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Bacteriophage research is gaining more importance due to increasing antibiotic resistance. However, for treatment with bacteriophages, diagnostics have to be improved. Bacteriophages carry adhesion proteins, which bind to the bacterial cell surface, for example tailspike proteins (TSP) for specific recognition of bacterial O-antigen polysaccharide. TSP are highly stable proteins and thus might be suitable components for the integration into diagnostic tools. We used the TSP of bacteriophage Sf6 to establish two applications for detecting Shigella flexneri (S. flexneri), a highly contagious pathogen causing dysentery. We found that Sf6TSP not only bound O-antigen of S. flexneri serotype Y, but also the glucosylated O-antigen of serotype 2a. Moreover, mass spectrometry glycan analyses showed that Sf6TSP tolerated various O-acetyl modifications on these O-antigens. We established a microtiter plate-based ELISA like tailspike adsorption assay (ELITA) using a Strep-tag®II modified Sf6TSP. As sensitive screening alternative we produced a fluorescently labeled Sf6TSP via coupling to an environment sensitive dye. Binding of this probe to the S. flexneri O-antigen Y elicited a fluorescence intensity increase of 80% with an emission maximum in the visible light range. The Sf6TSP probes thus offer a promising route to a highly specific and sensitive bacteriophage TSP-based Shigella detection system.
Bacteriophage research is gaining more importance due to increasing antibiotic resistance. However, for treatment with bacteriophages, diagnostics have to be improved. Bacteriophages carry adhesion proteins, which bind to the bacterial cell surface, for example tailspike proteins (TSP) for specific recognition of bacterial O-antigen polysaccharide. TSP are highly stable proteins and thus might be suitable components for the integration into diagnostic tools. We used the TSP of bacteriophage Sf6 to establish two applications for detecting Shigella flexneri (S. flexneri), a highly contagious pathogen causing dysentery. We found that Sf6TSP not only bound O-antigen of S. flexneri serotype Y, but also the glucosylated O-antigen of serotype 2a. Moreover, mass spectrometry glycan analyses showed that Sf6TSP tolerated various O-acetyl modifications on these O-antigens. We established a microtiter plate-based ELISA like tailspike adsorption assay (ELITA) using a Strep-tag®II modified Sf6TSP. As sensitive screening alternative we produced a fluorescently labeled Sf6TSP via coupling to an environment sensitive dye. Binding of this probe to the S. flexneri O-antigen Y elicited a fluorescence intensity increase of 80% with an emission maximum in the visible light range. The Sf6TSP probes thus offer a promising route to a highly specific and sensitive bacteriophage TSP-based Shigella detection system.
Communication is key to a wide variety of animal behaviours and multiple modalities are often involved in this exchange of information from sender to receiver. The communication of African weakly electric fish, however, is thought to be predominantly unimodal and is mediated by their electric sense, in which species-specific electric organ discharges (EODs) are generated in a context-dependent and thus variable sequence of pulse intervals (SPI). While the primary function of the electric sense is considered to be electrolocation, both of its components likely carry information regarding identity of the sender. However, a clear understanding of their contribution to species recognition is incomplete. We therefore analysed these two electrocommunication components (EOD waveform and SPI statistics) in two sympatric mormyrid Campylomormyrus species. In a set of five playback conditions, we further investigated which components may drive interspecific recognition and discrimination. While we found that both electrocommunication components are species-specific, the cues necessary for species recognition differ between the two species studied. While the EOD waveform and SPI were both necessary and sufficient for species recognition in C. compressirostris males, C. tamandua males apparently utilize other, non-electric modalities. Mapped onto a recent phylogeny, our results suggest that discrimination by electric cues alone may be an apomorphic trait evolved during a recent radiation in this taxon.
Communication is key to a wide variety of animal behaviours and multiple modalities are often involved in this exchange of information from sender to receiver. The communication of African weakly electric fish, however, is thought to be predominantly unimodal and is mediated by their electric sense, in which species-specific electric organ discharges (EODs) are generated in a context-dependent and thus variable sequence of pulse intervals (SPI). While the primary function of the electric sense is considered to be electrolocation, both of its components likely carry information regarding identity of the sender. However, a clear understanding of their contribution to species recognition is incomplete. We therefore analysed these two electrocommunication components (EOD waveform and SPI statistics) in two sympatric mormyrid Campylomormyrus species. In a set of five playback conditions, we further investigated which components may drive interspecific recognition and discrimination. While we found that both electrocommunication components are species-specific, the cues necessary for species recognition differ between the two species studied. While the EOD waveform and SPI were both necessary and sufficient for species recognition in C. compressirostris males, C. tamandua males apparently utilize other, non-electric modalities. Mapped onto a recent phylogeny, our results suggest that discrimination by electric cues alone may be an apomorphic trait evolved during a recent radiation in this taxon.
