wolbachia a heritable endosymbiont

1: Wolbachia � a Heritable Endosymbiont Patricia Sidelsky Symbiosis Wolbachia � A Heritable Endosymbiont. Wolbachia is a Bacterium that has an obligatory relationship with many arthropod hosts. The bacterium is dependent upon the host and cannot exist outside of the host organism. The bacterium resides in the host�s cytoplasm and exerts influence on the reproductive biology of its host from its cytoplasmic position. Wolbachia � A Heritable Endosymbiont Patricia Sidelsky This symbiotic relationship demonstrates some interesting interactions between nuclear and cytoplasmic factors. How can an endosymbiont influence the reproductive biology of its host ? Wolbachia bacteria infect the reproductive tissues of their arthropod hosts. They maintain symbiotic relationships with a variety of arthropod hosts. The bacteria exist in the cytoplasm of the host eggs and are maternally transmitted to the next generation providing the progeny with a selective advantage.Wolbachia � A Heritable Endosymbiont. Wolbachia is a Bacterium that has an obligatory relationship with many arthropod hosts. The bacterium is dependent upon the host and cannot exist outside of the host organism. The bacterium resides in the host�s cytoplasm and exerts influence on the reproductive biology of its host from its cytoplasmic position. Wolbachia � A Heritable Endosymbiont Patricia Sidelsky This symbiotic relationship demonstrates some interesting interactions between nuclear and cytoplasmic factors. How can an endosymbiont influence the reproductive biology of its host ? Wolbachia bacteria infect the reproductive tissues of their arthropod hosts. They maintain symbiotic relationships with a variety of arthropod hosts. The bacteria exist in the cytoplasm of the host eggs and are maternally transmitted to the next generation providing the progeny with a selective advantage.

2: Wolbachia Classified in a monophyletic clade in Domain Bacteria A member of Alphaproteobacteria � Related to Rickettsia Sequencing of 16sRNA connects Wolbachia to Ehrlichia and Rickettsia 16srRNA sequences used to determine relationships between Rickettsia Estimated to infect 15-20% of arthropods. New estimates include as many as 50% of insect species Broad host range � Insects, isopods, mites, and arachnids. They have been isolated from crustaceans. More recently found in Nematodes Manipulate the reproduction in their hosts in a number of unusual ways Wolbachia are members of the Alphaproteobacteria. They infect a broad spectrum of arthropods including mosquitoes, fruit flies, wasps, ants, butterflies, and mites. They have also been isolated from crustaceans such as the ancient Isopod line. They are have more recently identified the presence of Wolbachia has also been documented in nematodes.Wolbachia are members of the Alphaproteobacteria. They infect a broad spectrum of arthropods including mosquitoes, fruit flies, wasps, ants, butterflies, and mites. They have also been isolated from crustaceans such as the ancient Isopod line. They are have more recently identified the presence of Wolbachia has also been documented in nematodes.

Slide 3:The Proteobacteria are a group of bacteria with diverse characteristics. The Alphaproteobacteria include Rickettsia, Ehrlichia, and Wolbachia. The Ricketssia are maintained in the insects as a reservoir for infection of vertebrate hosts. Wolbachia has lost the ability to infect its vertebrate hosts and in doing so has developed its own strategy and life cycle. The classification of the Alphaproteobacteria is based on chaperonings( proteins) and 16sRNAThe Proteobacteria are a group of bacteria with diverse characteristics. The Alphaproteobacteria include Rickettsia, Ehrlichia, and Wolbachia. The Ricketssia are maintained in the insects as a reservoir for infection of vertebrate hosts. Wolbachia has lost the ability to infect its vertebrate hosts and in doing so has developed its own strategy and life cycle. The classification of the Alphaproteobacteria is based on chaperonings( proteins) and 16sRNA

