Identification of Belgian mosquito species (Diptera: Culicidae) by DNA barcoding.

Since its introduction in 2003, DNA barcoding has proven to be a promising method for the identification of many taxa, including mosquitoes (Diptera: Culicidae). Many mosquito species are potential vectors of pathogens, and correct identification in all life stages is essential for effective mosquito monitoring and control. To use DNA barcoding for species identification, a reliable and comprehensive reference database of verified DNA sequences is required. Hence, DNA sequence diversity of mosquitoes in Belgium was assessed using a 658 bp fragment of the mitochondrial cytochrome oxidase I (COI) gene, and a reference data set was established. Most species appeared as well-supported clusters. Intraspecific Kimura 2-parameter (K2P) distances averaged 0.7%, and the maximum observed K2P distance was 6.2% for Aedes koreicus. A small overlap between intra- and interspecific K2P distances for congeneric sequences was observed. Overall, the identification success using best match and the best close match criteria were high, that is above 98%. No clear genetic division was found between the closely related species Aedes annulipes and Aedes cantans, which can be confused using morphological identification only. The members of the Anopheles maculipennis complex, that is Anopheles maculipennis s.s. and An. messeae, were weakly supported as monophyletic taxa. This study showed that DNA barcoding offers a reliable framework for mosquito species identification in Belgium except for some closely related species.

Population genetic structure of the malaria vector Anopheles minimus A in Vietnam.

Anopheles minimus A, a major malaria vector in Southeast Asia, is the main target of vector control in this area. The impact of these control measures can be influenced by the population structure of the target species. In rural areas, An. minimus breeds along the banks of small clear-water streams, yet in the suburbs of Hanoi, northern Vietnam, there is an An. minimus population whose immature stages develop in water tanks. This study uses allozyme data (1) to assess the population structure of An. minimus A and (2) to evaluate the taxonomic status of the urban An. minimus population from Hanoi. The population from the suburbs of Hanoi was identified as An. minimus A. Although significant genetic differentiation was observed between rural and urban An. minimus A populations, they have not differentiated substantially by genetic drift. Limited macrogeographical differentiation was observed between two rural populations at distances of more than 1000 km. Consequently, geographical distance is not the primary factor in differentiating An. minimus A populations having the typical breeding ecology. The estimated effective population size is consistent with the moderate macrogeographical differentiation. Furthermore, no genetic structuring was observed between adult mosquitoes having different behaviour. The macrogeographical population structure indicates that genes may spread over large areas, whereas the presence of an 'urban' An. minimus A population shows the ability of this species to adapt to anthropogenic environmental changes.

Presence of Anopheles culicifacies B in Cambodia established by the PCR-RFLP assay developed for the identification of Anopheles minimus species A and C and four related species.

A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay developed for identification of five species of the Anopheles minimus Theobald group and a related mosquito species of the Myzomyia Series (Diptera: Culicidae) was applied to morphologically identified adult female specimens collected in Ratanakiri Province, north-eastern Cambodia. In addition to finding An. aconitus Dönitz, An. minimus species A and An. pampanai Büttiker & Beales, some specimens showed a new restriction banding pattern. Siblings of specimens that exhibited this new PCR-RFLP pattern were morphologically identified as An. culicifacies James sensu lato. Based on nucleotide sequences of the ribonuclear DNA internal transcribed spacer 2 region (ITS2) and the mitochondrial cytochrome oxidase I gene (COI), these specimens were recognized as An. culicifacies species B (sensu Green & Miles, 1980), the first confirmed record of the An. culicifacies complex from Cambodia. This study shows that the PCR-RFLP assay can detect species not included in the initial set-up and is capable of identifying at least seven species of the Myzomyia Series, allowing better definition of those malaria vector and non-vector anophelines in South-east Asia.

Confirmation of Anopheles varuna in vietnam, previously misidentified and mistargeted as the malaria vector Anopheles minimus.

