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[This corrects the article DOI: 10.1371/journal.ppat.1007705.].
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Comparative studies using non-parasitic model species such as Caenorhabditis elegans, have been very helpful in investigating the basic biology and evolution of parasitic nematodes. However, as phylogenetic distance increases, these comparisons become more difficult, particularly when outside of the nematode clade to which C. elegans belongs (V). One of the reasons C. elegans has nevertheless been used for these comparisons, is that closely related well characterized free-living species that can serve as models for parasites of interest are frequently not available. The Clade IV parasitic nematodes Strongyloides are of great research interest due to their life cycle and other unique biological features, as well as their medical and veterinary importance. Rhabditophanes, a closely related free-living genus, forms part of the Strongyloidoidea nematode superfamily. Rhabditophanes diutinus (= R. sp. KR3021) was included in the recent comparative genomic analysis of the Strongyloididae, providing some insight into the genomic nature of parasitism. However, very little is known about this species, limiting its usefulness as a research model. Here we provide a species description, name the species as R. diutinus and investigate its life cycle and subsequently gene expression in multiple life stages. We identified two previously unreported starvation induced life stages: dauer larvae and arrested J2 (J2A) larvae. The dauer larvae are morphologically similar to and are the same developmental stage as dauers in C. elegans and infective larvae in Strongyloides. As in C. elegans and Strongyloides, dauer formation is inhibited by treatment with dafachronic acid, indicating some genetic control mechanisms are conserved. Similarly, the expression patterns of putative dauer/infective larva control genes resemble each other, in particular between R. diutinus and Strongyloides spp. These findings illustrate and increase the usefulness of R. diutinus as a non-parasitic, easy to work with model species for the Strongyloididae for studying the evolution of parasitism as well as many aspects of the biology of Strongyloides spp, in particular the formation of infective larvae.
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Strongyloidea/fisiología , Animales , Larva , Estadios del Ciclo de Vida , PartenogénesisRESUMEN
Host-seeking behaviour and how a parasite identifies the correct host to infect remains a poorly understood area of parasitology. What is currently known is that host sensation and seeking behaviour is formed from a complex mixture of chemo-, thermo- and mechanosensory behaviours, of which chemosensation is the best studied. Previous studies of olfaction in parasitic nematodes suggested that this behaviour appears to be more closely related to target host and infection mode than phylogeny. However, there has not yet been a study comparing the chemotactic and temperature-dependent behaviours of very closely related parasitic and non-parasitic nematodes. To this end, we examined the temperature-dependent and chemotactic responses of the Strongyloidoidea superfamily of nematodes. We found differences in temperature response between the different species and within infective larvae. Chemotactic responses were highly divergent, with different attraction profiles between all species studied. When examining direct stimulation with fur, we found that it was insufficient to cause an attractive response. Overall, our results support the notion that olfactory sensation is more closely related to lifestyle and host range than phylogeny, and that multiple cues are required to initiate host-seeking behaviour.
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Nematodos , Rabdítidos , Animales , Interacciones Huésped-Parásitos/fisiología , Larva/fisiología , Nematodos/fisiología , TemperaturaRESUMEN
The recent progress in sequencing technology allowed the compilation of gene lists for a large number of organisms, though many of these organisms are hardly experimentally tractable when compared with well-established model organisms. One popular approach to further characterize genes identified in a poorly tractable organism is to express these genes in a model organism, and then ask what the protein does in this system or if the gene is capable of replacing the homologous endogenous one when the latter is mutated. While this is a valid approach for certain questions, I argue that the results of such experiments are frequently wrongly interpreted. If, for example, a gene from a parasitic nematode is capable of replacing its homologous gene in the model nematode Caenorhabditis elegans, it is often concluded that the gene is most likely involved in the same biological process in its own organism as the C. elegans gene is in C. elegans. This conclusion is not valid. All this experiment tells us is that the chemical properties of the parasite protein are similar enough to the ones of the C. elegans protein that it can perform the function of the C. elegans protein in C. elegans. Here I discuss this misconception and illustrate it using the analog of similar electric switches (components) controlling various devices (processes).
