scholarly journals Transgenic Testing Does Not Support a Role for Additional Candidate Genes in Wolbachia Male Killing or Cytoplasmic Incompatibility

mSystems ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Jessamyn I. Perlmutter ◽  
Jane E. Meyers ◽  
Seth R. Bordenstein
Keyword(s):  
Drosophila Melanogaster ◽  
Copy Number ◽  
Cytoplasmic Incompatibility ◽  
Functional Study ◽  
Content Type ◽  
Transgenic Expression ◽  
Additional Gene ◽  
Reproductive Parasitism ◽  
Transcript Length ◽  
Male Killing

ABSTRACT Endosymbiotic bacteria in the genus Wolbachia remarkably infect nearly half of all arthropod species. They spread in part because of manipulations of host sexual reproduction that enhance the maternal transmission of the bacteria, including male killing (death of infected males) and unidirectional cytoplasmic incompatibility (CI; death of offspring from infected fathers and uninfected mothers). Recent discoveries identified several genes in prophage WO of Wolbachia (wmk, cifA, and cifB) that fully or partially recapitulate male killing or CI when transgenically expressed in Drosophila melanogaster. However, it is not yet fully resolved if other gene candidates contribute to these phenotypes. Here, we transgenically tested 10 additional gene candidates for their involvement in male killing and/or CI. The results show that despite sequence and protein architecture similarities or comparative associations with reproductive parasitism, transgenic expression of the candidates does not recapitulate male killing or CI. Sequence analysis across Wmk and its closest relatives reveals amino acids that may be important to its function. In addition, evidence is presented to propose new hypotheses regarding the relationship between wmk transcript length and its ability to kill a given host, as well as copy number of wmk homologs within a bacterial strain, which may be predictive of host resistance. Together, these analyses continue to build the evidence for identification of wmk, cifA, and cifB as the major genes that have thus far been shown to cause reproductive parasitism in Wolbachia, and the transgenic resources provide a basis for further functional study of phage WO genes. IMPORTANCE Wolbachia are widespread bacterial endosymbionts that manipulate the reproduction of diverse arthropods to spread through a population and can substantially shape host evolution. Recently, reports identified three prophage WO genes (wmk, cifA, and cifB) that transgenically recapitulate many aspects of reproductive manipulation in Drosophila melanogaster. Here, we transgenically tested 10 additional gene candidates for CI and/or male killing in flies. The results yield no evidence for the involvement of these gene candidates in reproductive parasitism, bolstering the evidence for identification of the cif and wmk genes as the major factors involved in their phenotypes. In addition, evidence supports new hypotheses for prediction of male-killing phenotypes or lack thereof based on wmk transcript length and copy number. These experiments inform efforts to understand the full basis of reproductive parasitism for basic and applied purposes and lay the foundation for future work on the function of an interesting group of Wolbachia and phage WO genes.

scholarly journals Genome Sequence of the Drosophila melanogaster Male-Killing Spiroplasma Strain MSRO Endosymbiont

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Juan C. Paredes ◽  
Jeremy K. Herren ◽  
Fanny Schüpfer ◽  
Ray Marin ◽  
Stéphane Claverol ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Genome Sequence ◽  
Molecular Mechanisms ◽  
Germ Line ◽  
Ex Vivo ◽  
Experimental Studies ◽  
Content Type ◽  
Modes Of Transmission ◽  
Male Killing ◽  
High Level

