The hermit crab Clibanarius erythropus actively chooses between shells of different quality

The hermit crab Cilbanarius erythropus lacks protection of his sensitive peon and therefore needs robust shells of gastropodous to live in and get the possibility of retraction. This function of protection is only warranted in intact shells. In nature there is no oversupply of eligible shells and therefore damaged shells have to be used, too. Two kinds of damage are found in the filed, first shells that are reduced convolution which hinders retraction, and second perforated shells, which leave the peon uncovered. In a test of voice we determined the preference between those kinds of damage. The results show that with shells of Monoton lineata the C. erythropus prefers perforated shells, whereas with Nucella lapillus the shells with reduced convolution are preferred.Dem Einsiedlerkrebs Cilbanarius erythropus fehlt der Schutz seines empfindlichen Peons und braucht daher robuste Schalen von Schnecken, um darin zu leben und die Möglichkeit zu haben, sich zurückzuziehen. Diese Schutzfunktion ist nur bei intakten Schalen gewährleistet. In der Natur gibt es kein Überangebot an geeigneten Schalen und daher müssen auch beschädigte Schalen verwendet werden. Zwei Arten von Beschädigungen werden in dem Feld gefunden, erstens Schalen mit reduzierter Faltung, die das Zurückziehen behindern, und zweitens perforierte Schalen, die den Peon unbedeckt lassen. In einem Stimmtest haben wir die Präferenz zwischen diesen Schadensarten ermittelt. Die Ergebnisse zeigen, dass C. erythropus bei Schalen von Monoton lineata perforierte Schalen bevorzugt, während bei Nucella lapillus die Schalen mit reduzierter Faltung bevorzugt werden.Peer Reviewe

Simultaneous Identification of DNA and RNA Viruses Present in Pig Faeces Using Process-Controlled Deep Sequencing

Background: Animal faeces comprise a community of many different microorganisms including bacteria and viruses. Only scarce information is available about the diversity of viruses present in the faeces of pigs. Here we describe a protocol, which was optimized for the purification of the total fraction of viral particles from pig faeces. The genomes of the purified DNA and RNA viruses were simultaneously amplified by PCR and subjected to deep sequencing followed by bioinformatic analyses. The efficiency of the method was monitored using a process control consisting of three bacteriophages (T4, M13 and MS2) with different morphology and genome types. Defined amounts of the bacteriophages were added to the sample and their abundance was assessed by quantitative PCR during the preparation procedure. Results: The procedure was applied to a pooled faecal sample of five pigs. From this sample, 69,613 sequence reads were generated. All of the added bacteriophages were identified by sequence analysis of the reads. In total, 7.7 % of the reads showed significant sequence identities with published viral sequences. They mainly originated from bacteriophages (73.9%) and mammalian viruses (23.9%); 0.8 % of the sequences showed identities to plant viruses. The most abundant detected porcine viruses were kobuvirus, rotavirus C, astrovirus, enterovirus B, sapovirus and picobirnavirus. In addition, sequences with identities to the chimpanzee stool-associated circular ssDNA virus were identified. Whole genome analysis indicates that this virus, tentatively designated as pig stool-associated circular ssDNA virus (PigSCV), represents a novel pi

High diversity of picornaviruses in rats from different continents revealed by deep sequencing

Outbreaks of zoonotic diseases in humans and livestock are not uncommon, and an important component in containment of such emerging viral diseases is rapid and reliable diagnostics. Such methods are often PCR-based and hence require the availability of sequence data from the pathogen. Rattus norvegicus (R. norvegicus) is a known reservoir for important zoonotic pathogens. Transmission may be direct via contact with the animal, for example, through exposure to its faecal matter, or indirectly mediated by arthropod vectors. Here we investigated the viral content in rat faecal matter (n=29) collected from two continents by analyzing 2.2 billion next-generation sequencing reads derived from both DNA and RNA. Among other virus families, we found sequences from members of the Picornaviridae to be abundant in the microbiome of all the samples. Here we describe the diversity of the picornavirus-like contigs including near-full-length genomes closely related to the Boone cardiovirus and Theiler's encephalomyelitis virus. From this study, we conclude that picornaviruses within R. norvegicus are more diverse than previously recognized. The virome of R. norvegicus should be investigated further to assess the full potential for zoonotic virus transmission

Metagenomanalyse von Viren aus Kot von Hausschweinen und wildlebenden urbanen Ratten

