Aetiology and epidemiology of human cryptosporidiosis cases in Galicia (NW Spain), 2000-2008.

Cryptosporidium infects millions of people worldwide causing acute gastroenteritis, but despite its remarkable epidemiological and economic impact, information on the epidemiological trends of human cryptosporidiosis is still scarce in most countries. Here we investigate a panel of 486 cases collected in Galicia (NW Iberian Peninsula) between 2000 and 2008, which sheds new light on the epidemiology in this region of the South Atlantic European façade. Incidence rates in Galicia are one order of magnitude higher than those reported in other regions of Spain, suggesting that this parasite remains largely underdiagnosed in this country, and are also larger than those typical of other European countries with available data. Two species dominate our dataset, Cryptosporidium hominis (65%) and C. parvum (34%). The sex ratio of patients infected by either species was 0·5, but C. hominis was significantly more common in younger males. C. parvum infections were more acute and required more specialized medical attention, which suggests a differential adaptation of each species to human hosts. The parasites display strong seasonal and geographical variation. C. parvum incidence peaked during summer and was mainly detected in rural areas while C. hominis infections were more frequent in autumn and exhibited a more even geographical distribution. Such differences probably reflect their distinct sources of infection - C. parvum is mainly zoonotic and C. hominis anthroponotic - and the effects of climatic variables, like temperature and rainfall.

Is Acarapis woodi a single species? A new PCR protocol to evaluate its prevalence.

Acarapisosis is a disease of the adult honey bee Apis mellifera L., caused by the tracheal mite Acarapis woodi (Rennie), that affects the prothoracic tracheas of worker honey bees. Although it is not usually considered a real problem for honey bee colonies in southern Europe (mainly Spain and Greece), where the majority of professional beekeepers are located in Europe, recent works have reported the constant presence of this mite in this area, making it a potential cofactor for colony losses. In this study, we developed a specific PCR diagnostic tool that improves the techniques used so far and allowed us to confirm the presence of this parasite in Spain, urging the need to monitor its prevalence and implications in the health of the colonies. Indeed, in a total of 635 apiaries analysed, the prevalence of A. woodi in 2010 was 8.3 and 4 % in 2011. The mite is present in bee colonies over time and should not be underestimated as a possible cofactor in the collapse of bee colonies. Additionally, some positive samples were cloned so a genetic analysis on the diversity within A. woodi isolates was also approached. This allowed us to identify different genetic variants within an isolate, even when they were present at low frequencies. And this genetic analysis revealed the existence of a different clade of Acarapis sequences that could represent a new species or subspecies, although more research is required to verify the identity of this novel lineage at genetic and morphological level.

High-throughput genotyping assay for the large-scale genetic characterization of Cryptosporidium parasites from human and bovine samples.

The epidemiological study of human cryptosporidiosis requires the characterization of species and subtypes involved in human disease in large sample collections. Molecular genotyping is costly and time-consuming, making the implementation of low-cost, highly efficient technologies increasingly necessary. Here, we designed a protocol based on MALDI-TOF mass spectrometry for the high-throughput genotyping of a panel of 55 single nucleotide variants (SNVs) selected as markers for the identification of common gp60 subtypes of four Cryptosporidium species that infect humans. The method was applied to a panel of 608 human and 63 bovine isolates and the results were compared with control samples typed by Sanger sequencing. The method allowed the identification of species in 610 specimens (90·9%) and gp60 subtype in 605 (90·2%). It displayed excellent performance, with sensitivity and specificity values of 87·3 and 98·0%, respectively. Up to nine genotypes from four different Cryptosporidium species (C. hominis, C. parvum, C. meleagridis and C. felis) were detected in humans; the most common ones were C. hominis subtype Ib, and C. parvum IIa (61·3 and 28·3%, respectively). 96·5% of the bovine samples were typed as IIa. The method performs as well as the widely used Sanger sequencing and is more cost-effective and less time consuming.

Multilocus patterns of genetic variation across Cryptosporidium species suggest balancing selection at the gp60 locus.