ABSTRACTSpiroplasmas are helical and motile members of a cell wall-less eubacterial group calledMollicutes. Although all spiroplasmas are associated with arthropods, they exhibit great diversity with respect to both their modes of transmission and their effects on their hosts; ranging from horizontally transmitted pathogens and commensals to endosymbionts that are transmitted transovarially (i.e., from mother to offspring). Here we provide the first genome sequence, along with proteomic validation, of an endosymbiotic inheritedSpiroplasmabacterium, theSpiroplasma poulsoniiMSRO strain harbored byDrosophila melanogaster. Comparison of the genome content ofS. poulsoniiwith that of horizontally transmitted spiroplasmas indicates thatS. poulsoniihas lost many metabolic pathways and transporters, demonstrating a high level of interdependence with its insect host. Consistent with genome analysis, experimental studies showed thatS. poulsoniimetabolizes glucose but not trehalose. Notably, trehalose is more abundant than glucose inDrosophilahemolymph, and the inability to metabolize trehalose may preventS. poulsoniifrom overproliferating. Our study identifies putative virulence genes, notably, those for a chitinase, the H2O2-producing glycerol-3-phosphate oxidase, and enzymes involved in the synthesis of the eukaryote-toxic lipid cardiolipin.S. poulsoniialso expresses on the cell membrane one functional adhesion-related protein and two divergent spiralin proteins that have been implicated in insect cell invasion in other spiroplasmas. These lipoproteins may be involved in the colonization of theDrosophilagerm line, ensuringS. poulsoniivertical transmission. TheS. poulsoniigenome is a valuable resource to explore the mechanisms of male killing and symbiont-mediated protection, two cardinal features of many facultative endosymbionts.IMPORTANCEMost insect species, including important disease vectors and crop pests, harbor vertically transmitted endosymbiotic bacteria. These endosymbionts play key roles in their hosts’ fitness, including protecting them against natural enemies and manipulating their reproduction in ways that increase the frequency of symbiont infection. Little is known about the molecular mechanisms that underlie these processes. Here, we provide the first genome draft of a vertically transmitted male-killingSpiroplasmabacterium, theS. poulsoniiMSRO strain harbored byD. melanogaster. Analysis of theS. poulsoniigenome was complemented by proteomics andex vivometabolic experiments. Our results indicate thatS. poulsoniihas reduced metabolic capabilities and expresses divergent membrane lipoproteins and potential virulence factors that likely participate inSpiroplasma-host interactions. This work fills a gap in our knowledge of insect endosymbionts and provides tools with which to decipher the interaction betweenSpiroplasmabacteria and their well-characterized hostD. melanogaster, which is emerging as a model of endosymbiosis.

scholarly journals Paternal Grandmother Age Affects the Strength of Wolbachia-Induced Cytoplasmic Incompatibility in Drosophila melanogaster

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Emily M. Layton ◽  
Jungmin On ◽  
Jessamyn I. Perlmutter ◽  
Seth R. Bordenstein ◽  
J. Dylan Shropshire
Keyword(s):  
Drosophila Melanogaster ◽  
Cytoplasmic Incompatibility ◽  
Genetic Model ◽  
Relative Fitness ◽  
Content Type ◽  
Adult Sons ◽  
Fitness Advantage ◽  
Arthropod Species ◽  
Obligate Intracellular Bacteria ◽  
Methodological Advance

ABSTRACT Wolbachia are obligate intracellular bacteria that are globally distributed in half of all arthropod species. As the most abundant maternally inherited microbe in animals, Wolbachia manipulate host reproduction via reproductive parasitism strategies, including cytoplasmic incompatibility (CI). CI manifests as embryonic death when Wolbachia-modified sperm fertilize uninfected eggs but not maternally infected eggs. Thus, CI can provide a relative fitness advantage to Wolbachia-infected females and drive the infection through a population. In the genetic model Drosophila melanogaster, the Wolbachia strain wMel induces variable CI, making mechanistic studies in D. melanogaster cumbersome. Here, we demonstrate that sons of older paternal D. melanogaster grandmothers induce stronger CI than sons of younger paternal grandmothers, and we term this relationship the “paternal grandmother age effect” (PGAE). Moreover, the embryos and adult sons of older D. melanogaster grandmothers have higher Wolbachia densities, correlating with their ability to induce stronger CI. In addition, we report that Wolbachia density positively correlates with female age and decreases after mating, suggesting that females transmit Wolbachia loads that are proportional to their own titers. These findings reveal a transgenerational impact of age on wMel-induced CI, elucidate Wolbachia density dynamics in D. melanogaster, and provide a methodological advance to studies aimed at understanding wMel-induced CI in the D. melanogaster model. IMPORTANCE Unidirectional cytoplasmic incompatibility (CI) results in a postfertilization incompatibility between Wolbachia-infected males and uninfected females. CI contributes to reproductive isolation between closely related species and is used in worldwide vector control programs to drastically lower arboviral vector population sizes or to replace populations that transmit arboviruses with those resistant to transmission. Despite decades of research on the factors that influence CI, penetrance is often variable under controlled laboratory conditions in various arthropods, suggesting that additional variables influence CI strength. Here, we demonstrate that paternal D. melanogaster grandmother age influences the strength of CI induced by their sons. Older D. melanogaster females have higher Wolbachia densities and produce offspring with higher Wolbachia densities that associate with stronger CI. This work reveals a multigenerational impact of age on CI and expands our understanding of host-Wolbachia interactions and the biology of CI induced by the Wolbachia strain infecting the most widely used arthropod model, D. melanogaster.

