Remarkable richness of trypanosomes in tsetse flies (Glossina morsitans morsitans and Glossina pallidipes) from the Gorongosa National Park and Niassa National Reserve of Mozambique revealed by fluorescent fragment length barcoding (FFLB).

Trypanosomes of African wild ungulates transmitted by tsetse flies can cause human and livestock diseases. However, trypanosome diversity in wild tsetse flies remains greatly underestimated. We employed FFLB (fluorescent fragment length barcoding) for surveys of trypanosomes in tsetse flies (3086) from the Gorongosa National Park (GNP) and Niassa National Reserve (NNR) in Mozambique (MZ), identified as Glossina morsitans morsitans (GNP/NNR=77.6%/90.5%) and Glossina pallidipes (22.4%/9.5%). Trypanosomes were microscopically detected in 8.3% of tsetse guts. FFLB of gut samples revealed (GNP/NNR): Trypanosoma congolense of Savannah (27%/63%), Kilifi (16.7%/29.7%) and Forest (1.0%/0.3%) genetic groups; T. simiae Tsavo (36.5%/6.1%); T. simiae (22.2%/17.7%); T. godfreyi (18.2%/7.0%); subgenus Trypanozoon (20.2%/25.7%); T. vivax/T. vivax-like (1.5%/5.2%); T. suis/T. suis-like (9.4%/11.9%). Tsetse proboscises exhibited similar species composition, but most prevalent species were (GNP/NNR): T. simiae (21.9%/28%), T. b. brucei (19.2%/31.7%), and T. vivax/T. vivax-like (19.2%/28.6%). Flies harboring mixtures of trypanosomes were common (~ 64%), and combinations of more than four trypanosomes were especially abundant in the pristine NNR. The non-pathogenic T. theileri was found in 2.5% while FFLB profiles of unknown species were detected in 19% of flies examined. This is the first report on molecular diversity of tsetse flies and their trypanosomes in MZ; all trypanosomes pathogenic for ungulates were detected, but no human pathogens were detected. Overall, two species of tsetse flies harbor 12 species/genotypes of trypanosomes. This notable species richness was likely uncovered because flies were captured in wildlife reserves and surveyed using the method of FFLB able to identify, with high sensitivity and accuracy, known and novel trypanosomes. Our findings importantly improve the knowledge on trypanosome diversity in tsetse flies, revealed the greatest species richness so far reported in tsetse fly of any African country, and indicate the existence of a hidden trypanosome diversity to be discovered in African wildlife protected areas.

The role of relatedness in structuring the social network of a wild guppy population.

The role of relatedness in structuring animal societies has attracted considerable interest. Whilst a significant number of studies have documented kin recognition in shoaling fish under laboratory conditions, there is little evidence that relatedness plays a significant role in structuring social interactions in wild populations that are characterised by fission-fusion dynamics. Previous work has tended to compare relatedness within and among entire shoals. Such an approach however, does not have the ability to detect social sub-structuring within groups, which appears to be a major factor driving the social organisation of fission-fusion animal societies. Here, we use social network analysis combined with DNA microsatellite genotyping to examine the role of relatedness in structuring social relationships in a wild population of guppies (Poecilia reticulata). Consistent with previous findings, female-female dyads formed the strongest social relationships, which were stable over time. Interestingly, we also observed significant co-occurrence of male-male interactions, which is in contrast to previous work. Although we observed social sub-structuring in the population, we found no evidence for relatedness playing a significant role in underpinning this structure. Indeed, only seven first-degree relative dyads were identified among the 180 fish genotyped, indicating that the majority of individuals do not have a first-degree relative in the population. The high genetic diversity observed in this population is indicative of a large effective population size typical of lowland guppy populations. We discuss our findings in the context of the evolution of social organisation and the mechanisms and constraints that may drive the observed patterns in wild populations.

Identification and lineage genotyping of South American trypanosomes using fluorescent fragment length barcoding.

