Isoenzyme variation in Aedes aegypti correlated with Dirofilaria immitis infectability

From the Vero Beach strain of the mosquito Aedes (Stegomyia) aegypti (L.) (Diptera: Culicidae), substrains were selected for susceptibility (SS) and refractoriness (RR) to the dog heartworm Dirofilaria immitis (Leidy) (Filarioidea: Onchocercidae). These two lines and their reciprocal F1 hybrids were analysed for genetic variation at 14 enzyme loci, using polyacrylamide gel electrophoresis. Six of the enzyme loci showed variation (sample size 48 alleles/locus/line). Three of these were monomorphic in the refractory line but polymorphic in the susceptible, i.e. aconitase hydratase (Acoh), isocitrate dehydrogenase-1 (Idh-1) and phosphoglucomutase (Pgm). The other three loci, glucose-6-phosphate isomerase (Gpi), hexokinase-1 (Hk-1) and isocitrate dehydrogenase-2 (Idh-2), were polymorphic in both SS and RR lines and their hybrids. At two loci (Hk-1, Pgm) three alleles were detected, whereas the other polymorphic loci had only two alleles. For Hk-1, the most frequent allele was Hk-1(80) (0.563) in refractory and Hk-1(100) in the susceptible (0.521) and F1 hybrids. For Pgm the most frequent alleles were Pgm125 in the susceptible line (0.646) and Pgm100 in the F1 hybrids (0.563 and 0.604) and refractory line (1.000). The mean observed heterozygosity (Ho), the mean Hardy-Weinberg expected heterozygosity (He) and the mean number of alleles per locus in the refractory line were lower, but not significantly so, than in the susceptible line and their reciprocal F1 hybrids; the proportion of polymorphic loci was significantly lower in the refractory than in the susceptible line and their F1 hybrids. Within both lines all polymorphisms were in Hardy-Weinberg equilibrium, whereas significant departures from predicted frequencies were observed in SS x RR hybrids at four polymorphic loci (Acoh, Gpi, Hk-1, Pgm) and at three polymorphic loci (Acoh, Hk-1, Pgm) in RR x SS hybrids. The average Nei's and modified Rogers' genetic distances between the lines were 0.024 and 0.139, respectively. These electrophoretic data show that the refractory line (putatively lacking fi allele) can be distinguished from the susceptible line (fi/fi) and their hybrids (heterozygous fi) by isozyme marker frequencies, but it remains to be seen whether this difference is causal or chance linkage. In any case, this model system of Ae. aegypti/D. immitis provides opportunities to better understand and manipulate the molecular biology of filariasis transmission.     

中华稻蝗四个种群的遗传分化

采用水平淀粉凝胶电泳技术研究中华稻蝗(Oxya chinensis)四个种群的12个基因座位,探讨其遗传分化.这四个种群分别采自内蒙古呼和浩特、山西代县、山西太原和陕西西安.Ck和Mdh-2在四个种群中均为单态,其余的基因座位至少在一个种群内有两个以上的等位基因;在Ldh和Mdh-1的等位基因频率呈现梯度分布趋势.多态基因座位百分率(P)和平均每个基因座位的等位基因数目(A)分别为58.3%-66.7%和2.2-2.8,平均杂合度为Ho=0.173-0.240.除Gpi,Hk-2,Idh,Ldh和Mdh-1在部分蝗虫种群符合Hardy-Weinberg平衡外,其余绝大多数基因座位的基因型频率显著偏离Hardy-Weinberg平衡.四个种群间FST平均值不存在显著差异(FST=0.0510,P＞0.05),结合高的Nei's遗传一致度(I＞0.97)可知,种群之间遗传分化不明显.我们认为人类的农业活动可能促进了种群间的基因交流,从而降低了分化程度[动物学报50(2)187-192,2004].     

Identification and linkage relationships of three hexokinase genes in Aedes aegypti

Four regions of hexokinase activity are detected by starch gel electrophoresis of adult Aedes aegypti. Three of the regions, Hk-2, Hk-3, and Hk-4, are produced by three tightly linked loci, located on the third chromosome 7.25 map units from the locus fuzzy. The three loci show developmental differences as well as differences in substrate specificity.This work was supported by grants from the National Institutes of Health, AI 05118-02 and AI 12016.     

Isocitrate dehydrogenase in Aedes aegypti: formal genetics, preliminary linkage data and study of natural populations

Two loci for isocitrate dehydrogenase (Idh-1 ad Idh-2) are described in Ae. aegypti, both polymorphic with two codominant alleles. Crosses made to test linkage relationships of Idh-1 indicate that this locus is independent from sex (chromosome 1) and from Sod-1 and Hk-1 loci (chromosome 3), while it is linked to Pgm on the second chromosome. Average percent of recombination is 11.37, but significant differences have been found among strains. Data on genetic variability of Idh-1 and Idh-2 in three domestic african field populations are presented.     