The gut of humans and animals comprises a high number of microorganisms, which exert important functions during food digestion and defense of infections. Some of the microorganisms can cause diseases. The gut flora is comprised of bacteria, fungi and protozoa, but a large number of viruses is also present in the gut. Viruses can also be agents of disease. In addition, the viruses of bacteria – the bacteriophages – can regulate the growth of bacteria and transfer genes. The composition of the bacterial gut flora has recently been analysed using metagenomic methods. In contrast, only little is known about the composition of viruses (the so-called virome) in the gut. This is mainly due to methodological problems to identify the large variety of viruses present in a sample. However, the development of high-throughput sequencing techniques – summarized as Next Generation Sequencing (NGS) – has simplified the metagenome analyses during the last years and enables the analysis of viromes. Although first data on human viromes are available now, the viromes of animals have been only scarcely analysed. The aim of the investigations presented here is therefore the analysis of viromes from animals. As the enteric flora is complex and many pathogenic viruses are transmitted by faeces, the metagenomic analyses was conducted using faecal samples. By this, the general composition of the faecal viromes was assessed, but also the possible influence of factors like age and diet was considered. To this end, an NGS-based method for the reliable and reproducible analysis of the faecal virome was developed first. Thereafter, the method was applied to two animal species. Pigs were selected as this animal species represents an important farm animal in Germany and its gut health has a high economic impact. The composition of the viruses in the faeces of pigs should be analysed first. The influence of feeding probiotic bacteria und the age of pigs was investigated. The second selected animal species were wild rats, because these animals are known as reservoirs for pathogens for humans. The viruses detected in these faeces should be mainly analysed regarding their potential for zoonotic transmission to humans. The developed analytic method is based on filtrations and centrifugations to purify and concentrate all virus particles from the faeces. Whole nucleic acid was extracted from the virus particles, amplified and sequenced using NGS. Bioinformatic analyses were used to determine the composition of viruses in the sample based of the sequence data. A control system, which consisted of three bacteriophages added to the samples, was developed and used for optimization of the method and as quality control. In total, 205 different virus species from 36 different virus families were detected in eight pooled faecal sample each derived from six piglets or six young pigs. The main virus groups were pig viruses and bacteriophages. Plant viruses, which are frequently detected in human faecal viromes and which presumably originate from spices of the diet, comprised only a very small fraction in the pig samples. Using the metagenome analysis, a so far unknown pig virus (designated PigSCV) was also identified. In comparative investigations of faecal viromes from a feeding trail of sows and their piglets with the probiotic bacterium Enterococcus faecium NCIMB 10415 a large variability of their composition could generally be identified. However, the faecal virome was nearly not influenced by the probiotic feeding, but strongly by the age of the animals. In contrast to the youngest piglets, which showed a high proportion of pig viruses and a lower proportion of bacteriophages, the sows showed the opposite picture. In addition, the diversity of viral communities increased significantly with the age. Despite bacteriophages, the faecal viromes of 20 wild rats, which originated from the city of Berlin, mainly consisted of known rat viruses from the virus families Parvoviridae and Picobirnaviridae. However, so far unkown picorna-, bocaparvo- and sapoviruses as well as novel circular DNA viruses were also identified. Group A rotaviruses were detected for the first time in rats. Rotaviruses are known as pathogens causing gastroenteritis in animals and humans. The analysis of the whole genome of the rat rotavirus showed that it is closely related to rotaviruses from humans and animals and therefore it may possibly transmissible to them. In conclusion, the investigations show that the developed method is well suited for the analysis of faecal viromes. The application of the method shows the high variability of the virome, which is mainly dependent on the age of the animals. Using the method, we were able to describe viral communities and to detect pathogenic viruses as well as novel viruses identified. Further investigations should simplify the method in order to enable the analysis of higher sample numbers. In addition, the bioinformatic analysis of sequence data should be further developed in order to determine the viromes in more broadness and detail and to enable the analysis of interactions with other microorganisms and the host.Im Darm von Menschen und Tieren lebt eine Vielzahl von Mikroorganismen, die wichtige Funktionen bei der Verdauung und bei der Abwehr von Infektionen besitzen. Einige der Mikroorganismen können allerdings auch zu Erkrankungen führen. Die Darmflora setzt sich aus Bakterien, Pilzen und Protozoen zusammen, aber auch eine Vielzahl von Viren ist im