Biomedical subjects

F H Collins

Publications and source records attributed to F H Collins.

At least 37 records · Page 2Linked to original sources

PubMed1999

Population structure in the malaria vector, Anopheles arabiensis patton, in East Africa.

The population structure of the malaria vector Anopheles arabiensis was investigated using data from six microsatellite loci in samples from localities in Mozambique and Tanzania. Genotype frequencies were neither significantly different between houses in a village in Tanzania nor between villages within a 20-km radius in Mozambique. Thus a deme has an area greater than 20 km in radius. At five of the six loci the heterozygosity of the population from Mozambique was lower than that from Tanzania, implying a lower effective population size (Ne) at this southern edge of the species range. There were significant differences in genotype frequencies between the Tanzanian and Mozambique populations at five of the six loci (P<0.05). Values for both FST (mean=0.069) and RST (mean=0.025) were significantly different from zero (P<0.05) at four and three out of five loci, respectively, but there was no significant correlation between the two statistics. The wide variation in values of FST and RST across loci suggests that care should be taken in interpreting values derived from averaging across loci. Whether the variation results from sampling effects or selectional constraints on some loci is unclear. Although there is evidence for significant differentiation between these populations, estimates of gene flow (Nm) calculated from mean FST and RST statistics were relatively high, 3.4 and 4.9, respectively. We argue that this is more likely to reflect recent separation of these populations and/or large effective population size rather than large-scale present day migrations.

Animals↗

PubMed1999

Analysis of genetic variability in Anopheles arabiensis and Anopheles gambiae using microsatellite loci.

We analysed genetic variability in Anopheles arabiensis and Anopheles gambiae populations using microsatellite loci to determine whether the Rift Valley restricts the flow of genes. Deviations from Hardy-Weinberg expectations were significant, and were most likely to be due to the high frequency of null alleles observed. An. arabiensis populations occurring between 40 and 700 km apart across the Eastern arm of the Rift Valley were not differentiated (pair-wise F(ST) range: 0.0033-0.0265, P > 0.05). Neither were An. gambiae populations from Asembo Bay and Ghana (F(ST): 0.0063, P > 0.05) despite a geographical separation of about 5000 km. In contrast, significant differentiation was observed between An. gambiae populations from Asembo Bay and Kilifi (about 700 km apart; F(ST) = 0.1249, P < 0.01), suggesting the presence of a barrier to gene flow.

Alleles↗

PubMed1999

Identification of five species of the Anopheles dirus complex from Thailand, using allele-specific polymerase chain reaction.

The Anopheles dirus complex of mosquitoes contains some of the most important vectors of malaria in Southeast Asia. To distinguish five species of the complex that occur in Thailand, a method using the polymerase chain reaction (PCR) was developed. The method utilizes allele-specific amplification to detect fixed differences between the species in the DNA sequence of the ribosomal DNA internal transcribed spacer 2. Primers were designed to amplify fragments of diagnostic length from the DNA of the different species. The method was tested on 179 mosquitoes of the An. dirus complex from many parts of Thailand and shown to be effective. Every specimen was unambiguously identified as species A, B, C, D or F (i.e. An. dirus s.s. species B, C, D or An. nemophilous, respectively) by the PCR method, with confirmation of 58/61 identifications from polytene chromosome characteristics. For the other three specimens (3/44 from Kanchanaburi 5 locality), there was disagreement between the PCR and chromosomal methods of species identification (probably due to errors in the chromosomal identifications). Primers can be combined in a single PCR reaction providing a rapid, sensitive and straightforward method of species identification. Only small quantities of DNA are required, leaving most of the mosquito to be used for other analyses.

Alleles↗

PubMed1999

High amounts of genetic differentiation between populations of the malaria vector Anopheles arabiensis from West Africa and eastern outer islands.