4: Host - Endosymbiont Wolbachia are found in the reproductive tissues of their hosts The bacterial obligatory endosymbionts are passed vertically in the cytoplasm of the eggs of their hosts The Wolbachia affects sex determining factors in insects � affects the sex of the insect progeny The host exerts population control of bacteria A force in speciation in arthropods Viewed as a possibility for the evolution of insects The picture shows the presence of Wolbachia in the ovaries of an insect. They are present in ovaries and testes. Wolbachia can influence the development of ova and sperm, but they are only passed vertically in the cytoplasm of the eggs. This is referred to as maternal transmission. This process is compared to the mitochondrial inheritance pattern in eukaryotes. The bacterium exerts different effects on arthropods. The inheritance pattern in insects differs for instance Hymenopterans like wasps have a haplodiploid pattern for the determination of males and females, while flies( Drosophila melanogaster) have an XY ratio more comparable to our own. The presence of Wolbachia in different insect species suggests that the bacterium may contribute to population control. In the more distance past these bacteria are regarded by scientists as a force in speciation of coinfecting organisms.The picture shows the presence of Wolbachia in the ovaries of an insect. They are present in ovaries and testes. Wolbachia can influence the development of ova and sperm, but they are only passed vertically in the cytoplasm of the eggs. This is referred to as maternal transmission. This process is compared to the mitochondrial inheritance pattern in eukaryotes. The bacterium exerts different effects on arthropods. The inheritance pattern in insects differs for instance Hymenopterans like wasps have a haplodiploid pattern for the determination of males and females, while flies( Drosophila melanogaster) have an XY ratio more comparable to our own. The presence of Wolbachia in different insect species suggests that the bacterium may contribute to population control. In the more distance past these bacteria are regarded by scientists as a force in speciation of coinfecting organisms.

5: Wolbachia and Reproduction Vertical transmission � cytoplasmic inheritance Causes male killing and sterility in males Induces parthenogenesis Cytoplasmic incompatability( conflict between cytoplasmic and nuclear components) Wolbachia are transmitted in the cytoplasmic content of ova. They are distributed at the animal pole. It is theorized that they are positioned in the egg relative to their future position in the reproductive tissues. The maternal inheritance of cytoplasmic Wolbachia can cause the death of male embryos( Male Killing). If the male passes through embryological development, Wolbaachia can cause either sterility or feminization. In some species Wolbachia induces parthenogenesis. In most species the nuclear arthropod genes and the cytoplasmic genes of the Wolbachia cause an incompatability which is expressed as a bias in favor of female progen.Wolbachia are transmitted in the cytoplasmic content of ova. They are distributed at the animal pole. It is theorized that they are positioned in the egg relative to their future position in the reproductive tissues. The maternal inheritance of cytoplasmic Wolbachia can cause the death of male embryos( Male Killing). If the male passes through embryological development, Wolbaachia can cause either sterility or feminization. In some species Wolbachia induces parthenogenesis. In most species the nuclear arthropod genes and the cytoplasmic genes of the Wolbachia cause an incompatability which is expressed as a bias in favor of female progen.

6: Symbiont related Sexual Anomalies Thelytoky occurs in Trichogramma wasps In wasps there is a haplofiploid situation with males haploid and females diploid. Virgin Females produce diploid females without fertilization. The n( haploid) is converted to 2n( diploid) This occurs early in the first mitotic division Feminization of males Occurs in Isopods Suppresses the androgenic gland and causes males to become functional females Virgin females are capable of producing diploid females from a haploid egg. The transition from haploid state( this would become a male) to female occurs in the first mitotic division.Virgin females are capable of producing diploid females from a haploid egg. The transition from haploid state( this would become a male) to female occurs in the first mitotic division.

