Evidence of genetic selection for growth in new recruits of a marine fish.

Abstract A cohort of Diplodus sargus, a coastal marine fish abundant in the Mediterranean Sea, has been surveyed from its settlement following the pelagic larval stage up to 4 months of age, when the juveniles are moving to adult habitats in order to assess selective processes. We followed the mortality by looking at the decrease in population abundance and, simultaneously, the genetic structure using allozymes and the growth associated with each genotype to test for a relationship between genotype and phenotype. The recruitment survey demonstrated that 80% of individuals arrived within a single night and that they show very similar age providing a discrete pulse of new recruits that we followed for changes in survival and allele frequencies. After 4 months, there was a total mortality of 80.8%, with the disappearance of 181 of 224 fish that initially colonized the rocky barrier. The decrease in number followed a logarithmic model with a maximum decrease in the early period (first 30 days). The model derived from the 4 months of data demonstrates that most of the mortality in the cohort occurs over the first 120 days following settlement and the model predicted a final abundance of 10 individuals after 1 year. Within the same period of 4 months, we observed significant decrease in multilocus heterozygosity. Such a decrease in heterozygosity partly resulted from a purge of the Pgm-80* allele. Together with this major change in a natural population, an aquarium experiment demonstrated that individuals with Pgm-80* alleles show significantly lower growth than other new recruits. We propose that the decrease in frequency of Pgm-80* in the natural environment is the result of targeted predation that eliminates smaller individuals and therefore individuals bearing Pgm-80*. The potential metabolic effect as well as a scenario that could lead to the maintenance of polymorphism is discussed.

Effects of malaria infection on vitellogenesis in Anopheles gambiae during two gonotrophic cycles.

We report changes in the abundance of vitellogenin (Vg) mRNA, and concentration of haemolymph Vg and ovarian vitellin (Vn) in Anopheles gambiae following infection with Plasmodium yoelii nigeriensis. A parasite-induced reduction in Vg mRNA abundance was first detected 24 h after feeding on an infective blood meal, when ookinetes were invading the midgut. During a second gonotrophic cycle post-infection, developing oocysts reduced Vg mRNA abundance by up to 33% and the effect was detected from 2 h post blood meal. Concentrations of Vg were initially reduced by infection during the second cycle, as predicted from Vg mRNA measurements. However, after 24 h, excess Vg had accumulated in the haemolymph. This accumulation may be due to impaired uptake, since ovarian vitellin accumulation was significantly decreased by infection during both gonotrophic cycles.

Ikirara insertions reveal five new Anopheles gambiae transposable elements in islands of repetitious sequence.

Characterization of Anopheles gambiae genomic clones containing Ikirara inverted repeats revealed five novel sequences related to known transposable elements (TEs). One TE is related to the mariner/Tc1 superfamily of class II (DNA-to-DNA) transposons, while four are related to class I (RNA-mediated transposition) elements. Crusoe, the class II element; is most similar to the Caenorhabditis elegans transposon Tc1-like TEs. Vash elements, represented twice in our clones, are related to the Q/T1 family of A. gambiae non-LTR retrotransposable elements. Guildenstern is a member of the RT1 and RT2 non-LTR retrotransposon family. Although RT1 and RT2 elements normally have a highly stereotyped insertion preference for sequences within ribosomal genes, Guildenstern is not located in ribosomal sequence. JuanAg is the first anopheline member of the mosquito non-LTR retrotransposon family of Juan elements that previously had included just the culicine elements JuanA and JuanC. Approximately 753 bp is missing from the central portion of the JuanAg reverse transcriptase gene, where an Ikirara inverted repeat is found in its stead. Ozymandias, the only LTR retrotransposon found in the clones, is most similar to the Drosophila melanogaster 412 element. Single Ikirara inverted repeats were also found adjacent to nontransposable element repetitious sequences. Our analysis suggests that the A. gambiae genome organization could best be described as islands of short-period interspersion repetitious DNA in a sea of long-period interspersion, mostly unique sequence DNA.

Linkage of a gene causing malaria refractoriness to Diphenol oxidase-A2 on chromosome 3 of Anopheles gambiae.

