DNA probes to identify members of the Anopheles farauti complex

DNA probes have been constructed to distinguish between the members of the Anopheles farauti complex of mosquitoes known as species numbers 1, 2 and 3. Partial genomic libraries of the three known species were exposed to labelled total genomic DNA from each species. Colonies showing differential hybridization were selected for further testing. These probes were found which allow identification of the three known species: probe pAf1 (160 bp fragment) hybridizes to DNA from An. farauti nos. 1 and 2; probe pAf2 (95 bp fragment) hybridizes to DNA from An. farauti no. 2 only; and probe pAf3 (1.3 kb fragment) hybridizes strongly to DNA from An. farauti no. 3, less to no. 1 and faintly to no. 2. Increasing the stringency of hybridization reduced the cross-hybridization of probes pAf1 and pAf3. Only radioactively labelled probes were tested. Males and females and individuals from diverse habitats and localities showed the same species/probe hybridization characteristics. This technique allows faster identification of the sibling species than previous methods, and has the added advantage that it allows air-dried and alcohol stored specimens to be identified.     

Comparison of three nonradioactive and a radioactive DNA probe for the detection of target DNA by DNA hybridization

The specificity and sensitivity of three methods for the preparation and detection of nonradioactive probe DNA (biotin-nick translation, biotin-photolabel, and antigen-chemical linkage) were evaluated and compared with a nick-translated³²P-labeled DNA probe in DNA hybridization studies. The DNA probes were prepared from a restriction fragment (HindIII-3) from bacteriophage P1 DNA, and target DNA consisted of purified phage P1 DNA or P1 prophage DNA in lysogens ofEscherichia coli. A probe concentration of 50 ng/ml resulted in clear detection with the three nonradioactiveHindIII-3 DNA probes, whereas the specificity of the³²P-HindIII-3 DNA probe was satisfactory at a concentration of 25 ng/ml. However, the detection of false positives was greater with the³²P-labeled probe. The sensitivity of the radiolabeled DNA probe was marginally greater than that of the nonradioactive probes in dot blot hybridizations with purified phage P1 DNA. However, when the preparation time, ease of use, safety, duration of storage, and expense were compared for the four methods of labeling, the nonradiolabeled probes were generally superior to the radiolabeled probe.     

Differential ecology of Anopheles punctulatus and three members of the Anopheles farauti complex of mosquitoes on Guadalcanal, Solomon Islands, identified by PCR-RFLP analysis

From a series of larval collections made across northern Guadalcanal during the dry season, October–November 1997, four members of the Anopheles punctulatus group of mosquitoes (Diptera: Culicidae) were identified using PCR‐RFLP analysis. Anopheline larvae were found in 54/57 (95%) of the sites sampled, comprising An. farauti Laveran sensu stricto (32 sites), An. farauti species no. 2 (39 sites), An. farauti no. 7 (36 sites) and An. punctulatus Dönitz (10 sites). Anopheles punctulatus occurred only on the coastal plain, where it was associated with the more transient sites. Anopheles farauti sensu lato was more widespread throughout the survey region, with similar proportions of all three sibling species in both transient and permanent sites. Two members of the An. farauti complex, An. farauti s.s. and species no. 2, were found in brackish water. All breeding sites of An. punctulatus were cohabited by An. farauti s.l., sometimes by all three sibling species. Anopheles farauti s.s. was the only species collected on human bait, with a much higher biting rate early in the evening (57 bites/human/hour at 18.30–20.00 hours) than later (0.8 bites/human/hour at 21.00–24.00 hours).     

Shared salinity tolerance invalidates a test for the malaria vector Anopheles farauti s.s. on Guadalcanal, Solomon Islands [corrected

Among the Punctulatus Group of Anopheles mosquitoes (Diptera: Culicidae), first-instar larvae of the medically unimportant freshwater Anopheles farauti species No. 7 survives a seawater tolerance test (STT) that was previously thought to be diagnostic for the saltwater-tolerant malaria vector species, An. farauti Laveran s.s. Salt tolerance in these two closely related isomorphic species appears to be a shared derived character within the Farauti Complex. Failure to differentiate An. farauti s.s. from An. farauti No.7 will overestimate potential malaria vector numbers and waste limited larval control resources. Use of the STT should therefore be discontinued on Guadalcanal and other techniques such as allozyme electrophoresis used instead [corrected].     

