Niche evolution and thermal adaptation in the temperate species Drosophila americana.

The study of ecological niche evolution is fundamental for understanding how the environment influences species' geographical distributions and their adaptation to divergent environments. Here, we present a study of the ecological niche, demographic history and thermal performance (locomotor activity, developmental time and fertility/viability) of the temperate species Drosophila americana and its two chromosomal forms. Temperature is the environmental factor that contributes most to the species' and chromosomal forms' ecological niches, although precipitation is also important in the model of the southern populations. The past distribution model of the species predicts a drastic reduction in the suitable area for the distribution of the species during the last glacial maximum (LGM), suggesting a strong bottleneck. However, DNA analyses did not detect a bottleneck signature during the LGM. These contrasting results could indicate that D. americana niche preference evolves with environmental change, and thus, there is no evidence to support niche conservatism in this species. Thermal performance experiments show no difference in the locomotor activity across a temperature range of 15 to 38 °C between flies from the north and the south of its distribution. However, we found significant differences in developmental time and fertility/viability between the two chromosomal forms at the model's optimal temperatures for the two forms. However, results do not indicate that they perform better for the traits studied here in their respective optimal niche temperatures. This suggests that behaviour plays an important role in thermoregulation, supporting the capacity of this species to adapt to different climatic conditions across its latitudinal distribution.

Two distinct genomic regions, harbouring the period and fruitless genes, affect male courtship song in Drosophila montana.

Acoustic signals often have a significant role in pair formation and in species recognition. Determining the genetic basis of signal divergence will help to understand signal evolution by sexual selection and its role in the speciation process. An earlier study investigated quantitative trait locus for male courtship song carrier frequency (FRE) in Drosophila montana using microsatellite markers. We refined this study by adding to the linkage map markers for 10 candidate genes known to affect song production in Drosophila melanogaster. We also extended the analyses to additional song characters (pulse train length (PTL), pulse number (PN), interpulse interval, pulse length (PL) and cycle number (CN)). Our results indicate that loci in two different regions of the genome control distinct features of the courtship song. Pulse train traits (PTL and PN) mapped to the X chromosome, showing significant linkage with the period gene. In contrast, characters related to song pulse properties (PL, CN and carrier FRE) mapped to the region of chromosome 2 near the candidate gene fruitless, identifying these genes as suitable loci for further investigations. In previous studies, the pulse train traits have been found to vary substantially between Drosophila species, and so are potential species recognition signals, while the pulse traits may be more important in intra-specific mate choice.

A case of canine ocular onchocercosis in Portugal.

Onchocercosis is a newly recognized disease in dogs that has been reported with higher frequency in Europe and in the United States. We report a case of a 3-year-old male mongrel stray dog from the Algarve region (South Portugal) who had a retrobulbar granuloma containing a filaroid nematode of the genus Onchocerca. A gravid adult female parasite was embedded in a granulomatous inflammation adjacent to the sclera beyond the retina. The parasite was 191 to 267 mum in diameter (mean = 225 mum), surrounded by a cuticule and owing a uterus that was filled with small unsheated microfilariae. The cuticule consisted of two separated layers in longitudinal sections. The external layer had cuticular ridges and the internal layer contained striations. Sequencing of the COI and ND5 mitochondrial genes confirmed the identity of this parasite as Onchocerca lupi. Furthermore, the first sequence of the 12S mitochondrial gene is reported in this study.

The species delimitation problem in the Simulium damnosum complex, blackfly vectors of onchocerciasis.

The Simulium damnosum Theobald complex (Diptera: Simuliidae) comprises 57 cytoforms grouped into six subcomplexes. Previous phylogenetic studies using gene sequences have not completely resolved the evolutionary relationships of the cytoforms. The present study investigated the systematics of the complex using a phylogeographic approach. The differentiation between eastern and western forms observed in the phylogenetic studies is confirmed in the estimated haplotype networks. However, haplotypes tend to group in geographical clades and not according to cytoforms. Spatial analyses of the molecular variance also resulted in optimal groupings of sequences that did not correspond to cytoform boundaries. Moreover, Mantel tests showed significant correlations, although not strong, between genetic and geographical distances. This suggests an isolation-by-distance model of differentiation. Furthermore, there are instances in which genetic differentiation between cytoforms is low and not significant. These results indicate a lack of clear genetic differentiation between the cytoforms, which may be explained either by a separation of the taxa recent enough to allow the accumulation of few genetic differences or by recombination between the genomes of the cytoforms, which may be the result of hybridization with introgression or of non-independent evolutionary lineages. The results also emphasize the need for further sampling and for the use of more variable markers in order to clarify the evolutionary history of the group.

