Comparative genome analysis of the yellow fever mosquito Aedes aegypti with Drosophila melanogaster and the malaria vector mosquito Anopheles gambiae.

An in silico comparative genomics approach was used to identify putative orthologs to genetically mapped genes from the mosquito, Aedes aegypti, in the Drosophila melanogaster and Anopheles gambiae genome databases. Comparative chromosome positions of 73 D. melanogaster orthologs indicated significant deviations from a random distribution across each of the five A. aegypti chromosomal regions, suggesting that some ancestral chromosome elements have been conserved. However, the two genomes also reflect extensive reshuffling within and between chromosomal regions. Comparative chromosome positions of A. gambiae orthologs indicate unequivocally that A. aegypti chromosome regions share extensive homology to the five A. gambiae chromosome arms. Whole-arm or near-whole-arm homology was contradicted with only two genes among the 75 A. aegypti genes for which orthologs to A. gambiae were identified. The two genomes contain large conserved chromosome segments that generally correspond to break/fusion events and a reciprocal translocation with extensive paracentric inversions evident within. Only very tightly linked genes are likely to retain conserved linear orders within chromosome segments. The D. melanogaster and A. gambiae genome databases therefore offer limited potential for comparative positional gene determinations among even closely related dipterans, indicating the necessity for additional genome sequencing projects with other dipteran species.

Physical mapping of barley genes using an ultrasensitive fluorescence in situ hybridization technique.

The primary objective of this study was to elucidate gene organization and to integrate the genetic linkage map for barley (Hordeum vulgare L.) with a physical map using ultrasensitive fluorescence in situ hybridization (FISH) techniques for detecting signals from restriction fragment length polymorphism (RFLP) clones. In the process, a single landmark plasmid, p18S5Shor, was constructed that identified and oriented all seven of the chromosome pairs. Plasmid p18S5Shor was used in all hybridizations. Fourteen cDNA probes selected from the linkage map for barley H. vulgare 'Steptoe' x H. vulgare 'Morex' (Kleinhofs et al. 1993) were mapped using an indirect tyramide signal amplification technique and assigned to a physical location on one or more chromosomes. The haploid barley genome is large and a complete physical map of the genome is not yet available; however, it was possible to integrate the linkage map and the physical locations of these cDNAs. An estimate of the ratio of base pairs to centimorgans was an average of 1.5 Mb/cM in the distal portions of the chromosome arms and 89 Mb/cM near the centromere. Furthermore, while it appears that the current linkage maps are well covered with markers along the length of each arm, the physical map showed that there are large areas of the genome that have yet to be mapped.

Characterization of three Toll-like genes from mosquito Aedes aegypti.

Three Toll-related genes (AeToll1A, AeToll1B and AeToll5) were cloned and characterized from the yellow fever vector mosquito, Aedes aegypti. All three genes exhibited high levels of amino acid sequence similarity with Drosophila melanogaster (Dm)Toll1 and DmTehao (Toll5). AeToll1A and AeToll1B are 1124 and 1076 amino acid residues long, respectively. Both contain a carboxyl extension downstream of the Toll/interleukin-1 receptor (TIR) domain. AeToll5 is 1007 residues long and, like DmTehao, lacks the carboxyl terminal extension. Expression of these three genes was examined throughout development and after immune challenge. Both AeToll1A and AeToll5, like their Drosophila counterparts, activate transcription of drosomycin promoter in both Aedes and Drosophila cell lines. Deletion of the carboxyl extension of AeToll1A did not result in a further elevated level of the antifungal response. The intracellular signalling process appears to be species specific based on two observations. (1) DmToll is completely inactive in an Aedes cell line, suggesting a higher specificity requirement for DmToll in the intracellular signalling process. (2) Only one of three amino acid residues essential for DmToll function is required for AeToll1A function.

Integration of the Aedes aegypti mosquito genetic linkage and physical maps.

Two approaches were used to correlate the Aedes aegypti genetic linkage map to the physical map. STS markers were developed for previously mapped RFLP-based genetic markers so that large genomic clones from cosmid libraries could be found and placed to the metaphase chromosome physical maps using standard FISH methods. Eight cosmids were identified that contained eight RFLP marker sequences, and these cosmids were located on the metaphase chromosomes. Twenty-one cDNAs were mapped directly to metaphase chromosomes using a FISH amplification procedure. The chromosome numbering schemes of the genetic linkage and physical maps corresponded directly and the orientations of the genetic linkage maps for chromosomes 2 and 3 were inverted relative to the physical maps. While the chromosome 2 linkage map represented essentially 100% of chromosome 2, approximately 65% of the chromosome 1 linkage map mapped to only 36% of the short p-arm and 83% of the chromosome 3 physical map contained the complete genetic linkage map. Since the genetic linkage map is a RFLP cDNA-based map, these data also provide a minimal estimate for the size of the euchromatic regions. The implications of these findings on positional cloning in A. aegypti are discussed.