Trypanosoma cruzi and Trypanosoma rangeli are human-infective blood parasites, largely restricted to Central and South America. They also infect a wide range of wild and domestic mammals and are transmitted by a numerous species of triatomine bugs. There are significant overlaps in the host and geographical ranges of both species. The two species consist of a number of distinct phylogenetic lineages. A range of PCR-based techniques have been developed to differentiate between these species and to assign their isolates into lineages. However, the existence of at least six and five lineages within T. cruzi and T. rangeli, respectively, makes identification of the full range of isolates difficult and time consuming. Here we have applied fluorescent fragment length barcoding (FFLB) to the problem of identifying and genotyping T. cruzi, T. rangeli and other South American trypanosomes. This technique discriminates species on the basis of length polymorphism of regions of the rDNA locus. FFLB was able to differentiate many trypanosome species known from South American mammals: T. cruzi cruzi, T. cruzi marinkellei, T. dionisii-like, T. evansi, T. lewisi, T. rangeli, T. theileri and T. vivax. Furthermore, all five T. rangeli lineages and many T. cruzi lineages could be identified, except the hybrid lineages TcV and TcVI that could not be distinguished from lineages III and II respectively. This method also allowed identification of mixed infections of T. cruzi and T. rangeli lineages in naturally infected triatomine bugs. The ability of FFLB to genotype multiple lineages of T. cruzi and T. rangeli together with other trypanosome species, using the same primer sets is an advantage over other currently available techniques. Overall, these results demonstrate that FFLB is a useful method for species diagnosis, genotyping and understanding the epidemiology of American trypanosomes.

New Trypanosoma (Duttonella) vivax genotypes from tsetse flies in East Africa.

Salivarian trypanosomes pose a substantial threat to livestock, but their full diversity is not known. To survey trypanosomes carried by tsetse in Tanzania, DNA samples from infected proboscides of Glossina pallidipes and G. swynnertoni were identified using fluorescent fragment length barcoding (FFLB), which discriminates species by size polymorphisms in multiple regions of the ribosomal RNA locus. FFLB identified the trypanosomes in 65 of 105 (61.9%) infected proboscides, revealing 9 mixed infections. Of 7 different FFLB profiles, 2 were similar but not identical to reference West African Trypanosoma vivax; 5 other profiles belonged to known species also identified in fly midguts. Phylogenetic analysis of the glycosomal glyceraldehyde phosphate dehydrogenase gene revealed that the Tanzanian T. vivax samples fell into 2 distinct groups, both outside the main clade of African and South American T. vivax. These new T. vivax genotypes were common and widespread in tsetse in Tanzania. The T. brucei-like trypanosome previously described from tsetse midguts was also found in 2 proboscides, demonstrating a salivarian transmission route. Investigation of mammalian host range and pathogenicity will reveal the importance of these new trypanosomes for the epidemiology and control of animal trypanosomiasis in East Africa.

Genetic variation in strains of zebrafish (Danio rerio) and the implications for ecotoxicology studies.

There is substantial evidence that genetic variation, at both the level of the individual and population, has a significant effect on behaviour, fitness and response to toxicants. Using DNA microsatellites, we examined the genetic variation in samples of several commonly used laboratory strains of zebrafish, Danio rerio, a model species in toxicological studies. We compared the genetic variation to that found in a sample of wild fish from Bangladesh. Our findings show that the wild fish were significantly more variable than the laboratory strains for several measures of genetic variability, including allelic richness and expected heterozygosity. This lack of variation should be given due consideration for any study which attempts to extrapolate the results of ecotoxicological laboratory tests to wild populations.

Phylogenetic analysis reveals the presence of the Trypanosoma cruzi clade in African terrestrial mammals.

Despite the impact of some trypanosome species on human and livestock health, the full diversity of trypanosomes in Africa is poorly understood. A recent study examined the prevalence of trypanosomes among a wide variety of wild vertebrates in Cameroon using species-specific PCR tests, but six trypanosome isolates remained unidentified. Here they have been re-examined using fluorescent fragment length barcoding (FFLB) and phylogenetic analysis of glycosomal glyceraldehyde phosphate dehydrogenase gGAPDH and 18S ribosomal RNA (rDNA) genes. Isolates from a monkey (Cercopithecus nictitans) and a palm civet (Nandinia binotata) belonged to the Trypanosoma cruzi clade, known previously only from New World and Australian terrestrial mammals, and bats from Africa, Europe and South America. Of the four other isolates, three from antelope were identified as Trypanosoma theileri, and one from a crocodile as T. grayi. This is the first report of trypanosomes of the T. cruzi clade in African terrestrial mammals and expands the clade's known global distribution in terrestrial mammals. Previously it has been hypothesized that African and New World trypanosomes diverged after continental separation, dating the divergence to around 100 million years ago. The new evidence instead suggests that intercontinental transfer occurred well after this, possibly via bats or rodents, allowing these trypanosomes to establish and evolve in African terrestrial mammals, and questioning the validity of calibrating trypanosome molecular trees using continental separation.

