1. A Bug’s Life: Wolbachia Donna C. Sullivan, PhD Division of Infectious Diseases February 10, 2012

2. Base Pair Program

3. They All Go to College, But Then What? M.D./M.D.-Ph.D. = 21 Ph.D. = 6 Masters (Science) = 22 Law = 5 Nurse = 5 TOTAL= 59 (of 89 college graduates)

4. “O for a Muse of fire, that would ascend The brightest heaven of invention…” Henry V, Act 1, Prologue William Shakespeare

5. Collaboration Partners: HHMI, UMMC, Woods Hole Marine Biological Laboratories and Princeton University Discover the Microbes Within! The Wolbachia Project Kathy McKone, RBI Lead Teacher and HHMI/Princeton Workshop Leader

6. Wolbachia Rodeo Six high schools sent teams for a day long competition. Positive Wolbachia samples were sent to Woods Hole for sequencing

7. Phylogeny of Wolbachia NATURE REVIEWS | microbiology Vol OCT 2008, p741

8. R. Stouthamer, DAPI stained wasp embryo Wolbachia pipientis 1924: Hertig and Wolbach Host range: Arthropoda and Nematoda; reproductive tissues Maternally transmitted w/in species ◦ Horizontally transmitted between species Obligate intracellular lifestyle Important to invertebrate evolution (speciation, sex determination, sexual selection) Potential tool for pest bio-control and curing human filariasis Considered relatively obscure until the 90’s

9. Number Of Scientific Papers With Wolbachia In Title Nobel prize awarded for PCR PCR detection of Wolbachia in ~20% of all insects

10. The Wolbachia Pandemic At least 920,000 described species 85% of all known animal species 10 quintillion insects alive at any given time Up to 30 million estimated species (~22.5 million infected species!) Wolbachia = 1 of Life’s great pandemics Phylum Arthropoda Insects: up to 67% of species Isopods: 35% of terrestrial isopods Mites: 43% of mites Lice: ~100% of species Phylum Nematoda Filarial nematodes: 90% of species Nonfilarial: ~0% Purvis and Hector (2000) Insecta

11. Ways That Vertically Transmitted Microbes Can Increase In Frequency Increase host survival & reproduction (mutualism) ◦ Very common Most famous cases are the lineages leading to organelles ◦ Mitochondria evolved from the alpha- Proteobacteria about 2 billion years ago ◦ Chloroplasts evolved from cyanobacteria about 1 billion years ago But Wolbachia aren’t mitochondria….they are widow makers

12. By JOHN TRAVIS Undesirable Sex Partners Bacteria manipulate reproduction of insects and other species Imagine… …physicians across the United States…documenting similar events. In each case, an unfertilized egg in a woman had spontaneously begun to develop, ultimately producing a healthy female baby. One young researcher, who had analyzed the timing and locales of the virgin births, suggested a spreading infection might be causing the incidents. The Federal Centers for Disease Control and Prevention in Atlanta quickly dismissed the idea, calling it "ridiculous." Several months later came a well-publicized report in the Journal of the American Medical Association concluding that the number of infertile couples was rising rapidly worldwide. The international uproar intensified when physicians began to observe another reproductive curiosity: Some newborns that were genetically male appeared to be female. One week, the New England Journal of Medicine and the National Enquirer ran articles with the headline, "Is this the end of mankind, or just men?"

13. Maternal Inheritance Is Imperfect There is a direct physiological cost to infection. Infections can only be maintained where they increase either the survival or production of female hosts. ◦ Symbionts become parasites (manipulate reproduction of hosts to enhance their own transmission).  Distort the sex ratio of the host towards females. ◦ Some become mutualists, increasing the survival or reproductive success of their hosts  Essential for the host to survive and reproduce (primary symbionts).  Non-essential facultative roles and typically only infect a subset of the population (secondary symbionts).

14. Wolbachia are Infectious Widow makers! Parthenogenesis Feminization Male-Killing Reproductive Parasitism: each of these reproductive distortions leads to more infected females in the insect host species

15. Wolbachia Induced Phenotypes NATURE REVIEWS | microbiology OCT 2008, p741

16. Wolbachia Strains and Their Characteristics

17. How Wolbachia Might Be Used As a vector for the expression of genes of interest. As a tool to drive desirable genotypes into arthropod populations. To directly suppress arthropod populations.