scholarly journals Cytoplasmic incompatability in Drosophila melanogaster due to different Wolbachia genotypes

2009 ◽  
Vol 7 (2) ◽  
pp. 11-18
Author(s):  
Yury Y Ilinsky ◽  
Ilya K Zakharov
Keyword(s):  
Drosophila Melanogaster ◽  
Host Species ◽  
Cytoplasmic Incompatibility ◽  
Male Killing ◽  
Do So

Wolbachia is an endosymbiont prevalent among different arthropoda and in some Nematoda species. The bacteria ads across the populations of host species via induction of reproductive abnormalities: cytoplasmic incompatability, parthenogenesis, feminization, and male killing. we estimated the level of cytoplasmic incompatability in Drosophila melanogaster caused by three most prevalent Wolbachia genotypes, namely wmel, wmelCS и wmelCS2. wmel and wmelCS genotypes were shown to cause mild cytoplasmic incompatibility (< 10 %), while wmelCS Wolbachia were unable to do so. Possible mechanisms of sustainability of Wolbachia in the populations of Drosophila melanogaster are discussed.<table border="0" cellpadding="3" cellspacing="0"> <tbody> </tbody></table>

scholarly journals Loss of reproductive parasitism following transfer of male-killing Wolbachia to Drosophila melanogaster and Drosophila simulans

Heredity ◽  
2012 ◽  
Vol 109 (5) ◽  
pp. 306-312 ◽  
Author(s):  
Z Veneti ◽  
S Zabalou ◽  
G Papafotiou ◽  
C Paraskevopoulos ◽  
S Pattas ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Drosophila Simulans ◽  
Reproductive Parasitism ◽  
Male Killing

scholarly journals Effect of heritable symbionts on maternally-derived embryo transcripts

2019 ◽  
Author(s):  
Mariana Mateos ◽  
Nadisha O. Silva ◽  
Paulino Ramirez ◽  
Victor M. Higareda-Alvear ◽  
Rodolfo Aramayo ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Cytoplasmic Incompatibility ◽  
Developmental Stages ◽  
Early Embryo ◽  
Male Embryo ◽  
Ribosome Inactivating Proteins ◽  
Male Killing ◽  
Early Embryos ◽  
Early Developmental ◽  
Mrna Transcriptome

AbstractMaternally-transmitted endosymbiotic bacteria are ubiquitous in insects. Among other influential phenotypes, many heritable symbionts of arthropods are notorious for manipulating host reproduction through one of four reproductive syndromes, which are generally exerted during early developmental stages of the host: male feminization; parthenogenesis induction; male killing; and cytoplasmic incompatibility (CI). Major advances have been achieved in understanding mechanisms and identifying symbiont factors involved in reproductive manipulation, particularly male killing and cytoplasmic incompatibility. Nonetheless, whether cytoplasmically-transmitted bacteria influence the maternally-loaded components of the egg or early embryo has not been examined. In the present study, we investigated whether heritable endosymbionts that cause different reproductive phenotypes in Drosophila melanogaster influence the mRNA transcriptome of early embryos. We used mRNA-seq to evaluate differential expression in Drosophila embryos lacking endosymbionts (control) to those harbouring the male-killing Spiroplasma poulsonii strain MSRO-Br, the CI-inducing Wolbachia strain wMel, or Spiroplasma poulsonii strain Hyd1; a strain that lacks a reproductive phenotype and is naturally associated with Drosophila hydei. We found no consistent evidence of influence of symbiont on mRNA composition of early embryos, suggesting that the reproductive manipulation mechanism does not involve alteration of maternally-loaded transcripts. In addition, we capitalized on several available mRNA-seq datasets derived from Spiroplasma-infected Drosophila melanogaster embryos, to search for signals of depurination of rRNA, consistent with the activity of Ribosome Inactivating Proteins (RIPs) encoded by Spiroplasma poulsonii. We found small but statistically significant signals of depurination of Drosophila rRNA in the Spiroplasma treatments (both strains), but not in the symbiont-free control or Wolbachia treatment, consistent with the action of RIPs. The depurination signal was slightly stronger in the treatment with the male-killing strain. This result supports a recent report that RIP-induced damage contributes to male embryo death.