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Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genéticaRESUMEN
The gene daf-12 has long shown to be involved in the dauer pathway in Caenorhabditis elegans (C. elegans). Due to the similarities of the dauer larvae of C. elegans and infective larvae of certain parasitic nematodes such as Strongyloides spp., this gene has also been suspected to be involved in the development of infective larvae. Previous research has shown that the application of dafachronic acid, the steroid hormone ligand of DAF-12 in C. elegans, affects the development of infective larvae and metabolism in Strongyloides. However, a lack of tools for either forward or reverse genetics within Strongyloides has limited studies of gene function within these important parasites. After determining whether Strongyloides had the requisite proteins for RNAi, we developed and report here the first successful RNAi by soaking protocol for Strongyloides ratti (S. ratti) and use this protocol to study the functions of daf-12 within S. ratti. Suppression of daf-12 in S. ratti severely impairs the formation of infective larvae of the direct cycle and redirects development towards the non-infective (non-dauer) free-living life cycle. Further, daf-12(RNAi) S. ratti produce slightly but significantly fewer offspring and these offspring are developmentally delayed or incapable of completing their development to infective larvae (L3i). Whilst the successful daf-12(RNAi) L3i are still able to infect a new host, the resulting infection is less productive and shorter lived. Further, daf-12 knockdown affects metabolism in S. ratti resulting in a shift from aerobic towards anaerobic fat metabolism. Finally, daf-12(RNAi) S. ratti have reduced tolerance of temperature stress.
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Proteínas de Caenorhabditis elegans/genética , Técnicas de Silenciamiento del Gen/métodos , Receptores Citoplasmáticos y Nucleares/genética , Strongyloides ratti/genética , Secuencia de Aminoácidos/genética , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Colestenos , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas del Helminto , Larva , Estadios del Ciclo de Vida , Filogenia , Interferencia de ARN/fisiología , ARN Interferente Pequeño/genética , Ratas , Ratas Wistar , Receptores Citoplasmáticos y Nucleares/metabolismo , Homología de Secuencia de Aminoácido , Strongyloides ratti/metabolismoRESUMEN
Nematodes are found in virtually all habitats on earth. Many of them are parasites of plants and animals, including humans. The free-living nematode, Caenorhabditis elegans, is one of the genetically best-studied model organisms and was the first metazoan whose genome was fully sequenced. In recent years, the draft genome sequences of another six nematodes representing four of the five major clades of nematodes were published. Compared to mammalian genomes, all these genomes are very small. Nevertheless, they contain almost the same number of genes as the human genome. Nematodes are therefore a very attractive system for comparative genetic and genomic studies, with C. elegans as an excellent baseline. Here, we review the efforts that were made to extend genetic analysis to nematodes other than C. elegans, and we compare the seven available nematode genomes. One of the most striking findings is the unexpectedly high incidence of gene acquisition through horizontal gene transfer (HGT).
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Genoma de los Helmintos , Estadios del Ciclo de Vida , Nematodos/genética , Animales , Evolución Biológica , Femenino , Regulación del Desarrollo de la Expresión Génica , Transferencia de Gen Horizontal , Variación Genética , Masculino , Nematodos/clasificación , Nematodos/crecimiento & desarrollo , Nematodos/patogenicidad , Filogenia , Especificidad de la EspecieRESUMEN
Onchocerca ochengi is a nodule-forming filarial nematode parasite of cattle. It is the closest known relative of the human parasite Onchocerca volvulus, with which it shares the black fly vector Simulium damnosum. Onchocerca sp. "Siisa" was described in black flies and in cattle and, based on limited mitochondrial sequence information, appeared to be about equally phylogenetically distant from O. ochengi and O. volvulus. Based on molecular genetic markers and apparent interbreeding, we later proposed that O. sp. "Siisa" belongs to the species O. ochengi. However, we did not demonstrate directly that the hybrids were fertile, and we were still unable to resolve the phylogenetic relationship of O. ochengi, O. sp. "Siisa," and O. volvulus, leaving some concerns with the conclusion mentioned above. Here, we present fully assembled, manually curated mitochondrial genomes of O. ochengi and O. sp. "Siisa," and we compare multiple individuals of these two taxa with respect to their whole mitochondrial and nuclear genomes. Based on the mitochondrial genomes, O. ochengi and O. sp. "Siisa" are phylogenetically much closer to each other than to O. volvulus. The differences between them are well within the range of what is expected for within-species variation. The nuclear genome comparison provided no indication of genetic separation of O. ochengi and O. sp. "Siisa." From this, in combination with the earlier literature, we conclude that O. ochengi and O. sp. "Siisa" should be considered one species.