18. Human Health: Wolbachia May Help Control The Transmission Of Arthropod-borne Diseases (Malaria, Dengue fever, Filariasis, Trypanosomiasis, West Nile, Chagas) Isolate and culture symbiont Reinfect vector with transgenic symbiont Transform symbiont Transmits Disease Does Not Transmit Disease Anti-pathogen gene

19. Human Health: Wolbachia May Help Control The Transmission Of Arthropod-borne Diseases (Malaria, Dengue fever, Filariasis, Trypanosomiasis, West Nile, Chagas) Early- mortality Wolbachia strain Reinfect vector with transgenic symbiont Old Females Transmit Disease Females Die Too Early to Transmit Disease

20. Drive via Cytoplasmic Incompatibility X MaleFemaleProgeny X X X None (Incompatible)

21. Population Replacement Transmits Disease Does Not Transmit Disease

22. Wolbachia as Means for Insect Pest Control: Medfly Introduced Wolbachia into medfly Ceratitis capitata population using cherry fruit fly Rhagoletis cerasi as donor Induced complete cytoplasmic incompatibility Results encouraged idea of introducing Wolbachia into vector species Zabalou et al., 2004. PNAS 101:15042-15045 Ratios of uninfected females to uninfected males to trans-infected males (cannot mate with uninfected females).

23. Successful Establishment Of Wolbachia In Aedes Populations To Suppress Dengue Transmission wMel Wolbachia strain from Drosophila melanogaster stably introduced into A. aegypti Introduction through open releases of mosquitoes with the wMel Wolbachia strain into wild Australian A. aegypti populations Hoffman et al., 2011. Nature 476: 454-259

24. Occurrence Of Aedes aegypti And Wolbachia Infection In Ovitraps Outside Release Areas Successfully invaded two natural A. aegypti populations in Australia, reaching near-fixation in a few months following releases of wMel infected A. aegypti adults.

25. Wolbachia and Nematodes The relationship between Wolbachia and filarial nematodes is different. If Wolbachia is removed from host nematodes, the host organism loses its fertility and/or dies. Wolbachia are housed in the lateral chords and oocytes of their filarial nematode hosts. 25

26. Human Health: Bill and Melinda Gates Foundation

27. Wolbachia are Chemotherapy Targets for Curing River Blindness & Elephantiasis Caused By Filarial Nematodes Horeauf et al, 2003 11 months post-treatment Untreated Onchocerca volvlulus

28. Tissue Nematode (Roundworm): Onchocerca volvulus and River Blindness Transmitted by biting black flies Larvae develop into adults in subcutaneous tissues Adult females migrate via the blood to the eyes, provoking inflammatory reactions Females produce millions of microfilariae that migrate through the body and cause a manifestation of symptoms 28

29. Onchocerciasis: Cutaneous and Ocular Pathology Skin pathologyOcular pathology Subclinical or intermittent dermatitis Hyperpigmentation or depigmentation (leopard skin) Lichenified onchodermatitis with pruritic hyperpigmented/ hyperkeratotic plaques (asymetrical, localized) with local lymphadenopathy Visual impairment (500,000 cases) Blindness (270,000 cases) Varies between geographical locations, more common in savannah areas of West/ Central Africa, Latin America Involves cornea (punctate keratitis, sclerosing keratitis) Anterior chamber where dead microfilaria cause uveitis

30. Factors Affecting Visual Pathology Localization of nodules in upper part of the body Vector species Microfilarial burdens Parasite strain Higher Wolbachia load in more virulent savannah strain

31. Wolbachia-infected Onchocerca volvulus All individual worms and all life cycle stages contain the endosymbiont Wolbachia ◦ Inhabit lateral chords of adult worms and reproductive system of females Clearance of endosymbionts by antibiotic treatment causes inhibition of worm development, blocks embryogenesis and fertility, reduces worm viability

32. Immunopathology Of Onchocerciasis Obviously, host inflammatory responses to microfilariae, including autoimmune processes driven by cross-reaction between retinal and parasite protein Wolbachia and Wolbachia-derived molecules released from worms elicit immune responses

33. Clin. Microbiol. Rev. 2011, 24(3):459. Wolbachia-induced Responses Of Specific Cell Types: In vitro Studies

34. Murine Model of Onchocercal Keratitis O. volvulus depleted of Wolbachia by antibiotic treatment does not induce corneal inflammation. Related filarial species containing Wolbachia (Brugia malayi) induce keratitis while species lacking Wolbachia (Acanthocheilonemma viteae and Loa loa) do not. Isolated Wolbachia bacteria induce neutrophil recruitment to the corneal stroma.

