Genomic DNA isolation methods from honey bee (Apis mellifera L.) Spermatheca

The honey bee queen (Apis mellifera L.) has a polyandrous mating system, meaning that the queen mates on average with 17 drones from the surroundings in a congregation area. After the mating event, the spermatozoa of the drones are stored in an organ called spermatheca. Genetic analysis of the spermathecal content can provide an estimate of the genetic diversity and purity of the surrounding honey bee populations. This can be particularly useful for conservation and mating centers that need to monitor their populations’ genetic backgrounds. However, isolating enough DNA for genomic applications from such a small and complex matrix can be a challenge. Here, we compared the quantity and quality of DNA isolated using five methods: (i) phenol-chloroform-isopropanol, (ii) QIAamp DNA Minikit, (iii) QIAamp DNA Microkit, (iv) Macherey- Nagel Nucleospin Tissue, and (v) NEB Monarch Genomic DNA Purification Tissue. For each kit, when appropriate, variations including different isolation protocols, lysis incubation times, and the addition of RNA carrier were assayed. The quantity and quality of DNA extracted was assessed by spectrophotometric (SpectroStar®Nano LVis Plate) and fluorometric methods (Quantus ™ Fluorometer). Spectrophotometric quantification indicated nucleic acid concentrations ranging from 2.00 to 55.58 ng/μL, and in 91.43% of the cases, the A260/280 ratios were over 2.00, indicating an elevated presence of RNA. The fluorometric quantification, specific for double-stranded DNA, provided values ranging from 0.02 to 2.30 ng/μL. From the five methods, two alternative protocols of the commercial kit QIAamp DNA Microkit produced a sufficient DNA quantity (≥1.7 ng/μL measured by Quantus) for applications involving SNP genotyping, namely: the Tissue protocol with 6 hours of lysis incubation and the Tissue protocol with 3 hours of incubation, both with addition of RNA carrier. In contrast, overnight lysis decreased the DNA yield. The other methods generally produced low and/or inconsistent DNA recovery. According to our results, QIAamp DNA Microkit with the use of RNA carrier and lysis incubation times between 3 to 6 hours produce the required DNA quantities for SNP genotyping.Contributions of JW and EM were financed through the financial support of the German Federal Ministry for Food and Agriculture, through the intermediary of the Federal Office for Agriculture and Food (project no. 2818BM040).info:eu-repo/semantics/publishedVersio

A molecular tool to detect genetic introgression from Spermatheca content

The genetic integrity of Apis mellifera mellifera is threatened by introgression in many places of its native distribution, after recurrent importations of commercial queens typically belonging to the divergent C-lineage. A growing interest in keeping and protecting A. m. mellifera has motivated the development of conservation programs in many places of Europe. As part of the conservation efforts, isolated mating stations are set to avoid unwanted crosses, but these are not always effective as matings with unwanted drones are frequently reported. An interesting method to monitor the degree of isolation of mating stations could be through genetic analysis of the queen spermatheca contents. While this method implies that queens selected for monitoring are sacrificed, it can be a powerful way of assessing the effectiveness of mating stations because it would allow easy detection of unwanted alleles. Here, we developed an SNP-based tool suited to the analysis of DNA extracted from spermatheca or from pooled DNA of varying sources. To that end, we first designed an SNP panel from whole-genome sequence data generated from 228 drones, of which 148 belonged to the M-lineage (117 A. m. iberiensis and 31 A. m. mellifera) and 80 to the C-lineage (46 A. m. carnica and 34 A. m. ligustica). A total of 5,007 highly differentiated SNPs was found. Based on different criteria, 130 SNPs were selected to be included in the genotyping tool. This tool is based on the NEBNext Direct Genotyping Solution that allows high-throughput, sequence-based target genotyping of single-individual or pooled DNA. To assess the tool’s sensitivity and accuracy, 142 samples (DNA extracted from spermatheca and tissue, as well as known DNA mixtures) were genotyped. After removing the problematic SNPs, 81 were retained and these were able to provide an estimate of the pool introgression level with great accuracy. This tool represents a significant advance in the genetic analysis of honey bee colonies with a variety of applications, including breeding and conservation of A. m. mellifera.info:eu-repo/semantics/publishedVersio

The pesticidal Cry6Aa toxin from Bacillus thuringiensis is structurally similar to HlyE-family alpha pore-forming toxins