Evaluation of genetic variability of the Kamchatka crab Paralithodes camtschaticus (Tilesius) using allozyme markers

Interspecific genetic variation in populations of red king crab Paralithodes camtschaticus Tilesius (Litholidae, Decapoda: Crustacea) was examined using allozyme markers. The activity of 57 enzymes and the general protein presumably encoded by 92 loci was detected. The level of allozyme variability was low: the expected heterozygosity and the proportion of polymorphic loci were respectively 0.027 +/- 0.008 and 6.5%. This level of heterozygosity is three times lower than the average value for 122 crustacean species (0.082 +/- 0.007). Although genetic variants were found at 22 loci, their frequencies were generally low: only in loci 6-Pgd, Alp-1, and Pep-1 did the frequencies of the most common alleles not exceed 0.9. All polymorphic loci except one had two alleles; the exception was 6-Pgd, which had three alleles. The possible reasons for the low level of allozyme variability in red king crab are discussed.     

Heterozygosity in inbred strains of the tree-hole mosquitoAedes triseriatus

Inbred stocks of the tree-hole mosquito Aedes triseriatus from four localities were developed using full-sib mating. The progress of inbreeding was followed electrophoretically at eight variable and six less variable enzyme loci. Rates of fixation of several of these loci were substantially lower than expected. Discrepancies between observed and expected fixation values were evident in the early stages of inbreeding and became larger as inbreeding progressed. Odh, Hbd, Pgm, and Hk-4 were usually not fixed. By the F12 and F14 generations of brother-sister mating, most individuals in the two lines were heterozygotes (Odh and Hbd in the TK lines and Odh, Hbd, Pgm, and Hk-4 in the TV lines). The probability of maintaining heterozygosity at several selectively neutral and unlinked loci simultaneously is very low. Odh, Hbd, Pgm, and Hbd loci are linked to lethal recessives on chromosome 2, creating a balanced lethal system which in turn accounts for the heterozygosity in these inbred mosquitoes.     

The osmotic characteristics of human fetal liver-derived hematopoietic stem cell candidates

Hematopoietic stem cells derived from fetal liver have promising therapeutic potential for allotransplantation but require a specific protocol to minimize the damage produced by cryopreservation procedures. In this study, a fundamental approach was applied for designing a cell preservation protocol. To this end, the biophysical characteristics that describe the osmotic reaction of CD34(+)CD38(-) human fetal liver stem cell candidates were studied using fluorescent microscopy. The osmotically inactive volume of the stem cell candidates was determined as 48% of the isotonic volume. The permeability coefficients for water and Me(2)SO were determined at T = +22 degree C: L(p) = 0.27 +/- 0.03 microm x min(-1)atm(-1), P(Me(2)SO)) = 2.09 +/- 0.85 x 10 (-4) cm x min(-1), sigma (Me(2)SO)) = 0.63 +/- 0.03 and at T = +12 degree C: L(p) = 0.15 +/-0.02 microm x min(-1)atm(-1), P(Me(2)SO)) = 6.44 +/-1.42 x 10 (-5) cm x min(-1), sigma (Me(2)SO)) = 0.46 +/- 0.05. The results obtained suggest that post-hypertonic and hypotonic stress are the possible reasons for damage to a CD34(+)CD38(-) cell during the cryopreservation procedure.     

Population genetic studies of the Philippine Negritos. I. A pilot survey of red cell enzyme and serum protein groups

Electrophoretic surveys of red cell enzyme and serum protein systems representing 21 genetic loci were carried out on 129 blood samples of the Negritos of Pampanga, Central Luzon, the Philippines. Nine (out of 16) red cell enzyme loci and four (out of five) serum protein loci showed polymorphic variation. Low frequencies of ACP 1A, GPTs1, ESD2, and Hp1, and a markedly high frequency of PGM12 were contrasted to those in non-Negrito Filipinos. Variant ESD phenotypes with a slowly migrating isozyme occurred in high frequency. The new allele designated as ESD3Negrito (ESD3N) had a frequency of .10 +/- .019. In AK, a variant phenotype indistinguishable from AK 2-1 was observed in 14% of the sample. In the Gc system, a fast migrating variant was discovered in high frequency which was distinct from Gc Ab and Gc J. The variant allele, denoted GcNegrito (GcN), had a frequency of .21 +/- .025. A relatively high degree of allelic diversity in the Negrito sample was also suggested by the average heterozygosity for 21 loci screened (.165), which is compared to that of the Japanese population (.140).     