Synergistic action of propolis with levodopa in the management of Parkinsonism in Drosophila melanogaster

2020 ◽  
Vol 17 (3) ◽  
Author(s):  
Emmanuel Tiyo Ayikobua ◽  
Josephine Kasolo ◽  
Keneth Iceland Kasozi ◽  
Ejike Daniel Eze ◽  
Abass Safiriyu ◽  
...  
Keyword(s):  
Antioxidant Activity ◽  
Drosophila Melanogaster ◽  
Side Effects ◽  
Motor Activity ◽  
Ethanolic Extract ◽  
Synergistic Action ◽  
Content Type ◽  
Treatment Groups ◽  
Ethanolic Extract Of Propolis ◽  
Different Doses

AbstractBackgroundThe Phosphatase and tensin-induced putative kinase 1 (PINK1B9) mutant for Drosophila melanogaster is a key tool that has been used in assessing the pathology of Parkinsonism and its possible remedy. This research was targeted toward determining the effects of ethanolic extract of propolis, with levodopa therapy in the management of Parkinsonism.MethodThe PINK1B9 flies were divided into groups and fed with the different treatment doses of ethanoic extract of propolis. The treatment groups were subjected to 21 days of administration of propolis and the levodopa at different doses after which percentage climbing index, antioxidant activity and lifespan studies were done.ResultsPropolis alone improved motor activity, antioxidant and lifespan in Drosophila melanogaster than in PINK1 flies. Propolis in combination with levodopa significantly (P<0.05) improved physiological parameters at higher than lower concentrations in Parkinsonism Drosophila melanogaster demonstrating its importance in managing side effects associated with levodopa.ConclusionPropolis is a novel candidate as an alternative and integrative medicinal option to use in the management of Parkinsonism in both animals and humans at higher concentrations.

Phenotype and transmission efficiency of artificial and natural male-killing Spiroplasma infections in Drosophila melanogaster

2012 ◽  
Vol 109 (2) ◽  
pp. 243-247 ◽  
Author(s):  
Kate J. Hutchence ◽  
Rémi Padé ◽  
Heather L. Swift ◽  
Daimark Bennett ◽  
Gregory D.D. Hurst
Keyword(s):  
Drosophila Melanogaster ◽  
Transmission Efficiency ◽  
Male Killing

scholarly journals Stoichiometry and Turnover of the Bacterial Flagellar Switch Protein FliN

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Nicolas J. Delalez ◽  
Richard M. Berry ◽  
Judith P. Armitage
Keyword(s):  
Copy Number ◽  
Motor Proteins ◽  
Fluorescent Protein ◽  
Protein Complexes ◽  
Content Type ◽  
Rotation Direction ◽  
Cell Measurement ◽  
Biological Complexes ◽  
And Function

