ABSTRACT: Transcriptional profile of inbreeding depression expressing as extreme cold-sensitivity. The experiment is a crossdesign of a conditional lethal line and an inbred control line, at permissive and a restrictive conditions (the latter involving a cold shock)
Project description:Transcriptional profile of inbreeding depression expressing as extreme cold-sensitivity. The experiment is a crossdesign of a conditional lethal line and an inbred control line, at permissive and a restrictive conditions (the latter involving a cold shock)
Project description:Inbreeding depression is a widespread phenomenon of central importance to agriculture, medicine, conservation biology and evolutionary biology. Although the population genetic principles of inbreeding depression are well understood, we know little about its functional genomic causes. To provide insight into the molecular interplay between intrinsic stress responses, inbreeding depression and temperature tolerance, we performed a proteomic characterization of a well-defined conditional inbreeding effect in a single line of Drosophila melanogaster, which suffers from extreme cold sensitivity and lethality. We identified 48 differentially expressed proteins in a conditional lethal line as compared to two control lines. These proteins were enriched for proteins involved in hexose metabolism, in particular pyruvate metabolism, and many were found to be associated with lipid particles. These processes can be linked to known cold tolerance mechanisms, such as the production of cryoprotectants, membrane remodeling and the build-up of energy reserves. We checked mRNA-expression of seven genes with large differential protein expression. Although protein expression poorly correlated with gene expression, we found a single gene (CG18067) that, after cold shock, was upregulated in the conditional lethal line both at the mRNA and protein level. Expression of CG18067 also increased in control flies after cold shock, and has previously been linked to cold exposure and chill coma recovery time. Many differentially expressed proteins in our study appear to be involved in cold tolerance in non-inbred individuals. This suggest the conditional inbreeding effect to be caused by misregulation of physiological cold tolerance mechanisms.
Project description:Inbreeding is typically detrimental to individual fitness, with negative effects being often exaggerated in stressful environments. However, the causal mechanisms underlying inbreeding depression in general and the often increased susceptibility to stress in particular are not well understood. We here test whether inbreeding interferes with the heat-shock response, comprising an important component of the stress response which may therefore underscore sensitivity to stress. To this end we subjected the tropical butterfly Bicyclus anynana to a full-factorial design with three temperatures and three levels of inbreeding, and measured the expression of heat-shock protein (HSP) 70 via qPCR. HSP70 expression increased after exposure to heat as compared with cold or control conditions. Most strikingly, inbreeding strongly interfered with the heat-shock response, with inbred individuals showing a very weak upregulation of HSP70 only. Our results thus indicate that, in our study organism, interference with the heat-shock response may be one mechanism underlying reduced fitness of inbred individuals, especially when exposed to stressful conditions. However, these indications need to be corroborated using a broader range of different temperatures, genes and taxa.
Project description:While the population genetics of inbreeding is fairly well understood, the effects of inbreeding on the physiological and biochemical levels are not. Here we have investigated the effects of inbreeding on the Drosophila melanogaster metabolome. Metabolite fingerprints in males from five outbred and five inbred lines were studied by nuclear magnetic resonance spectroscopy after exposure to benign temperature, heat stress, or cold stress. In both the absence and the presence of temperature stress, metabolite levels were significantly different among inbred and outbred lines. The major effect of inbreeding was increased levels of maltose and decreased levels of 3-hydroxykynurenine and a galactoside [1-O-(4-O-(2-aminoethyl phosphate)-beta-d-galactopyranosyl)-x-glycerol] synthesized exclusively in the paragonial glands of Drosophila species, including D. melanogaster. The metabolomic effect of inbreeding at the benign temperature was related to gene expression data from the same inbred and outbred lines. Both gene expression and metabolite data indicate that fundamental metabolic processes are changed or modified by inbreeding. Apart from affecting mean metabolite levels, inbreeding led to an increased between-line variation in metabolite profiles compared to outbred lines. In contrast to previous observations revealing interactions between inbreeding and environmental stress on gene expression patterns and life-history traits, the effect of inbreeding on the metabolite profile was similar across the different temperature treatments.
Project description:The Exopalaemon carinicauda could be a useful crustacean laboratory animal in many research fields. We newly established an inbred line of Exopalaemon carinicauda named EC4 inbred line by brother×sister mating and keeping to F 11 generation. Trends in heterozygosity in the process of producing EC4 inbred line were examined through the characterization of polymorphisms based on gene frequencies of SNP and EST-SSR loci. The results demonstrated that the number of alleles (N), observed heterozygosity (Ho), expected heterozygosity (He), and polymorphism information content (PIC) gradually decreased with the increase of inbreeding generations. The genetic detection results indicated that 9 (29.03%, 9/31) of the SNP loci and 15 (32.61%, 15/46) of the EST-SSR loci were homozygous in F 11 generation of EC4 inbred line. The variation of the growth-related traits, the immune responses, and antioxidant status were described in experimental full-sibling inbred populations of E. carinicauda at five levels of inbreeding coefficient (F = 0.785, F = 0.816, F = 0.859, F = 0.886, F = 0.908) under controlled laboratory conditions. The body weight, body length, and survival rate in EC4 inbred line of all generations were less than the control population. Inbreeding affected the antibacterial activity, phenoloxidase (PO) activity, and superoxide dismutase (SOD) which decreased at the eleventh generation of EC4 inbred line. This study demonstrated that inbreeding had a negative effect on the economic traits and immune response, but our inbred line was established successfully until F 11 and confirmed by genetic detection using SNP and EST-SSR loci.
