A key for hypoxia genetic adaptation in alpaca could be a HIF1A truncated bHLH protein domain / Una clave para la adaptacion genética de la hypoxia en las alpacas podria ser un dominio de proteina bHLH truncado con HIF1A

Animals exposed to hypobaric hypoxia triggers a physiological hypoxia response via Hypoxia Inducible Factor (HIF) proteins that functions as transcriptional complexes. As the South American camelids inhabit at high Andean altitudes we have asked if they have developed genetic adaptations to live at high altitudes. In the present study we investigate genetic structures of the HIF1A proteins carried by members of the superorder Cetartiodactyla. During our investigation we discovered the existence of a genetic event that caused the loss of most of the bHLH domain in the proteins borne by the Alpaca and other members of the Cetartiodactyla superorder; we designate them as bHLH short sequences. Further analysis at the nucleotide level revealed in the 12 short sequences included in the study the presence at the 5´end of the bHLH domains stop codons. Seven out of the 12 short HIF1A proteins, have an identical or almost identical nucleotide sequence at their 5´end with a same TAA stop codon and at the same position. As the mutations affects to both the Artiodactyls and Cetaceans, we postulate that the mutation(s) occurred before their divergence about 55 million years ago. The relevance of these findings for genetic adaptation of Alpacas to hypobaric hypoxia of high altitude conditions is discussed.

Los animales expuestos a hypoxia hipobárica generan una respuesta hipóxica fisiológica debido a unas proteinas de Factor-Hipoxia Inducible (HIF) que funcionan como complejos transcripcionales. Debido a que los camelidos Americanos habitan en las grandes alturas andinas, nos hemos preguntado si han desarrollado una adaptación genética para vivir a grandes alturas. Eneste estudio hemos investigado la estructura genética de las proteinas HIF1A que llevan consigo los miembros de la superorden de los cetartiodáctilos. Durante nuestra investigación, descubrimos la existencia de un evento genético que causó la perdida de la mayoría del dominio bHLH en las proteinas transmitidas por la alpaca y otros miembros de la superorden de los cetartiodáctilos; las hemos designado como secuencias cortas de bHLH. Análisis posteriores a nivel nucleótido revelaron que en la doceava secuencia corta incluida en el studio, hubo presencia de codones de terminación en el extreme 5' del dominio de bHLH. Siete de las doce proteinas cortas HIF1A, tiene una secuencia idéntica o casi idéntica de nucleotidos en su extremo 5', con el mismo codón de terminación TAA y en la misma posición. Debido a que la mutación afecta tanto a Artiodáctilos como Cetáceos, proponemos que la mutación(es) ocurrió antes de su divergencia hace unos 55 millones de años. Analizamos la relevancia de estos descubrimientos sobre la adaptación genética de las alpacas a la hipoxia hipobárica en condiciones de grandes alturas.

Función ventricular derecha en residentes nativos a gran altura / Right ventricular function in native residents at high altitude