Comparative analysis of five immunity-related genes reveals different levels of adaptive evolution in the virilis and melanogaster groups of Drosophila.

Five immunity-related genes previously reported to be evolving under positive selection in Drosophila melanogaster and D. simulans have been analysed across the Drosophila genus using two types of approaches, random-site and branch-site likelihood models as well as the proportion of synonymous and non-synonymous variation within and between species. Different selective pressures have been detected in the sample of genes, one showing evidence for adaptive evolution across the phylogeny of Drosophila and two showing lineage-specific positive selection. Furthermore, amino-acid sites identified as being under positive selection in the melanogaster and the virilis groups are different, suggesting that the evolution of the proteins in these two divergent groups may have been shaped by different pathogens.

Molecular phylogeny of the filaria genus Onchocerca with special emphasis on Afrotropical human and bovine parasites.

Filarial parasites of the genus Onchocerca are found in a broad spectrum of ungulate hosts. One species, O. volvulus, is a human parasite that can cause severe disease (onchocerciasis or 'river blindness'). The phylogenetic relationships and the bionomics of many of the nearly 30 known species remain dubious. Here, the phylogeny of 11 species representing most major lineages of the genus is investigated by analysing DNA sequences from three mitochondrial genes (ND5, 12S and 16S rRNA) and portions of the intergenic spacer of the nuclear 5s rRNA. Special emphasis is given to a clade containing a yet unassigned specimen from Uganda (O. sp. 'Siisa'), which appears to be intermediate between O. volvulus and O. ochengi. While the latter can be differentiated by the O-150 tandem repeat commonly used for molecular diagnostics, O. volvulus and O. sp.'Siisa' cannot be differentiated by this marker. In addition, a worm specimen from an African bushbuck appears to be closely related to the bovine O. dukei and represents the basal taxon of the human/bovine clade. At the base of the genus, our data suggest O. flexuosa (red deer), O. ramachandrini (warthog) and O. armillata (cow) to be the representatives of ancient lineages. The results provide better insight into the evolution and zoogeography of Onchocerca. They also have epidemiological and taxonomic implications by providing a framework for more accurate molecular diagnosis of filarial larvae in vectors.

Molecular systematics of five Onchocerca species (Nematoda: Filarioidea) including the human parasite, O. volvulus, suggest sympatric speciation.

The genus Onchocerca (Nematoda: Filarioidea) consists of parasites of ungulate mammals with the exception of O. volvulus, which is a human parasite. The relationship between O. volvulus, O. ochengi and O. gibsoni remains unresolved. Based on morphology of the microfilariae and infective larvae, vector transmission and geographical distribution, O. ochengi and O. volvulus have been placed as sister species. Nevertheless, the cuticle morphology and chromosomal data (O. volvulus and O. gibsoni have n=4 while O. ochengi is n=5) suggest that O. gibsoni could be more closely related to O. volvulus than O. ochengi. Sequences from the 12S rRNA, 16S rRNA and ND5 mitochondrial genes have been used to reconstruct the phylogeny of five Onchocerca species including O. volvulus. Analyses with maximum likelihood and maximum parsimony showed that O. ochengi is the sister species of O. volvulus, in accordance with the classification based on morphology and geographical location. The separate specific status of the species O. gutturosa and O. lienalis was supported, although their phylogenetic relationship was not well resolved. The analyses indicated that the basal species was O. gibsoni, a South-East Asian and Australasian species, but this result was not statistically significant. The possible involvement of sympatric speciation in the evolution of this group of parasites is discussed.

Two new mutations in cis on (G)gamma chain of fetal hemoglobin: Hb F-Madrid [G gamma 50(D1)Ser-->Cys] and [G gamma 75(E19)Ile-->Thr].

We describe a new structural hemoglobin variant of (G)gamma with two amino acid replacements in cis found in the umbilical cord blood of a neonate in Madrid, Spain. The substitutions were identified on exon 2 of the (G)gamma globin gene, at codon 50 (T CT-->T GT) and at codon 75 (A TA-->A CA). We have named it Hb F-Madrid. The father of the propositus was the carrier of the same (G)gamma chain variant and, moreover, molecular study of alpha genes revealed the loss of an alpha gene (-alpha(3.7)/alpha alpha) both in the propositus and his mother.