Malaria control programs in Southeast Asia are faced with several questions concerning vector behavior and species identification, which need to be answered to consolidate and further improve the results of control practices. The vector system in Southeast Asia is complex because of the number of species potentially involved in malaria transmission. Additionally, the follow-up and evaluation of preventive control measures are hampered by the misidentification of vectors due to overlapping morphological characters of the female mosquitoes. In central Vietnam, control practices are aimed at 2 main species, Anopheles dirus s.l. and Anopheles minimus s.l. These reputed vectors were studied in an area of Binh Thuan Province of south-central Vietnam. Different collection methods were used to capture mosquitoes quarterly during a 1-year period. Mosquitoes were identified in the field and later subjected to detailed morphological examination and polymerase chain reaction-restriction fragment length polymorphism analysis. What was thought to be an unusual morphotype of An. minimus was shown to be Anopheles varuna, and most specimens identified as the former species in the field proved to be the latter species. Very few An. minimus individuals were found during the study period. The population of An. varuna was found to be highly zoophilic, and based on this behavior, it cannot be considered a vector in Vietnam. Because this species was previously being misidentified as An. minimus, a nonvector was mistargeted as a malaria vector in Binh Thuan Province. Anopheles dirus, which was found positive for Plasmodium falciparum circumsporozoite via enzyme-linked immunosorbent assay, is clearly the main vector in this area. Despite the fact that several potential secondary vectors were found during the study, the primary target for vector control in the region should be An. dirus.

Molecular identification of Anopheles minimus s.l. beyond distinguishing the members of the species complex.

Correct species identification is the starting point for studying the epidemiological role of vectors. Identification is mostly achieved using morphological criteria, but this fails when sibling species and species with overlapping morphological characters are involved. The problem with the identification of Anopheles minimus s. l., one of the most widespread malaria vectors in South-East Asia, is twofold: it is a complex of at least two isomorphic species, and based on morphology, members of the complex are difficult to distinguish from closely related species. An identification method was developed for An. minimus species A and C, and four related species, An. aconitus, An. pampanai, An. varuna and An. jeyporiensis. PCR-amplified internal transcribed spacer 2 (ITS2) ribonuclear DNA (rDNA) fragments were digested with restriction endonuclease BsiZI. Clear diagnostic banding patterns for the six species were obtained on agarose gels. Testing field-collected specimens from different regions in South-East Asia indicated that the technique will be applicable over a wide geographical area. From this it is clear that molecular identification has to focus not only on the species of complexes, but also on related species if they hamper the morphological identification of the 'sensu lato species'.

Isozymic comparison of five laboratory lines of tsetse flies belonging to the two subspecies of Glossina palpalis (Diptera: Glossinidae).

Three laboratory colonies of Glossina palpalis palpalis and two of G. p. gambiensis have been characterized by means of 14 polymorphic enzyme loci. The presence/absence of some alleles for three enzymes (octanol dehydrogenase, phosphoglucomutase and aldehyde oxidase) distinguished the two subspecies. Other differences in allozymes could not be used to discriminate between subspecies but could be used to distinguish populations within each of the subspecies. The genetic differences between populations of a given subspecies are briefly discussed.

Identification of two species within the Anopheles minimus complex in northern Vietnam and their behavioural divergences.

Elucidating the complex taxonomic status of the major malaria vector taxa and characterising the individual species within each complex is important for understanding the complexity of the vector system in the south-east Asian region and will allow to estimate the impact of vector control measures. This applies to countries such as Laos, Cambodia and Vietnam that spend about 60% of their malaria control budget on implementing vector control activities. We used isozyme electrophoresis to clarify the Anopheles minimus s.l. species composition in northern Vietnam and identify behavioural divergences of individual species. Using different collection methods, adult mosquitoes were caught at monthly intervals from June to November 1995 in four villages. An. minimus s.l. could be distinguished from closely related species, An. aconitus and An. jeyporiensis, at the Octanol dehydrogenase (Odh) enzyme locus. Significant positive Fis values gave clear evidence of nonrandom mating within the An. minimus s.l. population. The highest heterozygote deficiency was observed at locus Odh, which was diagnostic for 2 sympatric An. minimus species in Vietnam similar to the An. minimus A and C species known from Thailand. We found no evidence for restricted gene flow between monthly samples, villages, or collection methods in either of the two An. minimus species. They occurred in sympatry, but in different proportions depending on the collection site, and had dissimilar resting and biting behaviours. Thus a vector control strategy will have a nonuniform effect on the various components of this diverse vector system.