Los adaptados genéticamente a la altura son los tibetanos, sherpas y etíopes; los aymaras y quechuas están aclimatados (Bolivia, Perú y norte de Chile). En Bolivia el mal crónico de montaña afecta 10% de la población masculina. El objetivo fue determinar la función ventricular derecha en residentes sanos y con mal crónico de montaña mediante ecocardiografía transtoráxica. Se utilizaron participantes sanos y con mal crónico de montaña admitidos por el IBBA, desde el año 2012 al 2013. Las variables tomadas son: demográficas, espirometria forzada, gasometría arterial en reposo e hiperoxia, ECG y ECCTT. Los controles (n 40), la edad promedio (44,13±9,69 años), predominio masculino y sobrepeso (IMC 26,27±6,68kg /m2), procedentes de La Paz 3.600 msnm (54%), Potosí 4.000 msnm (22%), El Alto 4.100 msnm (15%) y Oruro 3.800 msnm (9%), el promedio de Hematocrito 51,34±2,91%, hemoglobina 17,15±0,89gr/ dl, Espirometria forzada y Gasometría arterial en reposo e hiperoxia normales, la ECCTT muestra hipertensión pulmonar leve (35,85±3,64mmHg), aumento de grosor del VD (0,51±0,08), TAPSE (2,94±3,85mmHg) y el índice de Tei (0,44±0,22) normales. Los casos (n 40), la edad promedio (48,43±8,08 años), predominio masculino y sobrepeso (IMC 29,54±3,41kg / m2), procedente de La Paz 3.600 msnm (56%), Potosí 4.000 msnm (24%), El Alto 4.100 msnm (13%) y Oruro 3.800 msnm (7%), Hematocrito 63,08±6,2%, Hemoglobina 21,01±2,01gr/dl con eritrotrocitosis, espirometría forzada normal, gasometría arterial en reposo con hipoxemia moderada (PaO2 51,73±4,68mmHg), hipocapnia (PaCO2 27,62±2,04mmHg) y gradiente Alveolo-arterial aumentado (7,61±3,15). Gasometría arterial en hiperoxia descarta shunt (PaO2 308,9±52,58mmHg), el ECG muestra 2 de 11 criterios de crecimiento VD, la ECCTT con hipertensión pulmonar moderada (PSAP 45,22±5,69mmHg), aumento de grosor del VD (0,73±0,22), TAPSE (2,08±0,18cm), normal e índice de Tei (0,51±0,10) ligeramente aumentado. Se concluyó que la función ventricular derecha se encuentra conservada, a pesar de tener hipertensión pulmonar leve (controles) y moderada (casos), con aumento del grosor del ventrículo derecho.

Those genetically adapted to the height are the Tibetans, Sherpas, and Ethiopians; the Aymara and Quechuas are acclimatized (Bolivia, Peru, and northern Chile). In Bolivia, chronic mountain sickness affects 10% of the male population. The objective was to determine the right ventricular function in healthy residents with chronic mountain sickness using transthoracic echocardiography. Use the healthy and chronically ill mountain participants admitted by the IBBA, from 2012 to 2013. The variables taken are demographic, forced spirometry, arterial blood gas at rest and hyperoxia, ECG, and ECCTT. Controls (n 40), average age (44.13 ± 9.69 years), male predominance and overweight (BMI 26.27 ± 6.68kg / m2), frequency from La Paz 3,600 masl (54%), Potosí 4,000 masl (22%), El Alto 4,100 masl (15%) and Oruro 3,800 masl (9%), the average Hematocrit 51.34 ± 2.91%, hemoglobin 17.15 ± 0.89gr / dl, Forced spirometry y Resting arterial blood gas and normal hyperoxia, ECCTT shows mild pulmonary hypertension (35.85 ± 3.64 mmHg), increased RV thickness (0.51 ± 0.08), TAPSE (2.94 ± 3.85 mmHg ) and the Tei index (0.44 ± 0.22) normal. The cases (n 40), the average age (48.43 ± 8.08 years), male predominance and overweight (BMI 29.54 ± 3.41kg / m2), derived from La Paz 3,600 masl (56%), Potosí 4,000 masl (24%), El Alto 4,100 masl (13%) and Oruro 3,800 masl (7%), Hematocrit 63.08 ± 6.2%, Hemoglobin 21.01 ± 2.01gr / dl with erythrocytosis, normal forced spirometry , resting arterial blood gas with moderate hypoxemia (PaO2 51.73 ± 4.68mmHg), hypocapnia (PaCO2 27.62 ± 2.04mmHg) and increased Alveolo-arterial gradient (7.61 ± 3.15). Arterial blood gas in hyperoxia rules out shunt (PaO2 308.9 ± 52.58mmHg), ECG shows 2 of 11 RV growth criteria, ECCTT with moderate pulmonary hypertension (PSAP 45.22 ± 5.69mmHg), increased RV thickness (0.73 ± 0.22), TAPSE (2.08 ± 0.18cm), normal and Tei index (0.51 ± 0.10) slightly increased. It was concluded that the right ventricular function is preserved, a weight of having mild pulmonary hypertension (controls) and moderate (cases), with increased thickness of the right ventricle.