Completion of the sequence of the nuclear ribosomal DNA subunit of Simulium sanctipauli, with descriptions of the 18S, 28S genes and the IGS.

We describe the IGS-ETS, 18S and 28S ribosomal gene sequences of Simulium sanctipauli Vajime & Dunbar, a member of the S. damnosum Theobald (Diptera: Simuliidae) complex of blackflies (Diptera: Simuliidae). These regions, together with the ITS-1, ITS-2 and 5.8S rDNA presented elsewhere (accession number U36206), constitute the composite sequence of the entire rDNA unit, making S. sanctipauli the second dipteran species of medical importance for which the entire rDNA has been sequenced. Despite the lack of sequence identity, the IGS of S. sanctipauli showed some structural similarities to other Diptera, i.e. the mosquito Aedes albopictus Skuse (Culicidae), the fruitfly Drosophila melanogaster Meigen (Drosophilidae) and the tsetse Glossina (Glossinidae). Two blocks of tandemly repeated subunits were present in the IGS of S. sanctipauli and, unlike other species of Diptera, they contained no duplications of promoter-like sequences. However, two promoter-like sequences were identified in the unique DNA stretches of the IGS by their sequence similarity to the promoter of Aedes aegypti L. (Diptera: Culicidae). The observed sequence variation can be explained, as in the case of Drosophila spp., by the occurrence of slippage-like and point mutation processes, with unequal crossing-over homogenizing (to a certain extent) the region throughout the gene family and blackfly population. The 18S and 28S rDNA genes show more intraspecific variability within the expansion segments than in the core regions. This is also the case in the interspecific comparison of these genes from S. sanctipauli with those of Simulium vittatum, Ae. albopictus and D. melanogaster. This pattern is typical of many eukaryotes and likely to be the result of a more relaxed functional selection in the expansion segments than on the core regions. The A + T content of the S. sanctipauli genes is high and similar to those of other Diptera. This could be the result of a change in the mutation pressure towards AT in the Diptera lineage.

Assessment of rDNA IGS as a molecular marker in the Simulium damnosum complex.

For five cytospecies of the Simulium damnosum Theobald complex of blackflies (Diptera: Simuliidae) from West Africa, both ends of the intergenic spacer region (IGS) of the rDNA have been sequenced with the aim of developing specific molecular markers. No specific differences in these two regions were detected between Simulium sanctipauli V. & D., Simulium sirbanum V. & D., Simulium soubrense V. & D., Simulium squamosum Enderlein and Simulium yahense V. & D., except in the number of A subrepeats at the 5' end of the IGS (two in S. squamosum and four or five in the others) and in position 310 of the 3' end (a C in S. squamosum and a G in the others). However, genetic distances within and between species overlapped. These DNA sequences had no strong phylogenetic signal, and the trees obtained were mostly unresolved. Although most sequences from S. squamosum clustered together, a few of them were more similar to those in other cytospecies. These results could be explained either by hybridization with genetic introgression or by ancestral polymorphism and recent speciation.

Molecular heterogeneity of myophosphorylase deficiency (McArdle's disease): a genotype-phenotype correlation study.

We report on 54 Spanish patients with McArdle's disease from 40 unrelated families. Molecular analysis revealed that the most common R49X mutation was present in 70% of patients and 55% of alleles. The G204S mutation was less frequent and found in 14.8% of patients and 9% of mutant alleles. The W797R mutation was observed in 16.5% of patients, accounting for 13.7% of mutant alleles. Moreover, 78% of mutant alleles among Spanish patients can be identified by using polymerase chain reaction-restriction fragment length polymorphism analysis for the R49X, G204S, and W797R mutations, which makes noninvasive diagnosis possible through molecular genetic analysis of blood DNA. Six novel mutations were found. Three were missense mutations, E348K, R601W, and A703V; two nonsense mutations, E124X and Q754X; and one single base pair deletion, 533 delA. No clear genotype-phenotype correlation emerges from our study. Most of the mutations of uncharged and solvent inaccessible residues and the truncations must disrupt the basic structure of the protein. The mutations of charged residues would be expected to interfere with internal hydrogen bonding networks, introducing severe incompatible partnering that is caused by poor packing or electrostatic repulsions.