7: Cytoplasmic Incompatability and vertical transmission If both male and female insects are infected with Wolbachia � the progeny will be infected If the female is infected and the male is not infected, the progeny will all be infected. If the female is not infected and the male is infected there will not be any progeny Why does a conflict exist between the cytoplasmic factors and the nuclear genes? How does this influence the determination of sex? CI( cytoplasmic incompartibility is the incompatibility between the sperm of infected males and the eggs of uninfected females or females infected with a different Wolbachia strain. If both male and female insects are infected with the same Wolbachia strain, the progeny will be infected. If the female is infected with Wolbachia and the male is not infected, all the progeny will be infected. If the female is not infected and the male is infected there will be no viable progeny. CI � is a modificaton rescule phenomenon. If a strain of Wolbachia is presnet in the sperm, the same strain must be present in the egg to rescue the progeny. The biological mechanism is undknown but it has been related to different proteins such as cytoskeletal elements and heat shock proteins.Why does a conflict exist between the cytoplasmic factors and the nuclear genes? How does this influence the determination of sex? CI( cytoplasmic incompartibility is the incompatibility between the sperm of infected males and the eggs of uninfected females or females infected with a different Wolbachia strain. If both male and female insects are infected with the same Wolbachia strain, the progeny will be infected. If the female is infected with Wolbachia and the male is not infected, all the progeny will be infected. If the female is not infected and the male is infected there will be no viable progeny. CI � is a modificaton rescule phenomenon. If a strain of Wolbachia is presnet in the sperm, the same strain must be present in the egg to rescue the progeny. The biological mechanism is undknown but it has been related to different proteins such as cytoskeletal elements and heat shock proteins.

8: Examples of symbiont � host interactions Male killing mechanism has been studied in two species of insect In Adalia bipunctata and Acraea encedon the presence of Wolbachia halves the hatch size and changes the female/male ratio Microbial nature of infection has been verified by treatment with rifampin and sulphamethoxazole After treatment with antibiotics the hatch size returns to normal and the with a normal sex ratio Condition can be artificially induced by injection of macerated infected insect into pupae of uninfected ( Hurst et al) Wolbachia has a broad range of hosts. The presence of Wolbachia in male hosts kills the male early in development in a diverse range of hosts. This has been observed in two species called the two-spot Ladybird called, Adalia bipunctata ( Coleoptora)and the butterfly Acraea encedon( Lepidoptera). Using DNA sequence scientists have located the presence of the wsp gene. In butterflies the eggs produced had half the normal hatch rate. The microbial nature of the trait was confirmed by testing sensitivity to antibiotics and microinjection experiments. The sex ratio and hatch rate returned to normal after treatment with rifampin and suphamethoxazole. The trait could be artificially introduced into lines when a macerate from infected lines was injected into uninfected pupae Wolbachia has a broad range of hosts. The presence of Wolbachia in male hosts kills the male early in development in a diverse range of hosts. This has been observed in two species called the two-spot Ladybird called, Adalia bipunctata ( Coleoptora)and the butterfly Acraea encedon( Lepidoptera). Using DNA sequence scientists have located the presence of the wsp gene. In butterflies the eggs produced had half the normal hatch rate. The microbial nature of the trait was confirmed by testing sensitivity to antibiotics and microinjection experiments. The sex ratio and hatch rate returned to normal after treatment with rifampin and suphamethoxazole. The trait could be artificially introduced into lines when a macerate from infected lines was injected into uninfected pupae

9: Why Male Killing ? Reduces competition for food ( H) Reduces cannibalism in siblings (H) Improves female fitness for breeding- selective advantage (H) Insures propagation of Wolbachia endosymbionts to future generations ( S + H) The selective advantages for male killing are theorized to be the following: It reproduces the competition for food among progeny. It reduced cannabilism in siblings. My reducing the competitions between siblings this increases the reproductive fitness of females. They are capable of producing more eggs in the next generation. In turn the increase in females insure the propagation of Wolbachia and its continuance for future generations.The selective advantages for male killing are theorized to be the following: It reproduces the competition for food among progeny. It reduced cannabilism in siblings. My reducing the competitions between siblings this increases the reproductive fitness of females. They are capable of producing more eggs in the next generation. In turn the increase in females insure the propagation of Wolbachia and its continuance for future generations.