An inbred line of the African malaria vector Anopheles gambiae is refractory to development of malaria parasites. It is homozygous for a 4.3-kb Sal I restriction fragment at the Dox-A2 locus, whereas the parent population is polymorphic at this locus, and a susceptible line is homozygous for an alternate 3.85-kb fragment. The Dox-A2 locus is located in the middle of chromosome 3R, in division 33B, and is tightly linked to a cluster of genes including Dopa decarboxylase that are involved in the production of melanin. Because the refractoriness phenotype, melanotic encapsulation of ookinete/oocysts, might involve activation of or alteration in one or more of these genes, we performed genetic crosses to determine whether a previously identified Plasmodium cynomolgi Ceylon refractoriness gene, Pif-C, is linked to Dox-A2. Backcross mosquitoes fed on one infected monkey developed infections of < or = 100 oocysts. About 50% of these mosquitoes appeared phenotypically refractory, as expected for the backcross performed, but gave slight evidence of linkage between a refractoriness gene and Dox-A2. In contrast, females fed on a monkey that yielded higher infection levels, up to > 300 oocysts, showed clear evidence of linkage between a refractoriness gene and Dox-A2. We conclude that this Dox-A2-linked refractoriness gene is expressed under conditions particular to the higher infection levels, or that environmental factors obscured the genetic effect of this gene at lower infection levels.

Excisions of the Ikirara1 transposon in an Anopheles gambiae cell line.

In order to determine whether there are active genomic copies of the Anopheles gambiae transposon Ikirara, we developed an excision assay based on an internally deleted copy, Ikirara1. This element has 216 bp perfect inverted repeats at its termini, apparently caused a duplication of the dinucleotide TA at its insertion site between vitellogenin genes, and is thought to have been inserted recently at this location. The firefly luciferase gene on the E. coli tac promoter was inserted into Ikirara1 and used as a reporter to assess whether activities in an A. gambiae cell line could cause Ikirara excision. Excisions were observed at a rate of 0.038% in these experiments, but none was detected in controls. The five independent excision products examined gave identical sequences. Excisions were nearly precise, but left behind a footprint of 15 bp of the 3' inverted repeat of Ikirara1 between duplicated TAs. These excisions can be explained by a mechanism formally similar to that proposed for excision of mariner/Tc1 elements with cuts at the transposon ends staggered by 15 bases.

Ikirara, a novel transposon family from the malaria vector mosquito, Anopheles gambiae.

Members of a novel transposon family, Ikirara, were found in the genome of the malaria vector Anopheles gambiae. They are most abundant in A. gambiae sensu stricto, but related sequences were found in all four other tested members of this species complex. No relatives were found in A. funestus or A. stephensi. Ikirara1, the first isolated family member, was found between two of the tandem Vitellogenin (Vg) genes. Because it was found at this location in G3 and only one of nine other A. gambiae s.s. strains examined, and because its 216 bp inverted terminal repeats are 100% identical, transposition to this locus may have been recent. Ikirara1 inverted repeat terminal sequences are similar to those of DNA to DNA transposons of the mariner/Tc1 and hAT superfamilies. Also similar to mariner/Tc1 elements, insertion of Ikirara1 apparently created a duplication of the dinucleotide TA at the target site.

Analysis of a lysozyme gene from the malaria vector mosquito, Anopheles gambiae.

A genomic DNA sequence encoding a basic lysozyme was isolated from the malaria vector mosquito Anopheles gambiae by screening a library with a probe prepared by PCR of reverse transcribed adult RNA. The sequence consists of an upstream region of about 2 kb, a coding region containing three exons and two introns, and a short 3' untranslated region. The coding region indicates that this mosquito lysozyme consists of a signal peptide of 20 residues followed by an 120 aa mature protein which is very similar to other basic lysozymes. The two small introns, 67 and 76 bp, are located at evolutionarily conserved sites. RT-PCR indicated that this gene is expressed abundantly in sugar-fed adults, and at considerably lower levels when females have fed on blood. Although it remains to be seen whether this gene is induced by bacterial infection, the surrounding sequence contains six sequence motifs very similar to the consensus binding sites for a transcription factor similar to NF-kappa B that are found associated with most insect immune response genes. This lysozyme gene maps to division 27 on the left arm of polytene chromosome 2L. An ORF unrelated to any animal protein in current data bases was found at the 5' end of the clone.

Analysis of a vitellogenin gene of the mosquito, Aedes aegypti and comparisons to vitellogenins from other organisms.