Cryopreservation of European eel (Anguilla anguilla) spermatozoa: effect of dilution ratio, foetal bovine serum supplementation, and cryoprotectants

The main aim of the present study was to investigate the effect of sperm freezing medium dilution ratio (1:1, 1:2, and 1:5 v/v), two cryoprotectants: dimethyl sulphoxide (Me(2)SO) and methanol (MeOH), and the addition of foetal bovine serum (FBS) on the cryopreservation of European eel sperm. The effect of these factors was evaluated comparing post-thawing viability with fluorescent staining (Hoechst bisbenzimide 33258) and the spermatozoa head morphometry, determined with computer-assisted morphology analysis (ASMA). The 1:5 (v/v) dilution ratio resulted in a lower viability in comparison with 1:1 and 1:2 (52.8+/-2.3% vs. 67.4+/-2.3% and 65.1+/-2.3%, respectively, p=0.0001), but without effects on the head morphology. Although the viability was not significantly different between Me(2)SO and MeOH (60.4+/-1.9 vs. 63.2+/-1.9%, respectively, p=0.305), a decrease of spermatozoa head area and perimeter was found when spermatozoa were frozen with methanol (6.19+/-0.01 vs. 6.36+/-0.01 microm(2) and 17.28+/-0.05 vs. 17.49+/-0.05 microm, for area and perimeter and MeOH and Me(2)SO, respectively, p=0.0001). Finally, a higher viability (75.1+/-1.7 vs. 48.5+/-1.7, with or without FBS, respectively, p=0.0001) and higher spermatozoa head size (6.40+/-0.01 vs. 6.15+/-0.01microm(2) and 17.88+/-0.05 vs. 16.89+/-0.05 microm, for area and perimeter, with or without FBS, respectively, p=0.0001) were found when cells were frozen-thawed in freezing media supplemented with FBS. Based on the above findings, dilution ratios lower than 1:5 (v/v) and the addition of serum improved the viability results after cryopreservation. Future studies are required in order to understand the spermatozoa membrane interchange mechanisms in response to the changes in spermatozoa head size caused by cryoprotectants and freezing media supplements.     

X-linkage of malic enzyme in Anopheles culicifacies species B

A survey for malic enzyme (Me) in laboratory strains of species A and species B of Anopheles culicifacies had uncovered two electrophoretic variants, slow and fast, in two strains of species B. Genetic analysis revealed the two variants to be codominant alleles segregating at a locus, Me, which is sex linked. Because of the XX-XY sex determining mechanism, in F1 females, two electromorphs, viz., slow and fast, were observed, whereas in males only one electromorph of maternal origin was seen. Linkage experiments with another X-linked mutant, white eye (w), indicated the map distance between the two loci to be 9.52 +/- 0.86.     

Assignment of two enzyme loci to the X chromosome of Anopheles quadrimaculatus species A

Analysis of isozyme variability in four natural populations of Anopheles quadrimaculatus Species A indicated that the loci for Malic enzyme (Me) and Mannose phosphate isomerase-1 (Mpi-1) are on the X chromosome. There were female heterozygotes, but no male heterozygotes were observed. Strains fixed for fast- and slow-migrating allozymes were devised and crossed. Progeny phenotypes conformed to expectations for sex linkage; female progeny were heterozygous, and male progeny were hemizygous for the maternal allele. The three-point cross, using the Me and Mpi-1 loci with the sex-linked mutant rose eye (ro), established the gene sequence Mpi-1-11.1-Me-40.8-ro.     

Recombination and nucleotide diversity in the sex chromosomal pseudoautosomal region of the emu, Dromaius novaehollandiae

Pseudoautosomal regions (PARs) shared by avian Z and W sex chromosomes are typically small homologous regions within which recombination still occurs and are hypothesized to share the properties of autosomes. We capitalized on the unusual structure of the sex chromosomes of emus, Dromaius novaehollandiae, which consist almost entirely of PAR shared by both sex chromosomes, to test this hypothesis. We compared recombination, linkage disequilibrium (LD), GC content, and nucleotide diversity between pseudoautosomal and autosomal loci derived from 11 emu bacterial artificial chromosome (BAC) clones that were mapped to chromosomes by fluorescent in situ hybridization. Nucleotide diversity (pi = 4N(e)mu) was not significantly lower in pseudoautosomal loci (14 loci, 1.9 +/- 2.4 x 10(-3)) than autosomal loci (8 loci, 4.2 +/- 6.1 x 10(-3)). By contrast, recombination per site within BAC-end sequences (rho = 4Nc) (pseudoautosomal, 3.9 +/- 6.9 x 10(-2); autosomal, 2.3 +/- 3.7 x 10(-2)) was higher and average LD (D') (pseudoautosomal, 4.2 +/- 0.2 x 10(-1); autosomal, 4.7 +/- 0.5 x 10(-1)) slightly lower in pseudoautosomal sequences. We also report evidence of deviation from a simple neutral model in the PAR and in autosomal loci, possibly caused by departures from demographic equilibrium, such as population growth. This study provides a snapshot of the population genetics of avian sex chromosomes at an early stage of differentiation.     