Shared salinity tolerance invalidates a test for the malaria vector Anopheles farauti s.s. on Guadalcanal, Solomon Islands

Among the Punctulatus Group of Anopheles mosquitoes (Diptera: Culicidae), first-instar larvae of the medically unimportant freshwater Anopheles farauti species No. 7 survives a seawater tolerance test (SST) that was previously thought to be diagnostic for the saltwater-tolerant malaria vector species, An. farauti Laveran s.s. Salt tolerance in these two closely related isomorphic species appears to be a shared derived character within the Farauti Complex. Failure to differentiate An. farauti s.s. from An. farauti No. 7 will overestimate potential malaria vector numbers and waste limited larval control resources. Use of the SST should therefore be discontinued on Guadalcanal and other techniques such as allozyme electrophoresis used instead.     

A sulfone group-labeled TEM-DNA probe: comparison with a 32P-labeled probe in dot-hybridization

A non-radioactive DNA probe for the TEM-type beta-lactamase gene was obtained by using the 'Chemiprobe' system. It was used along with a 32P-labeled TEM probe to screen for TEM beta-lactamase gene in 107 bacterial isolates representing 7 Gram-negative genera and previously classified as TEM-positive or negative. The DNA to be tested was extracted from these bacterial isolates by the Birnboim-Doly method and, after blotting into charged nylon membranes, it was submitted to hybridization with either the TEM 'Chemiprobe' or the 32P-TEM probe. The TEM 'Chemiprobe' could detect as few as 25 pg specific DNA if it was used at a concentration of 5 ng per cm2 of membrane. The results obtained by both probes were concordant in 93.5% of the entire sample. The TEM 'Chemiprobe' was specific since only one false positive was observed. Furthermore, it appeared at least as sensitive as the 32P-labeled TEM probe. As the dot-hybridization with the sulfone-labeled probe was sensitive, simple and easy to perform, it will be useful for large-scale screening in clinical laboratory.     

Responses of mosquitoes of the Anopheles farauti complex to 1-octen-3-ol and light in combination with carbon dioxide in northern Queensland, Australia

Abstract. In northern Queensland, Australia, three experiments were conducted to determine the response of mosquitoes of the Anopheles farauti complex to CDC traps baited with four attractant combinations: octenol + C0₂ and light; octenol and light; CO, and light; or C0₂ and octenol without light. A CDC-modified updraft light-trap was also trialled, but did not significantly enhance collections of An.farauti sensu lato. The combination of light, octenol and C0₂ caught significantly more An.farauti s.l. (both An.farauti No. 1 and No. 2 sibling species) when compared to C0₂ and light alone. Only small numbers of the An.farauti complex were captured when CDC traps were baited with octenol alone, i.e. no light or C0₂.     

Construction and applications of DNA probes for detection of polychlorinated biphenyl-degrading genotypes in toxic organic-contaminated soil environments

Several DNA probes for polychlorinated biphenyl (PCB)-degrading genotypes were constructed from PCB-degrading bacteria. These laboratory-engineered DNA probes were used for the detection, enumeration, and isolation of specific bacteria degrading PCBs. Dot blot analysis of purified DNA from toxic organic chemical-contaminated soil bacterial communities showed positive DNA-DNA hybridization with a 32P-labeled DNA probe (pAW6194, cbpABCD). Less than 1% of bacterial colonies isolated from garden topsoil and greater than 80% of bacteria isolated from PCB-contaminated soils showed DNA homologies with 32P-labeled DNA probes. Some of the PCB-degrading bacterial isolates detected by the DNA probe method did not show biphenyl clearance. The DNA probe method was found to detect additional organisms with greater genetic potential to degrade PCBs than the biphenyl clearance method did. Results from this study demonstrate the usefulness of DNA probes in detecting specific PCB-degrading bacteria, abundance of PCB-degrading genotypes, and genotypic diversity among PCB-degrading bacteria in toxic chemical-polluted soil environments. We suggest that the DNA probe should be used with caution for accurate assessment of PCB-degradative capacity within soils and further recommend that a combination of DNA probe and biodegradation assay be used to determine the abundance of PCB-degrading bacteria in the soil bacterial community.     