A genomic clone of the Aedes aegypti vitellogenin A1 gene was sequenced including 2015 bp of 5' untranscribed sequence, 6369 bp of open reading frame interrupted by two introns, and a short 3' untranslated region. Primer extension was used to identify the transcription initiation site. The amino termini of the large and small subunits were located by N-terminal sequencing of vitellin purified from eggs. The length of the signal sequence and the position of the cleavage site between the two subunits were also determined. Three sequential imperfect repeats were found near the beginning of the small subunit. The sequence of the coding region appears to be polymorphic. Comparison of the signal sequences of seven insect vitellogenin genes revealed several conserved leucines, and a conserved position of an intron. However, the signal sequences are not conserved between these genes and the yolk protein genes of Cyclorraphid Dipteran insects. The cleavage sites between the small and large subunits in the vitellogenins of the mosquito, A. aegypti, sawfly, Athalia rosae, boll weevil, Anthonomus grandis, and silkworm, Bombyx mori are flanked by sequences rich in serine. Pairwise dot matrix analysis at the protein level showed that the mosquito, boll weevil and silkworm vitellogenins are significantly related with approx. 50% similarity. One region of the three insect vitellogenin genes, near the N-terminal of the large subunit, showed the highest levels of similarity, from 57.5 to 64.4%. The position of cysteines in insect vitellogenins is conserved, particularly in the C-terminus of the large subunit. Dot matrix comparison of the mosquito vitellogenin with that of Xenopus laevis and Caenorhabditis elegans showed much lower, but still significant degrees of relationship. Pairwise comparisons of the mosquito vitellogenin and the Drosophila melanogaster yolk proteins did not show significant similarities. Potential regulatory regions in the mosquito VgA1 gene were identified by comparison to regulatory elements known from other organisms, especially D. melanogaster, which could provide useful information for further functional analysis.

Characterization of Anopheles pseudopunctipennis sensu lato from three countries of neotropical America from variation in allozymes and ribosomal DNA.

Enzyme electrophoresis and restriction fragment length polymorphism (RFLP) analysis of Anopheles pseudopunctipennis sensu lato from nine isolated populations in neotropical America confirmed previous observations that it constitutes a species complex. Electrophoretic studies showed fixed differences at two enzyme loci, glycerol dehydrogenase (Gcd) and phosphoglucomutase (Pgm), suggesting limited or no gene flow between populations from Mexico and South America. In addition, analysis of genetic distance showed two distinctive clusters, one from Mexico and the other from South America, separated at a Nei's distance level of 0.13, a value consistent in magnitude with that of other anopheline sibling species. The RFLP analysis revealed the presence of a ribosomal DNA fragment in Mexican strains that was absent in strains from South America. Two species have been identified through these studies, one provisionally named An. pseudopunctipennis A, a species from central Mexico, and the other An. pseudopunctipennis B, for the species found in the interAndean valleys and Andean slopes in regions of Peru and Bolivia. This research provides information required to elucidate the status of the different species of the An. pseudopunctipennis complex as vectors of malaria in the Americas.

Use of a restriction fragment length polymorphism (RFLP) as a genetic marker in crosses of Anopheles gambiae (Diptera: Culicidae): independent assortment of a diphenol oxidase RFLP and an esterase locus.

Analysis of restriction fragment length polymorphism (RFLP) is a powerful tool for analyzing linkage relationships in species where few genetic markers have been described and where conduct of crosses is difficult. It also permits integration of genetic and physical (cytogenetic) data when the probes have been mapped by in situ hybridization. To illustrate the utility of the method, and because some mutations of a diphenol oxidase gene might conceivably produce the malaria refractoriness phenotype of ookinete-oocyst encapsulation, backcrosses between two inbred lines of Anopheles gambiae Giles were carried out to determine the linkage relationship between the diphenol oxidase A2 (Dox) gene and the esterase locus associated with refractoriness to Plasmodium cynomolgi NIH. The Dox alleles were a Sal I restriction fragment length polymorphism visualized by probing Southern blotted DNA from portions of individual mosquitoes with a cloned Dox gene probe. The two genes were shown to segregate independently.

Method for in situ hybridization to polytene chromosomes from ovarian nurse cells of Anopheles gambiae (Diptera: Culicidae).