Polymorphism at nine microsatellite loci was examined to assess the level of genetic differentiation between four Anopheles arabiensis populations from Senegal, the high plateau of Madagascar, and Reunion and Mauritius islands. Eight of nine loci showed great polymorphism (2-16 alleles/locus) and significant genetic differentiation was revealed between all four populations by F- and R-statistics, with Fst estimates ranging from 0.080 to 0.215 and equivalent Rst values ranging between 0.022 and 0.300. These high amounts of genetic differentiation are discussed in relation to geographic distance including large bodies of water, and history of mosquito settlement, and insecticide use on the islands. The results suggest that historical events of drift rather than mutation are probably the forces generating genetic divergence between these populations, with homogenization of the gene pool by migration being drastically restricted across the ocean.

Animals↗

PubMed1999

A new marker, black, a useful recombination suppressor, In(2)2, and a balanced lethal for chromosome 2 of the mosquito Anopheles gambiae.

A new marker for the second chromosome of Anopheles gambiae, black, was isolated from progeny of 60Co-irradiated mosquitoes. The black mutation increases melanization of larval setae and portions of the cuticle that are heavily sclerotized such as the saddle and head capsule. Adults have a sooty color that almost completely eliminates white banding on wings, tarsi, and palps. Fertility and general vigor of black individuals is reduced relative to wild-type; however, this does not prevent routine use for genetic crossing. The black marker was mapped to an interval on chromosome 2 between collarless and Dieldrin resistance 22 centiMorgans (cM) from collarless and 39 cM from Dieldrin resistance. We also isolated from 60Co-irradiated mosquitoes a pericentric inversion, In(2)2, that was marked with dominant alleles of the independently assorting genes collarless and Dieldrin resistance. This inversion is in coupling with the pericentric inversion 2Rd and covers approximately two-thirds of chromosome 2 from divisions 9 to 22. While inbreeding In(2)2 heterozygotes, we isolated a stock in which the inversion was in repulsion to a chromosome marked with c b DlS and an unidentified recessive lethal. This arrangement produced a useful and stable chromosome 2 balancer system that has remained intact for 26 generations without selection. These genetic tools will reduce the effort requires to isolate, among other things, the genetic factors affecting malaria parasite interactions with the mosquito host.

Animals↗

PubMed1999

Genetics in the study of mosquito susceptibility to Plasmodium.

Within the past several years, a number of powerful genetic and genomic tools have been developed for use in research on the African malaria vector Anopheles gambiae. While these tools have been developed with a broad range of potential applications in mind, they have been particularly useful in advancing the effort to clone a set of An. gambiae genes that enable a refractory strain of this mosquito to encapsulate and kill a wide variety of different malaria parasites to which this mosquito is normally fully susceptible. This paper describes the latest progress in this map-based cloning research, which involves the collaborative contributions of a number of different laboratories in Europe and the United States.

Animals↗

PubMed1998

Stable transformation of the yellow fever mosquito, Aedes aegypti, with the Hermes element from the housefly.

The mosquito Aedes aegypti is the world's most important vector of yellow fever and dengue viruses. Work is currently in progress to control the transmission of these viruses by genetically altering the capacity of wild Ae. aegypti populations to support virus replication. The germ-line transformation system reported here constitutes a major advance toward the implementation of this control strategy. A modified Hermes transposon carrying a 4.7-kb fragment of genomic DNA that includes a wild-type allele of the Drosophila melanogaster cinnabar (cn) gene was used to transform a white-eyed recipient strain of Ae. aegypti. Microinjection of preblastoderm mosquito embryos with this construct resulted in 50% of the emergent G0 adults showing some color in their eyes. Three transformed families were recovered, each resulting from an independent insertion event of the cn+-carrying transposon. The cn+ gene functioned as a semidominant transgene and segregated in Mendelian ratios. Hermes shows great promise as a vector for efficient, heritable, and stable transformation of this important mosquito vector species.

Aedes↗

PubMed1998

A family of pupal-specific cuticular protein genes in the mosquito Anopheles gambiae.

We have cloned and sequenced members of a cuticular protein multi-gene family from the mosquito Anopheles gambiae. Three genes (agcp2a-c), each approximately 1 kb in length, were found in a 17.4 kb genomic phage clone. Analysis of ten cDNAs revealed that at least four related genes are present. The open reading frame of the genes and cDNAs showed 95% sequence identity. Divergence was observed in the sequence of the 3' ends and the number of copies of two repeated coding sequences. In situ hybridizations with a probe prepared from one of these circular protein genes physically mapped to two loci, 26B on chromosome 2L and 37A on 3R. Transcription of these An. gambiae cuticular protein genes appears to be limited to pharate pupae and the expressed protein(s) is found in early pupae. The deduced amino acid sequence of these proteins contains a hydrophilic region with significant similarity to other cuticular proteins including the pupal-specific cuticular protein, EDG84, of Drosophila melanogaster (Apple and Fristrom).