Genetic and physical mapping in mosquitoes: molecular approaches.

The genetic background of individual mosquito species and populations within those species influences the transmission of mosquito-borne pathogens to humans. Technical advances in contemporary genomics are contributing significantly to the detailed genetic analysis of this mosquito-pathogen interaction as well as all other aspects of mosquito biology, ecology, and evolution. A variety of DNA-based marker types are being used to develop genetic maps for a number of mosquito species. Complex phenotypic traits such as vector competence are being dissected into their discrete genetic components, with the intention of eventually using this information to develop new methods to prevent disease transmission. Both genetic- and physical-mapping techniques are being used to define and compare genome architecture among and within mosquito species. The integration of genetic- and physical-map information is providing a sound framework for map-based positional cloning of target genes of interest. This review focuses on advances in genome-based analysis and their specific applications to mosquitoes.

Diagnosis of duck plague in waterfowl by polymerase chain reaction.

A recently developed polymerase chain reaction (PCR) assay was used for diagnosis of duck plague in waterfowl tissues from past and current cases of waterfowl mortality and to identify duck plague virus in combined cloacal/oral-pharyngeal swab samples from healthy mallards (Anas platyrhynchos) after a disease outbreak. The PCR was able to detect viral DNA from all the individual or pooled tissues assayed from 10 waterfowl, including liver and spleen samples from three Muscovy ducks (Cairina moschata domesticus) that did not yield virus isolates. The strong staining intensity of the PCR products from the waterfowl tissues indicated that large amounts of virus were present, even when virus was not isolated. Duck plague DNA was also detected in a cloacal swab sample from a wood duck (Aix sponsa) carcass submitted for diagnosis. The PCR assay identified duck plague DNA in 13 swab samples that produced virus isolates from carrier mallards sampled in 1981 after a duck plague die-off. The duck plague PCR clearly demonstrated the ability to quickly diagnose duck plague in suspect mortality cases and to detect virus shed by carrier waterfowl.

Structure and location of a ferritin gene of the yellow fever mosquito Aedes aegypti.

We have isolated and sequenced a genomic clone encoding the 24- and 26-kDa ferritin subunits in the mosquito Aedes aegypti (Rockefeller strain). The A. aegypti gene differs from other known ferritin genes in that it possesses an additional intron and an unusually large second intron. The additional intron is located within the 5' untranslated region, between the CAP site and the start codon. The second intron contains numerous putative transposable elements. In addition, unlike the human and rat ferritin genes, the A. aegypti ferritin gene is a single copy gene, located at 88.3% FLpter on the q-arm of chromosome 1. Primer extension analysis indicates that the A. aegypti ferritin gene has multiple transcriptional start sites. A differential usage of these sites is observed with varied cellular iron concentrations.

First-generation physical map of the Culicoides variipennis (Diptera: Ceratopogonidae) genome.

Recombinant cosmids labeled with biotin-11-dUTP or digoxigenin by nick translation were used as in situ hybridization probes to metaphase chromosomes of Culicoides variipennis (Coquillett). Paired fluorescent signals were detected on each arm of sister chromatids and were ordered along the 3 chromosomes. Thirty-three unique probes were mapped to the 3 chromosomes of C. variipennis (2n = 6): 7 to chromosome 1, 20 to chromosome 2, and 6 to chromosome 3. This work represents the first stage in generating a physical map of the genome of C. variipennis.

Vertebrate exon trapping methods: implications for transcript mapping with mosquito DNA.

Exon trapping methods have played an important role in the development of transcript maps. In one in vivo vertebrate method, exons in a genomic DNA clone are transcribed, and they are recovered without any a priori information on the nature of the expressed transcript. The only requirement is that the genomic DNA clone contains exons separated by intervening introns that are removed by splicing during mRNA transcription and that the splice donor and acceptor site sequences follow those used by vertebrates. It is not known whether invertebrate splice donor and acceptor sites from genes that contain short introns will be processed correctly using an in vivo vertebrate exon trapping method. In this report, an analysis of mosquito splice sites using software designed to identify exons in genomic DNA sequence suggested that the vertebrate exon trapping method could recognize mosquito introns and exons. When a mosquito genomic DNA clone containing the D7 gene was tested experimentally, this method failed to recognize and process small introns (< 63 bp) faithfully. In spite of this failure, exons and exon fragments were recovered. The implications of these findings and their application to map-based positional cloning in mosquito genomics is discussed.