Construction and applications of DNA probes for detection of polychlorinated biphenyl-degrading genotypes in toxic organic-contaminated soil environments.

Several DNA probes for polychlorinated biphenyl (PCB)-degrading genotypes were constructed from PCB-degrading bacteria. These laboratory-engineered DNA probes were used for the detection, enumeration, and isolation of specific bacteria degrading PCBs. Dot blot analysis of purified DNA from toxic organic chemical-contaminated soil bacterial communities showed positive DNA-DNA hybridization with a 32P-labeled DNA probe (pAW6194, cbpABCD). Less than 1% of bacterial colonies isolated from garden topsoil and greater than 80% of bacteria isolated from PCB-contaminated soils showed DNA homologies with 32P-labeled DNA probes. Some of the PCB-degrading bacterial isolates detected by the DNA probe method did not show biphenyl clearance. The DNA probe method was found to detect additional organisms with greater genetic potential to degrade PCBs than the biphenyl clearance method did. Results from this study demonstrate the usefulness of DNA probes in detecting specific PCB-degrading bacteria, abundance of PCB-degrading genotypes, and genotypic diversity among PCB-degrading bacteria in toxic chemical-polluted soil environments. We suggest that the DNA probe should be used with caution for accurate assessment of PCB-degradative capacity within soils and further recommend that a combination of DNA probe and biodegradation assay be used to determine the abundance of PCB-degrading bacteria in the soil bacterial community.     

Distribution and evolution of the Anopheles punctulatus group (Diptera: Culicidae) in Australia and Papua New Guinea

The members of the Anopheles punctulatus group are major vectors of malaria and Bancroftian filariasis in the southwest Pacific region. The group is comprised of 12 cryptic species that require DNA-based tools for species identification. From 1984 to 1998 surveys were carried out in northern Australia, Papua New Guinea and on islands in the southwest Pacific to determine the distribution of the A. punctulatus group. The results of these surveys have now been completed and have generated distribution data from more than 1500 localities through this region. Within this region several climatic and geographical barriers were identified that restricted species distribution and gene flow between geographic populations. This information was further assessed in light of a molecular phylogeny derived from the ssrDNA (18S). Subsequently, hypotheses have been generated on the evolution and distribution of the group so that future field and laboratory studies may be approached more systematically. This study suggested that the ability for widespread dispersal was found to have appeared independently in species that show niche-specific habitat preference (Anopheles farauti s.s. and A. punctulatus) and conversely in species that showed diversity in their larval habitat (Anopheles farauti 2). Adaptation to the monsoonal climate of northern Australia and southwest Papua New Guinea was found to have appeared independently in A. farauti s.s., A. farauti 2 and Anopheles farauti 3. Shared or synapomorphic characters were identified as saltwater tolerance (A. farauti s.s. and Anopheles farauti 7) and elevational affinities above 1500 m (Anopheles farauti 5, Anopheles farauti 6 and A. farauti 2).     

Comparison of radio-labeled DNA probe with a nonisotopic probe for assay of serum hepatitis B virus DNA

A biotin-labeled DNA probe was compared to a 32P radio-labeled DNA probe for the detection of serum hepatitis B virus (HBV) DNA. Serum specimens were treated with proteolytic enzyme and detergent. DNA was extracted using phenol, denatured in sodium hydroxide and applied to a nitrocellulose filter paper using a vacuum filter device. The nitrocellulose filters were then incubated with either the biotin-labeled or the radio-labeled probe. Annealing of the probe, indicating the presence of HBV-DNA in the sample, was detected either by autoradiography for the 32P-labeled probe or by measuring the presence of an acid phosphatase attached to a streptavidin molecule for the biotin-labeled probe. Using the same 2-day time to complete the assays, excellent correlation of the qualitative and semiquantitative measurements were obtained using 20 HBsAg-positive and 9 HBsAg-negative sera. The nonisotopic assay detected 1.0 pg of HBV-DNA, a sensitivity comparable to reported sensitivities of 32P-labeled HBV-DNA probes when similar assay times are used. 0.02 pg/microliter of HBV-DNA was detected in a normal serum to which HBV-DNA in a recombinant plasmid was added. Our results indicate that the biotin-labeled HBV-DNA probe is approximately as sensitive as the radio-labeled probe for the detection of HBV-DNA using a similar assay time. Isotopic probe assays are more sensitive with longer assay times. The biotin-labeled probe offers the advantage of a longer shelf life and a nonisotopic assay procedure.     

