Scott P. Lawton

Molecular phylogenetics of tsetse flies (Diptera: Glossinidae) based on mitochondrial (COI, 16S, ND2) and nuclear ribosomal DNA sequences, with an emphasis on the palpalis group

Relationships of 13 species of the genus Glossina (tsetse flies) were inferred from mitochondrial (cytochrome oxidase 1, NADH dehydrogenase 2 and 16S) and nuclear (internal transcribed spacer 1 of rDNA) sequences. The resulting phylogeny confirms the monophyly of the morphologically defined fusca, morsitans and palpalis subgenera. Genetic distances between palpalis and morsitans subspecies suggest that their status needs revision. In particular, cytochrome oxidase 1 sequences showed large geographical differences within G. palpalis palpalis, suggesting the existence of cryptic species within this subspecies. The morphology of palpalis group female genital plates was examined, and individuals were found varying outside the ranges specified by the standard identification keys, making definitive morphological classification impossible. A diagnostic PCR to distinguish G. palpalis palpalis, G. tachinoides and G. palpalis gambiensis based on length differences of internal transcribed spacer 1 sequences is presented.

Genomes and geography: genomic insights into the evolution and phylogeography of the genus Schistosoma

Blood flukes within the genus Schistosoma still remain a major cause of disease in the tropics and subtropics and the study of their evolution has been an area of major debate and research. With the advent of modern molecular and genomic approaches deeper insights have been attained not only into the divergence and speciation of these worms, but also into the historic movement of these parasites from Asia into Africa, via migration and dispersal of definitive and snail intermediate hosts. This movement was subsequently followed by a radiation of Schistosoma species giving rise to the S. mansoni and S. haematobium groups, as well as the S. indicum group that reinvaded Asia. Each of these major evolutionary events has been marked by distinct changes in genomic structure evident in differences in mitochondrial gene order and nuclear chromosomal architecture between the species associated with Asia and Africa. Data from DNA sequencing, comparative molecular genomics and karyotyping are indicative of major constitutional genomic events which would have become fixed in the ancestral populations of these worms. Here we examine how modern genomic techniques may give a more in depth understanding of the evolution of schistosomes and highlight the complexity of speciation and divergence in this group.

Morphological and molecular characterization of a marine fish trypanosome from South Africa, including its development in a leech vector

BackgroundTrypanosomes are ubiquitous blood parasites of marine and freshwater fishes, typically transmitted by aquatic leeches. Phylogenetic studies have been dominated by examples derived from freshwater fishes, with few marine representatives. Furthermore, life cycle studies on marine fish trypanosomes have focused on those of the northern hemisphere. In this investigation, we have examined the life cycle and molecular taxonomy of a marine fish trypanosome from South Africa.MethodsTo locate trypanosome stages, leeches were removed from fishes captured on the west and south coasts of South Africa, and fish blood films and leech squashes were Giemsa-stained and screened; leeches were also examined histologically. To determine whether trypanosome stages in fishes and leeches were of the same genotype, DNA was extracted from Giemsa-stained fish blood films and leech squashes, and from fish whole blood. Fragments of the 18S rRNA gene were amplified by PCR using trypanosome-specific primers and sequenced. Resulting sequence data were compared with each other and with published trypanosome 18S rDNA sequences, and used for phylogenetic analysis.ResultsTrypanosomes were detected in blood films from fishes of the families Clinidae, Blenniidae and Gobiidae. The flagellates ranged in size and staining properties within the films and across fish hosts. In squashes and histological sections of adult and juvenile leeches, identified as Zeylanicobdella arugamensis, trypanosome developmental stages were predominantly slender epimastigotes. Sequence data showed that trypanosomes derived from fishes were identical, irrespective of whether they were small or large forms; sequences derived largely from leech epimastigotes were also identical to those obtained from fish trypanosomes. Fish and leech trypanosome sequences fell into a marine fish aquatic clade, and aligned most closely with two trypanosome sequences from marine fishes off Norway.ConclusionsCombined morphological and molecular methods indicate that the trypanosomes examined here represent a single pleomorphic species, rather than the three species described originally. This species is identified as Trypanosoma nudigobii Fantham, 1919 with the leech Z. arugamensis as its vector, and T. capigobii Fantham, 1919 and T. blenniclini Fantham, 1930 are regarded as junior synonyms of the species. Phylogenetic analysis establishes its affinity with marine fish trypanosomes off Norway.