Amino Acid Sequence↗

PubMed1998

Use of short tandem repeats for the analysis of genetic variability in sympatric populations of Anopheles gambiae and Anopheles arabiensis.

Anopheles gambiae and An. arabiensis were analysed at 30 short tandem repeat (STR) loci originally developed for use in An. gambiae. All specimens were collected from the same village in Kilifi district, coastal Kenya. All 30 loci were amplified in the An. gambiae specimens, whereas 25 out of 30 loci (83.3%) were successfully amplified in the An. arabiensis specimens. Both species had similar levels of polymorphism for the loci that were amplified (93.3% for An. gambiae and 92% for An. arabiensis). Median FST and RST values between the two species were 0.249 and 0.197, respectively, corresponding to Nm values of 0.75 and 0.51, respectively, and suggesting limited interchange of genes between these species. These, together with the relatively high Nei unbiased genetic distance (0.202) between the two sibling species, are consistent with the occurrence of sympatric species with limited gene flow. FST/RST values for individual loci varied greatly (FST range 0.00-0.87; RST range 0.00-0.73), indicating that the loci differ in their ability to measure levels of differentiation between these two species. Location of loci within paracentric inversions seems to be an important factor affecting levels of differentiation measured by the different loci.

Alleles↗

PubMed1998

The effective population size of Anopheles gambiae in Kenya: implications for population structure.

We estimated current and long-term effective population size (Ne) of two Anopheles gambiae (savanna cytotype) populations in Kenya. Temporal variation at nine microsatellite loci in each population sampled 7 and 9 years apart and genetic diversity in each sample were analyzed to answer the following questions. (1) Do bottlenecks occur in Kenyan populations of A. gambiae? (2) How variable are different populations with respect to their current and long-term Ne values? (3) What are the implications of these results on population structure and history? The estimates of Ne of Asembo and Jego were 6,359 and 4,258, respectively, and the lower 95% limits were 2,455 and 1,669, respectively. Thus, despite the typical observation of low density at the village level during the dry season, large populations are maintained annually. Large current Ne is consistent with previous studies showing low differentiation across the continent, especially under Wright's isolation-by-distance model. Current Ne in Asembo was 1.5-fold higher than in Jego, but this difference was not significant. Long-term Ne in Asembo (22,667) was 2.9-fold higher than that in Jego (7,855) based on the stepwise mutation model. The difference between populations was significant at both time points regardless of whether long-term Ne values were calculated based on the stepwise mutation model or the infinite-alleles model. Heterozygosity in Jego declined significantly between 1987 (59%) and 1996 (54%), whereas heterozygosity in Asembo was stable (66%-65%). Despite the relatively high and significant differentiation between Asembo and Jego (FST = 0.072-0.10, RST = 0.037-0.038), all alleles in Jego were found in Asembo but not vice versa. All of these findings suggest that lower Ne in Jego magnifies differentiation between the two populations. The long-term Ne was biased downward, because its calculation was based on an upper bound estimate of microsatellite mutation rate. Ne values based on mtDNA and allozymes were an order of magnitude higher. Long-term Ne therefore, is probably measured in hundreds of thousands and hence does not support a recent expansion of this species from a small population.

Animals↗

PubMed1998

Microgeographic genetic differentiation of Anopheles gambiae mosquitoes from Asembo Bay, western Kenya: a comparison with Kilifi in coastal Kenya.