A procedure for repeated usage of 33P-labeled DNA probes in hybridization experiments.

We describe a technique for repeated use of 33P-labeled DNA probes in Southern hybridization experiments. A nick-translated 33P-labeled DNA probe in a volume of 0.5-1.0 ml of hybridization mixture (final concentration, 10-100 ng/ml) is used to wet a sheet of filter paper (approx 10 microliters/cm2), which covers a nylon membrane with DNA transferred by Southern blotting, and both are set between two washed X-ray films. The "sandwich" is placed in a plastic bag for hybridization for 16-24 h at 42 degrees C. This very simple procedure using 33P-labeled DNA probes has a number of advantages over the standard method using 32P-labeled probes: (a) a significantly lower biohazard (body/arms exposure); (b) a very small volume of hybridization mixture in contact with a DNA-containing membrane and the higher probe concentrations attainable, causing some increase in sensitivity, and, finally, (c) repeated use of the probe-containing filter (over approx 3 days for unique sequences and up to 2 weeks for reiterated sequences) due to a relatively long 33P half-life (25.3 days).     

Comparative effect of microwaves and boiling on the denaturation of DNA

The effect of heat and microwave denaturation of small volumes of double-stranded plasmid DNA has been compared. Samples of intact plasmid DNA had plasmid DNA linearized by digestion with EcoRI were conventionally denatured in a boiling water bath or denatured by 2450 MHz of microwave energy for 0-300 s. Heat denaturation for periods longer than 120 s caused breakdown of linearized plasmid DNA; however, microwave denaturation for 10-300 s caused no apparent degradation of linearized DNA. Breakdown of DNA forms II and III was noted in plasmid DNA subjected to 300 s of either heat or microwave denaturation but breakdown of forms II and III occurred more quickly with heat than with microwave treatment. Microwave treatment was also found to be better than heat to denature 32P-labeled DNA probes subsequently used to detect homologous DNA samples immobilized on nitrocellulose filters. A microwave-treated 32P-labeled DNA probe was able to hybridize to DNA samples 20 times more dilute than a heat-treated 32P-labeled DNA probe. Depending on the form of DNA to be analyzed, these results indicate that small volumes of DNA solutions and radiolabeled DNA probes can be effectively denatured in a conventional microwave oven.     

Ribosomal DNA-probes differentiate five cryptic species in the Anopheles gambiae complex

This study describes the use of ribosomal DNA probes to identify the species of individual mosquitoes in the Anopheles gambiae complex, a group of six morphologically identical mosquito species among which are two of the principal vectors of malaria in Africa. The DNA probes are sequences of DNA derived from the ribosomal genes of An. gambiae. Each probe reveals a different sized restriction enzyme fragment specific to each of the five species in the complex that were examined in this study: An. gambiae, An. arabiensis, An. quadriannulatus, An. melas and An. merus. The probes detect highly repeated sequences of DNA, thus the method is sufficiently sensitive to be applied to a small portion of a mosquito. Furthermore, because the DNA can be extracted from desiccated or alcohol preserved specimens, the test is compatible with other mosquito assays performed on dried specimens such as blood meal and malaria sporozoite antigen ELISAs. Determination of the nucleotide sequences that underlie the species-specific restriction enzyme site differences detected by these probes will lead to the development of synthetic DNA probes that can be used to identify an individual mosquito to species on the basis of a simple dot-blot or squash-blot.     

