Mating Experiments Research Articles

Generation of TALEN-mediated FH knockout rat model.

Transcription activator-like effector nucleases (TALENs) are valuable tools for precise genome engineering of laboratory animals. Here we utilized this technique for efficient site-specific gene modification to create a fumarate hydratase (FH) gene knockout rat model, in which there was an 11 base-pair deletion in the first exon of the FH gene in 111 rats. 18 live-born targeted mutation offsprings were produced from 80 injected zygotes with 22.5% efficiency, indicating high TALEN knockout success in rat zygots. Only heterozygous deletion was observed in the offsprings. Sixteen pairs of heterozygous FH knockout (FH+/−) rats were arranged for mating experiments for six months without any homozygous KO rat identified. Sequencing from the pregnant rats embryo samples showed no homozygous FH KO, indicating that homozygous FH KO is embryonically lethal. Comparatively, the litter size was decreased in both male and female FH+/− KO rats. There was no behaviour difference between the FH+/− KO and the control rats except that the FH+/− KO male rats showed significantly higher body weight in the 16-week observation period. Clinical haematology and biochemical examinations showed hematopoietic and kidney dysfunction in the FH+/− KO rats. Small foci of anaplastic lesions of tubular epithelial cells around glomeruli were identified in the FH+/− kidney, and these anaplastic cells were comparatively positive for Ki67, p53 and Sox9, and such findings are most probably related to the kidney dysfunction reflected by the biochemical examinations of the rats. In conclusion, we have successfully established an FH+/− KO rat model, which will be useful for further functional FH studies.

Sublethal Concentration of Beta-Cypermethrin Influences Fecundity and Mating Behavior of Carposina sasakii (Lepidoptera: Carposinidae) Adults.

The purpose of this study was to evaluate the sublethal effects of the beta-cypermethrin on calling behavior and fecundity of a major fruit-boring pest of apple, Carposina sasakii Matsumura. The mating rate, fertility (total number of eggs laid per female), and adult longevity of adults were remarkably decreased as compared with that in control when the adults were exposed to 10% lethal concentrations (LC10) of beta-cypermethrin (LC10♀ × LC10♂), and the age-specific survival rate (lx) was also negatively affected by sublethal beta-cypermethrin especially for the LC10♀× LC10♂ mating combination. However, the age-specific fecundity (mx) was stimulated particularly in the combination of CK♀× LC10♂. Furthermore, the oviposition period was prolonged and the number of eggs was significantly increased for combinations of CK♀× LC10♂ and LC10♀× CK♂. In the mating experiments, males in control or LC10-beta-cypermethrin treatments preferred to mate with females in control. It might be because of lower calling rate of female survivors treated with sublethal beta-cypermethrin. Our data indicate that treatment of beta-cypermethrin had a sublethal effect on the development and production of C. sasakii, and their mating behavior changes in surviving adults that may contribute to assortative mating.

Taxonomy and developmental morphology of Rogersiomyces malaysianus comb. nov. (Cantharellales, Agaricomycetes)

A basidiomycete fungus from Vietnam, grown in a moist chamber on bark (as substrate) was identified as Hyphobasidiofera malaysiana. The isolation of this fungus into axenic culture allowed for investigations such as culture morphology, large and small subunit molecular phylogenies, life cycle karyology, and mating experiments. Comparative morphological data revealed that this species belongs to Rogersiomyces and the name Rogersiomyces malaysianus comb. nov. is proposed. Molecular studies revealed close relationships of Rogersiomyces, Sistotrema sensu lato, and Hydnum sensu stricto (Cantharellales, Agaricomycetes). The fungus produces binucleate basidiospores that germinate to form clamped and dikaryotic mycelium. The cultural characters and reproductive structures of this unusual sistotremoid fungus are described. It was concluded that the characteristic of dikaryotization into basidium, ephemeral basidiomata, and statismosporic basidia represent an adaptation of this fungus to irregular, but plentiful moistening during monsoon climate.

Co-occurrence of ecologically equivalent cryptic species of spider wasps.