Minor temperature shifts do not affect chromosomal ploidy but cause transcriptomic changes in Leishmania braziliensis promastigotes in vitro

BACKGROUND The leishmaniases are complex neglected diseases caused by protozoan parasites of the genus Leishmania. Leishmania braziliensis is the main etiological agent of cutaneous leishmaniasis in the New World. In recent studies, genomic changes such as chromosome and gene copy number variations (CNVs), as well as transcriptomic changes have been highlighted as mechanisms used by Leishmania species to adapt to stress situations. OBJECTIVES The aim of this study was to determine the effect of short-term minor temperature shifts in the genomic and transcriptomic responses of L. braziliensis promastigotes in vitro. METHODS Growth curves, genome and transcriptome sequencing of L. braziliensis promastigotes were conducted from cultures exposed to three different temperatures (24ºC, 28ºC and 30ºC) compared with the control temperature (26ºC). FINDINGS Our results showed a decrease in L. braziliensis proliferation at 30ºC, with around 3% of the genes showing CNVs at each temperature, and transcriptomic changes in genes encoding amastin surface-like proteins, heat shock proteins and transport proteins, which may indicate a direct response to temperature stress. MAIN CONCLUSIONS This study provides evidence that L. braziliensis promastigotes exhibit a decrease in cell density, and noticeable changes in the transcriptomic profiles. However, there were not perceptible changes at chromosome CNVs and only ~3% of the genes changed their copies in each treatment.

Coping with drought: stress and adaptive mechanisms, and management through cultural and molecular alternatives in cotton as vital constituents for plant stress resilience and fitness

Increased levels of greenhouse gases in the atmosphere and associated climatic variability is primarily responsible for inducing heat waves, flooding and drought stress. Among these, water scarcity is a major limitation to crop productivity. Water stress can severely reduce crop yield and both the severity and duration of the stress are critical. Water availability is a key driver for sustainable cotton production and its limitations can adversely affect physiological and biochemical processes of plants, leading towards lint yield reduction. Adaptation of crop husbandry techniques suitable for cotton crop requires a sound understanding of environmental factors, influencing cotton lint yield and fiber quality. Various defense mechanisms e.g. maintenance of membrane stability, carbon fixation rate, hormone regulation, generation of antioxidants and induction of stress proteins have been found play a vital role in plant survival under moisture stress. Plant molecular breeding plays a functional role to ascertain superior genes for important traits and can offer breeder ready markers for developing ideotypes. This review highlights drought-induced damage to cotton plants at structural, physiological and molecular levels. It also discusses the opportunities for increasing drought tolerance in cotton either through modern gene editing technology like clustered regularly interspaced short palindromic repeat (CRISPR/Cas9), zinc finger nuclease, molecular breeding as well as through crop management, such as use of appropriate fertilization, growth regulator application and soil amendments.

Purification and characterization of cold-adapted beta-agarase from an Antarctic psychrophilic strain

An extracellular β-agarase was purified from Pseudoalteromonas sp. NJ21, a Psychrophilic agar-degrading bacterium isolated from Antarctic Prydz Bay sediments. The purified agarase (Aga21) revealed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular weight of 80 kDa. The optimum pH and temperature of the agarase were 8.0 and 30 °C, respectively. However, it maintained as much as 85% of the maximum activities at 10 °C. Significant activation of the agarase was observed in the presence of Mg²⁺, Mn²⁺, K⁺; Ca²⁺, Na⁺, Ba²⁺, Zn²⁺, Cu²⁺, Co²⁺, Fe²⁺, Sr²⁺ and EDTA inhibited the enzyme activity. The enzymatic hydrolyzed product of agar was characterized as neoagarobiose. Furthermore, this work is the first evidence of cold-adapted agarase in Antarctic psychrophilic bacteria and these results indicate the potential for the Antarctic agarase as a catalyst in medicine, food and cosmetic industries.