Deforestation and vector-borne disease: Forest conversion favors important mosquito vectors of human pathogens

The global burden of vector-borne diseases accounts for more than 17% of infectious diseases in humans. Rapid global expansion of previously obscure pathogens, such as Zika and chikungunya viruses in recent years highlights the importance of understanding how anthropogenic changes influence emergence and spillover of vector-borne diseases. Deforestation has been identified as one anthropogenic change that influences vector-borne disease prevalence, although contrasting pictures of the effects of deforestation on vector-borne disease transmission have been reported. These conflicting findings are likely attributable to the inherent complexity of vector-borne disease systems, which involve diverse groups of vectors, hosts and pathogens, depending on geography. The current study represents a quantitative exploration of the link between deforestation and mosquitoes, the most important common constituents of vector-borne disease systems. Analysis of data compiled from published field studies for 87 mosquito species from 12 countries revealed that about half of the species (52.9%) were associated with deforested habitats. Of these species that are favored by deforestation, a much larger percentage (56.5%) are confirmed vectors of human pathogens, compared to those negatively impacted by deforestation (27.5%). Moreover, species that serve as vectors of multiple human pathogens were all favored by deforestation, including Anopheles bancroftii, Anopheles darlingi, Anopheles farauti, Anopheles funestus s.l., Anopheles gambiae s.l., Anopheles subpictus, Aedes aegypti, Aedes vigilax, Culex annulirostris, and Culex quinquefasciatus. Our quantitative analysis of vector and non-vector species, demonstrates that the net effect of deforestation favors mosquitoes that serve as vectors of human disease, while the obverse holds true for non-vectors species. These results begin to unify our understanding of the relationship between deforestation and vector mosquitoes, an important step in quantifying how land use change, specifically deforestation, affects human risk of vector-borne disease.     

两种DNA探针杂交检测结核分支杆菌方法的研究

为改进结核杆菌DNA探针的特异性与实用性,研制了以生物素标记的两种对结核分支杆菌特异的DNA探针:一个5’端标记的20bp的寡核苷酸探针和一个采用PCR方法合成的188bp长链探针。两种探针分别与结核分支杆菌的全染色体DNA,以及基因组上IS6110序列的一段317bp的PCR扩增产物进行斑点杂交,以碱性磷酸酶(AP)催化的染色反应检测,测试了两个探针的敏感性和特异性。系统地比较研究了两种探针杂交检测条件:探针的浓度选择,杂交温度与洗膜温度的选择,以及杂交与洗膜温度对检测的敏感性与特异性的影响。寡核苷酸探针和188bp探针杂交检测纯化结核分支杆菌基因组DNA的敏感性分别为100ng与6ng,杂交检测PCR产物的敏感性分别是400pg与50pg。两探针的最佳杂交浓度均为40～160ng/ml,最佳杂交温度分别是42℃与68℃,最佳洗膜温度分别是60℃与60～68℃之间。两种探针均仅与结核分支杆菌及BCG有杂交信号,而与其它受试分支杆菌及非分支杆菌杂交结果都呈阴性。它们的特异性都很强,但188bp探针的敏感性约是寡核苷酸探针的7～16倍,而且188bp探针检测本底较低,是检测结核分支杆菌的较佳选择     

Commercial detection methods for biotinylated gene probes: Comparison with32P-labeled DNA probes

This study had two objectives: (a) to determine whether biotinylated DNA probes could be substituted for³²P-labeled DNA probes to detect the presence of the TEM-1 -lactamase gene in crude bacterial preparations, and (b) to evaluate two commercial detection systems for biotinylated probes—an alkaline phosphatase kit produced by Bethesda Research Laboratories (BRL) and an acid phosphatase kit produced by Enzo Biochem. Both the kits produced nonspecific reactions with TEM-1-negative organisms. Treatment with chloroformphenol and proteinase K did not remove these nonspecific reactions. When plasmid DNA was purified by electrophoresis and transferred to nitrocellulose filters by the Southern blot method, there was no qualitative difference between the biotinylated and radioactive probes. However, the³²P-labeled probes were quantitatively 100 times more sensitive than the biotinylated probes. In addition, the Enzo Biochem kit and the³²P-labeled probes could be used with charged nylon membranes, whereas the BRL kit could be used only with nitrocellulose filters.     