FISH landmarks for barley chromosomes (Hordeum vulgare L.).

Barley metaphase chromosomes (2n = 14) can be identified by fluorescence in situ hybridization (FISH) and digital imaging microscopy using heterologous 18S rDNA and 5S rDNA probe sequences. When these sequences are used together, FISH landmark signals were seen so that all 7 chromosomes were uniquely identified and unambiguously oriented. The chromosomal location of the landmark signals was determined by FISH to a barley trisomic series using the 18S and 5S probes labeled with different fluorophores. The utility of these FISH landmarks for barley physical mapping was also demonstrated when an Amy-2 cDNA clone and a BAC clone were hybridized with the FISH landmark probes.

Identification of duck plague virus by polymerase chain reaction.

A polymerase chain reaction (PCR) assay was developed for detecting duck plague virus. A 765-bp EcoRI fragment cloned from the genome of the duck plague vaccine (DP-VAC) virus was sequenced for PCR primer development. The fragment sequence was found by GenBank alignment searches to be similar to the 3' ends of an undefined open reading frame and the gene for DNA polymerase protein in other herpesviruses. Three of four primers sets were found to be specific for the DP-VAC virus and 100% (7/7) of field isolates but did not amplify DNA from inclusion body disease of cranes virus. The specificity of one primer set was tested with genome templates from other avian herpesviruses, including those from a golden eagle, bald eagle, great horned owl, snowy owl, peregrine falcon, prairie falcon, pigeon, psittacine, and chicken (infectious laryngotracheitis), but amplicons were not produced. Hence, this PCR test is highly specific for duck plague virus DNA. Two primer sets were able to detect 1 fg of DNA from the duck plague vaccine strain, equivalent to five genome copies. In addition, the ratio of tissue culture infectious doses to genome copies of duck plague vaccine virus from infected duck embryo cells was determined to be 1:100, making the PCR assay 20 times more sensitive than tissue culture for detecting duck plague virus. The speed, sensitivity, and specificity of this PCR provide a greatly improved diagnostic and research tool for studying the epizootiology of duck plague.

Fluorescence in situ hybridization landmarks for chromosomes of culicoides variipennis (Diptera: Ceratopogonidae).

Because the three chromosomes of Culicoides variipennis (Coquillett) are morphologically indistinguishable, physical landmarks were needed so that the chromosomes can be identified uniquely and oriented unambiguously before initiating the construction of a physical map based on FISH (fluorescence in situ hybridization). When repetitive sequence clones p1887 and K8.1a8 were labeled with digoxigenin and probe K8a.2G2 was labeled with biotin-11-dUTP and digoxigenin, the 3 chromosomes could be differentiated unambiguously when visualized with specific band-pass filters. This provided the basis for C. variipennis FISH landmark probes that enabled the identification and orientation of all 3 pairs of chromosomes. Using this multicolor FISH labeling strategy, probes that provide unique landmarks for the C. variipennis FISH physical map have been found and may be used in all FISH reactions where an unknown probe is placed to metaphase chromosomes of C. variipennis. A physical map of the C. variipennis genome will provide the foundation for map-based positional cloning of the gene(s) that control vector competence for the bluetongue viruses.

Cytological and molecular characterization of repetitive DNA sequences of Solanum brevidens and Solanum tuberosum.

The chromosomal distribution, copy numbers, and nucleotide sequences were determined for four repetitive DNA clones, pSB1 and pSB7 of Solanum brevidens and pST3 and pST10 of Solanum tuberosum. Using fluorescence in situ hybridization (FISH), pSB1 and pSB7 were localized near the telomeres and in some centromeric and interstitial sites of S. brevidens chromosomes, but not in S. tuberosum chromosomes, after high stringency washes. The clone pST3 showed signals in the telomeric areas of a few chromosomes in S. tuberosum, but signals were not detected in S. brevidens. All three repeated sequences (pSB1, pSB7, and pST3) were detected in chromosomal areas that are typically known to contain tandemly repeated sequences. The S. tuberosum clone pST10 did not show signals in either species even at low stringency conditions. The estimated copy numbers of the four clones were 1500, 6750, 300, and 400 for pSB1, pSB7, pST3, and pST10, respectively, in the corresponding haploid genomes (S. brevidens and S. tuberosum). The inserts of the four clones pSB1, pSB7, pST3, and pST10 were 322, 167, 845 and 121 bp, respectively. After sequencing, no significant sequence homologies were found among the four clones. A homology search in sequence data bases showed that pSB7 has variable homology (78-100%) with another repetitive sequence of S. brevidens Sb4/2 depending on its subrepeat. It also showed some homology with one repeat of tomato (pLEG15) and one repeat of Solanum circaeifolium (pSC15).