Many cryptic species have been discovered in various taxonomic groups based on molecular phylogenetic analyses and mating experiments. Some sympatric cryptic species share equivalent resources, which contradicts the competitive exclusion principle. Two major theories have been proposed to explain the apparent lack of competitive exclusion, i.e. niche-based coexistence and neutral model, but a conclusive explanation is lacking. Here, we report the co-occurrence of cryptic spider wasp species appearing to be ecologically equivalent. Molecular phylogenetic analyses and mating experiments revealed that three phylogenetically closely related species are found sympatrically in Japan. These species share the same resources for larval food, and two of the species have the same niche for nesting sites, indicating a lack of competitive exclusion. This evidence may suggest that ecologically equivalent species can co-occur stably if their shared resources are sufficiently abundant that they cannot be over-exploited.

Last male wins the egg fertilization fight: A case study in ladybird, Menochilus sexmaculatus

Sexual selection and the mechanisms involved in sperm competition have not been greatly explored in ladybird beetles. The present study was conducted to investigate the processes of sperm competition and the role of mate guarding behaviour in its regulation in ladybird beetles. We investigated these questions in polyandrous females of the ladybird, Menochilus sexmaculatus (Coleoptera: Coccinellidae) using a phenotypic marker (typical and intermediate morph) to assess paternity of offspring; to determine sperm competition. We conducted two double mating experiments: (i) complete first and second matings, and (ii) disrupted first and complete second matings each using homomorphic and heteromorphic pairing in alternation. Males which mated last were found to sire up to 72% of the offspring produced, indicating last male sperm precedence. Morph itself, independent of mating order, did not have a significant effect on proportion of offspring sired. Paternity share of the last male was negatively associated with mating duration of the first male; mating duration of the first male being indicative of mate guarding. This therefore indicates that prolonged matings by first males are essentially examples of post-copulatory mate guarding to prevent last male sperm precedence.

Multidrug resistance and transferability of blaCTX-M among extended-spectrum β-lactamase-producing enteric bacteria in biofilm.

This study aimed to investigate the occurrence of biofilm-forming extended-spectrum β-lactamase (ESBL)-producing enteric bacteria in hospital wastewater and to evaluate their antibiotic resistance behaviour and transferability of the plasmid-encoded blaCTX-M gene in biofilm. ESBL production was confirmed using the combined disc test and Etest. Amplification of blaCTX-M was performed by PCR. Antibiotic susceptibility was evaluated using the disc diffusion assay and broth dilution method. Transfer of blaCTX-M in planktonic and biofilm state was performed by broth mating and filter mating experiments, respectively. Among 110 enteric bacteria, 24 (21.8%) isolates belonging to Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae were found to produce ESBL and formed varying levels of biofilm in vitro. Presence of blaCTX-M was detected in 18 (75%) ESBL-producing isolates. A many fold increase in resistance to antibiotics was observed in biofilm. Among ESBL-producers, seven isolates could transfer the blaCTX-M gene by conjugation, with transfer frequencies ranging from 2.22×10(-4) to 7.14×10(-2) transconjugants/recipient cell in the planktonic state and from 3.04×10(-3) to 9.15×10(-1) in biofilm. The transfer frequency of blaCTX-M was significantly higher in biofilm compared with the planktonic state, and co-transfer of ciprofloxacin resistance was also detected in five isolates. This study demonstrates that biofilm-forming ESBL-producing enteric bacteria with a greater transfer frequency of resistance genes will lead to frequent dissemination of β-lactam and fluoroquinolone resistance genes in environmental settings. The emergence and spread of such multidrug resistance is a serious threat to animal and public health.

Detection of Self Incompatibility Genotypes in Prunus africana: Characterization, Evolution and Spatial Analysis.

In flowering plants, self-incompatibility is an effective genetic mechanism that prevents self-fertilization. Most Prunus tree species exhibit a homomorphic gametophytic self-incompatibility (GSI) system, in which the pollen phenotype is encoded by its own haploid genome. To date, no identification of S-alleles had been done in Prunus africana, the only member of the genus in Africa. To identify S-RNase alleles and hence determine S-genotypes in African cherry (Prunus africana) from Mabira Forest Reserve, Uganda, primers flanking the first and second intron were designed and these amplified two bands in most individuals. PCR bands on agarose indicated 26 and 8 different S-alleles for second and first intron respectively. Partial or full sequences were obtained for all these fragments. Comparison with published S-RNase data indicated that the amplified products were S-RNase alleles with very high interspecies homology despite the high intraspecific variation. Against expectations for a locus under balancing selection, frequency and spatial distribution of the alleles in a study plot was not random. Implications of the results to breeding efforts in the species are discussed, and mating experiments are strongly suggested to finally prove the functionality of SI in P. africana.