Background The Cry6 family of proteins from Bacillus thuringiensis represents a group of powerful toxins with great potential for use in the control of coleopteran insects and of nematode parasites of importance to agriculture. These proteins are unrelated to other insecticidal toxins at the level of their primary sequences and the structure and function of these proteins has been poorly studied to date. This has inhibited our understanding of these toxins and their mode of action, along with our ability to manipulate the proteins to alter their activity to our advantage. To increase our understanding of their mode of action and to facilitate further development of these proteins we have determined the structure of Cry6Aa in protoxin and trypsin-activated forms and demonstrated a pore-forming mechanism of action. Results The two forms of the toxin were resolved to 2.7 Å and 2.0 Å respectively and showed very similar structures. Cry6Aa shows structural homology to a known class of pore-forming toxins including hemolysin E from Escherichia coli and two Bacillus cereus proteins: the hemolytic toxin HblB and the NheA component of the non-hemolytic toxin (pfam05791). Cry6Aa also shows atypical features compared to other members of this family, including internal repeat sequences and small loop regions within major alpha helices. Trypsin processing was found to result in the loss of some internal sequences while the C-terminal region remains disulfide-linked to the main core of the toxin. Based on the structural similarity of Cry6Aa to other toxins, the mechanism of action of the toxin was probed and its ability to form pores in vivo in Caenorhabditis elegans was demonstrated. A non-toxic mutant was also produced, consistent with the proposed pore-forming mode of action. Conclusions Cry6 proteins are members of the alpha helical pore-forming toxins – a structural class not previously recognized among the Cry toxins of B. thuringiensis and representing a new paradigm for nematocidal and insecticidal proteins. Elucidation of both the structure and the pore-forming mechanism of action of Cry6Aa now opens the way to more detailed analysis of toxin specificity and the development of new toxin variants with novel activities

Within-Household Transmission and Bacterial Diversity of Staphylococcus pseudintermedius.

Staphylococcus pseudintermedius can be transmitted between dogs and their owners and can cause opportunistic infections in humans. Whole genome sequencing was applied to identify the relatedness between isolates from human infections and isolates from dogs in the same households. Genome SNP diversity and distribution of plasmids and antimicrobial resistance genes identified related and unrelated isolates in both households. Our study shows that within-host bacterial diversity is present in S. pseudintermedius, demonstrating that multiple isolates from each host should preferably be sequenced to study transmission dynamics

Genomic dynamics of antimicrobial resistance in canine and human derived Staphylococcus pseudintermedius

Staphylococcus pseudintermedius is a bacterium found on the skin and mucous membranes of healthy dogs. Generally, the bacteria is harmless, but the bacteria can also cause infections, such as ear and skin infections. Occasionally, infections with Staphylococcus pseudintermedius occur in humans. We compared Staphylococcus pseudintermedius bacteria originating from infections in dogs with Staphylococcus pseudintermedius bacteria originating from infections in humans. This comparison showed no difference between S. pseudintermedius bacteria from dogs and humans, thereby confirming our suspicion that the Staphylococcus pseudintermedius bacteria pass from the dog to its owner through intensive contact between them. Infections caused by Staphylococcus pseudintermedius can usually be treated effectively with antibiotics, but Staphylococcus pseudintermedius can become resistant to an antibiotic. If so, the Staphylococcus pseudintermedius bacterium is no longer sensitive to the antibiotic and an infection is difficult to treat. In our research, we looked at the best way to measure resistance in Staphylococcus pseudintermedius bacteria in the laboratory. In addition, we investigated how Staphylococcus pseudintermedius becomes resistant to antibiotics. We showed that Staphylococcus pseudintermedius becomes resistant by picking up pieces of DNA from nearby bacteria. Staphylococcus pseudintermedius can even pick up multiple different pieces of DNA and thus become multi-resistant. This means that the bacterium is insensitive to a large number of antibiotics, which makes treatment even more difficult. That is not just unpleasant for the dog with an infection caused by a multi-resistant Staphylococcus pseudintermedius, but may also pose a risk for its owner

Genomic dynamics of antimicrobial resistance in canine and human derived Staphylococcus pseudintermedius