Darm zu finden. Viren können ebenfalls als Krankheitserreger fungieren. Die Viren der Bakterien – die Bakteriophagen – können darüber hinaus das Wachstum von Bakterien regulieren und Gene übertragen. Während die Zusammensetzung der bakteriellen Darmflora in der letzten Zeit durch Metagenom-Analysen schon gut untersucht wurde, ist über die Zusammensetzung der Viren (das sogenannte Virom) im Darm bisher nur wenig bekannt. Dies ist vor allem auf methodische Schwierigkeiten zurückzuführen, die große Vielfalt der in einer Probe enthaltenen Viren zu identifizieren. Die Entwicklung neuer Technologien zur Hochdurchsatz-Sequenzierung von DNA - zusammenfassend als Next Generation Sequencing (NGS) bezeichnet – hat aber in den letzten Jahren Metagenomanalysen stark vereinfacht und damit auch die Analyse von Viromen möglich gemacht. Während erste Daten zur Zusammensetzung von Viromen des Menschen vorliegen, sind die Virome von Tieren bisher nur wenig untersucht worden. Ziel der hier vorgestellten Untersuchungen sollte deshalb die Analyse von Viromen von Tieren sein. Da die Darmflora besonders komplex aufgebaut ist und viele krankmachende Viren über den Kot übertragen werden, sollten die Metagenomanalysen an Kotproben durchgeführt werden. Hierbei sollte sowohl die generelle Zusammensetzung des fäkalen Viroms ermittelt werden, als auch der mögliche Einfluss von Faktoren wie Alter und Ernährung auf diese berücksichtigt werden. Zunächst sollte hierfür eine NGS- basierte Methode zur sicheren und reproduzierbaren Analyse des fäkalen Viroms entwickelt werden. Danach sollte die Methode an zwei ausgewählten Tierarten angewendet werden. Schweine wurden ausgewählt, weil diese Tierart ein wichtiges landwirtschaftliches Nutztier in Deutschland darstellt, deren Darmgesundheit große ökonomische Bedeutung hat. Zunächst sollte die Zusammensetzung der Viren in Fäzes von Schweinen ermittelt werden. Danach sollte der Einfluss einer Fütterung mit probiotischen Bakterien und des Alters der Schweine untersucht werden. Als zweite Tierart wurden wildlebende Ratten ausgewählt, weil diese als Reservoire von Krankheitserregern des Menschen bekannt sind. Die in den Fäzes erhaltenen Viren wurden vor allem hinsichtlich ihrer möglichen zoonotischen Übertragbarkeit auf den Menschen analysiert. Die entwickelte Analyse-Methode beruht auf Filtrationen und Zentrifugationen, die alle Viruspartikel aus den Fäzes reinigen und konzentrieren. Anschließend wurde die Gesamt-Nukleinsäure aus den Viruspartikeln extrahiert, vermehrt und mittels NGS sequenziert. Bioinformatische Analysen ermittelten aus den Sequenzen die Zusammensetzung der Viren in der Probe. Ein Kontrollsystem, bestehend aus drei Bakteriophagen, die der Probe zugegeben wurden, wurde zur Optimierung der Methode und als Qualitätskontrolle entwickelt und benutzt. Im Ergebnis konnten in acht Pool-Proben aus Fäzes von jeweils sechs Ferkeln beziehungsweise jungen Schweinen etwa 205 unterschiedliche Virusarten aus 36 verschiedenen Virusfamilien nachgewiesen werden. Die hauptsächlichen Virusgruppen waren Schweineviren und Bakteriophagen. Pflanzenviren, die im menschlichen fäkalen Virom häufig detektiert wurden und die wahrscheinlich aus Gewürzen aus der Nahrung stammen, wurden in den Schweineproben kaum vorgefunden. Mit Hilfe der Metagenom-Analyse konnte auch ein bisher unbekanntes Schweinevirus (als PigSCV bezeichnet) identifiziert werden. Bei vergleichenden Untersuchungen von fäkalen Viromen aus einem Fütterungsversuch von Sauen und ihren Ferkeln mit dem probiotischen Bakterium Enterococcus faecium NCIMB 10415 konnte generell eine große Variabilität ihrer Zusammensetzungen festgestellt werden. Das fäkale Virom wurde allerdings kaum von der probiotischen Fütterung beeinflusst, sondern war vor allem stark vom Alter der Tiere abhängig. Während die jüngsten Ferkel einen hohen Anteil von Schweineviren und einen geringen Anteil von Bakteriophagen aufwiesen, war dies bei den Sauen genau umgekehrt. Darüber hinaus nahm die Diversität der Zusammensetzung der enthaltenen Viren mit dem Alter deutlich zu. Bakteriophagen ignorierend, bestanden die fäkalen Virome von 20 wildlebenden Ratten, die aus der Innenstadt von Berlin stammten, hauptsächlich aus bekannten Rattenviren aus den Virusfamilien Parvoviridae und Picobirnaviridae. Es wurden jedoch auch bisher unbekannte Picorna-, Bocaparvo- und Sapoviren sowie neue zirkuläre DNA-Viren identifiziert. Erstmals wurde ein Gruppe A-Rotavirus in Ratten nachgewiesen. Rotaviren sind als Gastroenteritis-Erreger bei Tieren und Menschen bekannt. Die Analyse des Gesamtgenoms des Ratten- Rotavirus zeigt, dass es eng mit Rotaviren von Menschen und Tieren verwandt ist und deshalb möglicherweise auf diese übertragen werden kann. Zusammenfassend zeigen die Untersuchungen, dass die entwickelte Methode gut für die Analyse von fäkalen Viromen geeignet ist. Die Anwendung der Methode offenbart eine hohe Variabilität der Virome, die vor allem vom Alter der Tiere abhängig ist. Mit Hilfe der Methode konnten sowohl virale Gemeinschaften beschrieben werden als auch pathogene Viren detektiert und neue Viren identifiziert werden. Weiterführende Untersuchungen sollten einerseits die Methode vereinfachen, um größere Probenzahlen untersuchen zu können. Andererseits sollte insbesondere die bioinformatische Analyse der Sequenzdaten weiterentwickelt werden, um Virome in Zukunft noch umfassender und genauer bestimmen zu können und die Analyse von Wechselwirkungen mit deren Wirt und anderen Mikroorganismen zu ermögliche