Gram-negative bacteria protect themselves with an outermost layer containing lipopolysaccharide (LPS). O-antigen-specific bacteriophages use tailspike proteins (TSP) to recognize and cleave the O-polysaccharide part of LPS. However, O-antigen composition and structure can be highly variable depending on the environmental conditions. It is important to understand how these changes may influence the early steps of the bacteriophage infection cycle because they can be linked to changes in host range or the occurrence of phage resistance. In this work, we have analyzed how LPS preparations in vitro trigger particle opening and DNA ejection from the E. coli podovirus HK620. Fluorescence-based monitoring of DNA release showed that HK620 phage particles in vitro ejected their genome at velocities comparable to those found for other podoviruses. Moreover, we found that HK620 irreversibly adsorbed to the LPS receptor via its TSP at restrictive low temperatures, without opening the particle but could eject its DNA at permissive temperatures. DNA ejection was solely stimulated by LPS, however, the composition of the O-antigen dictated whether the LPS receptor could start the DNA release from E. coli phage HK620 in vitro. This finding can be significant when optimizing bacteriophage mixtures for therapy, where in natural environments O-antigen structures may rapidly change.
Gram-negative bacteria protect themselves with an outermost layer containing lipopolysaccharide (LPS). O-antigen-specific bacteriophages use tailspike proteins (TSP) to recognize and cleave the O-polysaccharide part of LPS. However, O-antigen composition and structure can be highly variable depending on the environmental conditions. It is important to understand how these changes may influence the early steps of the bacteriophage infection cycle because they can be linked to changes in host range or the occurrence of phage resistance. In this work, we have analyzed how LPS preparations in vitro trigger particle opening and DNA ejection from the E. coli podovirus HK620. Fluorescence-based monitoring of DNA release showed that HK620 phage particles in vitro ejected their genome at velocities comparable to those found for other podoviruses. Moreover, we found that HK620 irreversibly adsorbed to the LPS receptor via its TSP at restrictive low temperatures, without opening the particle but could eject its DNA at permissive temperatures. DNA ejection was solely stimulated by LPS, however, the composition of the O-antigen dictated whether the LPS receptor could start the DNA release from E. coli phage HK620 in vitro. This finding can be significant when optimizing bacteriophage mixtures for therapy, where in natural environments O-antigen structures may rapidly change.
Objective
We investigated the potential role of indirect benefits for female mate preferences in a highly promiscuous species of live-bearing fishes, the sailfin molly Poecilia latipinna using an integrative approach that combines methods from animal behavior, life-history evolution, and genetics. Males of this species solely contribute sperm for reproduction, and consequently females do not receive any direct benefits. Despite this, females typically show clear mate preferences. It has been suggested that females can increase their reproductive success through indirect benefits from choosing males of higher quality.
Results
Although preferences for large body size have been recorded as an honest signal for genetic quality, this particular study resulted in female preference being unaffected by male body size. Nonetheless, larger males did sire more offspring, but with no effect on offspring quality. This study presents a methodical innovation by combining preference testing with life history measurements—such as the determination of the dry weight of fish embryos—and paternity analyses on single fish embryos.
Objective
We investigated the potential role of indirect benefits for female mate preferences in a highly promiscuous species of live-bearing fishes, the sailfin molly Poecilia latipinna using an integrative approach that combines methods from animal behavior, life-history evolution, and genetics. Males of this species solely contribute sperm for reproduction, and consequently females do not receive any direct benefits. Despite this, females typically show clear mate preferences. It has been suggested that females can increase their reproductive success through indirect benefits from choosing males of higher quality.
Results
Although preferences for large body size have been recorded as an honest signal for genetic quality, this particular study resulted in female preference being unaffected by male body size. Nonetheless, larger males did sire more offspring, but with no effect on offspring quality. This study presents a methodical innovation by combining preference testing with life history measurements—such as the determination of the dry weight of fish embryos—and paternity analyses on single fish embryos.