Isozyme polymorphisms maintained by lethal loci in inbred strains of Aedes triseriatus

We analyzed the progeny of brother-sister matings of two inbred strains of the treehole mosquito, Aedes triseriatus, at several isozyme loci to determine which factors were maintaining heterozygosity at these loci after prolonged inbreeding. The triseriatus Kramer (TK) lines were polymorphic for the Odh and Hbd loci after 12 generations of full-sib mating, and the triseriatus Vero Beach (TV) lines were polymorphic for the Odh, Hk-4, Pgm, and Hbd loci after 14 generations of full-sib mating. Genetic analysis of 22 TK F13 matings and nine TV F15 matings showed that heterozygosity was enforced by lethal loci to which all the polymorphic isozyme loci were linked. The lethal loci formed a balanced lethal system. We determined the relative positions and map distances of lethals and enzyme loci.     

Mapping of FMR1, the gene implicated in fragile X-linked mental retardation, on the mouse X chromosome

A genetic map of the Cf-9 to Dmd region of the mouse X chromosome has been established by typing 100 offspring from a Mus musculus x Mus spretus interspecific backcross for the four loci Cf-9, Cdr, Gabra3, and Dmd. The following order and genetic distances in centimorgans were determined: (Cf-9)-2.4 +/- 1.7-(Cdr)-2.0 +/- 1.4-(Gabra3)-4.1 +/- 2.0-(Dmd). Six backcross offspring carrying X chromosomes with recombination events in the Cdr-Dmd region were identified. These recombination events were used to define the position of Fmr-1, the murine homologue of FMR1, which is the gene implicated in the fragile X syndrome in man, and that of DXS296h, the murine homologue of DXS296. Both Fmr-1 and DXS296h were mapped into the same recombination interval as Gabra3 on the mouse X chromosome. These findings provide strong support for the concept that the order of loci lying in the Cf-9 to Gabra3 segment of the X chromosome is highly conserved between human and mouse.     

Analysis of heterozygosity levels at P1,TF, PGM1, ACP1, HP, GC, GLO, C3, and ESD loci in pulmonary tuberculosis patients with different treatment outcomes]

Heterozygosity at nine genetic loci (PI, TF, PGM1, ACP1, HP, GC, GLO1, C3, and ESD) was analyzed in pulmonary tuberculosis patients with good (group 1, N = 71) and poor (group 2, N = 35) response to treatment. The observed heterozygosities were compared with the expected values, which were calculated from allele frequencies in a control sample of healthy individuals (N = 328 with all but one locus and 78 with ESD) according to Hardy-Weinberg expectations. The analysis showed that the observed heterozygosities gl of patients significantly differed from the expected values hl in the case of four loci (GC, PI, C3, and ACP1). The observed heterozygosity was higher than expected in three cases (PI, C3, and ACP1) and lower then expected (GC) in one case. When data on each individual locus were compared using Fisher's exact test, both groups of patients proved to significantly differ (PF < 0.05) from the control group in the same four loci. No difference in observed heterozygosity was detected between the two groups of patients. The mean expected heterozygosity was h = 0.386 +/- 0.00674; the mean observed heterozygosity was g = 0.415 +/- 0.02 in group 1, g = 0.402 +/- 0.026 in group 2, and g = 0.371 +/- 0.00955 in the control group. The t test did not reveal a significant difference between the mean values of expected observed heterozygosities. Heterozygosity at individual loci, rather than mean heterozygosity, was proposed as an integral nonspecific indicator of the genetic control of a disease, because the former directly implicates individual marker loci in the development of a disorder, whereas effects of individual loci may eliminate each other when mean heterozygosity is computed. Based on the results obtained, a genetic control was assumed for the development of the tuberculosis process in the lungs.     