Cryptosporidium is an apicomplexan protozoan that lives in most vertebrates, including humans. Its gp60 gene is functionally involved in its attachment to host cells, and its high level of genetic variation has made it the reference marker for sample typing in epidemiological studies. To understand the origin of such high diversity and to determine the extent to which this classification applies to the rest of the genome, we analysed the patterns of variation at gp60 and nine other nuclear loci in isolates of three Cryptosporidium species. Most loci showed low genetic polymorphism (πS <1%) and similar levels of between-species divergence. Contrastingly, gp60 exhibited very different characteristics: (i) it was nearly ten times more variable than the other loci; (ii) it displayed a significant excess of polymorphisms relative to between-species differences in a maximum-likelihood Hudson-Kreitman-Aguadé test; (iii) gp60 subtypes turned out to be much older than the species they were found in; and (iv) showed a significant excess of polymorphic variants shared across species from random expectations. These observations suggest that this locus evolves under balancing selection and specifically under negative frequency-dependent selection (FDS). Interestingly, genetic variation at the other loci clusters very well within the groups of isolates defined by gp60 subtypes, which may provide new tools to understand the genome-wide patterns of genetic variation of the parasite in the wild. These results suggest that gp60 plays an active and essential role in the life cycle of the parasite and that genetic variation at this locus might be essential for the parasite's long-term success.

Hidden effects of X chromosome introgressions on spermatogenesis in Drosophila simulans x D. mauritiana hybrids unveiled by interactions among minor genetic factors.

One of the most frequent outcomes of interspecific hybridizations in Drosophila is hybrid male sterility. Genetic dissection of this reproductive barrier has revealed that the number of responsible factors is very high and that these factors are frequently engaged in complex epistatic interactions. Traditionally, research strategies have been based on contrasting introgressions of chromosome segments that produce male sterility with those that allow fertility. Few studies have investigated the phenotypes associated with the boundary between fertility and sterility. In this study, we cointrogressed three different X chromosome segments from Drosophila mauritiana into D. simulans. Hybrid males with these three segments are usually fertile, by conventional fertility assays. However, their spermatogenesis shows a significant slowdown, most manifest at lower temperatures. Each of the three introgressed segments retards the arrival of sperm to the seminal vesicles. Other small disturbances in spermatogenesis are evident, which altogether lead to an overall reduction in the amount of motile sperm in their seminal vesicles. These results suggest that a delay in the timing of spermatogenesis, which might be brought about by the cumulative action of many different factors of minor segment, may be the primary cause of hybrid male sterility.

Recent worldwide expansion of Nosema ceranae (Microsporidia) in Apis mellifera populations inferred from multilocus patterns of genetic variation.

Nosema ceranae has been found infecting Apismellifera colonies with increasing frequency and it now represents a major threat to the health and long-term survival of these honeybees worldwide. However, so far little is known about the population genetics of this parasite. Here, we describe the patterns of genetic variation at three genomic loci in a collection of isolates from all over the world. Our main findings are: (i) the levels of genetic polymorphism (πS≈1%) do not vary significantly across its distribution range, (ii) there is substantial evidence for recombination among haplotypes, (iii) the best part of the observed genetic variance corresponds to differences within bee colonies (up to 88% of the total variance), (iv) parasites collected from Asian honeybees (Apis cerana and Apis florea) display significant differentiation from those obtained from Apismellifera (8-16% of the total variance, p<0.01) and (v) there is a significant excess of low frequency variants over neutral expectations among samples obtained from A. mellifera, but not from Asian honeybees. Overall these results are consistent with a recent colonization and rapid expansion of N. ceranae throughout A. mellifera colonies.

A long-term study on seasonal changes of gametic disequilibrium between allozymes and inversions in Drosophila subobscura.

Seasonal variation (spring, early summer, late summer, and autumn) of gametic disequilibrium between gene arrangements (OST and O3+4) of the O chromosome and Lap, Pept-1, and Acph allozyme loci, located inside these inversions, has been recorded in a natural population of Drosophila subobscura during seven years over a 15-year period. The length of the study allowed us to investigate the temporal variation of the allozyme-inversion associations by statistical methods of time series analysis. Cyclic seasonal changes of allozyme-inversion associations for both Lap and Pept-1 are detected in the natural population. In both cases, the patterns of seasonal change are due to the seasonal change of frequency of Lap and Pept-1 allozymes occurring exclusively within the OST gene arrangement. In contrast, the allozyme frequencies at these loci within the O3+4 gene arrangement are stable along seasons. The patterns of temporal variation of allozyme-inversion associations for Lap and Pept-1 in the natural population are contrasted with those previously published that correspond to gene arrangements of the O chromosome and nucleotide polymorphism at the rp49 region located inside these inversions, suggesting that natural selection is operating on these allozyme-inversion associations.