New molecular tools for the identification of trypanosome species.

Trypanosomes are the causative agents of many diseases of medical and veterinary importance, including sleeping sickness and nagana in Africa, and Chagas disease in South America. Accurate identification of trypanosome species is essential, as some species are morphologically indistinguishable, yet differ greatly in their pathogenicity. A range of molecular tools has been developed for identification of species and strains of trypanosomes. PCR, using primer sets designed to amplify a specific DNA fragment from each trypanosome species, is frequently used. More recently, generic systems have been developed that can potentially recognize all trypanosome species, such as amplification of the internal transcribed spacer and fluorescent fragment length barcoding, both of which use interspecies size variation in PCR fragments amplified from the ribosomal RNA locus. Loop-mediated isothermal amplification is a promising technique and is able to detect trypanosomes in blood, serum and cerebrospinal fluid. The advantages of these techniques for high-throughput and sensitive molecular identification will be discussed.

PERMANENT GENETIC RESOURCES: Identification of microsatellite loci for parentage analysis in roach Rutilus rutilus and eight other cyprinid fish by cross-species amplification, and a novel test for detecting hybrids between roach and other cyprinids.

In order to identify microsatellite loci for parentage analysis in roach Rutilus rutilus, 59 published primer sets were tested on roach and eight other cyprinid fish. Twenty polymorphic loci were identified for roach, of which the polymerase chain reaction products of seven could be pooled for sequencer analysis. Together, these seven loci have an exclusion probability of 0.997 for parentage, when no parents are known. We also describe a novel test for hybrids between roach and four other cyprinids, based on intraspecies length differences of internal transcribed spacer region 1.

The identification, diversity and prevalence of trypanosomes in field caught tsetse in Tanzania using ITS-1 primers and fluorescent fragment length barcoding.

We report on the development of two generic, PCR-based methods, which replace the multiple species-specific PCR tests used previously to identify the trypanosome species carried by individual tsetse flies. The first method is based on interspecies size variation in the PCR product of the ITS-1 region of the ribosomal RNA (rRNA) locus. In the second approach, length variation of multiple fragments within the 18S and 28S rRNA genes is assayed by PCR amplification with fluorescent primers; products are subsequently sized accurately and rapidly by the use of an automated DNA sequencer. Both methods were used to identify samples collected during large-scale field studies of trypanosome-infected tsetse in Tanzania in the National Parks of Tarangire and Serengeti, and the coastal forest reserve of Msubugwe. The fluctuations of trypanosome prevalence over time and two different field seasons are discussed. As well as facilitating the identification of trypanosome species with increased speed, precision and sensitivity, these generic systems have enabled us to identify two new species of trypanosome.

A novel, high-throughput technique for species identification reveals a new species of tsetse-transmitted trypanosome related to the Trypanosoma brucei subgenus, Trypanozoon.

We describe a novel method of species identification, fluorescent fragment length barcoding, based on length variation in regions of the 18S and 28Salpha ribosomal DNA. Fluorescently tagged primers, designed in conserved regions of the 18S and 28Salpha ribosomal DNA, were used to amplify fragments with inter-species size variation, and sizes determined accurately using an automated DNA sequencer. By using multiple regions and different fluorochromes, a barcode unique to each species was generated. The technique was developed for the identification of African tsetse-transmitted trypanosomes and validated using DNA from laboratory isolates representing known species, subspecies and subgroups. To test the methodology, we examined 91 trypanosome samples from infected tsetse fly midguts from Tanzania, most of which had already been identified by species-specific and generic PCR tests. Identifications were mainly in agreement, but the presence of an unknown trypanosome in several samples was revealed by its unique barcode. Phylogenetic analyses based on 18S rDNA and glycosomal glyceraldehyde phosphate dehydrogenase gene sequences confirmed that this trypanosome is a new species and it is within the Trypanosoma brucei clade, as a sister group of subgenus Trypanozoon. The overall identification rate of trypanosome-infected midgut samples increased from 78 to 96% using FFLB instead of currently available PCR tests. This was due to the high sensitivity of FFLB as well as its capacity to identify previously unrecognised species. FFLB also allowed the identification of multiple species in mixed infections. The method enabled high-throughput and accurate species identification and should be applicable to any group of organisms where there is length variation in regions of rDNA.