A procedure for in situ hybridization to polytene chromosomes from the ovarian nurse cells of Anopheles gambiae Giles has been developed. This procedure involves a modification of established methods for Drosophila larval salivary gland polytene chromosomes. Treatment of chromosome squashes with xylene followed by slow rehydration provides required resolution of chromosome banding patterns, possibly because fatty contaminants are removed from ovarian nurse cell preparations. Using this procedure, unique DNA sequences from a genomic library of An. gambiae have been mapped on adult mosquito polytene chromosomes. The ability to locate genetic markers on chromosomes will allow correlation of physical and genetic maps. Such maps will facilitate identification of genetic loci and expand our knowledge of the genomic organization of An. gambiae.

Use of chitinase to facilitate detection of protozoan, helminth and single copy genes in squashed whole mosquitoes.

The application of DNA probes to detect foreign DNA in whole arthropods has been limited by the inability of the probes to distinguish between small quantities of target DNA and the background signal generated by non-specific hybridization of mosquito material. We report that treatment of nitrocellulose filters upon which mosquitoes have been squashed with chitinase and proteinase K eliminates non-specific hybridization of DNA probes to mosquito components. Using this technique we have been able to detect a single larva of Brugia malayi, sporozoites of Plasmodium falciparum and Plasmodium berghei, and a single-copy gene in directly squashed vector mosquitoes. Use of this simple, rapid technique should facilitate the successful use of DNA probes in field studies.

Stable integration and expression of a bacterial gene in the mosquito Anopheles gambiae.

Foreign DNA was successfully introduced into the germline of the African mosquito vector of malaria Anopheles gambiae. Stable integration of genes into the germlines of insects had been achieved previously only in Drosophila melanogaster and related species and required the use of the P element transposon. In these experiments with Anopheles gambiae, the plasmid pUChsneo was used, which contains the selectable marker neo gene flanked by P element inverted repeats. Mosquitoes injected with this plasmid were screened for resistance to the neomycin analog G-418. A single event of plasmid insertion was recovered. Integration appears to be stable and, thus far, resistance to G-418 has been expressed for eight generations. The transformation event appears to be independent of P.

The use of restriction fragment length polymorphisms and DNA duplications to study the organization of the actin multigene family in Dictyostelium discoideum.

The techniques of restriction fragment length polymorphism analysis and examination of gene copy number in duplication-bearing Dictyostelium discoideum strains have been used to map four actin genes of the wild-type strain NC4 to specific linkage groups. In part, this was accomplished by identification of restriction fragments corresponding to particular cloned actin genes using gene-specific probes from unique sequence 5' and 3' untranslated regions. Cloned gene Actin 8 (designation act-8) maps to linkage group I; Actins 12 (act-12) and M6 (actM6) to linkage group II. An uncloned gene (act-100) also maps to linkage group II in the same region as actM6, as defined by a chromosomal duplication. From analysis of other wild isolates of D. discoideum, it was determined that in these isolates at least two actin genes map to linkage group I and at least four map to linkage group II. These results demonstrate the utility of molecular techniques in genetic analysis of Dictyostelium, particularly for developmentally regulated genes that have been cloned but that have no identified mutant phenotypes.

Gene-specific expression of the actin multigene family of Dictyostelium discoideum.

We have investigated the expression of 14 cloned genes of the 20-member actin multigene family of Dictyostelium discoideum using gene-specific mRNA complementary probes and an RNase protection assay. Actin gene expression was studied in vegetative cells and in cells at a number of developmental stages chosen to represent the known major shifts in actin mRNA and protein synthesis. At least 13 of these genes are expressed. A few genes are expressed very abundantly at 10% or more of total actin mRNA; however, the majority are maximally expressed at 1 to 5% of actin message. Although all of the genes are transcribed in vegetative cells, most genes appear to be independently regulated. Actin 8 appears to be transcribed at constant, high levels throughout growth and development. Actin 12 mRNA is maximally expressed in vegetative cells but the level is reduced appreciably by the earliest stage of development examined, while Actin 7 mRNA is specifically induced approximately sevenfold at this time. The rest of the genes appear to be induced 1.5 to 2-fold early in development, coincident with the increase in total actin mRNA. Since 12 of the genes code for extremely homologous proteins, it is possible that the large number of actin genes in Dictyostelium is utilized for precise regulation of the amount of actin produced at any stage of development, even though individual gene expression appears in some cases to be very stage-specific. In addition to these 13 actin genes, at least two and possibly four more genes are known to be expressed, because they are represented by complementary DNA clones, and an additional one or two expressed genes are indicated by primer extension experiments. Only one known gene, Actin 2-sub 2, is almost certainly a pseudogene. Thus the vast majority of Dictyostelium actin genes are expressed.