Progression of the load of waterborne and intestinal parasitic diseases in the State of Amazonas

In the State of Amazonas, Brazil, urban expansion together with precarious basic sanitation conditions and human settlement on river banks has contributed to the persistence of waterborne and intestinal parasitic diseases. Time series of the recorded cases of cholera, typhoid fever, hepatitis A and leptospirosis are described, using data from different levels of the surveillance systems. The sources for intestinal parasitosis prevalence data (non-compulsory reporting in Brazil) were Medical Literature Analysis and Retrieval System Online (MEDLINE), Literatura Latino-Americana (LILACS) and the annals of major scientific meetings. Relevant papers and abstracts in all languages were accessed by two independent reviewers. The references cited by each relevant paper were scrutinized to locate additional papers. Despite its initial dissemination across the entire State of Amazonas, cholera was controlled in 1998. The magnitude of typhoid fever has decreased; however, a pattern characterized by eventual outbreaks still remains. Leptospirosis is an increasing cause of concern in association with the annual floods. The overall prevalence of intestinal parasites is high regardless of the municipality and the characteristics of areas and populations. The incidence of hepatitis A has decreased over the past decade. A comparison of older and recent surveys shows that the prevalence of intestinal parasitic diseases has remained constant. The load of waterborne and intestinal parasitic diseases ranks high among the health problems present in the State of Amazonas. Interventions aiming at basic sanitation and vaccination for hepatitis A were formulated and implemented, but assessment of their effectiveness in the targeted populations is still needed.

The remarkable journey of adaptation of the Plasmodium falciparum malaria parasite to New World anopheline mosquitoes

Plasmodium falciparum originated in Africa, dispersed around the world as a result of human migration and had to adapt to several different indigenous anopheline mosquitoes. Anophelines from the New World are evolutionary distant form African ones and this probably resulted in a more stringent selection of Plasmodium as it adapted to these vectors. It is thought that Plasmodium has been genetically selected by some anopheline species through unknown mechanisms. The mosquito immune system can greatly limit infection and P. falciparum evolved a strategy to evade these responses, at least in part mediated by Pfs47, a highly polymorphic gene. We propose that adaptation of P. falciparum to new vectors may require evasion of their immune system. Parasites with a Pfs47 haplotype compatible with the indigenous mosquito vector would be able to survive and be transmitted. The mosquito antiplasmodial response could be an important determinant of P. falciparum population structure and could affect malaria transmission in the Americas.

Variables perinatales de recién nacidos de madres Aymara sugieren adaptación genética a la altura / Perinatal variables from newborns of Aymara mothers suggest a genetic adaptation to high altitude

Background: Studies performed in Andean populations living in high altitude, indicate that the reduced availability of oxygen could be associated to both a fetal growth retardation and a lower birth weight. These variables are predictive of morbidity and mortality during the first year of life. Aim: To study perinatal variables of newborns of mothers living at contrasting altitudinal levels, harboring different degrees of Aymara ancestry. Subjects and Methods: Review of medical records of 5,295 women whose deliveries occurred between February 2004 and August 2010. Information was obtained on place of residence, grouped into two categories: coast (150 to 3,000 m) and high plateau (3,000 to 4,300 m), ancestry was estimated using number of Aymara surnames that were homologated to percentages of Amerindian admixture, gestational age, birth weight, height, head circumference and obstetric variables. Results: Gestational age showed a tendency to increase and birth weight, height and head circumference to decrease with altitude of residence. Only weight reached statistical significance. Women with Aymara ancestry gave birth to children with a significantly higher gestational age, weight and cranial circumference. Conclusions: Altitude of residence is related to a decrease in perinatal variables that proved to be less pronounced in newborns of mothers with a higher degree of Aymara ancestry. Results suggest a genetic adaptation to hypoxia that could be related to candidate genes linked to the capture, transport or utilization of oxygen.

DNA sequence variation of drought-response candidate genes in Austrocedrus chilensis

Background: Austrocedrus chilensis (D. Don) Pic. Ser. et Bizzarri commonly known as Patagonian cypress is a member of the Cupressaceae family, characterized by a high adaptive potential for growing in marginal areas and good timber quality. The species grows over a wide area and under a wide range of rainfall. This study assessed adaptive genetic variation at SNP level in candidate genes involved in response to drought stress. Results: A total of 18 single nucleotide polymorphisms (SNPs) were found among 1,428 bp. Average nucleotide diversity value (π = 0.00312) was similar to those previously reported in other Cupressaceae. The Fst average among genes and populations was 0.163 and the lowest differentiation was observed in continuous and humid populations. A number of neutrality tests were applied to find evidence of positive selection in our candidate gene set, but only AcAQP2 gene in Pedregoso and San Ramón populations revealed significant departures from neutrality with positive values suggesting balancing selection. Conclusions: In this study we report the levels of nucleotide diversity searched in some drought stress candidate genes in Austrocedrus chilensis and the selective factors that may be acting on this species.