10: Mechanism of Action Bacterial modification of sperm by bacterial interactions Bacteria present in testes interact with DNA binding proteins � These may be similar to H1, histones The same bacterial strain must be present in the female in order to rescue the egg Mitotic defects appear in the early divisions of the fertilized egg( during cleavage) The pressure on sperm and egg development may be related to the interaction of reproductive cells with the H1 histone involved with the maintenance of chromosomes and binds specifically to the DNA. In order for the fertilization to produce viable progeny, the same bacterial strain must be present in the female to rescue the egg and therefore produce viable progeny. The evidence for the incompatibility appears to be in the form of a problem with the first mitotic divisions during cleavage of the fertilized egg.The pressure on sperm and egg development may be related to the interaction of reproductive cells with the H1 histone involved with the maintenance of chromosomes and binds specifically to the DNA. In order for the fertilization to produce viable progeny, the same bacterial strain must be present in the female to rescue the egg and therefore produce viable progeny. The evidence for the incompatibility appears to be in the form of a problem with the first mitotic divisions during cleavage of the fertilized egg.

11: Wolbachia � Evidence for specificity of infection PCR for 16sRNA from infected lines Product purified and ligated into pGEM vector Plasmids were transformed into DH5a Colonies with insert were detected 39 /42 positive for Wolbachia insert and not other bacteria 16sRNA Experiments in Molecular Biology support the specificity of the infection of the Lady bugs and the butterflies with Wolbachia strains. In an expeirfment on the specificity of the infection G.D. Hurst at University College in London with his colleagues, extracted and purified DNA from infected lines. They then performed a polymerase chain reaction on the purified DNA. The primer for this experiment was a 16sRNA primer for Wolbachia. After amplification the product was inserted into a cloning vector pGEM- Y produced by Promega. E. coli bacteria were transformed and the colonies with the insert were detected. Subsequently the plasmid DNA was extracted and sequenced for the Wolbachia 16sRNA. It was present in 39/42 of the transformed colonies.Experiments in Molecular Biology support the specificity of the infection of the Lady bugs and the butterflies with Wolbachia strains. In an expeirfment on the specificity of the infection G.D. Hurst at University College in London with his colleagues, extracted and purified DNA from infected lines. They then performed a polymerase chain reaction on the purified DNA. The primer for this experiment was a 16sRNA primer for Wolbachia. After amplification the product was inserted into a cloning vector pGEM- Y produced by Promega. E. coli bacteria were transformed and the colonies with the insert were detected. Subsequently the plasmid DNA was extracted and sequenced for the Wolbachia 16sRNA. It was present in 39/42 of the transformed colonies.

12: Wolbachia and genetics Genome recently sequenced Loss of genes due to obligatory nature of relationship between bacteria and host Loss of genes over time due to dependency on host Many repetitive elements and insertion sequences in the genome Imply high level of intergenomic and intragenomic recombination The genome of Wolbachia was recently sequenced. The Genomic map is available online at the NCBI, National Center for Biotechnology Information. On this site, scientists can study the genes and the proteins in the Wolbachia genome of two species. One species has been isolated from Drosophila simulans the other from a filarial nematode. When compared to members of other bacteria in Alphaproteobacteria there appears to be a loss of genes that is reflective of the obligatory nature of the relationship between bacteria and arthropod host. Repetitive DNA, mobile elements including insertion sequences(IS) suggest recombinant events of intergenomic or intragenomic recombination. Comparable to the Elysia and its plastid endosymbionts some genes may have been transferred to insect hosts in the evolution of this symbiotic relationship.The genome of Wolbachia was recently sequenced. The Genomic map is available online at the NCBI, National Center for Biotechnology Information. On this site, scientists can study the genes and the proteins in the Wolbachia genome of two species. One species has been isolated from Drosophila simulans the other from a filarial nematode. When compared to members of other bacteria in Alphaproteobacteria there appears to be a loss of genes that is reflective of the obligatory nature of the relationship between bacteria and arthropod host. Repetitive DNA, mobile elements including insertion sequences(IS) suggest recombinant events of intergenomic or intragenomic recombination. Comparable to the Elysia and its plastid endosymbionts some genes may have been transferred to insect hosts in the evolution of this symbiotic relationship.