Intraspecific isozyme polymorphism of Anopheles gambiae in relation to environment, behavior, and malaria transmission in southwestern Burkina Faso.

A cellulose acetate electrophoresis system was used to study the isozyme polymorphism of the Anopheles gambiae complex in a rural village and a city in southwestern Burkina Faso. In both areas A. gambiae Giles was the dominant species (95%) whereas A. arabiensis Patton represented only 5%. Both species were separated readily by octanol dehydrogenase Odh and mannose phosphate isomerase (Mpi) even if they shared some alleles at these two loci. Polymorphism analysis (13 loci) at the intraspecific level of A. gambiae showed a significant difference between the specimens collected in the city from those collected in the village in their allelic and genotypic frequencies of isocitrate dehydrogenase-1 and malate dehydrogenase-1 and in their allelic frequencies for Mpi. No genetic difference was observed between the human biting A. gambiae collected inside or outside the houses in either the village or the city. The Plasmodium falciparum-infected A. gambiae differed from the noninfected ones in their allelic and genotypic frequencies at Mpi and acid phosphatase (Acp). A two-fold difference in infection rate was found for the genotype Mpi130/130 and Acp110/100 compared with other genotypes. However, infected anophelines were found in all genotypes that belonged to these two enzyme systems. Consequently, no refractory mechanism occurs in these natural populations.

Enzyme polymorphisms in the Anopheles gambiae (Diptera:Culicidae) complex related to feeding and resting behavior in the Imbo Valley, Burundi.

A cellulose acetate electrophoresis system was used to study the Anopheles gambiae complex at 2 villages in Central Imbo Valley and at 1 village in South Imbo Valley (Burundi). In South Imbo, only An. gambiae Giles sensu stricto was present. In the drier Central Imbo, the dominant species was An. arabiensis Patton (97.5%); An. gambiae s.s. represented only 2.5%. Both species were separated readily by Odh and Mpi, because they did not share alleles at those loci. Indoor resting An. gambiae s.s. from South Imbo differed significantly from outdoor resting females at 2 Ioci, Mpi, and Got-2. In Central Imbo, total An. arabiensis did not differ between adjacent villages. Endophagic An. arabiensis significantly differed at loci alpha-Gpd, and Idh-1 between the 2 villages, whereas no difference was observed between exophagic females. Allelic and genotypic frequencies at the locus Mdh-2 were significantly different between indoor and outdoor biting An. arabiensis. Active choice for the best place to bite or to rest seemed to be associated with specific genotypes. The Nei genetic distance values were typical of conspecific populations, ranging from 0.154 to 0.160 between An. gambiae s.s. and An. arabiensis. Even though vector populations were structured at spatial and behavioral levels, they were panmictic, and thus selection of exophilic or exophagic vectors, or both, by insecticide pressure is not likely to occur.

Cytogenetic and isozymic comparisons of two laboratory lines of Glossina palpalis gambiensis.

The genetics of two laboratory colonies of Glossina palpalis gambiensis were characterized by C-banding and isoenzyme studies. The colonies, derived from flies collected in the same locality, had different histories in the laboratory and different susceptibilities to trypanosome infection. Although the two lines were also found to differ in the frequencies of chromosome and isozyme variants, the variation was not enough to put their specific status in doubt; it was probably the result of genetic drift since the foundation of the colonies.

Several antigenic determinants exposed on the gp120 moiety of HIV-1 gp160 are hidden on the mature gp120.