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Genoma Mitocondrial/genética , Genoma de Protozoos/genética , Onchocerca/clasificación , Onchocerca/genética , Animales , Bovinos/parasitología , Enfermedades de los Bovinos/parasitología , Humanos , Insectos Vectores/parasitología , Mitocondrias/genética , Onchocerca/aislamiento & purificación , Oncocercosis/parasitología , Filogenia , Polimorfismo de Nucleótido Simple/genética , Simuliidae/parasitología , Piel/parasitologíaRESUMEN
Nematodes of the genus Strongyloides are important parasites of vertebrates including man. Currently, little is known about their germline organization or reproductive biology and how this influences their parasitic life strategies. Here, we analyze the structure of the germline in several Strongyloides and closely related species and uncover striking differences in the development, germline organization, and fluid dynamics compared to the model organism Caenorhabditis elegans. With a focus on Strongyloides ratti, we reveal that the proliferation of germ cells is restricted to early and mid-larval development, thus limiting the number of progeny. In order to understand key germline events (specifically germ cell progression and the transcriptional status of the germline), we monitored conserved histone modifications, in particular H3Pser10 and H3K4me3. The evolutionary significance of these events is subsequently highlighted through comparisons with six other nematode species, revealing underlying complexities and variations in the development of the germline among nematodes.
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Células Germinativas/metabolismo , Histonas/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Procesos de Determinación del Sexo/fisiología , Strongyloides/metabolismo , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismo , Diferenciación Celular , Proliferación Celular , Metilación , Reproducción/fisiología , Strongyloides/citología , Strongyloides/genéticaRESUMEN
Nematodes of the genus Strongyloides are intestinal parasites of vertebrates including man. Currently, Strongyloides and its sister genus Parastrongyloides are being developed as models for translational and basic biological research. Strongyloides spp. alternate between parthenogenetic parasitic and single free-living sexual generations, with the latter giving rise to all female parasitic progeny. Parastrongyloides trichosuri always reproduces sexually and may form many consecutive free-living generations. Although the free-living adults of both these species share a superficial similarity in overall appearance when compared to Caenorhabditis elegans, there are dramatic differences between them, in particular with respect to the organization of the germline. Here we address two such differences, which have puzzled investigators for several generations. First, we characterize a population of non-dividing giant nuclei in the distal gonad, the region that in C. elegans is populated by mitotically dividing germline stem cells and early meiotic cells. We show that in these nuclei, autosomes are present in higher copy numbers than X chromosomes. Consistently, autosomal genes are expressed at higher levels than X chromosomal ones, suggesting that these worms use differential chromatin amplification for controlling gene expression. Second, we address the lack of males in the progeny of free-living Strongyloides spp. We find that male-determining (nullo-X) sperm are present in P. trichosuri, a species known to produce male progeny, and absent in Strongyloides papillosus, which is consistent for a species that does not. Surprisingly, nullo-X sperm appears to be present in Strongyloides ratti, even though this species does not produce male progeny. This suggests that different species of Strongyloides employ various strategies to prevent the formation of males in the all-parasitic progeny of the free-living generation.
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Cromosomas , Células Germinativas , Ploidias , Reproducción/genética , Strongyloides/genética , Animales , Femenino , Regulación del Desarrollo de la Expresión Génica , Gónadas , Masculino , Strongyloides/fisiologíaRESUMEN
Classical and reverse genetics remain invaluable tools for the scientific investigation of model organisms. Genetic analysis of endoparasites is generally difficult because the sexual adults required for crossing and other manipulations are usually hidden within their host. Strongyloides spp. and Parastrongyloides spp. are notable exceptions to this and their free-living adults offer unique opportunities to manipulate these parasites experimentally. Here I review the modes of inheritance in the two generations of Strongyloides/Parastrongyloides and I discuss the opportunities and the limitations of the currently available methodology for the genetic analysis of these two genera.