Phenotypic plasticity in fish life-history traits in two neotropical reservoirs: Petit-Saut Reservoir in French Guiana and Brokopondo Reservoir in Suriname

Fish species are known for their large phenotypic plasticity in life-history traits in relation to environmental characteristics. Plasticity allows species to increase their fitness in a given environment. Here we examined the life-history response of fish species after an abrupt change in their environment caused by the damming of rivers. Two reservoirs of different age, both situated on the Guiana Shield, were investigated: the young Petit-Saut Reservoir in French Guiana (14 years) and the much older Brokopondo Reservoir in Suriname (44 years). Six life-history traits in 14 fish species were studied and compared to their value in the Sinnamary River prior to the completion of Petit-Saut Reservoir. The traits analyzed were maximum length, absolute and relative length at first maturation, proportion of mature oocytes in ripe gonad, batch fecundity and mean size of mature oocytes. The results revealed a general increase of reproductive effort. All species showed a decrease in maximum length. Compared to the values observed before the dam constructions, eight species had larger oocytes and three species showed an increased batch fecundity. These observed changes suggest a trend towards a pioneer strategy. The changes observed in Petit-Saut Reservoir also seemed to apply to the 30 years older Brokopondo Reservoir suggesting that these reservoirs remain in a state of immaturity for a long time.(AU)

Peixes são conhecidos pela grande plasticidade fenotípica com que respondem às características do meio, o que lhes permite aumentar as chances de sucesso frente a variações ambientais. No presente trabalho foram examinadas as respostas biológicas de teleósteos após uma abrupta modificação no ambiente provocada pelo represamento dos rios. Dois reservatórios de diferentes idades, situados no norte da América do Sul, foram investigados: um mais jovem (14 anos), Petit-Saut, na Guiana Francesa, e outro mais antigo (44 anos), Brokopondo, no Suriname. Em 14 espécies de peixes foram avaliados seis atributos biológicos, os quais foram comparados com a situação apresentada no rio Sinnamary antes do enchimento do Reservatório Petit-Saut. Avaliamos o tamanho máximo dos indivíduos, os comprimentos absoluto e relativo de primeira maturação, a proporção de ovócitos maduros em gônadas desovantes, a fecundidade por lote, e o tamanho médio dos ovócitos maduros. Os resultados indicam ter havido aumento do esforço reprodutivo com a formação dos reservatórios. Todas as espécies tiveram redução de tamanho. Comparados aos valores observados antes da formação dos reservatórios, oito espécies tiveram ovócitos maiores e três espécies mostraram aumento da fecundidade por lote. A constatação dessas mudanças aponta para a adoção de estratégias de ocupação pioneira. Aquelas observadas no Reservatório Petit-Saut parecem também aplicar-se ao Reservatório Brokopondo, 30 anos mais antigo, sugerindo que esses reservatórios mantêm-se em condição imatura por muito tempo(AU)

Energy-responsive timekeeping.

An essential component of energy homeostasis lies in an organism's ability to coordinate daily patterns in activity, feeding, energy utilization and energy storage across the daily 24-h cycle. Most tissues of the body contain the molecular clock machinery required for circadian oscillation and rhythmic gene expression. Under normal circumstances, behavioural and physiological rhythms are orchestrated and synchronized by the suprachiasmatic nucleus (SCN) of the hypothalamus, considered to be the master circadian clock. However, metabolic processes are easily decoupled from the primarily light-driven SCN when food intake is desynchronized from normal diurnal patterns of activity. This dissociation from SCN based timing demonstrates that the circadian system is responsive to changes in energy supply and metabolic status. There has long been evidence for the existence of an anatomically distinct and autonomous food-entrainable oscillator (FEO) that can govern behavioural rhythms, when feeding becomes the dominant entraining stimulus. But now rapidly growing evidence suggests that core circadian clock genes are involved in reciprocal transcriptional feedback with genetic regulators of metabolism, and are directly responsive to cellular energy supply. This close interaction is likely to be critical for normal circadian regulation of metabolism, and may also underlie the disruption of proper metabolic rhythms observed in metabolic disorders, such as obesity and type-II diabetes.