Microgeographic differentiation in Anopheles gambiae from seven villages less than 10 km apart in Asembo Bay, western Kenya was estimated by analysis of variability in seven microsatellite loci. Results from the Asembo Bay villages were compared with specimens collected in Kilifi, coastal Kenya, 700 km to the east. Allele frequency distribution was very similar in all villages in Asembo Bay, but differed for the Kilifi population. Genetic differentiation among villages was low with loci-specific Fst falling within the range of 0.0000-0.0085. These low estimates of differentiation correspond to among-village migration indices greater than 5.66, suggesting a high level of gene flow within the Asembo population. The Nm value between Kilifi and Asembo Bay was 1.54, indicating much lower levels of gene flow. Average observed heterozygosity among the seven villages was in all but one case less than the expected heterozygosity, most likely indicating the presence of null alleles, but possibly the presence of randomly mating units (demes) smaller than the village. We conclude that there is likely no genetic structure at the level of the village in Asembo Bay but that gene flow is restricted between western and coastal Kenya, probably by the high elevation rift.

Alleles↗

PubMed1997

Quantitative trait loci for refractoriness of Anopheles gambiae to Plasmodium cynomolgi B.

The severity of the malaria pandemic in the tropics is aggravated by the ongoing spread of parasite resistance to antimalarial drugs and mosquito resistance to insecticides. A strain of Anopheles gambiae, normally a major vector for human malaria in Africa, can encapsulate and kill the malaria parasites within a melanin-rich capsule in the mosquito midgut. Genetic mapping revealed one major and two minor quantitative trait loci (QTLs) for this encapsulation reaction. Understanding such antiparasite mechanisms in mosquitoes may lead to new strategies for malaria control.

Animals↗

PubMed1997

Cloning and localization of a glutathione S-transferase class I gene from Anopheles gambiae.

1,1,1-Trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) resistance in both adults and larvae of Anopheles gambiae is mediated by stage-specific glutathione S-transferases (GSTs). On the basis of their biochemical characteristics the larval resistance-associated GSTs are likely to be insect class I GSTs. Aggst1-2, a class I GST gene, which is expressed in larvae, has been cloned from the malaria vector A. gambiae. The gene was inserted into a bacterial expression system, and the detection of 1-chloro-2,4-dinitrobenzene (CDNB) conjugating activity in Eschericia coli expressing the recombinant enzyme confirmed that aggst1-2 encodes a catalytically active GST. The gene encodes a 209 amino acid protein with 46% sequence similarity to a Drosophila melanogaster class I GST (GST-D1), 44% similarity with a Musca domestica class I GST (MdGST-1), but only low levels of homology with class II insect GSTs, including the adult specific AgGST2-1 from A. gambiae. Southern analysis of genomic DNA indicated that A. gambiae has multiple class I GSTs. In situ hybridization of class I genomic and cDNA clones to polytene chromosomes identified a single region of complementarity on chromosome 2R division 18B, suggesting that these class I GSTs in A. gambiae are arranged sequentially in the genome. Three positive overlapping recombinant clones were identified from an A. gambiae genomic library. Mapping and partial sequencing of these clones suggests that there are several GSTs and truncated GST pseudogenes within the 30kb of DNA that these clones span.

Amino Acid Sequence↗

PubMed1997

Transient expression of the Drosophila melanogaster cinnabar gene rescues eye color in the white eye (WE) strain of Aedes aegypti.

The lack of eye pigment in the Aedes aegypti WE (white eye) colony was confirmed to be due to a mutation in the kynurenine hydroxylase gene, which catalyzes one of the steps in the metabolic synthesis of ommochrome eye pigments. Partial restoration of eye color (orange to red phenotype) in pupae and adults occurred in both sexes when first or second instar larvae were reared in water containing 3-hydroxykynurenine, the metabolic product of the enzyme kynurenine hydroxylase. No eye color restoration was observed when larvae were reared in water containing kynurenine sulfate, the precursor of 3-hydroxykynurenine in the ommochrome synthesis pathway. In addition, a plasmid clone containing the wild type Drosophila melanogaster gene encoding kynurenine hydroxylase, cinnabar (cn), was also able to complement the kynurenine hydroxylase mutation when it was injected into embryos of the A. aegypti WE strain. The ability to complement this A. aegypti mutant with the transiently expressed D. melanogaster cinnabar gene supports the value of this gene as a transformation reporter for use with A. aegypti WE and possibly other Diptera with null mutations in the kynurenine hydroxylase gene.