Unravelling the riddle of Radix: DNA barcoding for species identification of freshwater snail intermediate hosts of zoonotic digeneans and estimating their inter-population evolutionary relationships

Radix spp. are intermediate host snails for digenean parasites of medical and veterinary importance. Within this genus, species differentiation using shell and internal organ morphology can result in erroneous species identification, causing problems when trying to understand the population biology of Radix. In the present study, DNA barcoding, using cox1 and ITS2 sequences, identified populations of Radix auricularia and Radix balthica from specimens originally morphologically identified as Radix peregra from the UK. Assessment of cox1 and ITS2 as species identification markers showed that, although both markers differentiated species, cox1 possessed greater molecular diversity and higher phylogenetic resolution. Cox1 also proved useful for gaining insights into the evolutionary relationships of Radix species populations. Phylogenetic analysis and haplotype networks of cox1 indicated that R. auricularia appeared to have invaded the UK several times; some haplotypes forming a distinct UK specific clade, whilst others are more akin to those found on mainland Europe. This was in contrast to relationships between R. balthica populations, which had low molecular diversity and no distinct UK specific haplotypes, suggesting recent and multiple invasions from mainland Europe. Molecular techniques therefore appear to be crucial for distinguishing Radix spp., particularly using cox1. This barcoding marker also enables the population biology of Radix spp. to be explored, and is invaluable for monitoring the epidemiology of fluke diseases especially in the light of emerging diseases and food security.

Aporocotyle michaudi n. sp. (Digenea: Aporocotylidae) from the emerald rock cod, Trematomus bernacchii (Teleostei: Perciformes) in Antarctica

Aporocotyle michaudi n. sp. is described from the gill blood vessels of the emerald rock cod Trematomus bernacchii in the Ross Sea, Antarctica. It is distinguished from all other species of Aporocotyle by its body tegument showing single conical spines, spinous buccal capsule, and genital atrium positioned medially; all congeners described to date are characterized by clusters of tegumental spines, unspined buccal capsule and genital atrium located in the lateral part of the body. Aporocotyle michaudi n. sp. clearly differs from A. notothenia (the only other species of Aporocotyle found in a perciform fish) in its shape and arrangement of tegumental spines, buccal capsule features, location of genital atrium, body size, ratio of esophagus/body length, anterior caeca/posterior caeca ratio, number of testes, cirrus sac and ovary size and shape, and host. The new species is easily distinguished from A. argentinensis (the species that most closely resembles A. michaudi) by the shape and arrangement of tegumental spines, buccal capsule features, genital atrium location, left anterior caecum longer than right, esophagus/body length ratio, number of testes, cirrus sac size and shape, host and molecular analyses. Phylogenetic analyses of partial 28S rDNA genetic data showed that sequences representing the new species form a distinct clade with all other sequences for species of Aporocotyle and appear basal within the genus. Aporocotyle michaudi n. sp. represents the only species of genus described in Antarctica.

Adaptive radiation within the vaccine target tetraspanin-23 across nine Schistosoma species from Africa

High levels of polymorphism in DNA sequences of tetraspanin-23 (TSP-23) were revealed within and between nine different species of Schistosoma from Africa including Schistosoma mansoni, Schistosoma rodhaini, Schistosoma margrebowiei, Schistosoma mattheei, Schistosoma intercalatum, Schistosoma haematobium, Schistosoma guineensis, Schistosoma curassoni and Schistosoma bovis. The greatest levels of diversity coincided with evidence of positive selection (d(N)/d(S)>1) within regions that code for extracellular loops of TSP-23 believed to interact with the host immune system. Kolaskar and Tongaonkar antigenicity predictions of protein sequences were compared across species and high levels of variation in antigenicity were also identified with each species which possessed their own unique antigenic profile. Phylogenetic analysis of TSP-23 proteins suggested evidence of convergent evolution in antigenic lineages as no true inter-species phylogenetic relationships were seen. This could be indicative of host-specific evolution of antigens in different species of schistosomes, a factor that should be considered carefully when developing vaccine targets.

Identification of a major causative agent of human cercarial dermatitis, Trichobilharzia franki (Müller and Kimmig 1994), in southern England and its evolutionary relationships with other European populations