Staphylococcus pseudintermedius is a bacterium found on the skin and mucous membranes of healthy dogs. Generally, the bacteria is harmless, but the bacteria can also cause infections, such as ear and skin infections. Occasionally, infections with Staphylococcus pseudintermedius occur in humans. We compared Staphylococcus pseudintermedius bacteria originating from infections in dogs with Staphylococcus pseudintermedius bacteria originating from infections in humans. This comparison showed no difference between S. pseudintermedius bacteria from dogs and humans, thereby confirming our suspicion that the Staphylococcus pseudintermedius bacteria pass from the dog to its owner through intensive contact between them. Infections caused by Staphylococcus pseudintermedius can usually be treated effectively with antibiotics, but Staphylococcus pseudintermedius can become resistant to an antibiotic. If so, the Staphylococcus pseudintermedius bacterium is no longer sensitive to the antibiotic and an infection is difficult to treat. In our research, we looked at the best way to measure resistance in Staphylococcus pseudintermedius bacteria in the laboratory. In addition, we investigated how Staphylococcus pseudintermedius becomes resistant to antibiotics. We showed that Staphylococcus pseudintermedius becomes resistant by picking up pieces of DNA from nearby bacteria. Staphylococcus pseudintermedius can even pick up multiple different pieces of DNA and thus become multi-resistant. This means that the bacterium is insensitive to a large number of antibiotics, which makes treatment even more difficult. That is not just unpleasant for the dog with an infection caused by a multi-resistant Staphylococcus pseudintermedius, but may also pose a risk for its owner

Expression of reciprocity in Savosavo

Wegener C. Expression of reciprocity in Savosavo. In: Evans N, Gaby A, Levinson SC, Majid A, eds. Reciprocals and Semantic Typology. Amsterdam: John Benjamins; 2011: 213-224.This paper describes how reciprocity is expressed in the Papuan (i.e. non-Austronesian­) language Savosavo, spoken in the Solomon Islands. The main strategy is to use the reciprocal nominal mapamapa, which can occur in different NP positions and always triggers default third person singular masculine agreement, regardless of the number and gender of the referents. After a description of this as well as another strategy that is occasionally used (the ‘joint activity construction’), the paper will provide a detailed analysis of data elicited with set of video stimuli and show that the main strategy is used to describe even clearly asymmetric situations, as long as more than one person acts on more than one person in a joint activity

Comparative genomics of phenotypic antimicrobial resistances in methicillin-resistant Staphylococcus pseudintermedius of canine origin

Staphylococcus pseudintermedius is an important pathogen in dogs. Since 2004, methicillin- resistant S. pseudintermedius (MRSP) isolates, often multidrug resistant, have been observed in dogs in the Netherlands. This study aims to link the observed resistance phenotypes in canine MRSP to genotypic antimicrobial resistance markers, and to study the phylogeny of MRSP by genomic comparisons. The genomes of fifty clinical isolates of MRSP from dogs from the Netherlands were sequenced. The resistance genes were identified, and for twenty one different antimicrobials their presence and sequence were associated with the resistance phenotypes. In case of observed discrepancies, the genes were aligned with reference genes. Of the phenotypic resistances, 98.3% could be explained by the presence of an associated resistance gene or point mutation. Discrepancies were mainly resistance genes present in susceptible isolates; 43.8% (7/16) were explained by an insertion, deletion or mutation in the gene. In relation with the resistance gene presence or absence, a single-nucleotide polymorphism (SNP) based phylogeny was constructed to define the population dynamics. The resistance gene content differed according to clonal complex, from very conserved (CC45), to partly conserved (CC71) to highly diverse (CC258) resistance gene patterns. In conclusion, this study shows that the antimicrobial genotype from whole genome sequencing is highly predictive of the resistance phenotype in MRSP. Interestingly, the observed clonal complexes of MRSP isolates were linked with resistance gene patterns

Comparative genomics of phenotypic antimicrobial resistances in methicillin-resistant Staphylococcus pseudintermedius of canine origin

Staphylococcus pseudintermedius is an important pathogen in dogs. Since 2004, methicillin- resistant S. pseudintermedius (MRSP) isolates, often multidrug resistant, have been observed in dogs in the Netherlands. This study aims to link the observed resistance phenotypes in canine MRSP to genotypic antimicrobial resistance markers, and to study the phylogeny of MRSP by genomic comparisons. The genomes of fifty clinical isolates of MRSP from dogs from the Netherlands were sequenced. The resistance genes were identified, and for twenty one different antimicrobials their presence and sequence were associated with the resistance phenotypes. In case of observed discrepancies, the genes were aligned with reference genes. Of the phenotypic resistances, 98.3% could be explained by the presence of an associated resistance gene or point mutation. Discrepancies were mainly resistance genes present in susceptible isolates; 43.8% (7/16) were explained by an insertion, deletion or mutation in the gene. In relation with the resistance gene presence or absence, a single-nucleotide polymorphism (SNP) based phylogeny was constructed to define the population dynamics. The resistance gene content differed according to clonal complex, from very conserved (CC45), to partly conserved (CC71) to highly diverse (CC258) resistance gene patterns. In conclusion, this study shows that the antimicrobial genotype from whole genome sequencing is highly predictive of the resistance phenotype in MRSP. Interestingly, the observed clonal complexes of MRSP isolates were linked with resistance gene patterns</p