Lipomatosis simétrica múltiple: Aportaciónde un caso y revisión de la literatura / Multiple symmetrical lipomatosis. report of a case and review of the literature

La lipomatosis simétrica múltiple es una enfermedad que se caracteriza por presentar múltiples y simétricas masas de tejido adiposo no encapsulado, indoloras y localizadas frecuentemente en cuello, hombros y otras partes del tronco. Habitualmente se asocia a alcoholismo y trastornos metabólicos, y puede cursar con afectación mediastínica y neuropatía. Presentamos el caso de un paciente de 63 años de edad, con importante hábito enólico. Se describen las características clínico-morfológicas, los hallazgos histopatológicos de una biopsia de grasa subcutánea y músculo esquelético, además de otras pruebas complementarias y los posibles tratamientos electivos. Se incluye, además, una amplia revisión de la literatura sobre esta patología (AU)

Characterisation of nuclear ribosomal DNA sequences from Onchocerca volvulus and Mansonella ozzardi (Nematoda: Filarioidea) and development of a PCR-based method for their detection in skin biopsies.

The internal transcribed spacer region (ITS1, 5.8S gene and ITS2) of the two filarial nematodes Onchocerca volvulus and Mansonella ozzardi was sequenced, and two species-specific primers designed in the ITS2 to develop a PCR-based method for their specific detection and differentiation. When used with a universal reverse primer, the two species-specific primers gave amplification products of different size, which were readily separated in an agarose gel. The PCR was tested on skin biopsies from 51 people from three localities in Brazil where M. ozzardi is present, and results have been compared with those of parasitological examination of blood. The species-specific PCR gave a higher percentage of detection of infection by M. ozzardi than the parasitological examination of blood. No infection with O. volvulus was detected by PCR. This PCR-based assay may assist in determining the nature of infection in areas where both filarial species exist in sympatry.

Autosomal dominant limb-girdle muscular dystrophy: a large kindred with evidence for anticipation.

BACKGROUND: Fourteen genetically distinct forms of limb-girdle muscular dystrophy (LGMD) have been identified, including five types of autosomal dominant LGMD (AD-LGMD). OBJECTIVE: To describe clinical, histologic, and genetic features of a large Spanish kindred with LGMD and apparent autosomal dominant inheritance spanning five generations. METHOD: The authors examined 61 members of the family; muscle biopsies were performed on five patients. Linkage analysis assessed chromosomal loci associated with other forms of AD-LGMD. RESULTS: A total of 32 individuals had weakness of the pelvic and shoulder girdles. Severity appeared to worsen in successive generations. Muscle biopsy findings were nonspecific and compatible with MD. Linkage analysis to chromosomes 5q31, 1q11-q21, 3p25, 6q23, and 7q demonstrated that this disease is not allelic to LGMD forms 1A, 1B, 1C, 1D, and 1E. CONCLUSIONS: This family has a genetically distinct form of AD-LGMD. The authors are currently performing a genome-wide scan to identify the disease locus.

Evaluation of heart involvement in gamma-sarcoglycanopathy (LGMD2C). A study of ten patients.

Thorough non-invasive cardiovascular studies were conducted in a series of ten gamma-sarcoglycanopathy Gypsy patients with the founder C283Y mutation in 13q12. Results were compared with those obtained in an age-matched group of normal boys and girls. The studies included electrocardiographic and echocardiographic evaluations using pulsed wave Doppler tissue imaging to assess regional diastolic function and myocardial velocities at various levels. This study confirms the significant electrocardiographic abnormalities described in previous studies. Furthermore, measurement of myocardial velocity at different levels demonstrated an abnormal relaxation pattern in the tricuspid annulus in four of the oldest patients, which strongly suggests intrinsic myocardial involvement of the right ventricle. To our knowledge, these specific studies have not been previously performed in a clinically and genetically homogeneous group of gamma-sarcoglycanopathy patients and suggest primary myocardial involvement probably due to gamma-sarcoglycan deficiency in cardiac muscle fibres. Our results could be of interest in the follow-up of these patients and the prevention and treatment of late cardiological complications.

Regional genetic variation in the major sperm protein genes of Onchocerca volvulus and Mansonella ozzardi (Nematoda: Filarioidea).

Onchocerca volvulus and Mansonella ozzardi are two human filarial parasites present in South and Central America. In the Brazilian Amazonia they are found in sympatry, and the lack of clear morphological diagnostic characters in the microfilariae hinders their identification. The major sperm protein (MSP) gene of both species has been sequenced and characterised to determine its potential as a molecular diagnostic character. The length of the MSP gene is different in each species, and this could be used to detect and differentiate them by running the polymerase chain reaction (PCR) product in an agarose gel. Two major gene groups were identified in O. volvulus with a genetic distance of 6% between them. In M. ozzardi only one major group of genes was observed. The high similarity between the protein amino acid sequence of both filarial species confirms that the MSP has been highly conserved through nematode evolution.