Coadaptation and malaria control

Malaria emerges from a disequilibrium of the system 'human-plasmodium-mosquito' (HPM). If the equilibrium is maintained, malaria does not ensue and the result is asymptomatic plasmodium infection. The relationships among the components of the system involve coadaptive linkages that lead to equilibrium. A vast body of evidence supports this assumption, including the strategies involved in the relationships between plasmodium and human and mosquito immune systems, and the emergence of resistance of plasmodia to antimalarial drugs and of mosquitoes to insecticides. Coadaptive strategies for malaria control are based on the following principles: (1) the system HPM is composed of three highly complex and dynamic components, whose interplay involves coadaptive linkages that tend to maintain the equilibrium of the system; (2) human and mosquito immune systems play a central role in the coadaptive interplay with plasmodium, and hence, in the mainten-ance of the system's equilibrium; the under- or overfunction of human immune system may result in malaria and influence its severity; (3) coadaptation depends on genetic and epigenetic phenomena occurring at the interfaces of the components of the system, and may involve exchange of infectrons (genes or gene fragments) between the partners; (4) plasmodia and mosquitoes have been submitted to selective pressures, leading to adaptation, for an extremely long while and are, therefore, endowed with the capacity to circumvent both natural (immunity) and artificial (drugs, insecticides, vaccines) measures aiming at destroying them; (5) since malaria represents disequilibrium of the system HPM, its control should aim at maintaining or restoring this equilibrium; (6) the disequilibrium of integrated systems involves the disequilibrium of their components, therefore the maintenance or restoration of the system's equilibrium depend on the adoption of integrated and coordinated measures acting on all components,...

Insect resistance and risk assessment studies of advanced generations of basmati rice expressing two genes of Bacillus thuringiensis

Advanced generations of different transgenic lines of indica basmati rice (Basmati-370) expressing two unrelated Bt genes, cry1Ac and cry2A were evaluated for resistance to Yellow Stem Borer (YSB) and Rice Leaf Folder (RLF) under field conditions compared to control lines over three years (2003-2005). Homozygous lines were selected and analyzed for insect resistance, morphological, physiochemical properties and risk assessment studies. After artificial infestation of target insects, the transgenic plants showed significant resistance. Data were recorded in terms of dead hearts and white heads at vegetative and flowering stage respectively. Transgenic lines showed up to 100 and 96 percent resistance against yellow stem borer at vegetative and flowering stages, respectively. Natural damage of rice leaf folder was also observed during the year 2005. The transgenic plants were 98 percent more resistant as compared to untransformed control plants. Variations in some morphological characteristics, e.g., the average number of tillers, plant height and maturity were also observed. Transgenic lines produced 40 percent more grains than control plants. All these characteristics were stably inherited in advanced generations. The transgenic lines had no significant effect on non-target insects (insects belonging to orders other than Lepidoptera and Diptera) in field or under storage conditions. Chances of pollen-mediated gene flow were recorded at a rate of 0.14 percent.

Is premating isolation in Drosophila overestimated due to uncontrolled factors?

Sexual isolation in Drosophila is typically measured by multiple-choice mating tests. While many environmental variables during such tests are controlled by the researcher, there are some factors that are usually uncontrolled. We demonstrate, using Drosophila melanogaster and D. pseudoobscura flies, that the temperature of rearing, pre-adult density, and level of consanguinity, can all produce differences in mating propensity between genetically equivalent flies. These differences in mating propensity, in turn, can give rise to statistically significant results in multiple-choice mating tests, leading to positive isolation values and the artifactual inference of sexual isolation between populations. This fact agrees with a nonrandom excess of significant positive tests found in a review of the literature of Drosophila intraspecific mating choice. An overestimate of true cases of sexual isolation in Drosophila in the literature can, therefore, not be ruled out.

Differential display-mediated identification of three drought-responsive expressed sequence tags in tea [Camellia sinensis (L.) O. Kuntze].