ABSTRACTSome proteins in biological complexes exchange with pools of free proteins while the complex is functioning. Evidence is emerging that protein exchange can be part of an adaptive mechanism. The bacterial flagellar motor is one of the most complex biological machines and is an ideal model system to study protein dynamics in large multimeric complexes. Recent studies showed that the copy number of FliM in the switch complex and the fraction of FliM that exchanges vary with the direction of flagellar rotation. Here, we investigated the stoichiometry and turnover of another switch complex component, FliN, labeled with the fluorescent protein CyPet, inEscherichia coli. Our results confirm that,in vivo, FliM and FliN form a complex with stoichiometry of 1:4 and function as a unit. We estimated that wild-type motors contained 120 ± 26 FliN molecules. Motors that rotated only clockwise (CW) or counterclockwise (CCW) contained 114 ± 17 and 144 ± 26 FliN molecules, respectively. The ratio of CCW-to-CW FliN copy numbers was 1.26, very close to that of 1.29 reported previously for FliM. We also measured the exchange of FliN molecules, which had a time scale and dependence upon rotation direction similar to those of FliM, consistent with an exchange of FliM-FliN as a unit. Our work confirms the highly dynamic nature of multimeric protein complexes and indicates that, under physiological conditions, these machines might not be the stable, complete structures suggested by averaged fixed methodologies but, rather, incomplete rings that can respond and adapt to changing environments.IMPORTANCEThe flagellum is one of the most complex structures in a bacterial cell, with the core motor proteins conserved across species. Evidence is now emerging that turnover of some of these motor proteins depends on motor activity, suggesting that turnover is important for function. The switch complex transmits the chemosensory signal to the rotor, and we show, by using single-cell measurement, that both the copy number and the fraction of exchanging molecules vary with the rotational bias of the rotor. When the motor is locked in counterclockwise rotation, the copy number is similar to that determined by averaged, fixed methodologies, but when locked in a clockwise direction, the number is much lower, suggesting that that the switch complex ring is incomplete. Our results suggest that motor remodeling is an important component in tuning responses and adaptation at the motor.

scholarly journals Intracellular and Extracellular Expression of Bacillus thuringiensis Crystal Protein Cry5B in Lactococcus lactis for Use as an Anthelminthic

2015 ◽  
Vol 82 (4) ◽  
pp. 1286-1294 ◽  
Author(s):  
Evelyn Durmaz ◽  
Yan Hu ◽  
Raffi V. Aroian ◽  
Todd R. Klaenhammer
Keyword(s):  
Bacillus Thuringiensis ◽  
Lactococcus Lactis ◽  
Copy Number ◽  
Oral Delivery ◽  
Membrane Integrity ◽  
Biologically Active ◽  
High Copy Number ◽  
Cry Protein ◽  
Western Blots ◽  
Content Type

ABSTRACTTheBacillus thuringiensiscrystal (Cry) protein Cry5B (140 kDa) and a truncated version of the protein, tCry5B (79 kDa), are lethal to nematodes. Genes encoding the two proteins were separately cloned into a high-copy-number vector with a strong constitutive promoter (pTRK593) inLactococcus lactisfor potential oral delivery against parasitic nematode infections. Western blots using a Cry5B-specific antibody revealed that constitutively expressed Cry5B and tCry5B were present in both cells and supernatants. To increase production,cry5Bwas cloned into the high-copy-number plasmid pMSP3535H3, carrying a nisin-inducible promoter. Immunoblotting revealed that 3 h after nisin induction, intracellular Cry5B was strongly induced at 200 ng/ml nisin, without adversely affecting cell viability or cell membrane integrity. Both Cry5B genes were also cloned into plasmid pTRK1061, carrying a promoter and encoding a transcriptional activator that invoke low-level expression of prophage holin and lysin genes inLactococcuslysogens, resulting in a leaky phenotype. Cry5B and tCry5B were actively expressed in the lysogenic strainL. lactisKP1 and released into cell supernatants without affecting culture growth. Lactate dehydrogenase (LDH) assays indicated that Cry5B, but not LDH, leaked from the bacteria. Lastly, using intracellular lysates fromL. lactiscultures expressing both Cry5B and tCry5B,in vivochallenges ofCaenorhabditis elegansworms demonstrated that the Cry proteins were biologically active. Taken together, these results indicate that active Cry5B proteins can be expressed intracellularly in and released extracellularly fromL. lactis, showing potential for future use as an anthelminthic that could be delivered orally in a food-grade microbe.

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