13: Genetics of Wolbachia Wolbachian genome consists of one circular chromosome 1.26Mbp � 1195 proteins � GC content 35% Contains genes with Ankyrin repeats � Ankyrins connect endosymbionts to the cell cytoskleton which is an adaptation for intracellular existence Type Four Secretion System � required for persistence of endosymbiont in host � conserved in Rickettsia The chromosome of Wolbachia is a circular chromosome. It consists of 1.26 Mbp. The estimated number of proteins coded for is 1195. The base content is 35%, which is considered to be low. The repetitive Ankyrin genes appear to be important in maintaining the status of the symbiont in the cytoplasm of the host. It is a cytoskeletal element which suggests that it assists the bacterial in maintaining its position in the cell. The type four secretion system also required for persistence of the endosymbiont are membrane proteins consistent with Rickettsiae. These are translocation proteins that deliver DNA horizontally between microorganisms or interestingly deliver DNA and proteins to eukaryote cells during infection.The chromosome of Wolbachia is a circular chromosome. It consists of 1.26 Mbp. The estimated number of proteins coded for is 1195. The base content is 35%, which is considered to be low. The repetitive Ankyrin genes appear to be important in maintaining the status of the symbiont in the cytoplasm of the host. It is a cytoskeletal element which suggests that it assists the bacterial in maintaining its position in the cell. The type four secretion system also required for persistence of the endosymbiont are membrane proteins consistent with Rickettsiae. These are translocation proteins that deliver DNA horizontally between microorganisms or interestingly deliver DNA and proteins to eukaryote cells during infection.

14: Lateral Transfer of Genes Evidence of lateral( horizontal) transfer of genes in Wolbachia species in Drosophila melanogaster and Nasonia vitripennis. Drosophila and Nasonia can maintain infection by two strains of Wolbachia Exchange between bacteria that coinfect in the same cells ( density dependent) Exchange between endosymbiont and host( gene loss) IS sequences in Wolbachia suggest Bacteriophage transfer between Wolbachia strains. Recent research has detected 3 prophage sequences in the Wolbachia genome. ( Wu et al) Prophages � lateral transfer. One is a small nine gene group. Two are called WO- A and Wo-B. One of these is descended from a temperate lambda phage. WO-B has undergone rearrangement and now is inactive.Prophages � lateral transfer. One is a small nine gene group. Two are called WO- A and Wo-B. One of these is descended from a temperate lambda phage. WO-B has undergone rearrangement and now is inactive.

15: Importance of study Wolbachia does not infect humans � studying Wolbachia can lead to a better understanding of Rickettsial � host interactions for medical applciations Understanding Wolbachia can lead to vector control in infections such as Rocky Mountain Spotted Fever ( Rickettsia and tic) Since Wolbachia infects diverse species of insects including beetles, fruit flies, and mosquitoes, it could be evaluated for a form of biological control Used as a model to study speciation Why does the relationship of Wolbachia warrant additional research? Read slideWhy does the relationship of Wolbachia warrant additional research? Read slide