Interaction of Wolbachia and Bloodmeal Type in Artificially Infected Aedes albopictus (Diptera: Culicidae).

Maternally inherited Wolbachia bacteria are being introduced into vector mosquito populations, with the goal of reducing the transmission of diseases such as dengue fever. The infection dynamics of Wolbachia depends upon the ability of Wolbachia to manipulate host reproduction as well as any fitness costs imposed upon the host. Some vector mosquito species are opportunistic blood feeders, utilizing both human and nonhuman vertebrate hosts, and the effects of bloodmeal source on Wolbachia phenotype is not well understood. Here we transfer wMelPop Wolbachia from Drosophila melanogaster (Meigen) into wild-type Aedes albopictus (Skuse) and characterize the resulting triple infection by examining for an effect of human and mouse blood on the Wolbachia infection persistence and phenotypes. When provided with human blood, the triple Wolbachia infection was persistent, with high maternal inheritance and relatively little fecundity cost, and a pattern of imperfect unidirectional cytoplasmic incompatibility was observed in mating experiments between wild-type and triply infected individuals. With mouse blood, reduced female fecundity and low maternal inheritance were observed in wMelPop-infected females, which affected the typical pattern of unidirectional CI. Our findings indicate the interactive effects of Wolbachia infection and blood source drive distinct shifts in the Wolbachia-host symbiotic association.

Potential transfer of extended spectrum β-lactamase encoding gene, blashv18 gene, between Klebsiella pneumoniae in raw foods

This study investigated the transfer frequency of the extended-spectrum β-lactamase-encoding gene (blaSHV18) among Klebsiella pneumoniae in tryptic soy broth (TSB), pasteurized milk, unpasteurized milk, alfalfa sprouts and chopped lettuce at defined temperatures. All transconjugants were characterized phenotypically and genotypically. KP04ΔKM and KP08ΔKM isolated from seed sprouts and KP342 were used as recipients in mating experiments with K. pneumoniae ATCC 700603 serving as the donor. In mating experiments, no transconjugants were detected at 4 °C in liquid media or chopped lettuce, but detected in all media tested at 15 °C, 24 °C, and 37 °C. At 24 °C, the transfer of blaSHV18 gene occurred more frequently in alfalfa sprouts (5.15E-04 transconjugants per recipient) and chopped lettuce (3.85E-05) than liquid media (1.08E-05). On chopped lettuce, transconjugants were not detected at day 1 post-mating at 15 °C, but observed on day 2 (1.43E-05). Transconjugants carried the blaSHV18 gene transferred from the donor and the virulence gene harbored by recipient. More importantly, a class 1 integrase gene and resistance to tetracycline, trimethoprim/sulfamethoxazole were co-transferred during mating. These quantitative results suggest that fresh produce exposed to temperature abuse may serve as a competent vehicle for the spread of gene encoding for antibiotic resistance, having a potential negative impact on human health.

Acetylshikonin from Zicao exerts antifertility effects at high dose in rats by suppressing the secretion of GTH

Zicao is being highlighted as a promising Chinese medicine due to all the beneficial effects that have been associated with its use. Unfortunately, studies on the toxicity of Zicao in different species are still missing and should be carried out. In this study, we investigated whether Acetylshikonin (AS) from Zicao has an anti-fertility effect through mating experiments and explored its underling mechanism. Sprague-Dawley rats received no treatment or were treated with 120, 360 or 1080 mg/kg AS extract by intragastric administration for 2 weeks. The rat pregnancy rate of the 1080 mg/kg dose group was significantly decreased relative to control group, while it recovered after a month of drug withdrawal, which indicated that the effect of antifertility is reversible. Serum follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels in rat were significantly decreased by AS. The secretion of FSH in rat anterior pituitary cells was decreased but the synthesis was not affected. AS reduced the number of developing follicle and mature follicle in rat ovarian cortical. Maybe all of these resulted from AS decreased the expression of synaptotagmin-1 and SNAP-25 which were the critical proteins of exocytosis. Our data suggested that AS at high dose can suppress the ability of pregnancy of the rats through decreasing serum FSH and LH levels by affecting exocytosis process of gonadotropic hormone (GTH).