Evidence for control of mercury accumulation rates in Canadian High Arctic lake sediments by variations of aquatic primary productivity.

Climate warming in the 20th Century has had profound effects on the limnology of High Arctic lakes, including substantial increases in autochthonous primary productivity (APP). Here, we report organic carbon and Hg core profiles from two lakes which support the hypothesis that 20th Century increases in sedimentary Hg at these latitudes were largely driven by APP increases, via Hg scavenging by algae and/or suspended detrital algal matter. Hydrocarbons quantitatively released by thermal cracking of algal-derived organic matter ("S2" carbon) were used to reconstruct past APP. Variation of S2 flux accounted for 87-91% of the variance in total Hg flux in the study lakes since 1854. Mercury and S2 carbon were also associated during the pre-Industrial Period, co-varying by as much as 30% during past warm/cool periods. As a test of the hypothesis, predicted values for 20th Century [Hg] were derived from pre-1900 Hg-S2 relationships. Measured 20th Century [Hg] was on average only 6-11% higher than that predicted in one lake, and 33% higher in the other. S2-normalization of [Hg] in the latter lake suggested that 78% of the average increase in 20th Century [Hg] could be explained by scavenging. These findings suggest that the atmospheric contribution of long-range anthropogenic Hg to High Arctic lakes may have been overestimated by several-fold because of this climate-driven process, and was responsible for no more than 22% of the 20th Century [Hg] increase in the study lakes.

The inadvertent introduction into Australia of Trypanosoma nabiasi, the trypanosome of the European rabbit (Oryctolagus cuniculus), and its potential for biocontrol.

Wild rabbits (Oryctolagus cuniculus) in Australia are the descendents of 24 animals from England released in 1859. We surveyed rabbits and rabbit fleas (Spilopsyllus cuniculi) in Australia for the presence of trypanosomes using parasitological and PCR-based methods. Trypanosomes were detected in blood from the European rabbits by microscopy, and PCR using trypanosome-specific small subunit ribosomal RNA (SSU rRNA) gene primers and those in rabbit fleas by PCR. This is the first record of trypanosomes from rabbits in Australia. We identified these Australian rabbit trypanosomes as Trypanosoma nabiasi, the trypanosome of the European rabbit, by comparison of morphology and SSU rRNA gene sequences of Australian and European rabbit trypanosomes. Phylogenetic analysis places T. nabiasi in a clade with rodent trypanosomes in the subgenus Herpetosoma and their common link appears to be transmission by fleas. Despite the strict host specificity of trypanosomes in this clade, phylogenies presented here suggest that they have not strictly cospeciated with their vertebrate hosts. We suggest that T. nabiasi was inadvertently introduced into Australia in the 1960s in its flea vector Spilopsyllus cuniculi, which was deliberately introduced as a potential vector of the myxoma virus. In view of the environmental and economic damage caused by rabbits in Australia and other islands, the development of a virulent or genetically modified T. nabiasi should be considered to control rabbits.

Phylogenetic analysis of freshwater fish trypanosomes from Europe using ssu rRNA gene sequences and random amplification of polymorphic DNA.

The taxonomy and phylogenetic relationships of fish trypanosomes are uncertain. A collection of 22 cloned trypanosome isolates from 14 species of European freshwater fish and 1 species of African freshwater fish were examined by molecular phylogenetic analysis. The small subunit ribosomal RNA (ssu rRNA) genes of 8 clones were sequenced and compared with ssu rRNA gene sequences from a wider selection of vertebrate trypanosome isolates by phylogenetic analysis. All trypanosomes from freshwater fish fell in a single clade, subdivided into 3 groups. This clade sits within a larger, robust clade containing trypanosomes from marine fish and various amphibious vertebrates. All 22 trypanosome clones were analysed by random amplification of polymorphic DNA. The resulting dendrogram shows 3 groups, which are congruent with the groups identified in the ssu rRNA gene phylogeny. Two of the groups contain the majority of trypanosome isolates and within-group variation is slight. These groups do not separate purported trypanosome species distinguished by morphology or host origin, and thus these criteria do not appear to be reliable guides to genetic relationships among fish trypanosomes. However, we suggest that the 2 groups themselves may represent different species of fish trypanosomes. The polymorphic DNA markers we have identified will facilitate future comparisons of the biology of these 2 groups of fish trypanosomes.