There is no information on drought-modulated gene(s) in tea [Camellia sinensis (L.) O. Kuntze], a woody and perennial plant of commercial importance. Using differential display of mRNA, three drought-modulated expressed sequence tags (ESTs) were identified. Northern and BLAST analysis revealed that clone dr1 (drought-responsive), induced only by drought but not by ABA, showed significant scores with PR-5 (pathogenesis related) family of PR-protein gene. Another clone dr2, repressed by drought but not by ABA, had nucleotide repeats for polyasparate that are also present in chicken calsequestrin-like mRNA. Clone dr3, responded similarly to clone dr2 but did not show significant homology with the reported genes, hence appears to be novel. Identification of these ESTs is an initial step to clone the full length genes and their promoters.

Ecologically relevant stress resistance: from microarrays and quantitative trait loci to candidate genes - a research plan and preliminary results using Drosophila as a model organism and climatic and genetic stress as model stresses.

We aim at studying adaptation to genetic and environmental stress and its evolutionary implications at different levels of biological organization. Stress influences cellular processes, individual physiology, genetic variation at the population level, and the process of natural selection. To investigate these highly connected levels of stress effects, it is advisable - if not critical - to integrate approaches from ecology, evolution, physiology, molecular biology and genetics. To investigate the mechanisms of stress resistance, how resistance evolves, and what factors contribute to and constrain its evolution, we use the well-defined model systems of Drosophila species, representing both cosmopolitan species such as D. melanogaster with a known genome map, and more specialized and ecologically well described species such as the cactophilic D. buzzatii. Various climate-related stresses are used as model stresses including desiccation, starvation, cold and heat. Genetic stress or genetic load is modelled by studying the consequences of inbreeding, the accumulation of (slightly) deleterious mutations, hybridization or the loss of genetic variability. We present here a research plan and preliminary results combining various approaches: molecular techniques such as microarrays, quantitative trait loci (QTL) analyses, quantitative PCR, ELISA or Western blotting are combined with population studies of resistance to climatic and genetic stress in natural populations collected across climatic gradients as well as in selection lines maintained in the laboratory.

Tolerance to stress and environmental adaptability of Chromobacterium violaceum

Chromobacterium violaceum is a Gram-negative bacterium, abundant in a variety of ecosystems in tropical and subtropical regions, including the water and borders of the Negro River, a major component of the Amazon Basin. As a free-living microorganism, C. violaceum is exposed to a series of variable conditions, such as different sources and abundance of nutrients, changes in temperature and pH, toxic compounds and UV rays. These variations, and the wide range of environments, require great adaptability and strong protective systems. The complete genome sequencing of this bacterium has revealed an enormous number and variety of ORFs associated with alternative pathways for energy generation, transport-related proteins, signal transduction, cell motility, secretion, and secondary metabolism. Additionally, the limited availability of iron in most environments can be overcome by iron-chelating compounds, iron-storage proteins, and by several proteins related to iron metabolism in the C. violaceum genome. Osmotically inducible proteins, transmembrane water-channel, and other membrane porins may be regulating the movement of water and maintaining the cell turgor, activities which play an important role in the adaptation to variations in osmotic pressure. Several proteins related to tolerance against antimicrobial compounds, heavy metals, temperature, acid and UV light stresses, others that promote survival under starvation conditions, and enzymes capable of detoxifying reactive oxygen species were also detected in C. violaceum. All these features together help explain its remarkable competitiveness and ability to survive under different types of environmental stress