The transcriptional regulators SteA and StuA contribute to keratin degradation and sexual reproduction of the dermatophyte Arthroderma benhamiae.

Most superficial fungal infections are caused by dermatophytes, a specialized group of filamentous fungi which exclusively infect keratinized host structures such as hair, skin and nails. Since little is known about the molecular basis of pathogenicity and sexual reproduction in dermatophytes, here we functionally addressed two central transcriptional regulators, SteA and StuA. In the zoophilic species Arthroderma benhamiae a strategy for targeted genetic manipulation was recently established, and moreover, the species is teleomorphic and thus allows performing assays based on mating. By comparative genome analysis homologs of the developmental regulators SteA and StuA were identified in A. benhamiae. Knock-out mutants of the corresponding genes as well as complemented strains were generated and phenotypically characterized. In contrast to A. benhamiae wild type and complemented strains, both mutants failed to produce sexual reproductive structures in mating experiments. Analysis of growth on keratin substrates indicated that loss of steA resulted in the inability of ΔsteA mutants to produce hair perforation organs, but did not affect mycelia formation during growth on hair and nails. By contrast, ΔstuA mutants displayed a severe growth defect on these substrates, but were still able to produce hair perforations. Hence, formation of hair perforation organs and fungal growth on hair per se are differentially regulated processes. Our findings on the major role of SteA and StuA during sexual development and keratin degradation in A. benhamiae provide insights into their role in dermatophytes and further enhance our knowledge of basic biology and pathogenicity of these fungi.

Reproductive Incompatibility Involving Senegalese Aedes aegypti (L) Is Associated with Chromosome Rearrangements

Aedes aegypti, the primary vector of dengue, yellow fever and Zika flaviviruses, consists of at least two subspecies. Aedes aegypti (Aaa) is light in color, has pale scales on the first abdominal tergite, oviposits in artificial containers, and preferentially feeds on humans. Aedes aegypti formosus (Aaf), has a dark cuticle, is restricted to sub-Saharan Africa, has no pale scales on the first abdominal tergite and frequently oviposits in natural containers. Scale patterns correlate with cuticle color in East Africa but not in Senegal, West Africa where black cuticle mosquitoes display a continuum of scaling patterns and breed domestically indoors. An earlier laboratory study did not indicate any pre- or postzygotic barriers to gene flow between Aaa and Aaf in East Africa. However, similar attempts to construct F1 intercross families between Aaa laboratory strains and Senegal Ae. aegypti (SenAae) failed due to poor F1 oviposition and low F2 egg-to-adult survival. Insemination and assortative mating experiments failed to identify prezygotic mating barriers. Backcrosses were performed to test for postzygotic isolation patterns consistent with Haldane’s rule modified for species, like Aedes, that have an autosomal sex determining locus (SDL). Egg-pupal survival was predicted to be low in females mated to hybrid F1 males but average when a male mates with a hybrid F1 female. Survival was in fact significantly reduced when females mated to hybrid males but egg-pupal survival was significantly increased when males were mated to hybrid F1 females. These observations are therefore inconclusive with regards to Haldane’s rule. Basic cytogenetic analyses and Fluorescent In Situ Hybridization (FISH) experiments were performed to compare SenAae strains with the IB12 strain of Aaa that was used for genome sequencing and physical mapping. Some SenAae strains had longer chromosomes than IB12 and significantly different centromeric indices on chromosomes 1 and 3. DAPI staining was used to identify AT-rich regions, chromomycin A3 following pretreatment with barium hydroxide stained for GC-rich regions and stained the ribosomal RNA locus and YOYO-1 was used to test for differential staining. Chromosome patterns in SenAae strains revealed by these three stains differed from those in IB12. For FISH, 40 BAC clones previously physically mapped on Aaa chromosomes were used to test for chromosome rearrangements in SenAae relative to IB12. Differences in the order of markers identified two chromosomal rearrangements between IB12 and SenAae strains. The first rearrangement involves two overlapping pericentric (containing the centromere) inversions in chromosome 3 or an insertion of a large fragment into the 3q arm. The second rearrangement is close to the centromere on the p arm of chromosome 2. Linkage analysis of the SDL and the white-eye locus identified a likely chromosomal rearrangement on chromosome 1. The reproductive incompatibility observed within SenAae and between SenAae and Aaa may be generally associated with chromosome rearrangements on all three chromosomes and specifically caused by pericentric inversions on chromosomes 2 and 3.