16: Wolbachia and River Blindness Wolbachia has also been identified in filarial worms ( Nematoda) One of these worms causes Riverblindness(Onchoceriasis) Wolbachia provides metabolic support through many pathways for the worm. Contributes to inflammatory responses Instead of antihelminth drug protocols � will antibiotics be a cure?????? Wolbachia has been isolated from filarial worms( Nematoda) The Worms are transmitted to vertebrates by a specific simulian fly associated with rivers in Africa. That is why the disease is called River Blindness. The symbiotic relatiosnhip in this organism appears to be based on metabolic support for the presence of the worm in its host. It defintiely induces part of the inflammatory response in the vertebrate host iincluding blindness. The worms can actually be seen in the eyes. It has been a difficult task to erradicate this disease in Africa. Environmental controls are difficult. Attempts to disrupt the life cycle of the flies with pesticides and kill the worm with antihelminthic medications have not eliminated this debilitating disease. Researchers hae pointed to the treatment of this infection with an antibiotic such as Docycline which kills the Wolbachia. In this case the elimination of the Wolbachia will kills the worms because the worms are dependent upon the Wolbachia�s contributions to its metabolic processes. In summary Wolbachia is a fascinating bacteria. The endosymbiont- host relationshi8p in arthropods should be studied for its potential as a biological control for both biological pests, vector control in the transmission of disease, and population control of insects in the environment.Wolbachia has been isolated from filarial worms( Nematoda) The Worms are transmitted to vertebrates by a specific simulian fly associated with rivers in Africa. That is why the disease is called River Blindness. The symbiotic relatiosnhip in this organism appears to be based on metabolic support for the presence of the worm in its host. It defintiely induces part of the inflammatory response in the vertebrate host iincluding blindness. The worms can actually be seen in the eyes. It has been a difficult task to erradicate this disease in Africa. Environmental controls are difficult. Attempts to disrupt the life cycle of the flies with pesticides and kill the worm with antihelminthic medications have not eliminated this debilitating disease. Researchers hae pointed to the treatment of this infection with an antibiotic such as Docycline which kills the Wolbachia. In this case the elimination of the Wolbachia will kills the worms because the worms are dependent upon the Wolbachia�s contributions to its metabolic processes. In summary Wolbachia is a fascinating bacteria. The endosymbiont- host relationshi8p in arthropods should be studied for its potential as a biological control for both biological pests, vector control in the transmission of disease, and population control of insects in the environment.

17: References Anderson,Cort and Timothy Karr. Wolbachia:Evolutionary Novelty in a Rickettsial Bacteria. BMC Evolutionary Biology, 2001:1-10. Huigens,M.E. On the Evolution of Wolbachia Induced Parthenogenesis in Trichogramma Wasps. http://www.gcw.nl/dissertations/3389/dis3389.pdf Hurst, Gregory et al. Male-Killing Wolbachia in Two Species of Insect. National Center for Biotechnology Information � ncbi. http://www.ncbi.nlm.nih.gov/ Johanowicz,Denise et Marjorie Foy.Wolbachia Endosymbionts. Florida Entomologist.1998: 310-316. Protein Data Bank - http://www.rcsb.org/pdb/ Rigaud, T., Juchault, P., and Mocquard, J. P. 1991. Experimental study of the sex ratio of broods in terrestrial Crustacea Armadillium vulgare. Possible implications in natural populations. J. Evol. Biol. 1991: 603 - 607. Sun,Ling. Et al. Gene Organization in the dnaA Region of Wolbachia. Jornal of Bacterioology. 199.18115):4708-4710.

18: References( con) Wernegreen,Jennifer Endosymbiosis: Lessons in Conflict Resolution. Plos Biology. March 2004, 2:307-311. Werren,John H. Biology of Wolbachia. Annual Review of Entomology. 1997.42:587-609. Werren, John H. and Leo Beukboom. Sex Determination, Sex Ratios, and Genetic Conflict.Annual Review of Ecological Systematics. 1998,29:233-261. Werren, John H. and David Windsor. Wolbachia Infection Frequencies in Insects: Evidence of a Global Equilibrium. Proceedings of the Royal Society. 200 267: 1277-1285 Werren, John H. Wolbachia Run Amok. Proceedings of the National Academy of Science. 1997. 92: 11154-11155 Wu,Martin et al. Phylogenomics of the Reproductive Parasite Wolbachia pipientis wMeI: A Streamlined Genome Overrun by Mobile Genetic Elements. Plos Biology. 2004,2:327-333.