A new lineage of trypanosomes from Australian vertebrates and terrestrial bloodsucking leeches (Haemadipsidae).

Little is known about the trypanosomes of indigenous Australian vertebrates and their vectors. We surveyed a range of vertebrates and blood-feeding invertebrates for trypanosomes by parasitological and PCR-based methods using primers specific to the small subunit ribosomal RNA (SSU rRNA) gene of genus Trypanosoma. Trypanosome isolates were obtained in culture from two common wombats, one swamp wallaby and an Australian bird (Strepera sp.). By PCR, blood samples from three wombats, one brush-tailed wallaby, three platypuses and a frog were positive for trypanosome DNA. All the blood-sucking invertebrates screened were negative for trypanosomes both by microscopy and PCR, except for specimens of terrestrial leeches (Haemadipsidae). Of the latter, two Micobdella sp. specimens from Victoria and 18 Philaemon sp. specimens from Queensland were positive by PCR. Four Haemadipsa zeylanica specimens from Sri Lanka and three Leiobdella jawarerensis specimens from Papua New Guinea were also PCR positive for trypanosome DNA. We sequenced the SSU rRNA and glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) genes in order to determine the phylogenetic positions of the new vertebrate and terrestrial leech trypanosomes. In trees based on these genes, Australian vertebrate trypanosomes fell in several distinct clades, for the most part being more closely related to trypanosomes outside Australia than to each other. Two previously undescribed wallaby trypanosomes fell in a clade with Trypanosoma theileri, the cosmopolitan bovid trypanosome, and Trypanosoma cyclops from a Malaysian primate. The terrestrial leech trypanosomes were closely related to the wallaby trypanosomes, T. cyclops and a trypanosome from an Australian frog. We suggest that haemadipsid leeches may be significant and widespread vectors of trypanosomes in Australia and Asia.

Reactivation of a Trypanosoma cruzi infection in a rhesus monkey (Macaca mulatta) experimentally infected with SIV.

Trypanosoma cruzi-like flagellates were incidentally noted in blood smears of a routinely monitored rhesus monkey experimentally infected with the simian immunodeficiency virus (SIV). Immunodeficiency in the course of the SIV infection reactivated a chronic infection of Chagas' disease that had been unnoticed when the macaque was imported to Europe. The animal developed no specific clinical symptoms of American trypanosomiasis, but histologically a chagasic myocarditis was detected. Analysis of the small subunit rRNA gene of the trypanosome identified the protozoan as T. cruzi.

Effects of the tricothecene mycotoxin diacetoxyscirpenol on egg production of broiler breeders.

Three experiments were conducted to determine the effects of 4,15-diacetoxyscirpenol (DAS) on egg quality and egg production of broiler breeders. In Experiment 1, feed containing 0, 1.25, 2.5, or 5.0 mg DAS/ kg was fed from 67 to 69 wk of age followed by a 3-wk recovery period on a slat-litter floor. In Experiment 2, individually caged broiler breeder females were studied from 23 to 31 wk of age. The basal diet containing 0, 5, 10, or 20 mg DAS/kg was fed from 25 to 27 wk of age. In Experiment 3, individually caged broiler breeder hens were studied from 23 to 32 wk of age. DAS was fed at levels of 0 (basal), 5, 10, and 20 mg DAS/kg for 2 wk beginning at Week 24, followed by the basal breeder diet for 7 wk. Egg production was not affected by levels of up to 5 mg DAS/kg in the older hens of Experiment 1. When fed from 25 to 27 wk of age in Experiment 2, DAS decreased egg production at the 20 mg/kg level only. When fed from 24 to 25 wk of age in Experiment 3, DAS had no significant effect on egg production or egg quality. Short-term consumption of DAS at levels that might naturally occur appears to have little effect on broiler breeder egg production.