Detection and identification of Mycoplasma infections by DNA hybridization

Infection of cell cultures by mycoplasmas can be detected by hybridization of the DNA of suspected cell cultures with recombinant plasmids containing fragments of the mycoplasma DNA. The test is very sensitive and allows detection of as little as 1 ng of mycoplasmal DNA, roughly equivalent to the DNA amount of 10(6) mycoplasmas. This approach turns out to be effective for detection and identification of mycoplasmas in clinical material, plant and insect tissues. A set of DNA probes for detection of mycoplasmas infecting cell cultures by dot hybridization has been constructed. This set consists of specific DNA probes and universal DNA probe. Recombinant plasmids, pAl32, pMa13, pMh9, containing specific DNA fragments of Acholeplasma-laidlawii, Mycoplasma arginini, Mycoplasma hominis (the prevalent mycoplasma contaminants of home cell cultures) are species-specific DNA probes. Recombinant plasmid pMg16 containing rRNA genes of Mycoplasma gallisepticum is the universal DNA probe for detection of any mycoplasma (or any prokaryote) contaminations. These two classes of DNA probes may be considered as complementing each other. These 32P labeled probes do not hybridize with eukaryotic DNA. The set of DNA probes allows not only to detect infection of cell cultures by mycoplasmas but also to identify the species of mycoplasmas and to evaluate the multiplicity of mycoplasma infection.     

The Anopheles punctulatus group of mosquitoes in the Solomon Islands and Vanuatu surveyed by allozyme electrophoresis

Abstract. Four species within the Anopheles punctulatus group of mosquitoes (Diptera: Culicidae) were identified by allozyme analysis of samples collected from thirty-three localities in Guadalcanal, Makira, Malaita, Temotu and Western Provinces in the Solomon Islands and six localities on Efate, Espiritu Santo, Maewo and Malekula Islands in Vanuatu. Three of these species are members of the An. farauti complex. A key is given to identify five species of the An. punctulatus group known to occur in the Solomon Islands using their isoenzyme characteristics.
An. farauti No. 1 was widespread in coastal areas of the Solomon Islands and was the only species detected in Vanuatu, including Efate Island (where Faureville is the type locality of An. farauti Laveran sensu stricto). An. farauti No. 2 and An. punctulatus were common in the Solomon Islands in more inland areas. An. farauti No. 7, reported here for the first time, was found as larvae in freshwater at six localities on north Guadalcanal. Three other members of the An. punctulatus group which have been reported previously from the Solomon Islands: An. koliensis, An. renellensis and an electrophoretic variant of An. farauti sensu lato, were not found in our samples.
Previously recognized vectors of malaria and bancroftian filariasis in the Solomon Islands are An. farauti No. 1 (i.e. An. farauti s.s. ), An. koliensis and An. punctulatus s. s. Adult females of An. farauti No. 2 and An. farauti No. 7 were not attracted to human bait in areas where their larvae occurred, indicating that these two species are not anthropophilic and therefore unlikely to transmit human pathogens.     

Synthetic DNA probes for the identification of sibling species in the Anopheles gambiae complex

The cloned DNA sequences pAna1, pAnq1 and pAnm14, which may be used to distinguish between at least five of the six species in the Anopheles gambiae Giles complex of Afrotropical malaria vector mosquitoes, have been sequenced. Each clone was found to possess a series of repeated sequences of 41, 30 and 163 bases respectively. In pAnq1 and pAnm14 the repeats were in direct tandem array, whilst in pAna1 the repetitive sequence was found to be interspersed by 15-17 variable bases. A comparison of a number of copies of each of the repetitive sequences within the three clones enabled the definition of the consensus sequence for each repetitive element. Based on these consensus sequences, three oligonucleotides of 21, 23 and 26 bases were derived from pAna1, pAnq1 and pAnm14 respectively. When tested as probes against DNA dot-blots and squash-blots of mosquito specimens, each oligonucleotide retained the same species-specificity as the original clones from which they were derived. The radioactively labelled oligonucleotides were able to detect as little as 5 ng of target genomic DNA in an overnight autoradiographic exposure. The synthetic DNA probes will form the basis of a simplified system for the field identification of Anopheles gambiae sibling species specimens.