Ultimate Drivers and Proximate Correlates of Polyandry in Predatory Mites.

Polyandry is more widespread than anticipated from Bateman’s principle but its ultimate (evolutionary) causes and proximate (mechanistic) correlates are more difficult to pinpoint than those of polygyny. Here, we combined mating experiments, quantification of reproductive traits and microsatellite genotyping to determine the fitness implications of polyandry in two predatory mite species, where males are highly polygynous (up to 45 fertilized females during life), whereas females range from monandry to various polyandry levels. The medium-level polyandrous (up to eight male mates possible) Neoseiulus californicus received clear direct and indirect benefits: multiply mated females produced more offspring with higher survival chances over longer times than singly mated females. In contrast, singly and multiply mated females of the low-level polyandrous (commonly two male mates at maximum) Phytoseiulus persimilis produced similar numbers of offspring having similar survival chances. In both species, multiple mating resulted in mixed offspring paternities, opening the chance for indirect fitness benefits such as enhanced genetic compatibility, complementarity and/or variability. However, the female re-mating likelihood and the paternity chance of non-first male mates were lower in P. persimilis than in N. californicus. Regarding proximate factors, in both species first mating duration and female re-mating likelihood were negatively correlated. Based on occasional fertilization failure of first male mates in P. persimilis, and mixed offspring paternities in both species, we argue that fertilization assurance and the chance to gain indirect fitness benefits are the ultimate drivers of polyandry in P. persimilis, whereas those of N. californicus are higher offspring numbers coupled with enhanced offspring viability and possibly other indirect fitness benefits. Overall, the adaptive significance and proximate events well reflected the polyandry levels. Our study provides a key example for linking behavioral experiments, quantification of reproductive traits and paternity analysis via offspring genotyping to explain the evolution of differing levels of polyandry.

Morphological analysis of the female reproductive system of Stenorhynchus seticornis (Brachyura: Inachoididae) and comparisons with other Majoidea

AbstractWe examined the female reproductive system of the yellowline arrow crab Stenorhynchus seticornis by means of histological and histochemical techniques. Mature specimens obtained in the field were kept in the laboratory for mating experiments. After 24 h, newly mated females were dissected, and their reproductive trait routinely processed for embedding in historesin. The specimens examined each possessed a pair of kidney‐shaped seminal receptacles (SR), and these we classified as ventral type, based on the location of the oviduct opening. The mesodermal dorsal region (DR) of SR consisted of a stratified epithelium with scaly cells, while the ectodermal ventral region (VR) was composed of a simple epithelium covered by a cuticle. The oviduct opened at the transition region (TR) between DR and VR and had no velum. The simple epithelium of TR had more folds on the face of the oviduct opening. The vagina exhibited the same features as the TR epithelium and was contiguous to VR, anchored by muscles. In the lumen, from one to three strata of sperm packets were observed, the dorsal one containing free sperm, and the most ventral stratum, spermatophores. An acidophilic glycoprotein layer enclosed the strata. Spermatophores in the ventral stratum were enclosed in a voluminous secretion, composed by acid polysaccharides most likely from the last male mated. The ventral‐type receptacle, stratified sperm packets, and polyandry, usually observed in females of Majoidea, suggest the occurrence of sperm competition in S. seticornis, favoring the sperm of the last male mated, as its sperm mass is located near the opening of the female oviduct.