Project description:Understanding how inbreeding affects fitness is biologically important for conservation and pest management. Despite being a worldwide pest of many economically important cruciferous crops, the influence of inbreeding on diamondback moth, Plutella xylostella (L.), populations is currently unknown. Using age-stage-specific life tables, we quantified the inbreeding effects on fitness-related traits and demographic parameters of P. xylostella. Egg hatching rate, survival and fecundity of the inbred line significantly declined compared to those of the outbred line over time. The inbred P. xylostella line showed significantly lower intrinsic rate of increase (r), net reproduction rate (R0), and finite increase rate (λ), and increasing generation time (T). Inbreeding effects vary with developmental stages and the fitness-related traits can be profoundly affected by the duration of inbreeding. Our work provides a foundation for further studies on molecular and genetic bases of the inbreeding depression for P. xylostella.
Project description:Mouse embryonic stem (ES) cells are derived from the inner cell mass of blastocyst stage embryos and are used primarily for the creation of genetically engineered strains through gene targeting. While some inbred strains of mice are permissive to the derivation of embryonic stem cell lines and are therefore easily engineered, others are nonpermissive or recalcitrant. Genetic engineering of recalcitrant strain backgrounds requires gene targeting in a permissive background followed by extensive backcrossing of the engineered allele into the desired strain background. The inbred mouse strain DBA/2J is a recalcitrant strain that is used as a model of many human diseases, including glaucoma, deafness and schizophrenia. Here, we describe the generation of germ-line competent ES cell lines derived from DBA/2J mice. We also demonstrate the utility of DBA/2J ES cells with the creation of conditional knockout allele for Endothelin-2 (Edn2) directly on the DBA/2J strain background.
Project description:By monitoring the mitotic transmission of a marked chromosome bearing a defective centromere, we have identified conditional alleles of two genes involved in chromosome segregation (cse). Mutations in CSE1 and CSE2 have a greater effect on the segregation of chromosomes carrying mutant centromeres than on the segregation of chromosomes with wild-type centromeres. In addition, the cse mutations cause predominantly nondisjunction rather than loss events but do not cause a detectable increase in mitotic recombination. At the restrictive temperature, cse1 and cse2 mutants accumulate large-budded cells, with a significant fraction exhibiting aberrant binucleate morphologies. We cloned the CSE1 and CSE2 genes by complementation of the cold-sensitive phenotypes. Physical and genetic mapping data indicate that CSE1 is linked to HAP2 on the left arm of chromosome VII and CSE2 is adjacent to PRP2 on chromosome XIV. CSE1 is essential and encodes a novel 109-kDa protein. CSE2 encodes a 17-kDa protein with a putative basic-region leucine zipper motif. Disruption of CSE2 causes chromosome missegregation, conditional lethality, and slow growth at the permissive temperature.
Project description:Temperature is essential to the maintenance of optimal physiological functioning in aquatic organisms. Fish can manage natural fluctuations in temperature; however, in freshwater ecosystems acute and rapid temperature changes can originate from sources such as large dams and industrial effluents. These rapid temperature changes may induce several physiological and behavioural responses that can result in lethal and sub-lethal consequences. The present study assessed immediate sub-lethal and short-term (10 days) lethal responses of three species of Australian freshwater fish larvae and early-stage juveniles to a range of different 'field-relevant' cold shocks (-4, -6, -8 and -10°C). Murray cod (Maccullochella peelii), silver perch (Bidyanus bidyanus) and golden perch (Macquaria ambigua) were tested at two age groups to elucidate the interaction between ontogeny and sensitivity to cold shock. Cold shock caused mortality and reductions in swimming ability (time to exhaustion and lines crossed) in all species of fish at both age groups. Sensitivity was correlated to the magnitude of cold shock; a 10°C drop in temperature caused the highest mortalities. Ontogeny interacted with the severity of cold shock; the younger fish experienced higher mortalities and greater impairment to swimming ability. This study demonstrates the potential lethal and sub-lethal impacts of cold shock on freshwater fish at a critical life-history stage. Understanding the impacts of cold shock will aid management of freshwater ecosystems for the benefit of fish populations, with the current study identifying critical life stages to be considered in remediation and guiding thresholds necessary to reduce the impact of cold shock on native fish populations.
Project description:Anopheles gambiae is a major mosquito vector of malaria in Africa. Although increased use of insecticide-based vector control tools has decreased malaria transmission, elimination is likely to require novel genetic control strategies. It can be argued that the absence of an A. gambiae inbred line has slowed progress toward genetic vector control. In order to empower genetic studies and enable precise and reproducible experimentation, we set out to create an inbred line of this species. We found that amenability to inbreeding varied between populations of A. gambiae. After full-sib inbreeding for ten generations, we genotyped 112 individuals--56 saved prior to inbreeding and 56 collected after inbreeding--at a genome-wide panel of single nucleotide polymorphisms (SNPs). Although inbreeding dramatically reduced diversity across much of the genome, we discovered numerous, discrete genomic blocks that maintained high heterozygosity. For one large genomic region, we were able to definitively show that high diversity is due to the persistent polymorphism of a chromosomal inversion. Inbred lines in other eukaryotes often exhibit a qualitatively similar retention of polymorphism when typed at a small number of markers. Our whole-genome SNP data provide the first strong, empirical evidence supporting associative overdominance as the mechanism maintaining higher than expected diversity in inbred lines. Although creation of A. gambiae lines devoid of nearly all polymorphism may not be feasible, our results provide critical insights into how more fully isogenic lines can be created.