It is well-known that prey species often face trade-offs between defense against predation and competitiveness, enabling predator-mediated coexistence. However, we lack an understanding of how the large variety of different defense traits with different competition costs affects coexistence and population dynamics. Our study focusses on two general defense mechanisms, that is, pre-attack (e.g., camouflage) and post-attack defenses (e.g., weaponry) that act at different phases of the predator—prey interaction. We consider a food web model with one predator, two prey types and one resource. One prey type is undefended, while the other one is pre-or post-attack defended paying costs either by a higher half-saturation constant for resource uptake or a lower maximum growth rate. We show that post-attack defenses promote prey coexistence and stabilize the population dynamics more strongly than pre-attack defenses by interfering with the predator’s functional response: Because the predator spends time handling “noncrackable” prey, the undefended prey is indirectly
facilitated. A high half-saturation constant as defense costs promotes coexistence more and stabilizes the dynamics less than a low maximum growth rate. The former imposes high costs at low resource concentrations but allows for temporally high growth rates at predator-induced resource peaks preventing the extinction of the defended prey. We evaluate the effects of the different defense mechanisms and costs on coexistence under different enrichment levels in order to vary the importance of bottom-up and top-down control of the prey community.
It is well-known that prey species often face trade-offs between defense against predation and competitiveness, enabling predator-mediated coexistence. However, we lack an understanding of how the large variety of different defense traits with different competition costs affects coexistence and population dynamics. Our study focusses on two general defense mechanisms, that is, pre-attack (e.g., camouflage)and post-attack defenses (e.g., weaponry) that act at different phases of the predator—prey interaction. We consider a food web model with one predator, two prey types and one resource. One prey type is undefended, while the other one is pre-or post-attack defended paying costs either by a higher half-saturation constant for resource uptake or a lower maximum growth rate. We show that post-attack defenses promote prey coexistence and stabilize the population dynamics more strongly than pre-attack defenses by interfering with the predator’s functional response: Because the predator spends time handling “noncrackable” prey, the undefended prey is indirectly
facilitated. A high half-saturation constant as defense costs promotes coexistence more and stabilizes the dynamics less than a low maximum growth rate. The former imposes high costs at low resource concentrations but allows for temporally high growth rates at predator-induced resource peaks preventing the extinction of
the defended prey. We evaluate the effects of the different defense mechanisms and costs on coexistence under different enrichment levels in order to vary the importance of bottom-up and top-down control of the prey community.
The anatomically modern human Homo sapiens sapiens is distinguished by a high adaptability in physiology, physique and behaviour in short term changing environmental conditions. Since our environmental factors are constantly changing because of anthropogenic influences, the question arises as to how far we have an impact on the human phenotype in the very sensitive growth phase in children and adolescents. Growth and development of all children and adolescents follow a universal and typical pattern. This pattern has evolved as the result of trade-offs in the 6-7 million years of human evolution. This typically human growth pattern differs from that of other long-living social primate species. It can be divided into different biological age stages, with specific biological, cognitive and socio-cultural signs. Phenotypic plasticity is the ability of an organism to react to an internal or external environmental input with a change in the form, state, and movement rate of activity (West-Eberhard 2003). The plasticity becomes visible and measurable particularly when, in addition to the normal variability of the phenotypic characteristics within a population, the manifestation of this plasticity changes within a relatively short time. The focus of the present work is the comparison of age-specific dimensional changes. The basic of the presented studies are more than 75,000 anthropometric data-sets of children and adolescence from 1980 up today and historical data of height available in scientific literature. Due to reduced daily physical activity, today's 6-18 year-olds have lower values of pelvic and elbow breadths. The observed increase in body height can be explained by hierarchies in social networks of human societies, contrary to earlier explanations (influence of nutrition, good living conditions and genetics). A shift towards a more feminine fat distribution pattern in boys and girls is parallel to the increase in chemicals in our environment that can affect the hormone system. Changing environmental conditions can have selective effects over generations so that that genotype becomes increasingly prevalent whose individuals have a higher progeny rate than other individuals in this population. Those then form the phenotype which allows optimum adaptation to the changes of the environmental conditions. Due to the slow patterns of succession and the low progeny rate (Hawkes et al. 1998), fast visible in the phenotype due to changes in the genotype of a population are unlikely to occur in the case of Homo sapiens sapiens within short time. In the data sets on which the presented investigations are based, such changes appear virtually impossible. The study periods cover 5-30 to max.100 years (based on data from the body height from historical data sets).