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Biología Molecular/métodos , Reproducción , Strongyloides/genética , Strongyloides/fisiología , AnimalesRESUMEN
The nematode genus Strongyloides consists of fairly species-specific small intestinal parasites of various vertebrates, among them the human pathogen S. stercoralis. Between the parthenogenetic parasitic generations these worms can also form single facultative sexual free-living generations. In addition to their primary hosts, several species can also live more or less well in other permissive hosts, which are sometimes not very closely related with the normal host. For example, S. stercoralis can also infect dogs and non-human primates. Here we compare the infection and reproductive success over time and the gene expression profiles as determined by quantitative sequencing of S. ratti parasitizing in its natural host rat and in the permissive host gerbil. We show that in gerbils fewer infective larvae successfully establish in the host, but those that do accomplish this survive and reproduce for longer and produced a higher proportion of males during the first two month of infection. Globally, the gene expression profiles in the two hosts are very similar. Among the relatively few differentially expressed genes, astacin-like and acetylcholinesterase genes are prominently represented. In the future it will be interesting to see if these changes in the suboptimal host are indeed ecologically sensible responses to the different host.
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Gerbillinae/parasitología , Ratas Wistar/parasitología , Enfermedades de los Roedores/parasitología , Strongyloides ratti/fisiología , Estrongiloidiasis/parasitología , Transcriptoma , Animales , Heces/parasitología , Femenino , Expresión Génica , Especificidad del Huésped , Interacciones Huésped-Parásitos , Masculino , Ratas , Razón de Masculinidad , Regulación hacia ArribaRESUMEN
The rat parasitic nematode Strongyloides ratti (S. ratti) has recently emerged as a model system for various aspects of parasite biology and evolution. In addition to parasitic parthenogenetic females, this species can also form facultative free-living generations of sexually reproducing adults. These free-living worms are bacteriovorous and grow very well when cultured in the feces of their host. However, in fecal cultures the worms are rather difficult to find for observation and experimental manipulation. Therefore, it has also been attempted to raise S. ratti on Nematode Growth Media (NGM) plates with Escherichia coli OP50 as food, exactly as described for the model nematode Caenorhabditis elegans. Whilst worms did grow on these plates, their longevity and reproductive output compared to fecal cultures were dramatically reduced. In order to improve the culture success we tested other plates occasionally used for C. elegans and, starting from the best performing one, systematically varied the plate composition, the temperature and the food in order to further optimize the conditions. Here we present a plate culturing protocol for free-living stages of S. ratti with strongly improved reproductive success and longevity.
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Medios de Cultivo , Strongyloides ratti/crecimiento & desarrollo , Agar , Animales , Medios de Cultivo/química , Medios de Cultivo/normas , Escherichia coli , Heces/parasitología , Fertilidad , Alimentos , Longevidad , Oviposición , Reproducción , Strongyloides ratti/fisiología , TemperaturaRESUMEN
The diplogastrid nematode Pristionchus pacificus is a nematode model system for comparative studies to Caenorhabditis elegans and integrative evolutionary biology aiming for interdisciplinary approaches of evo-devo, population genetics, and ecology. For this, fieldwork can be combined with laboratory studies, and P. pacificus has a well-developed methodological toolkit of forward genetics, whole genome sequencing, DNA-mediated transformation, and various -omics platforms. Here, we establish CRISPR/Cas9-based gene inactivation and describe various boundary conditions of this methodology for P. pacificus. Specifically, we demonstrate that most mutations arise within the first 9 hours after injections. We systematically tested the efficiency of sgRNAs targeting different exons in Ppa-dpy-1 and characterized the molecular nature of the induced mutations. Finally, we provide a protocol that might also be useful for researchers working with other non-Caenorhabditis nematodes.
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Sistemas CRISPR-Cas , Técnicas de Inactivación de Genes , Nematodos/genética , Animales , Clonación Molecular , Nematodos/clasificaciónRESUMEN
Genetic analysis using experimentally induced mutations has been a most valuable tool in the analysis of various organisms. However, genetic analysis of endoparasitic organisms tends to be difficult because of the limited accessibility of the sexually reproducing adults, which are normally located within the host. Nematodes of the genera Strogyloides and Parastrongyloides represent an exception to this because they can form facultative free-living sexually reproducing generations in between parasitic generations. Here we present a protocol for the chemical mutagenesis of Strongyloides ratti. Further we evaluate the feasibility of identifying the induced mutations by whole genome re-sequencing.