Effect of culture density on sexual reproduction of Ardissonea crystallina (Bacillariophyta) inhabiting the Black Sea

One of the key factors that affect the sexual reproduction of diatoms is the cell concentration in the mating experiments. The concentration of pheromones, which probably initiate gametogenesis in the mixture of cells of opposite sexes, depends on the culture density. The influence of the cell concentration and inoculation pattern on the sexual reproduction has been studied in the experiments with the taxonomically important marine diatom Ardissonea crystallina (C. Agardh) Grunow. Several clones have been isolated from samples collected near Sevastopol (Black Sea). The cell concentration that is most favorable for the species reproduction has been estimated. A low initial density may also increase the time required to start heterothallic sexual reproduction. The optimum cell concentration that is most favorable for the species reproduction has been estimated. Larger volume of the medium allowed reproduction at higher cell concentration. If the initial concentration of cells was greater than the optimal density, reproduction often did not occur, probably due to the cell metabolism products accumulated in the culture.

Divergence before the host shift? Prezygotic reproductive isolation among three varieties of a specialist fly on a single host plant

1. Although divergence via host‐plant shifting is a common theme in the speciation of some phytophagous insects, it is not clear whether host shifts are typically initiators of speciation or if they instead contribute to divergence events already in progress. While host shifts appear to be generally associated with speciation events for flies in the genus Strauzia, three sympatric varieties of the sunflower fly [Strauzia longipennis (Wiedemann)] co‐occur on the same host plant in the Midwestern United States and may have evolved reproductive barriers without a host shift.2. The strength of two prezygotic reproductive barriers was compared among the three S. longipennis varieties: one barrier that is often associated with divergent ecological selection (allochronic isolation), and another that is more likely to be independent of ecological selection (pre‐copulatory sexual isolation). The presence and relative strength of each barrier between fly varieties were evaluated using microsatellites, no choice mating experiments, studies of allochronic isolation, and field collection data.3. Evidence for both allochronic isolation and pre‐copulatory sexual isolation was detected between the three varieties of S. longipennis. The measure of isolation calculated for each barrier between the three varieties was lower than measures calculated between different species of Strauzia found on different hosts, suggesting that subsequent host shifts may increase the degree of reproductive isolation. For Strauzia and other specialist insects, some reproductive isolation may evolve prior to, and indeed may facilitate, host shifts.

Enterococcus faecium ST17 from Coastal Marine Sediment Carrying Transferable Multidrug Resistance Plasmids.

The multidrug-resistant Enterococcus faecium 17i48, sequence type 17, from marine sediment, carrying erm(B), tet(M), and tet(L) genes, was analyzed for the presence of antibiotic resistance plasmids and for the ability to transfer resistance genes. The strain was found to harbor the replicon type (repA) of pRE25, pRUM, pHTβ, and the axe-txe toxin-antitoxin (TA) system. In mating experiments, tet(M) and tet(L) were cotransferred with the repApRE25, whereas erm(B) was consistently cotransferred with the axe-txe and repApRUM, suggesting that tetracycline and erythromycin resistance genes were carried on different elements both transferable by conjugation, likely via pHTβ-mediated mobilization. Hybridization and PCR mapping demonstrated that tet(M) and tet(L) were located in tandem on a pDO1-like plasmid that also carried the repApRE25, whereas erm(B) was carried by a pRUM-like plasmid. Sequencing of the latter plasmid showed a high nucleotide identity with pRUM and the presence of cat, aadE, sat4, and a complete aphA resistance genes. These findings show that the genetic features of E. faecium 17i48 are consistent with a hospital-adapted clone and suggest that antibiotic resistance may spread in the environment, also in the absence of antibiotic pressure, due to TA system plasmid maintenance.

Conjugation-Mediated Transfer of Antibiotic-Resistance Plasmids Between Enterobacteriaceae in the Digestive Tract of Blaberus craniifer (Blattodea: Blaberidae).