The sequencing of the human genome in the early 2000s led to an increased interest in cheap and fast sequencing technologies. This interest culminated in the advent of next generation sequencing (NGS). A number of different NGS platforms have arisen since then all promising to do the same thing, i.e. produce large amounts of genetic information for relatively low costs compared to more traditional methods such as Sanger sequencing. The capabilities of NGS meant that researchers were no longer bound to species for which a lot of previous work had already been done (e.g. model organisms and humans) enabling a shift in research towards more novel and diverse species of interest. This capability has greatly benefitted many fields within the biological sciences, one of which being the field of evolutionary biology. Researchers have begun to move away from the study of laboratory model organisms to wild, natural populations and species which has greatly expanded our knowledge of evolution. NGS boasts a number of benefits over more traditional sequencing approaches. The main benefit comes from the capability to generate information for drastically more loci for a fraction of the cost. This is hugely beneficial to the study of wild animals as, even when large numbers of individuals are unobtainable, the amount of data produced still allows for accurate, reliable population and species level results from a small selection of individuals.
The use of NGS to study species for which little to no previous research has been carried out on and the production of novel evolutionary information and reference datasets for the greater scientific community were the focuses of this thesis. Two studies in this thesis focused on producing novel mitochondrial genomes from shotgun sequencing data through iterative mapping, bypassing the need for a close relative to serve as a reference sequence. These mitochondrial genomes were then used to infer species level relationships through phylogenetic analyses. The first of these studies involved reconstructing a complete mitochondrial genome of the bat eared fox (Otocyon megalotis). Phylogenetic analyses of the mitochondrial genome confidently placed the bat eared fox as sister to the clade consisting of the raccoon dog and true foxes within the canidae family. The next study also involved reconstructing a mitochondrial genome but in this case from the extinct Macrauchenia of South America. As this study utilised ancient DNA, it involved a lot of parameter testing, quality controls and strict thresholds to obtain a near complete mitochondrial genome devoid of contamination known to plague ancient DNA studies. Phylogenetic analyses confidently placed Macrauchenia as sister to all living representatives of Perissodactyla with a divergence time of ~66 million years ago. The third and final study of this thesis involved de novo assemblies of both nuclear and mitochondrial genomes from brown and striped hyena and focussed on demographic, genetic diversity and population genomic analyses within the brown hyena. Previous studies of the brown hyena hinted at very low levels of genomic diversity and, perhaps due to this, were unable to find any notable population structure across its range. By incorporating a large number of genetic loci, in the form of complete nuclear genomes, population structure within the brown hyena was uncovered. On top of this, genomic diversity levels were compared to a number of other species. Results showed the brown hyena to have the lowest genomic diversity out of all species included in the study which was perhaps caused by a continuous and ongoing decline in effective population size that started about one million years ago and dramatically accelerated towards the end of the Pleistocene.
The studies within this thesis show the power NGS sequencing has and its utility within evolutionary biology. The most notable capabilities outlined in this thesis involve the study of species for which no reference data is available and in the production of large amounts of data, providing evolutionary answers at the species and population level that data produced using more traditional techniques simply could not.
High-throughput sequence data retrieved from ancient or other degraded samples has led to unprecedented insights into the evolutionary history of many species, but the analysis of such sequences also poses specific computational challenges. The most commonly used approach involves mapping sequence reads to a reference genome. However, this process becomes increasingly challenging with an elevated genetic distance between target and reference or with the presence of contaminant sequences with high sequence similarity to the target species. The evaluation and testing of mapping efficiency and stringency are thus paramount for the reliable identification and analysis of ancient sequences. In this paper, we present ‘TAPAS’, (Testing of Alignment Parameters for Ancient Samples), a computational tool that enables the systematic testing of mapping tools for ancient data by simulating sequence data reflecting the properties of an ancient dataset and performing test runs using the mapping software and parameter settings of interest. We showcase TAPAS by using it to assess and improve mapping strategy for a degraded sample from a banded linsang (Prionodon linsang), for which no closely related reference is currently available. This enables a 1.8-fold increase of the number of mapped reads without sacrificing mapping specificity. The increase of mapped reads effectively reduces the need for additional sequencing, thus making more economical use of time, resources, and sample material.
High-throughput sequence data retrieved from ancient or other degraded samples has led to unprecedented insights into the evolutionary history of many species, but the analysis of such sequences also poses specific computational challenges. The most commonly used approach involves mapping sequence reads to a reference genome. However, this process becomes increasingly challenging with an elevated genetic distance between target and reference or with the presence of contaminant sequences with high sequence similarity to the target species. The evaluation and testing of mapping efficiency and stringency are thus paramount for the reliable identification and analysis of ancient sequences. In this paper, we present ‘TAPAS’, (Testing of Alignment Parameters for Ancient Samples), a computational tool that enables the systematic testing of mapping tools for ancient data by simulating sequence data reflecting the properties of an ancient dataset and performing test runs using the mapping software and parameter settings of interest. We showcase TAPAS by using it to assess and improve mapping strategy for a degraded sample from a banded linsang (Prionodon linsang), for which no closely related reference is currently available. This enables a 1.8-fold increase of the number of mapped reads without sacrificing mapping specificity. The increase of mapped reads effectively reduces the need for additional sequencing, thus making more economical use of time, resources, and sample material.