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Genoma de los Helmintos/fisiología , Mutagénesis/fisiología , Strongyloides ratti/genética , Animales , Metanosulfonato de Etilo/farmacología , Estudios de Factibilidad , Femenino , Genoma de los Helmintos/efectos de los fármacos , Estudio de Asociación del Genoma Completo , Mutagénesis/efectos de los fármacos , Mutágenos/farmacología , Ratas , Ratas Wistar , Análisis de Secuencia de ADN , Strongyloides ratti/efectos de los fármacosRESUMEN
Parasitic lifestyles evolved many times independently. Just within the phylum Nematoda animal parasitism must have arisen at least four times. Switching to a parasitic lifestyle is expected to lead to changes in various life history traits including reproductive strategies. Parasitic nematode worms of the genus Strongyloides represent an interesting example to study these processes because they are still capable of forming facultative free-living generations in between parasitic ones. The parasitic generation consists of females only, which reproduce parthenogenetically. The sex in the progeny of the parasitic worms is determined by environmental cues, which control a, presumably ancestral, XX/XO chromosomal sex determining system. In some species the X chromosome is fused with an autosome and one copy of the X-derived sequences is removed by sex-specific chromatin diminution in males. Here I propose a hypothesis for how today's Strongyloides sp. might have evolved from a sexual free-living ancestor through dauer larvae forming free-living and facultative parasitic intermediate stages.
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Nematodos/genética , Cromosomas Sexuales/genética , Strongyloides/genética , Animales , Evolución Biológica , Femenino , Larva , Masculino , ReproducciónRESUMEN
BACKGROUND: About 600 million people are estimated to be infected with Strongyloides stercoralis, the species that causes most of the human strongyloidiasis cases. S. stercoralis can also infect non-human primates (NHPs), dogs and cats, rendering these animals putative sources for zoonotic human S. stercoralis infection. S. fuelleborni is normally found in old world NHPs but occasionally also infects humans, mainly in Africa. Dogs in southeast Asia carry at least two types of Strongyloides, only one of which appears to be shared with humans ("dog only" and "human and dog" types). For S. stercoralis with molecular taxonomic information, there is a strong sampling bias towards southeast and east Asia and Australia. METHODOLOGY/PRINCIPLE FINDINGS: In order to extend the geographic range of sampling, we collected human and dog derived Strongyloides spp. and hookworms from two locations in Bangladesh and subjected them to molecular taxonomic and genomic analysis based on nuclear and mitochondrial sequences. All hookworms found were Necator americanus. Contrary to earlier studies in Asia, we noticed a rather high incidence of S. fuelleborni in humans. Also in this study, we found the two types of S. stercoralis and no indication for genetic isolation from the southeast Asian populations. However, we found one genomically "dog only" type S. stercoralis in a human sample and we found two worms in a dog sample that had a nuclear genome of the "dog only" but a mitochondrial genome of the "human and dog" type. CONCLUSIONS/SIGNIFICANCE: S. fuelleborni may play a more prominent role as a human parasite in certain places in Asia than previously thought. The introgression of a mitochondria haplotype into the "dog only" population suggests that rare interbreeding between the two S. stercoralis types does occur and that exchange of genetic properties, for example a drug resistance, between the two types is conceivable.
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BACKGROUND: Strongyloidiasis is a neglected tropical disease (NTD) that is caused mainly by Strongyloides stercoralis, with an estimated 600 million people infected worldwide, and in fewer cases by Strongyloides fuelleborni fuelleborni and Strongyloides fuelleborni kellyi. A number of studies have been conducted on the genetic diversity of S. stercoralis in East and Southeast Asia; however, there is very limited corresponding information from West Asian countries, including Iran. METHODS: For Strongyloides worms collected from patients in southwestern Iran, the hypervariable regions I (HVR-I) and IV (HVR-IV) of the nuclear 18S ribosomal DNA (rDNA) locus (SSU) and a fragment of the subunit 1 mitochondrial cytochrome c oxidase gene (cox-1) were sequenced. For a subset of the worms, whole-genome sequencing data were generated. RESULTS: The cox-1 sequences of 136 worms isolated from 23 patients indicated that all isolates were S. stercoralis. Among the cox-1 sequences, 33 polymorphic sites and 13 haplotypes were found. The phylogenetic analysis demonstrated that some sequences clustered fairly closely with sequences from humans and dogs from other parts of the world, while others formed a separate, Iran-specific group. Among 64 S. stercoralis analyzed, we found three of the previously described SSU HVR-I haplotypes, with haplotype II being the most frequent haplotype. In contrast to Southeast Asia, where S. stercoralis heterozygous for different haplotypes at the HVR-I locus are rare, we found 20 worms to be heterozygous for two different HVR-I haplotypes, 18 of which fell into the Iran-specific cox-1 cluster. SSU-heterozygous worms also showed elevated heterozygosity at the whole-genome level. CONCLUSIONS: We conclude that the S. stercoralis population from the Khuzestan province shares much of the genetic diversity with the population in Southeast Asia, but there is an indication of additional genetic input. There appears to be some population structure with different subpopulations, which however do interbreed at least occasionally.