Cockroaches, insects of the order Blattodea, seem to play a crucial role in the possible conjugation-mediated genetic exchanges that occur among bacteria that harbor in the cockroach intestinal tract. The gut of these insects can be thought of as an effective invivo model for the natural transfer of antimicrobial resistance plasmids among bacteria. In our study, we evaluated the conjugation-mediated horizontal transfer of resistance genes between Escherichia coli and other microorganisms of the same Enterobacteriaceae family within the intestinal tract of Blaberus craniifer Burmeister, 1838 (Blattodea: Blaberidae). Different invivo mating experiments were performed using E. coli RP4 harboring the RP4 plasmid carrying ampicillin, kanamycin, and tetracycline resistance genes as the donor and E. coli K12 resistant to nalidixic acid or Salmonella enterica serovar Enteritidis IMM39 resistant to streptomycin as the recipients. The RP4 plasmid was successfully transferred to both recipients, producing E. coli K12-RP4 and S. Enteritidis IMM39-RP4 transconjugants. Conjugation frequencies invivo were similar to those previously observed invitro. The transfer of the RP4 plasmid in all transconjugants was confirmed by small-scale plasmid isolation and agar gel electrophoresis, suggesting that the intestinal tract of cockroaches is an effective invivo model for natural gene transfer. Our results confirm that cockroaches allow for the exchange of antimicrobial resistance plasmids among bacteria and may represent a potential reservoir for the dissemination of antibiotic-resistant bacteria in different environments. These findings are particularly significant to human health in the context of health care settings such as hospitals.

Editing of the Luteinizing Hormone Gene to Sterilize Channel Catfish, Ictalurus punctatus, Using a Modified Zinc Finger Nuclease Technology with Electroporation

Channel catfish (Ictalurus punctatus) is the most important freshwater aquaculture species in the USA. Genetically enhanced fish that are sterile could both profit the catfish industry and reduce potential environmental and ecological risks. As the first step to generate sterile channel catfish, three sets of zinc finger nuclease (ZFN) plasmids targeting the luteinizing hormone (LH) gene were designed and electroporated into one-cell embryos, different concentrations were introduced, and the Cel-I assay was conducted to detect mutations. Channel catfish carrying the mutated LH gene were sterile, as confirmed by DNA sequencing and mating experiments. The overall mutation rate was 19.7% for 66 channel catfish, and the best treatment was ZFN set 1 at the concentration 25μg/ml. To our knowledge, this is the first instance of gene editing of fish via plasmid introduction instead of mRNA microinjection. The introduction of the ZFN plasmids may have reduced mosaicism, as mutated individuals were gene edited in every tissue evaluated. Apparently, the plasmids were eventually degraded without integration, as they were not detectable in mutated individuals using PCR. Carp pituitary extract failed to induce spawning and restoration of fertility, indicating the need for developing other hormone therapies to achieve reversal of sterility upon demand. This is the first sterilization achieved using ZFN technology in an aquaculture species and the first successful gene editing of channel catfish. Our results will help understand the roles of the LH gene, purposeful sterilization of teleost fishes, and is a step towards control of domestic, hybrid, exotic, invasive, and transgenic fishes.

Genome sequence and virulence factors of a group G Streptococcus dysgalactiae subsp. equisimilis strain with a new element carrying erm(B).

A Streptococcus dysgalactiae subsp. equisimilis (SDSE) strain WCHSDSE-1, which caused an outbreak of tonsillopharyngitis among healthcare workers in China, was subjected to genome sequencing and analysis. WCHSDSE-1 belongs to the Lancefield group G, emm type stG211.1 and sequence type 44. WCHSDSE-1 has virulence factors for adherence, impairing the recruitment of neutrophils to infection sites and toxins including streptolysins O and S and exotoxin G. WCHSDSE-1 has a 45.4-kb element resembling a conjugative transposon. This element is absent from other known SDSE genomes and contains the macrolide-resistant gene erm(B). Conjugative transfer of erm(B) was not successful in mating experiments, suggesting that the element might have lost its ability of conjugation. An almost identical element, which contains the tetracycline-resistant gene tet(M) instead of erm(B), is present on the genome of Filifactor alocis ATCC 35896. The boundaries and insertion sites of the two elements were identified and both were flanked by a 3-bp direct repeat, which is characteristic of transposition. In conclusion, the spectrum of virulence factors of WCHSDSE-1 is similar to other SDSE strains causing invasive diseases. WCHSDSE-1 possesses a new transposable element encoding macrolide resistance, which could pick up different resistance genes and could be transferred across species in oral microflora.