On the difficulties of discriminating between major and minor hybrid male sterility factors in Drosophila by examining the segregation ratio of sterile and fertile sons in backcrossing experiments.

The observation of segregation ratios of sterile and fertile males in offspring samples from backcrossed hybrid females is, in principle, a valid method to unveil the genetic basis of hybrid male sterility in Drosophila. When the female parent is heterozygous (hybrid) for a sterility factor with major effects, equal proportions of fertile and sterile sons are expected in her offspring. However, intact (not recombined) chromosome segments of considerable length are expected to give segregation ratios that can not be easily differentiated from the 1:1 ratio expected from a single factor. When the phenotypic character under analysis can be determined by combinations of minor factors from the donor species spanning a certain chromosome length, very large offspring samples may be needed to test this alternative hypothesis against the null hypothesis of a single major factor. This is particularly the case of hybrid male sterility determinants in Drosophila.

Rates of movement of transposable elements on the second chromosome of Drosophila melanogaster.

The rates of movement of 11 families of transposable elements of Drosophila melanogaster were studied by means of in situ hybridization of probes to polytene chromosomes of larvae from a long-term mutation accumulation experiment. Replicate mutation-accumulation lines carrying second chromosomes derived from a single common ancestral chromosome were maintained by backcrosses of single males heterozygous for a balancer chromosome and a wild-type chromosome, and were scored after 116 generations. Twenty-seven transpositions and 1 excision were detected using homozygous viable and fertile second chromosomes, for a total of 235,056 potential sources of transposition events and a potential 252,880 excision events. The overall transposition rate per element per generation was 1.15 x 10(-4) and the excision rate was 3.95 x 10(-6). The single excision (of a roo element) was due to recombination between the element's long terminal repeats. A survey of the five most active elements among nine homozygous lethal lines revealed no significant difference in the estimates of transposition and excision rates from those from viable lines. The excess of transposition over excision events is in agreement with the results of other in situ hybridization experiments, and supports the conclusion that replicative increase in transposable element copy number is opposed by selection. These conclusions are compared with those from other studies, and with the conclusions from population surveys of element frequencies.

A polygenic basis of hybrid sterility may give rise to spurious localizations of major sterility factors.

A simple model is presented to illustrate how an underlying generalized polygenic basis of hybrid sterility is expected to lead to spurious localizations of factors with major effects, when a conventional experiment of recombination mapping is carried out. The model shows that a major gene will be detected at roughly the same distance from each of the chromosome markers used in the experiment. These expectations are contrasted with the results from several experiments on hybrid male sterility in Drosophila, which claimed to have mapped single sterility factors. It is concluded that, except for one report, all the evidence presented so far on the genetic basis of hybrid male sterility in Drosophila is in fact compatible with the generalized polygenic model.

Rates of movement and distribution of transposable elements in Drosophila melanogaster: in situ hybridization vs Southern blotting data.

Genomic copy numbers and the rates of movement of nine families of transposable elements (TEs) of Drosophila melanogaster were estimated in two sets of mutation accumulation lines: Beltsville and Madrid. Southern blotting was used to screen a large number of samples from both genetic backgrounds for TEs. The Madrid lines were also screened by in situ hybridization of TEs to polytene chromosomes, in order to obtain more detailed information about the behaviour of TEs in the euchromatin. Southern blotting data provided evidence of insertions and excision events in both genetic backgrounds, occurring at rates of approximately 10(-5) and 10(-6) per element copy per generation, respectively. In contrast, in situ data from the Madrid background presented a completely different picture, with no evidence for excisions, and a significantly higher rate of transposition (1.01 x 10(-4)). Direct comparison of the two data sets suggests that the Southern blotting technique had serious deficiencies: (i) it underestimated element abundance; (ii) it revealed less than 30% of the new insertions detected by in situ hybridization; and (iii) changes in the size of restriction fragments from any source were spuriously identified as simultaneous insertion-excision events. Our in situ data are consistent with previous studies, and suggest that selection is the main force controlling element spread by transposition.