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Strongyloides stercoralis , Estrongiloidiasis , Humanos , Animales , Perros , Strongyloides stercoralis/genética , Genotipo , Filogenia , Irán/epidemiología , Estrongiloidiasis/epidemiología , Estrongiloidiasis/veterinaria , Strongyloides , HecesRESUMEN
The Strongyloides genus of parasitic nematodes have a fascinating life cycle and biology, but are also important pathogens of people and a World Health Organization-defined neglected tropical disease. Here, a community of Strongyloides researchers have posed thirteen major questions about Strongyloides biology and infection that sets a Strongyloides research agenda for the future. This article is part of the Theo Murphy meeting issue 'Strongyloides: omics to worm-free populations'.
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Estadios del Ciclo de Vida , Strongyloides , Animales , HumanosRESUMEN
The parasitic roundworms Strongyloides stercoralis (in man) and Strongyloides ratti (in rats) employ environmentally controlled XX/XO sex determination with a pair of X chromosomes and two pairs of autosomes. Strongyloides papillosus (in sheep) has only two pairs of chromosomes, one of which combines the genetic material homologous to the S. ratti chromosomes X and I. This species creates males through the elimination of one copy of the portion related to the X chromosome (chromatin diminution). It is not clear which one of these two sex-determining mechanisms is ancestral. We demonstrate that Strongyloides vituli (in cattle) has two pairs of chromosomes like its very close relative S. papillosus whereas Parastrongyloides trichosuri, a closely related out-group to Strongyloides spp. in Australian brushtail possums, has three chromosome pairs and employs XX/XO sex determination. The X chromosome of P. trichosuri is homologous to the X chromosome of S. ratti. Our data strongly suggest that the last common ancestor of Strongyloides spp. and Parastrongyloides spp. had two pairs of autosomes along with two or one X chromosome in females and males, respectively. The situation with two pairs of chromosomes is likely derived and occurred through the fusion of the X chromosome with an autosome.
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Evolución Biológica , Rabdítidos/genética , Procesos de Determinación del Sexo/genética , Cromosoma X , Animales , Femenino , Cariotipo , MasculinoRESUMEN
Paramphistomidae and Gastrothylacidae are parasitic flatworms occurring in wild and domestic ruminants in different parts of the world especially in Asia and Africa. In Central Africa, few studies have been done using molecular techniques to resolve taxonomical groupings and understand the epizootiology of these parasites. In this study, we molecularly characterized two hundred adult flukes collected from the fore stomachs of cattle and sheep in the Adamawa region of the northern Cameroon. PCR and sequencing of the nuclear ITS-2 of the ribosomal DNA gene and a portion of the mitochondrial cox-1 locus revealed the presence of at least nine species belonging to the genera of Cotylophoron, Calicophorn, Orthocoelium and Carmyerius. In Zebu cattle, we identified Ca. microbothrium, Ca. clavula, Ca. phillerouxi, Co. cotylophorum, Co. fuelleborni, O. scoliocoelium, Car. gregarius, Car. graberi and Car. mancupatus and one yet unknown Paramphistomoidea sp, whereas in sheep, only Ca. microbothrium was found. The present study also strongly suggests cross-hybridization between the two Cotylophoron species coexisting in cattle. These results have implications for the diagnosis and control of rumen flukes in the region and point to the need for accurate species identification to understand parasite distribution and population genetics.