Background
The unisexual Amazon molly (Poecilia formosa) originated from a hybridization between two sexual species, the sailfin molly (Poecilia latipinna) and the Atlantic molly (Poecilia mexicana). The Amazon molly reproduces clonally via sperm-dependent parthenogenesis (gynogenesis), in which the sperm of closely related species triggers embryogenesis of the apomictic oocytes, but typically does not contribute genetic material to the next generation. We compare for the first time the gonadal transcriptome of the Amazon molly to those of both ancestral species, P. mexicana and P. latipinna.
Results
We sequenced the gonadal transcriptomes of the P. formosa and its parental species P. mexicana and P. latipinna using Illumina RNA-sequencing techniques (paired-end, 100 bp). De novo assembly of about 50 million raw read pairs for each species was performed using Trinity, yielding 106,922 transcripts for P. formosa, 115,175 for P. latipinna, and 133,025 for P. mexicana after eliminating contaminations. On the basis of sequence similarity comparisons to other teleost species and the UniProt databases, functional annotation, and differential expression analysis, we demonstrate the similarity of the transcriptomes among the three species. More than 40% of the transcripts for each species were functionally annotated and about 70% were assigned to orthologous genes of a closely related species. Differential expression analysis between the sexual and unisexual species uncovered 2035 up-regulated and 564 down-regulated genes in P. formosa. This was exemplary validated for six genes by qRT-PCR.
Conclusions
We identified more than 130 genes related to meiosis and reproduction within the apomictically reproducing P. formosa. Overall expression of these genes seems to be down-regulated in the P. formosa transcriptome compared to both ancestral species (i.e., 106 genes down-regulated, 29 up-regulated). A further 35 meiosis and reproduction related genes were not found in the P. formosa transcriptome, but were only expressed in the sexual species. Our data support the hypothesis of general down-regulation of meiosis-related genes in the apomictic Amazon molly. Furthermore, the obtained dataset and identified gene catalog will serve as a resource for future research on the molecular mechanisms behind the reproductive mode of this unisexual species.
Abstract
Background
The unisexual Amazon molly (Poecilia formosa) originated from a hybridization between two sexual species, the sailfin molly (Poecilia latipinna) and the Atlantic molly (Poecilia mexicana). The Amazon molly reproduces clonally via sperm-dependent parthenogenesis (gynogenesis), in which the sperm of closely related species triggers embryogenesis of the apomictic oocytes, but typically does not contribute genetic material to the next generation. We compare for the first time the gonadal transcriptome of the Amazon molly to those of both ancestral species, P. mexicana and P. latipinna.
Results
We sequenced the gonadal transcriptomes of the P. formosa and its parental species P. mexicana and P. latipinna using Illumina RNA-sequencing techniques (paired-end, 100 bp). De novo assembly of about 50 million raw read pairs for each species was performed using Trinity, yielding 106,922 transcripts for P. formosa, 115,175 for P. latipinna, and 133,025 for P. mexicana after eliminating contaminations. On the basis of sequence similarity comparisons to other teleost species and the UniProt databases, functional annotation, and differential expression analysis, we demonstrate the similarity of the transcriptomes among the three species. More than 40% of the transcripts for each species were functionally annotated and about 70% were assigned to orthologous genes of a closely related species. Differential expression analysis between the sexual and unisexual species uncovered 2035 up-regulated and 564 down-regulated genes in P. formosa. This was exemplary validated for six genes by qRT-PCR.
Conclusions
We identified more than 130 genes related to meiosis and reproduction within the apomictically reproducing P. formosa. Overall expression of these genes seems to be down-regulated in the P. formosa transcriptome compared to both ancestral species (i.e., 106 genes down-regulated, 29 up-regulated). A further 35 meiosis and reproduction related genes were not found in the P. formosa transcriptome, but were only expressed in the sexual species. Our data support the hypothesis of general down-regulation of meiosis-related genes in the apomictic Amazon molly. Furthermore, the obtained dataset and identified gene catalog will serve as a resource for future research on the molecular mechanisms behind the reproductive mode of this unisexual species.