Effects of the trichothecene mycotoxin diacetoxyscirpenol on feed consumption, body weight, and oral lesions of broiler breeders.

Three experiments were conducted to determine the effects of 4,15-diacetoxyscirpenol (DAS) on BW, feed consumption, and oral lesions of broiler breeders. In Experiment 1, caged broiler breeder hens were fed 0, 5, 10, or 20 mg DAS/kg diet from 24 to 25 wk of age. There were dose-related decreases in BW and feed consumption indicating feed refusal, as well as dose-related increases in the extent of mouth lesions. The areas of the mouth most sensitive to DAS were associated with the salivary glands and the tip of the tongue. In Experiment 2, individually caged male and female broiler breeders were fed a basal diet containing 0, 5, 10, or 20 mg DAS/kg from 25 to 27 wk of age. There were dose-related decreases in BW and feed consumption for the female broiler breeders, whereas there was a decrease in feed consumption for the male broiler breeders at the 10 and 20 mg DAS/kg levels. In Experiment 3, male broiler breeders were fed 0 or 10 mg DAS/kg diet from 23 to 25 wk of age on a litter floor. For this experiment the daily intake of feed was restricted, and the feed consumption rate was measured. There was an increased amount of unconsumed feed at 23 wk of age due to the presence of DAS. In summary, the experiments provided evidence that DAS caused decreased BW and feed consumption as well as cytotoxic injury including oral lesions in broiler breeders.

Ice shelf microbial ecosystems in the high arctic and implications for life on snowball earth.

The Ward Hunt Ice Shelf (83 degrees N, 74 degrees W) is the largest remaining section of thick (> 10 m) land-fast sea ice along the northern coastline of Ellesmere Island, Canada. Extensive meltwater lakes and streams occur on the surface of the ice and are colonized by photosynthetic microbial mat communities. This High Arctic cryo-ecosystem is similar in several of its physical, biological and geochemical features to the McMurdo Ice Shelf in Antarctica. The ice-mats in both polar regions are dominated by filamentous cyanobacteria but also contain diatoms, chlorophytes, flagellates, ciliates, nematodes, tardigrades and rotifers. The luxuriant Ward Hunt consortia also contain high concentrations (10(7)-10(8) cm-2) of viruses and heterotrophic bacteria. During periods of extensive ice cover, such as glaciations during the Proterozoic, cryotolerant mats of the type now found in these polar ice shelf ecosystems would have provided refugia for the survival, growth and evolution of a variety of organisms, including multicellular eukaryotes.

Effects of the tricothecene mycotoxin diacetoxyscirpenol on fertility and hatchability of broiler breeders.

Two experiments were conducted to determine the effects of 4,15-diacetoxyscirpenol (DAS) on fertility and hatchability of broiler breeders. In Experiment 1, naturally mated broiler breeders were studied. A limited daily allocation of feed containing 0 (basal), 1.25, 2.5, or 5.0 mg DAS/kg diet was provided from 67 to 69 wk of age in slat-litter floor pens. Fertility was consistently improved by the 5.0 mg/kg level of DAS and intermittently by the 1.25 and 2.50 mg/kg levels. The effect disappeared upon removal of DAS. In Experiment 2, individually caged broiler breeder males and females were fed a basal diet containing 0, 5, 10, or 20 mg DAS/kg diet from 25 to 27 wk of age. Semen was pooled from males within each treatment and used to inseminate females from each treatment in a 4 x 4 factorial design. Female-related fertility was increased at the 5 and 10 mg DAS/kg levels and male-related fertility was decreased by the 10 and 20 mg DAS/kg levels. Small, fluid-filled cysts were observed on the testes of many DAS-treated males upon necropsy. In summary, low levels of DAS (< or =10 mg DAS/kg) appeared to improve female-related fertility, presumably because of enhanced spermatozoal storage within the oviduct. Conversely, DAS (> or =10 mg DAS/kg) decreased male-related fertility, presumably by direct toxic effects on the testes. Overall, it appeared that levels of DAS below 5 mg DAS/kg feed would not be detrimental to fertility and hatchability.