Mouse mAb reactive to the HIV-1 envelope glycoprotein precursor gp160 of the HTLVIII(B) isolate were characterized in radioimmunoprecipitation and immunoblot tests with the use of HTLVIII(B) isolate as Ag. The reactivities of these mAb were also measured in a capture enzyme immunoassay and in radioimmunoprecipitation assay by using gp160 and gp120 expressed as vaccinia recombinants. Striking differences in exposure of specific epitopes were noted between the gp120 component of the gp160 precursor and the fully processed gp120 in both tests. These conformational rearrangements affecting the gp120 moiety of the HIV-1 envelope glycoprotein might have important implications on its immunogenicity.

[First demonstration of polytene chromosomes in adult Glossina]. / Première mise en évidence de chromosomes polytènes chez les glossines adultes.

The polytene chromosomes in adult Glossina are demonstrated for the first time. They are situated in the nuclei of the cells of the aorta. The technique to reveal them and the results are described and discussed.

Reactivity and immunogenicity of bivalent (AC) and tetravalent (ACW135Y) meningococcal vaccines containing O-acetyl-negative or O-acetyl-positive group C polysaccharide.

The immunogenicity and the reactivity of two bivalent (AC) and two tetravalent (ACW135Y) meningococcal vaccines containing either the O-acetyl-positive or the O-acetyl-negative group C polysaccharide were compared in healthy adolescent and adult volunteers. The vaccines contained high-molecular-weight, purified capsular meningococcal polysaccharides and were administered subcutaneously at a dose of 50 micrograms for each polysaccharide. Reactivity was low for all vaccines, and the tetravalent vaccines were not significantly more reactive than the bivalent vaccines. Immunogenicity was measured by assay of bactericidal antibodies in pre- and postvaccination sera. More than 90% of the vaccines had at least a fourfold increase in the bactericidal antibody titer against each group of meningococcus represented in the vaccines. Addition of polysaccharide W135 and polysaccharide Y to polysaccharides A and C did not alter the immunogenicity of the latter polysaccharides. Thus, there is no evidence of antigenic competition with the tetravalent vaccine. Comparison of the antibody response to the O-acetyl-positive and the O-acetyl-negative variants of group C polysaccharide in the bivalent vaccines, as measured by both bactericidal and enzyme-linked immunosorbent assays, indicates that in adults, the two types of group C polysaccharide are similarly immunogenic.

RIT 2214, a new biosynthetic penicillin produced by a mutant of Cephalosporium acremonium.

A number of lysine-requiring auxotrophs of Cephalosporium acremonium were investigated for incorporation of side-chain precursors and for accumulation of beta-lactam compounds. One of the auxotrophs, Acremonium chrysogenum ATCC 20389, producing cephalosporin C and penicillin N only if grown in media supplemented with DL-alpha-amino-adipic acid (DL-alpha-AAA), was found to use L-S-carboxymethylcysteine (L-CMC) as a side-chain precursor for the synthesis of a new penicillin (RIT 2214). No corresponding cephalosporin was detected. The penicillin present in the culture filtrate, was concentrated by adsorption on activated carbon and successive column chromatography on Amberlite IRA-68 and Amberlite XAD-4. Final purification was achieved by cellulose column chromatography. RIT 2214 was identified as 6-(D)-[(2-amino-2-carboxy)-ethylthio]-acetamido]-penicillanic acid by spectral analysis, bioactivity spectrum, elucidation of side-chain structure and finally by semisynthesis. Its biological properties were also evaluated.

DNA uptake by Streptomyces species.

Uptake of homologous and heterologous radioactively labelled DNA was studied in ten Streptomyces species. Among these, S. kasugaensis and S. virginiae were shown to take up 0.5-2.0% of the supplied donor DNA at a well-defined point in their growth cycle. The heterologous donor DNA taken up retained its original buoyant density in CsCl gradients. Other strains either extensively degraded the donor DNA or showed poor uptake efficiency.

Evaluation of a commercial air filter for removal of viruses from the air.

The effectiveness of a commercial absolute air filter for removal of viruses from air was tested with an actinophage, under the usual conditions of a laminar airflow clean room. A new method of dry phage dispersion is described. The filter showed an average reduction of 99.996% of airborne actinophage.