BackgroundTrichobilharzia is the most species rich and widely distributed genus of schistosomes and is known throughout Europe and North America as an agent of human cercarial dermatitis. The disease is caused by an acute allergic reaction in the skin that develops as a consequence of repeated contact with water containing schistosomatid cercariae. However, despite historical outbreaks of the disease, there are no published records of accurately identified Trichobilharzia species from the UK.MethodsTwo hundred Radix auricularia (L.) were sampled from a recreational fishing lake in Hampshire and emerging schistosomatid cercariae were collected for microscopy and DNA extraction. General morphological description of the cercariae was performed, alongside sequencing and phylogenetic analysis of the 28S ribosomal DNA for accurate species identification as well as comparisons of ITS1 in order to identify evolutionary affinities with other European populations. All molecular comparisons were performed using published sequences.ResultsThe phylogenetic analysis of 28S sequences identified the cercariae as Trichobilharzia franki. Two unique British ITS1 haplotypes were identified which were most closely related to haplotypes of T. franki populations from France. Haplotype network analysis indicated the mixing of T. franki populations throughout Europe. It is suggested that parasite distribution is the probable result of the movement of migratory waterfowl.ConclusionsThis is the first accurate record of T. franki in the UK. The movement of T. franki with waterfowl could pose a considerable human health risk, as in mainland Europe, and signifies T. franki-associated human cercarial dermatitis as a re-emerging disease in the UK.

A foreign invader or a reclusive native? DNA bar coding reveals a distinct European lineage of the zoonotic parasite Schistosoma turkestanicum (syn. Orientobilharzia turkestanicum (Dutt and Srivastava, 1955))

Natural foci of Schistosoma turkestanicum (syn. Orientobilharzia turkestanicum) has been identified in the Gemenc Forest regions of Hungary utilising red deer as the definitive host. In order to identify the origins of this parasite in Europe standard DNA bar coding techniques were employed to sequence fragments of the cytochrome oxidase 1 (cox1) and the nuclear ribosomal internal transcribed region (ITS) from 10 individual adult male worms. Phylogenetic reconstruction using maximum likelihood phylogenetic reconstruction and haplotype networks of the cox1 showed all the worms to be of a distinct unique Hungarian lineage although some ITS haplotypes were shared with worms from populations in China and Iran. Molecular clock analysis suggests an early divergence event around 270,000years before present (YBP) between all S. turkestanicum populations giving rise to the Chinese, Iranian and Hungarian lineages. However, divergence of the sequences within the Hungarian population appears to have occurred approximately 63,000 YBP suggesting a long established population of S. turkestanicum in Europe. This suggests that the Hungarian population of S. turkestanicum has been native since the Ice Age and probably established itself during the last interglacial period as red deer moved into Europe from North Africa and the Middle East. This may also indicate that the parasite may have unknown populations established in several other countries in Eastern, Central and Southern Europe.

Pathology and molecular analysis of Hapalotrema mistroides (Digenea: Spirorchiidae) infecting a Mediterranean loggerhead turtle Caretta caretta

Turtle blood flukes belonging to the family Spirorchiidae (Digenea) represent a major threat for sea turtle health and are considered the most important parasitic cause of turtle stranding and mortality worldwide. Despite the large diversity of spirorchiid species found globally, there are only 2 records for free-ranging Mediterranean sea turtles that date back to the late 1800s involving just Hapalotrema mistroides Monticelli, 1896. This study describes the first fatal confirmed case of spirorchiidiasis in a free-ranging Mediterranean loggerhead turtle Caretta caretta (Linnaeus) and, owing to the complexities of taxonomic identification of these parasites, provides the first molecular characterization and phylogenetic analysis of H. mistroides from the Mediterranean Sea. The loggerhead turtle showed cachexia and digestive disorders associated with severe damage to the pancreas and intestinal ganglia, caused by deposition of Hapalotrema eggs forming granulomas. Massive Hapalotrema egg emboli in several tissues and organs and encephalitis were the most probable contributions to the death of the turtle. The congruence between the phylogenetic analysis of both the ITS2 and 28S rDNA resolved the Italian and USA H. mistroides as the same species, confirming the parasite identification. The case here described clearly indicates that the blood flukes should be considered in the differential diagnosis of Mediterranean sea turtle diseases.

DNA barcoding of the medically important freshwater snail 'Physa acuta' reveals multiple invasion events into Africa

The medically important freshwater snail Physa acuta is highly invasive and has been reported in several freshwater environments across Africa. To identify species and provide initial insights into the origins of P. acuta into African freshwater environments standard molecular barcoding analyses, using the mitochondrial cytochrome c oxidase subunit I gene (COI), was performed on P. acuta isolates from Angola, Burundi and South Africa. Phylogenetic analyses of isolates from Africa could not be distinguished from P. acuta populations from other countries. Comparisons of COI sequences between isolates of P. acuta showed there to be no geographically specific clusters and the African isolates were distributed across four distinct unrelated clades suggesting several independent invasion events. Haplotype analyses indicated that there were a high number of haplotypes with low variation between them, which led to significant differences in AMOVA analyses between countries. This was further evidence of multiple invasion events suggesting multiple novel haplotypes being continually and independently introduced to each country. This approach not only provides initial insight into the invasion of Africa by P. acuta but a molecular method to monitor and manage the use of an agent of biological control.