Organization of the actin multigene family of Dictyostelium discoideum and analysis of variability in the protein coding regions.

There are 17 to 20 actin genes in the genome of the cellular slime mold Dictyostelium discoideum. Genomic clones of 15 of the genes have been isolated. Extensive nucleotide sequence within the protein-coding regions has been determined, including the complete nucleotide sequence of four genes representing the three distinct evolutionary groups of Dictyostelium actin genes. All are similar to mammalian cytoplasmic actins at diagnostic amino acid positions, and there is generally less variability among Dictyostelium actin genes than among Drosophila actin genes. Two genes, Actins 3-sub 1 and 3-sub 2 differ substantially from all the rest in terms of replacement amino acid substitutions and probably encode actin-related proteins rather than bona fide actins. Each contains several amino acid substitutions that should alter the secondary structure of the resulting proteins, and Actin 3-sub 2 encodes four additional amino acids at the C terminus. This gene is as divergent from other Dictyostelium actin genes as is the yeast or a soybean actin gene. At present, evidence suggests that all 15 genes examined are expressed, except the previously identified Actin 2-sub 2. We suggest that Dictyostelium might maintain a high number of functional actin genes for the purpose of regulating the level of actin synthesis within narrow limits, rather than because most genes perform different functions.

Organization of the Dictyostelium discoideum actin multigene family. Flanking sequences show subfamily homologies and unusual dyad symmetries.

Sequences flanking the protein-coding regions of 15 of the 17 to 20 actin genes in the cellular slime mold Dictyostelium discoidium have been determined. Comparison of sequences among genes shows that they contain extensive homologies at both the 5' and 3' ends of the coding regions. On the basis of these homologies, actin genes fall into three groups. Group I consists of Actin 8 alone. Group II consists of the two closely linked genes Actin 3-sub1 and Actin 3-sub2. These two genes differ from all other actin genes in the location of their TATA box and oligo(dT) run, and diverge substantially in their coding sequence as well. Group III contains all the rest of the genes we have studied. Within this group, there are two subgroups of genes, IIIA (Actins 5, 9 and 10) and IIIB (Actins M6, 2-sub1 and 2-sub2, 4, 6, 7, 11 and 12). Two actin cDNA clones, ITL-1 and III-12/A1, which have no cloned genomic counterparts, are members of groups IIIA and IIIB, respectively. Homologies at the 3' ends of genes do not extend beyond a short genomic poly(A) sequence, the probable termination of transcription. Homologies at the 5' ends may extend about 300 base-pairs 5' to the ATG but, in most cases, extend only about 150 base-pairs 5' to the ATG. We have identified a group of short, relatively G + C-rich sequences within the extremely A + T-rich sequence at the 5' ends of actin-coding regions, which are shared among different actin genes. Many of these sequences exhibit dyad symmetry, and their general location and order is conserved among the different actin genes. We suggest that they may have a role in regulation of the transcriptional patterns of individual actin genes.

Maternally influenced embryonic lethality: allele specific genetic rescue at a female fertility locus in Drosophila melanogaster.

A new locus, mel(1)R1, with a maternal effect on embryonic development, has been mapped at about 0.5 on the X chromosome of Drosophila melanogaster and localized cytologically between bands 2D6 and 3A1. Genotypically mutant embryos die if produced by homozygous mutant females but survive if produced by heterozygous females. Two mutant alleles have been isolated. One of these is genetically rescuable: when homozygous mutant females are mated to mutant males, all the embryos die, but when these females are mated to normal males, female offspring are produced. The other allele is not rescuable. Genetic rescue is dominant at this locus since females heterozygous for the two mutant alleles produce female offspring in crosses to normal males.

An effect of centromere function on the behavior of ring-X chromosomes in Drosophila melanogaster.

It is shown that under the influence of an autosomal meiotic mutant that causes abnormalities in meiotic centromere function (mei-S332), ring-X chromosomes are frequently nonrecoverable. Evidence is presented that this nonrecoverability is caused by a failure of sister ring-chromatids to successfully effect an equational separation with resultant dominant lethality. Because mei-S332 results in meiotic abnormalities only after replication has been completed, and because ring chromosomes are normally transmitted with approximately the same efficiency as rod chromosomes, it is suggested that during replication in normal meioses, sister ring-chromatids form mutually interlocked ring complexes that are resolved without genetic consequences at anaphase II, with the resolution owing at least in part to normal centromere function.