[Gamma-sarcoglycanopathy: clinico-pathological and genetic study of 11 cases]. / Gamma-sarcoglicanopatía: estudio clinicopatológico y genético de 11 casos.

INTRODUCTION: Limb Girdle Muscular Dystrophy type 2C (LGMD2C) is an autosomal recessive dystrophy due to the deficit of gamma-sarcoglycan, one of the proteins of the dystrophin-associated proteins complex (DAP). A new mutation in the gamma-sarcoglycan gene, 13q12, has been described recently and is exclusive of the gypsy community. OBJECTIVE: To describe the clinicopathological and the genetic findings of eleven cases from a Spanish gypsy family with LGMD2C and the mutation C283Y. MATERIAL AND METHODS: We describe a large gypsy family with the C283Y mutation and eleven affected patients. We have performed an extensive clinical and pathological study with immunohistochemistry and Western blot analyses in the eleven patients and a genetic study of a total of twenty-seven members of the family. RESULTS: The patients presented a severe muscular dystrophy with a dystrophic pattern in the muscle biopsy, normal immunolabeling for dystrophin, very weak for alpha-, beta- and delta-sarcoglycan and absent for gamma-sarcoglycan. These eleven patients were found to be homozygous for the mutation and twelve other members of the family, heterozygous. CONCLUSIONS: The clinical picture and the evolution of the disease herein described is similar to that observed in DMD. Two fundamental differences were found: the autosomal recessive mode of inheritance, and the normal immunohistochemistry and immunoblot for dystrophin in the skeletal muscle.

[Dystrophinopathies, congenital muscular dystrophy, limb-girdle dystrophies: updated classification]. / Distrofinopatías, distrofia muscular congénita y distrofias de cinturas: clasificación actualizada.

OBJECTIVES: To review the up-dated classification of limb girdle muscular dystrophies (LGMDs) in relation to the defective protein and the genetic abnormality. To explain how these proteins are related to dystrophin and to the proteins of the extracellular matrix. To show that an accurate diagnosis is necessary and that it can be adequately made in neuromuscular pathology laboratories. DEVELOPMENT: We present a study of the different types of LGMDs, dystrophinopathies and congenital muscular dystrophy. We emphasize the recent events which concluded in the identification of these disorders, the genetic alteration, the defective proteins and, briefly, the clinical features. CONCLUSIONS: The recent identification of numerous skeletal muscle proteins and of the codifying genes made possible a new classification of a large group of muscular dystrophies. The possibility to study these proteins on the muscle biopsy with immunohistochemistry and Western blot techniques indicates the need of an accurate diagnosis in specialized neuromuscular laboratories. Since there is a great number of genes discovered and of mutations within the same gene, and the clinical picture of different diseases can be similar, a previous study of the protein is advisable as a guide for a further genetic study.

[Portal venous flow (ultrasonography-Doppler) in patients with alcoholic liver cirrhosis]. / Flujo venoso portal (ultrasonografía-Doppler) en pacientes cirróticos alcohólicos.

To evaluate the clinical usefulness of the ultrasonographic (echo-Doppler) measurements of portal blood flow their results were compared with several clinical and biochemical parameters in alcoholic cirrhotic patients. The technique was standardized and its reproducibility was checked in 30 cirrhotics and in 20 control subjects. In controls, portal area was greater when measured on its transversal axis and in deep inspiration. In cirrhotics the area did not change neither according to the axis nor the respiratory movements. The estimated velocity of blood flow was dependent on the angle of insonation. Measurements performed on longitudinal axis, at 50 degrees in expiratory apnea showed, in the same subject, an interday variability of 7%. In cirrhotic patients portal blood flow was higher than in controls (0.93 +/- 0.32 L/min vs 0.64 +/- 0.12, p < 0.001) being the difference due to a greater area. Considering controls and Child's A and B cirrhotic patients, portal blood flow correlated with the severity of disease (r = 0.738, p < 0.001). In Child C, portal blood flow was decreased, compared to Child's B patients. The presence and size of esophageal varices was also correlated to portal blood flow (r = 0.461, p < 0.05). However no differences were observed between the groups with or without previous variceal bleeding. It is concluded that echo-doppler measurement of portal blood flow is a reproducible technic in standardized conditions, detecting changes related to global liver function and the presence and size of esophageal varices.