Selective feeding by three native North American freshwater mussels implies food competition with zebra mussels

The response of a benthic macroinvertebrate assemblage to whole-lake biomanipulation was studied in a small Finnish mesotrophic lake. From 1993 to 1997, over 200 kg ha^–1 of fish, mainly roach (Rutilus rutilus (L.)) and bream (Abramis brama (L.)) were caught and the fish biomass was reduced by nearly 80%. The biomass and density of benthic invertebrates were investigated during the years of fish removal and for the following three years. The decrease in benthivorous fish stock led to a higher biomass and density of all major groups of benthic invertebrates during the early years of fish removal. Non-biting midges (Chironomidae), water mites (Hydrachnellae), mayfly nymphs (Ephemeroptera), sphaeriid clams (Bivalvia: Sphaeridae) and biting midges (Ceratopogonidae) seemed to respond most profoundly to changes in fish biomass. The biomass of most invertebrate groups correlated negatively with the catch-per-unit-effort (CPUE). The total biomass and density of invertebrates had strong negative correlations with the CPUE (r= -0.85, p = 0.016, r = -0.84, p = 0.019, respectively), but they did not correlate significantly with total phosphorus, chlorophyll a, or temperature. However, the variation in total biomass that was not explained by the CPUE, was significantly associated with total phosphorus.The fish stock recovered to almost its initial level within three years after fish removal had been discontinued. As an apparent response to increased predation pressure, the biomasses of many invertebrate groups decreased again in the years 1999–2000. The strong relationship between macrozoobenthos and fish populations in the studied lake is likely to be a consequence of the open and sparsely vegetated bottom, which offers minimal shelter to invertebrate prey. An additional factor behind the recent low biomass levels may be changes in primary production. Phosphorus and chlorophyll a concentrations started to decrease markedly after three years of fishing and they have remained at a low level.     

Generation of whole genome sequences of new Cryptosporidium hominis and Cryptosporidium parvum isolates directly from stool samples

Background

Whole genome sequencing (WGS) of Cryptosporidium spp. has previously relied on propagation of the parasite in animals to generate enough oocysts from which to extract DNA of sufficient quantity and purity for analysis. We have developed and validated a method for preparation of genomic Cryptosporidium DNA suitable for WGS directly from human stool samples and used it to generate 10 high-quality whole Cryptosporidium genome assemblies. Our method uses a combination of salt flotation, immunomagnetic separation (IMS), and surface sterilisation of oocysts prior to DNA extraction, with subsequent use of the transposome-based Nextera XT kit to generate libraries for sequencing on Illumina platforms. IMS was found to be superior to caesium chloride density centrifugation for purification of oocysts from small volume stool samples and for reducing levels of contaminant DNA.

Results

The IMS-based method was used initially to sequence whole genomes of Cryptosporidium hominis gp60 subtype IbA10G2 and Cryptosporidium parvum gp60 subtype IIaA19G1R2 from small amounts of stool left over from diagnostic testing of clinical cases of cryptosporidiosis. The C. parvum isolate was sequenced to a mean depth of 51.8X with reads covering 100 % of the bases of the C. parvum Iowa II reference genome (Bioproject PRJNA 15586), while the C. hominis isolate was sequenced to a mean depth of 34.7X with reads covering 98 % of the bases of the C. hominis TU502 v1 reference genome (Bioproject PRJNA 15585).The method was then applied to a further 17 stools, successfully generating another eight new whole genome sequences, of which two were C. hominis (gp60 subtypes IbA10G2 and IaA14R3) and six C. parvum (gp60 subtypes IIaA15G2R1 from three samples, and one each of IIaA17G1R1, IIaA18G2R1, and IIdA22G1), demonstrating the utility of this method to sequence Cryptosporidium genomes directly from clinical samples. This development is especially important as it reduces the requirement to propagate Cryptosporidium oocysts in animal models prior to genome sequencing.

Conclusion

This represents the first report of high-quality whole genome sequencing of Cryptosporidium isolates prepared directly from human stool samples.     

Bayesian adaptive sequence alignment algorithms

The selection of a scoring matrix and gap penalty parameters continues to be an important problem in sequence alignment. We describe here an algorithm, the 'Bayes block aligner, which bypasses this requirement. Instead of requiring a fixed set of parameter settings, this algorithm returns the Bayesian posterior probability for the number of gaps and for the scoring matrices in any series of interest. Furthermore, instead of returning the single best alignment for the chosen parameter settings, this algorithm returns the posterior distribution of all alignments considering the full range of gapping and scoring matrices selected, weighing each in proportion to its probability based on the data. We compared the Bayes aligner with the popular Smith-Waterman algorithm with parameter settings from the literature which had been optimized for the identification of structural neighbors, and found that the Bayes aligner correctly identified more structural neighbors. In a detailed examination of the alignment of a pair of kinase and a pair of GTPase sequences, we illustrate the algorithm's potential to identify subsequences that are conserved to different degrees. In addition, this example shows that the Bayes aligner returns an alignment-free assessment of the distance between a pair of sequences.      

Evidence from nuclear sequences that invariable sites should be considered when sequence divergence is calculated

It has long been known, from the distribution of multiple amino acid replacements, that not all amino acids of a sequence are replaceable. More recently, the phenomenon was observed at the nucleotide level in mitochondrial DNA even after allowing for different rates of transition and transversion substitutions. We have extended the search to globin gene sequences from various organisms, with the following results: (1) Nearly every data set showed evidence of invariable nucleotide positions. (2) In all data sets, substitution rates of transversions and transitions were never in the ratio of 2/1, and rarely was the ratio even constant. (3) Only rarely (e.g., the third codon position of beta hemoglobins) was it possible to fit the data set solely by making allowance for the number of invariable positions and for the relative rates of transversion and transition substitutions. (4) For one data set (the second codon position of beta hemoglobins) we were able to simulate the observed data by making the allowance in (3) and having the set of covariotides (concomitantly variable nucleotides) be small in number and be turned over in a stochastic manner with a probability that was appreciable. (5) The fit in the latter case suggests, if the assumptions are correct and at all common, that current procedures for estimating the total number of nucleotide substitutions in two genes since their divergence from their common ancestor could be low by as much as an order of magnitude. (6) The fact that only a small fraction of the nucleotide positions differ is no guarantee that one is not seriously underestimating the total amount of divergence (substitutions). (7) Most data sets are so heterogeneous in their number of transition and transversion differences that none of the current models of nucleotide substitution seem to fit them even after (a) segregation of coding from noncoding sequences and (b) splitting of the codon into three subsets by codon position. (8) These frequently occurring problems cannot be seen unless several reasonably divergent orthologous genes are examined together.