The general composition of the faecal virome of pigs depends on age, but not on feeding with a probiotic bacterium.

BACKGROUND:The pig faecal virome, which comprises the community of viruses present in pig faeces, is complex and consists of pig viruses, bacteriophages, transiently passaged plant viruses and other minor virus species. Only little is known about factors influencing its general composition. Here, the effect of the probiotic bacterium Enterococcus faecium (E. faecium) NCIMB 10415 on the pig faecal virome composition was analysed in a pig feeding trial with sows and their piglets, which received either the probiotic bacterium or not. RESULTS:From 8 pooled faecal samples derived from the feeding trial, DNA and RNA virus particles were prepared and subjected to process-controlled Next Generation Sequencing resulting in 390,650 sequence reads. In average, 14% of the reads showed significant sequence identities to known viruses. The percentage of detected mammalian virus sequences was highest (55-77%) in the samples of the youngest piglets and lowest (8-10%) in the samples of the sows. In contrast, the percentage of bacteriophage sequences increased from 22-44% in the youngest piglets to approximately 90% in the sows. The dominating mammalian viruses differed remarkably among 12 day-old piglets (kobuvirus), 54 day-old piglets (boca-, dependo- and pig stool-associated small circular DNA virus [PigSCV]) and the sows (PigSCV, circovirus and "circovirus-like" viruses CB-A and RW-A). In addition, the Shannon index, which reflects the diversity of sequences present in a sample, was generally higher for the sows as compared to the piglets. No consistent differences in the virome composition could be identified between the viromes of the probiotic bacterium-treated group and the control group. CONCLUSION:The analysis indicates that the pig faecal virome shows a high variability and that its general composition is mainly dependent on the age of the pigs. Changes caused by feeding with the probiotic bacterium E. faecium could not be demonstrated using the applied metagenomics method

Detection of bacteriophages used as process control in the analysed samples.

<p>Equal amounts of the bacteriophages T4, MS2 and M13 were added to the pooled faecal samples prior to analysis and the generated reads were screened for the recovered genomic sequence reads of these bacteriophages. The percentage of the number of reads from these bacteriophages in relation to all detected virus reads is indicated. The samples are designated with the group letter (C – control, P – probiotic) and the day number (ap – ante partum, pp – post partum).</p

Relative abundance of mammalian virus genera among all animal viruses detected in the analyzed faecal viromes.

<p>The diagrams show the number of reads with sequence identities to a certain mammalian virus genus in relation to all animal virus reads. Different colours were used for different mammalian virus genera (see Legend). Mammalian viruses, which are so far not assigned to a certain genus, are indicated in apostrophes. Mammalian virus genera showing an abundance of less than 1% in a distinct faecal virome are subsumed in dark light grey colour (<1%). Viruses from non-mammalian hosts are subsumed in light grey colour. The group receiving the probiotic bacterium <i>E. faecium</i> NCIMB 10415 (group P) is shown in the upper row; the control group (group C) is in the lower row. Samples derived from piglets are shown left and those from the sows are shown right. The time-points of sampling are indicated below.</p