrays, the expression patterns of 13,440 clones representing 8,664 unique transcripts were revealed from six different developmental stages: early larvae (late third instar/early fourth instar), late larvae (late fourth instar), early pupae (<30 min after pupation), late pupae (after tanning), and adult female and male mosquitoes (24 h postemergence). After microarray analysis, 560 unique transcripts were identified to show at least a fourfold up- or down-regulation in at least one developmental stage. Based on the expression patterns, these gene products were clustered into 13 groups. In total, eight genes were analyzed by quantitative real-time polymerase chain reaction to validate microarray results. Among 560 unique transcripts, 446 contigs were assigned to respective genes from the An. gambiae genome. The expression patterns and annotations of the genes in the 13 groups are discussed in the context of development including metabolism, transport, protein synthesis and degradation, cellular processes, cellular communication, intra- or extra-cellular architecture maintenance, response to stress or immune-related defense, and spermatogenesis.

Application of free-flow electrophoresis/2-dimentional gel electrophoresis for fractionation and characterization of native proteome of Pseudomonas putida KT2440

Free Flow Electrophoresis (FFE) is a liquid-based isoelectric focusing method. Unlike conventional in-gel fractionation of proteins, FFE can resolve proteins in their native forms and fractionation of subcellular compartments of the cell is also possible. To test the efficacy of the FFE method, the native cytosol proteome of a bacterium, Pseudomonas putida KT2440 was fractionated by FFE and the spectrum of protein elutes was characterized in association with 2-dimentional gel electrophoresis (2-DE). Major native proteins of P. putida KT2440 were eluted in the range of pH 4.8∼6.0 in FFE, whereas the denatured proteome of P. putida KT2440 was widely distributed in the rage of pH 4∼10 in the 2-DE analysis. In addition, one of the three FFE major fractions, which was eluted at pH 5.0, was further analyzed using 2-DE/MS-MS. Then, the pH range of identified proteins eluted in 2-DE/MS-MS was 4.72∼5.89, indicating that observed pi values of native cytosolic proteomes in FFE were narrower than those of denatured cytosolic proteome. These results suggest that FFE fractionation and 2-DE/MS analysis may be useful tools for characterization of native proteomes of P. putida KT2440 and comparative analysis between denatured and native proteomes.

Bioinformatic analysis and annotation of expressed sequence tags (ESTs) generated from Anopheles sinensis mosquitoes challenged with apoptosis-inducing chemical, actinomycin-D

In 1993, after 10 years of absence, malaria re‐emerged in the Republic of Korea in Gyeonggi and Gangwon Provinces near the Demilitarized Zone (DMZ). Anopheline mosquitoes are known to play a critical role in malaria transmission. Several studies have used gene expression data to examine the transcription responses of Anopheles gambiae after microbial and Plasmodium infections; however, as of now, there have been no reports of studies concerning the innate immune response and apoptosis in Anopheles sinensis. We have constructed two independent cDNA libraries from An. sinensis challenged with actinomycin‐D (T, treated with apoptosis‐inducing chemical) and without actinomycin‐D (C, control). Analysis of the 5704 (C, 2848; T, 2856) expression sequence tags (ESTs) generated from these libraries identified 884 contigs (C, 421; T, 463) and 2180 singletons (C, 1038; T, 1142) with average lengths of 831 and 817 bp, respectively. Furthermore, comparison of our EST sequences against the eukaryotic orthologous group database showed that 4324 (75.82%) of 5704 sequences could be grouped into three major functional categories. These EST data provide the basis for further studies characterizing the molecular mechanisms of midgut apoptosis.

Optimization of double-stranded RNAi intrathoracic injection method in Aedes aegypti : Quantitative and temporal analysis of dsRNA

RNA interference is widely used to analyze gene functions via phenotypic knockdown of target transcripts in mosquitoes, which transmit numerous mosquito‐borne diseases. Functional analysis of mosquito genes is indispensable to understand and reduce transmission of mosquito‐borne diseases in mosquitoes. Intrathoracic injection of double‐stranded RNA (dsRNA) remains the simplest and most customizable method in mosquitoes for functional analysis of the genes of interest. However, achieving consistent and effective knockdown by dsRNAi is often elusive and may require extensive optimization. We tested the effectiveness of gene silencing by intrathoracic injection of four different quantities of dsRNA targeting two Aedes aegypti genes, cysteine desulfurylase (Nfs1) and short‐chain dehydrogenase (SDH). We found that Nfs1 gene has a lower expression level upon silencing than SDH gene. In the case of the gene that is easier to silence, Nfs1 gene expression was significantly silenced by all four tested quantities of dsRNA up to 21 days post infection (d.p.i.), but silencing of SDH, the gene that is difficult to silence, was less effective, with knockdown lasting up to 9 d.p.i. only when 1,000 ng of dsRNA was used. Based on our observation, intrathoracic injection of 500 ng of dsRNAs per mosquito is recommended to achieve effective knockdown for well‐silenced transcripts such as Nfs1 for up to 3 weeks. This includes most in vivo bioassays involving arboviral infections in Ae. aegypti. The estimated quantities of dsRNA described in this study should be applicable to most Ae. aegypti dsRNAi studies and thus provide a guideline to develop efficient dsRNAi in other experimental investigations.

High quality RNA isolation from Aedes aegypti midguts using laser microdissection microscopy

BackgroundLaser microdissection microscopy (LMM) has potential as a research tool because it allows precise excision of target tissues or cells from a complex biological specimen, and facilitates tissue-specific sample preparation. However, this method has not been used in mosquito vectors to date. To this end, we have developed an LMM method to isolate midgut RNA using Aedes aegypti.ResultsTotal RNA was isolated from Ae. aegypti midguts that were either fresh-frozen or fixed with histological fixatives. Generally, fresh-frozen tissue sections are a common source of quality LMM-derived RNA; however, our aim was to develop an LMM protocol that could inactivate pathogenic viruses by fixation, while simultaneously preserving RNA from arbovirus-infected mosquitoes. Three groups (10 - 15 mosquitoes per group) of female Ae. aegypti at 24 or 48-hours post-blood meal were intrathoracically injected with one of seven common fixatives (Bouins, Carnoys, Formoys, Cal-Rite, 4% formalin, 10% neutral buffered formalin, or zinc formalin) to evaluate their effect on RNA quality. Total RNA was isolated from the fixed abdomens using a Trizol® method. The results indicated that RNA from Carnoys and Bouins fixative samples was comparable to that of fresh frozen midguts (control) in duplicate experiments. When Carnoys and Bouins were used to fix the midguts for the LMM procedure, however, Carnoys-fixed RNA clearly showed much less degradation than Bouins-fixed RNA. In addition, a sample of 5 randomly chosen transcripts were amplified more efficiently using the Carnoys treated LMM RNA than Bouins-fixed RNA in quantitative real-time PCR (qRT-PCR) assays, suggesting there were more intact target mRNAs in the Carnoys fixed RNA. The yields of total RNA ranged from 0.3 to 19.0 ng per ~3.0 × 106 μm2 in the LMM procedure.ConclusionsCarnoys fixative was found to be highly compatible with LMM, producing high quality RNA from Ae. aegypti midguts while inactivating viral pathogens. Our findings suggest that LMM in conjunction with Carnoys fixation can be applied to studies in Ae. aegypti infected with arboviruses without compromising biosafety and RNA quality. This LMM method should be applicable to other mosquito vector studies.