Cryopreservation of isolated fish blastomeres: effects of cell stage, cryoprotectant concentration, and cooling rate on postthawing survival

The toxicity of the cryoprotectant dimethyl sulfoxide (Me(2)SO) to isolated blastomeres was examined in three fish species representative of distinct environments: marine (whiting, Sillago japonica); estuarine (pejerrey, Odontesthes bonariensis); and freshwater (medaka, Oryzias latipes). The effects of embryonic stage, Me(2)SO concentration, and cooling rate on the cryopreservation of blastomeres were also studied. Whiting sheds small planktonic eggs whereas the other two species shed large demersal eggs. Isolated blastomeres from the three species tolerated Me(2)SO concentrations up to 9% relatively well for over 5 h but lost viability rapidly at 18%. Cells from later embryonic stages (512 or 1024 cells) were more tolerant of Me(2)SO than those from earlier stages (128 or 256 cells). The three factors examined, alone or in combination, had a significant effect on the survival of blastomeres after freezing and thawing, but the extent of the effect and the optimum conditions varied with the species. In general, the highest rates of successful cryopreservation were observed with older rather than younger blastomeres, slower rather than faster cooling, and with 9-18% rather than 0% Me(2)SO. Survival rates for blastomeres cryopreserved under the most effective combination of the three factors examined for each species were 19.9 +/- 10.1% for whiting, 34.1 +/- 8.5% for medaka, and 67.4 +/- 12.8% for pejerrey. Copyright 1999 Academic Press.     

Antibodies to granulocytic Ehrlichia in moose, red deer, and roe deer in Norway

Serum samples from 104 moose (Alces alces), 124 red deer (Cervus elaphus) and 114 roe deer (Capreolus capreolus), collected from different counties in southern Norway from 1994 to 2000, were analysed by an indirect immunofluorescent antibody staining method for antibodies to Ehrlichia equi. The overall seroprevalences for granulocytic Ehrlichia spp. in moose, red deer, and roe deer from Ixodes ricinus infested counties were 43%, 55%, and 96%, respectively. Antibody prevalence was significantly higher in roe deer than in moose and red deer (P < 0.001). Mean antibody titers (log10 +/- SD) to E. equi in sera from moose, red deer, and roe deer were 1:1,497 (3.17 +/- 0.646), 1:234 (2.37 +/- 0.424) and 1:676 (2.83 +/- 0.404), respectively. The present work indicates that all these wild ruminant species are exposed to granulocytic Ehrlichia in Norway.     

Genetic studies of blood markers in Przewalski's horses

Ninety-six Przewalski's horses (Equus przewalskii) were blood typed using systems of inherited blood variants known to be highly effective for parentage testing of domestic horses (E. caballus). Sixteen red cell antigenic factors detected using sera prepared by alloimmunization of domestic horses were shown to be inherited in six systems (A, C, D, P, Q, and U) and in the same patterns as domestic horses. Family data confirmed autosomal, codominant inheritance at five loci of serum protein variants (Al, Tf, Xk, Pi, and Es) and three loci of red cell proteins (PGM, PHI, and Hb). One serum protein locus (Gc) and two red cell protein loci (PGD and CA) appeared to be monomorphic. Despite the narrow genetic base and high inbreeding coefficients of captive Przewalski's horses, average heterozygosity calculated over 18 loci was estimated to be 0.320 +/- 0.05, which was similar to that found in five breeds of domestic horses.     

Genetic linkage between loci for a red cell alloantigen (U) and serum protease inhibitor (Pi) in the horse

Preliminary evidence for the fifth autosomal linkage group in the horse, comprised of the loci for a red cell alloantigen (U) and serum protease inhibitor (Pi), was demonstrated by means of paternal half-sib groups in thoroughbred, standardbred and Arabian breeds. Recombination frequency in males was estimated to be 0.125 +/- 0.019.     

Analysis of isozyme loci in the face fly,Musca autumnalis DeGeer

Vertical polyacrylamide gel electrophoresis was used to resolve enzymes at 50 putative loci in Iowa face flies, a recently arrived, colonizing, Palearctic species. Sixty-two percent of the 50 loci were polymorphic. The mean number of alleles per polymorphic locus was 3.2 +/- 1.38; the mean among all loci was 2.38 +/- 1.62 alleles. The effective number of alleles among 50 loci was 1.4 +/- 0.62. Mean observed and expected heterozygosities for the 50 face fly loci were 0.167 +/- 0.037 and 0.186 +/- 0.031, respectively. Comparison of the electrophoretic data for Musca autumnalis and for M. domestica L. showed similar high levels of gene diversity. A survey of gene diversity at 12 loci among six geographically independent laboratory colonies demonstrated statistically significant genetic differentiation that was probably due to drift after colonization.