Gene expression in Chromobacterium violaceum

The repertoire of 4,431 open reading frames (ORFs), eight rRNA operons and 98 tRNA genes of Chromobacterium violaceum must be expressed in a regulated manner for successful adaptation to a wide variety of environmental conditions. To accomplish this feat, the organism relies on protein machineries involved in transcription, RNA processing and translation. Analysis of the C. violaceum genome showed that transcription initiation, elongation and termination are performed by the five well-known RNA polymerase subunits, five categories of sigma 70 factors, one sigma 54 factor, as well as six auxiliary elongation and termination factors. RNA processing is performed by a variety of endonucleases and exonucleases, such as ribonuclease H, ribonuclease E, ribonuclease P, and ribonuclease III, in addition to poly(A) polymerase and specific methyltransferases and pseudouridine synthases. ORFs for all ribosomal proteins, except S22, were found. Only 19 aminoacyl-tRNA synthetases were found, in addition to three aminoacyl-tRNA synthetase-related proteins. Asparaginyl-tRNA (Asn) is probably obtained by enzymatic modification of a mischarged aminoacyl-tRNA. The translation factors IF-1, IF-2, IF-3, EF-Ts, EF-Tu, EF-G, RF-1, RF-2 and RF-3 are all present in the C. violaceum genome, although the absence of selB suggests that C. violaceum does not synthesize selenoproteins. The components of trans-translation, tmRNA and associated proteins, are present in the C. violaceum genome. Finally, a large number of ORFs related to regulation of gene expression were also found, which was expected, considering the apparent adaptability of this bacterium

Thermal adaptation in Drosophila serrata under conditions linked to its southern border: unexpected patterns from laboratory selection suggest limited evolutionary potential.

To investigate the ability of Drosophila serrata to adapt to thermal conditions over winter at the species southern border, replicate lines from three source locations were held as discrete generations over three years at either 19 degrees C (40 generations) or temperatures fluctuating between 7 degrees C and 18 degrees C (20 generations). Populations in the fluctuating environment were maintained either with an adult 0 degrees C cold shock or without a shock. These conditions were expected to result in temperature-specific directional selection for increased viability and productivity under both temperature regimes, and reduced development time under the fluctuating-temperature regime. Selection responses of all lines were tested under both temperature regimes after controlling for carry-over effects by rearing lines in these environments for two generations. When tested in the 19 degrees C environment, lines evolving at 19 degrees C showed a faster development time and a lower productivity relative to the other lines, while cold shock reduced development time and productivity of all lines. When tested in the fluctuating environment, productivity of the 7-18 degrees C lines selected with a cold shock was relatively lower than that of lines selected without a shock, but this pattern was not observed in the other populations. Viability and body size as measured by wing length were not altered by selection or cold shock, although there were consistent effects of source population on wing length. These results provide little evidence for temperature-specific adaptation in D. serrata-although the lines had diverged for some traits, these changes were not consistent with a priori predictions. In particular, there was no evidence for life-history changes reflecting adaptation to winter conditions at the southern border. The potential for D. serrata to adapt to winter conditions may therefore be limited.

The contribution of ancestry, chance, and past and ongoing selection to adaptive evolution.

The relative contributions of ancestry, chance, and past and ongoing selection to variation in one adaptive (larval feeding rate) and one seemingly nonadaptive (pupation height) trait were determined in populations of Drosophila melanogaster adapting to either low or high larval densities in the laboratory. Larval feeding rates increased rapidly in response to high density, and the effects of ancestry, past selection and chance were ameliorated by ongoing selection within 15-20 generations. Similarly, in populations previously kept at high larval density, and then switched to low larval density, the decline of larval feeding rate to ancestral levels was rapid (15-20 generations) and complete, providing support for a previously stated hypothesis regarding the costs of faster feeding in Drosophila larvae. Variation among individuals was the major contributor to variation in pupation height, a trait that would superficially appear to be nonadaptive in the environmental context of the populations used in this study because it did not diverge between sets of populations kept at low versus high larval density for many generations. However, the degree of divergence among populations (F(ST)) for pupation height was significantly less than expected for a selectively neutral trait, and we integrate results from previous studies to suggest that the variation for pupation height among populations is constrained by stabilizing selection, with a flat, plateau-like fitness function that, consequently, allows for substantial phenotypic variation within populations. Our results support the view that the genetic imprints of history (ancestry and past selection) in outbreeding sexual populations are typically likely to be transient in the face of ongoing selection and recombination. The results also illustrate the heuristic point that different forms of selection-for example directional versus stabilizing selection-acting on a trait in different populations may often not be due to differently shaped fitness functions, but rather due to differences in how the fitness function maps onto the actual distribution of phenotypes in a given population. We discuss these results in the light of previous work on reverse evolution, and the role of ancestry, chance, and past and ongoing selection in adaptive evolution.