FISH landmarks for Aedes aegypti chromosomes.

Aedes aegypti metaphase chromosome landmarks have been developed so that each chromosome of the haploid genome can be unambiguously identified and oriented by fluorescence in situ hybridization (FISH) and digital imaging microscopy. The FISH tags were derived from three cosmids that contain repetitive Ae. aegypti sequences and their unique FISH tagging characteristics are demonstrated. The sequence of the three chromosomal tags revealed that the chromosome 1 tag is an 18S fragment from the ribosomal cistron, and the other two chromosomal tags are repeats found in Ae. aegypti with no apparent similarity to known sequences. A single plasmid that contains the three chromosomes tag sequences has been constructed to simplify future FISH physical mapping.

Metaphase chromosomes of Culicoides variipennis Diptera:Ceratopogonidae).

The cytogenetics of Culicoides variipennis (Coquillett) tissue derived from a continuous cell line is presented. The karyotype consisted of 68.5% diploid (2n = 6), 30% tetraploid (4n = 12), and 1.5% octaploid (8n = 24) for the metaphase spreads examined. Distinguishing cytological features were not seen, and the 3 pairs of homomorphic chromosomes were not distinguished from each other.

FISH digital imaging microscopy in mosquito genomics.

The yellow fever mosquito, Aedes aegypti, transmits pathogens that affect both humans and livestock, and has been the focus of extensive research to identify genetic loci that may be useful in control strategies. Fluorescence in situ hybridization (FISH) and digital imaging microscopy have provided a rapid mechanism to populate the physical map with probes derived from genetic markers, cDNAs and recombinant genomic libraries. When the physical and genetic linkage maps are aligned, map-based cloning will allow the rapid isolation of target genomic sequences. The strategy of FISH mapping and the results of initial hybridization studies are reviewed here by Martin Ferguson, Susan Brown and Dennis Knudson. An Ae. aegypti-specific genomic database, which collates data from mapping studies, sequences, references and other relevant information, is also discussed.

Toward a physical map of Aedes aegypti.

Labelled recombinant cosmids were used as in situ hybridization probes to Aedes aegypti metaphase chromosomes. The cosmid probes yielded paired signals, one on each arm of sister chromatids, and they were ordered along the three chromosomes. In total, thirty-seven different probes were mapped to the three chromosomes of Ae. aegypti (2n = 6): twenty-eight to chromosome 1, six to chromosome 2, and six to chromosome 3. These results represent an initial stage in the generation of a physical map of the Ae. aegypti genome.

Coltiviruses isolated from mosquitoes collected in Indonesia.

Viruses with genomes consisting of 12 segments of double-stranded (ds) RNA were isolated from mosquito pools collected in Indonesia in 1980 and 1981. The genome segments of 11 virus isolates exhibited two distinct electrophoretic patterns in agarose gels, and the viruses probably represent distinct groups of Coltiviruses. Ten of the 11 virus isolates produced recognizably similar PAGE profiles of two groups of 6 segments (6-6) with 3 distinct profile variants, and the remaining virus isolate displayed a unique 6-5-1 profile. These profiles were distinct from that of Colorado tick fever (CTF) virus RNA. The genetic relatedness of the isolates was examined by RNA-RNA hybridization. The viruses with a 6-6 profile were closely related in the majority of their segments, with segments 7, 11, and 12 being variant; and segment 9 the most divergent. The segments of the virus with the unique PAGE profile (6-5-1) did not cross-hybridize with any of the other viruses. Similarly, RNA from virus isolates from Indonesian did not cross-hybridize with the segments of CTF virus. These data indicate that two additional genotypes are present in the Coltivirus genus.

Isolation of bluetongue and epizootic hemorrhagic disease viruses from mosquitoes collected in Indonesia.

Three viruses isolated from anopheline mosquitoes in Indonesia have been identified as bluetongue and epizootic hemorrhagic disease viruses. Another virus isolate showed no relationship to other orbiviruses tested and should be regarded as a new virus; the name Golok is proposed for it. The mosquitoes were collected in 1980 and 1981 in a program designed to isolate flaviviruses infecting humans. It is apparent that such collections of arthropods which feed on large mammals could be screened for other viruses which may infect domestic livestock.