Aedes↗

PubMed1997

The Anopheles albimanus white gene: molecular characterization of the gene and a spontaneous white gene mutation.

We have cloned and characterized the white gene of Anopheles albimanus. Comparison of the deduced amino acid sequence of this white gene with its homologs from six species of Diptera show that the An. albimanus gene is most similar to the white gene of An. gambiae (92% identity). A spontaneous white-eyed mutant An. albimanus was caused by an approximately 10 kb insertion into a CT dinucleotide repeat region of intron 2 of the white locus. The flanks of this insertion are long (at least 400 bp), nearly perfect inverted terminal repeat sequences. This cloned white gene should be useful as a marker for germ line transformation of An. albimanus.

ATP-Binding Cassette Transporters↗

PubMed1997

The white gene from the yellow fever mosquito, Aedes aegypti.

We report the cloning and primary characterization of both cDNA and genomic fragments from the white gene of the yellow fever mosquito, Aedes aegypti. Comparisons of the conceptual translation product with white genes from four other species within the order Diptera show that the Ae. aegypti gene is most similar to the white gene of the mosquito vector of human malaria, Anopheles gambiae (86% identity and 92% similarity). The analysis of the primary sequence of genomic DNA at the 5'-end of the coding region revealed the presence of an intron that is also present in An. gambiae, but not in the vinegar fly, Drosophila melanogaster. The isolated clones of the Ae. aegypti white gene will enable the construction of a marker gene for use in the development of a germline transformation system for this species.

ATP-Binding Cassette Transporters↗

PubMed1997

Cloning and characterization of a serine protease from the human malaria vector, Anopheles gambiae.

The nucleotide and deduced amino acid sequence of a serine protease (AgSp24D) from the human malaria vector, Anopheles gambiae, is presented. The gene product is a 271 amino acid protein that contains the conserved serine, histidine and aspartic acid residues found in serine proteases, and has the highest identity to a serine protease of unknown function from Drosophila melanogaster. In situ hybridization to the polytene chromosomes detects a single band at 24D. Northern analysis reveals only low levels of transcripts in larvae and pupae, but more abundant transcription products occur in adults. Interestingly, this analysis also shows that adult males express much higher levels of AgSp24D mRNA than females. In addition, Plasmodium-refractory mosquitoes express higher levels of AgSp24D mRNA than susceptible mosquitoes although the biological significance of this remains to be examined. The thorax is the primary site for expression in the adults. The lack of a dramatic increase in AgSp24D mRNA levels following blood feeding suggests that this protease is not involved in digestive processes. Transcriptional induction does not follow cold shock, septic wounding, bacterial injection, laminarin injection or CM-Sephadex bead injection.

Amino Acid Sequence↗

PubMed1997

Microgeographic structure of Anopheles gambiae in western Kenya based on mtDNA and microsatellite loci.

The population genetic structure of the Anopheles gambiae in western Kenya was studied using length variation at five microsatellite loci and sequence variation in a 648-nt mtDNA fragment. Mosquitoes were collected from houses in villages spanning up to 50 km distance. The following questions were answered. (i) Are mosquitoes in a house more related genetically to each other than mosquitoes between houses? (ii) What degree of genetic differentiation occurs on these geographical scales? (iii) How consistent are the results obtained with both types of genetic markers? At the house level, no differentiation was detected by FST and RST, and the band sharing index test revealed no significant associations of alleles across loci. Likewise, indices of kinship based on mtDNA haplotypes in houses were even lower than in the pooled sample. Therefore, the hypothesis that mosquitoes in a house are more related genetically was rejected. At increasing geographical scales, microsatellite allele distributions were similar among all population samples and no subdivision of the gene pool was detected by FST or RST. Likewise, estimates of haplotype divergence of mtDNA between populations were not higher than the within population estimates, and mtDNA-based FST values were not significantly different from zero. That sequence variation in mtDNA provided matching results with microsatellite loci (while high genetic variation was observed in all loci), suggested that this pattern represents the whole genome. The minimum area associated with a deme of A. gambiae in western Kenya is therefore larger than 50 km in diameter.

Alleles↗