35. Role Of Wolbachia In River Blindness Wolbachia release in the cornea causes corneal edema and opacity by inducing neutrophil and macrophage infiltration and activation in the corneal stroma. This induces cutaneous and systemic side effects such as fever, tachycardia, hypotension, lymphadenopathy, and pruritus. Adverse events correlate with microfilarial loads and are associated with Wolbachia DNA and whole bacterial levels in blood, proinflammatory cytokines, neutrophilia, and antibacterial peptides (calprotectin and calgranulin). Clin. Microbiol. Rev. 2011, 24(3):459. TLR, Toll-like receptor MyD88, myeloid differentiation primary response gene (88) TNF-, tumor necrosis factor alpha DEC, diethylcarbamazine

36. Predicted Sequence Of Events In O. volvulus/Wolbachia-induced Keratitis Clin. Microbiol. Rev. 2011, 24(3):459. KC, keratinocyte-derived chemokine; MIP, macrophage-inflammatory protein 2

37. Microfilaridermia (Mf) After Treatment With Doxycycline Plus Ivermectin In Comparison To Ivermectin Alone Hoerauf et al. 2001. Lancet 357:1415.

38. Tissue Nematode (Roundworm): Wuchereria bancrofti Tropical infection spread by mosquitoes Vector deposits larvae which move into lymphatics and develop Chronic infection causes blockage of lymphatic circulation and elephantitis, massive swelling in the extremities 38

39. Tissue Nematode (Roundworm): Filariasis due to W. bancrofti Endemic in central Africa, Mediterranean coast, parts of Asia (China, Korea, Japan, the Philippines) Blood specimens may show microfilariae Acute symptoms include: fever, lymphangitis/lymphadenitis Result of inflammatory response to molting adolescent worms and dead adults in lymphatic vessels May involve any part of body, blocking lymphatic system 39

40. Tissue Nematode (Roundworm): Elephantitis 40

41. Elephantiasis: Wucheria bancrofti 41

42. Lancet 2005; 365: 2116–21

43. Wolbachia and Virus Protection in Insects (The “Not In My House” Idea) Wolbachia infection of D. melanogaster has been shown to increase the fly’s resistance to some viral infections, resulting in infections with lower virus titers and less associated pathology. The resistance phenotype appears to be limited to RNA viruses, with the strength of resistance varying substantially among the viruses tested thus far.

46. Wolbachia transfected: inbred PGYP1 line Wolbachia transfected backcrossed with wild type Cairns mosquito (outbred): PGYP1.out line Treat with Tetracycline (kill the Wolbachia) PGYP1.tet linePGYP1.out.tet line Wild Type Cairns Mosquito: Wolbachia Negative Mosquitos With and Without Wolbachia

47. DENV-2 In Whole Mosquito Bodies (ELISA Assay) Mosquitoes were fed an artificial blood meal spiked with DENV-2 No Wolbachia-infected mosquitoes (PGYP1.out) tested positive for DENV-2 DENV-2 infection rates in Wolbachia-uninfected mosquitoes (PGYP1.out.tet and Cairns3) ranged from 30%–100% Cell 139, 1268–1278, December 24, 2009

48. Quantitative PCR Analysis of Dengue Virus in Mosquitoes Cell 139, 1268–1278, December 24, 2009

49. Localization of Wolbachia and Dengue Virus in A. aegypti Mosquitoes Sections were probed simultaneously with polyclonal anti-wsp antibody (Wolbachia) and monoclonal anti-DENV antibody Wolbachia-uninfected control mosquitoes showed DENV-2 infection predominantly in mosquito fat body, ommatidia and nervous system. DENV-2 was not detected in any of these tissues in Wolbachia- infected mosquitoes. Cell 139, 1268–1278, December 24, 2009 PGYP1.tet (- Wolb) PGYP1. (+ Wolb)

50. Does This Phenomenon Extend to Other Viruses? Day 14 infection rates were 87% and 79% for the PGYP1.out.tet and Cairns3 controls (Wolbachia negative) and 17% for the Wolbachia infected PGYP1.out line (p < 0.001, chi- square). Viral interference effect appeared robust for two unrelated arboviruses Cell 139, 1268–1278, December 24, 2009

51. Plasmodium gallinaceum Detection in Aedes spp. Mosquitoes Fed mosquitos on infected chickens Reduced the frequency of mosquitoes in the population infected with malaria Reduced number potentially able to transmit Plasmodium from 74% (tet controls) down to 42%. Plasmodium genomic DNA was 26-fold less abundant Wolbachia infected cells Cell 139, 1268–1278, December 24, 2009

52. Immune Gene Regulation in Response to Wolbachia Infection The expression levels of eleven immune pathway genes in the Wolbachia wMelPop-CLA infected PGYP1.out and its uninfected control line were compared for two independently reared cohorts of mosquitoes. Four genes encoding representatives of immune effector molecules were significantly up-regulated in the presence of Wolbachia. Cell 139, 1268–1278, December 24, 2009

53. Conclusions wMelPop-CLA Wolbachia infection reduces the ability of two distantly related arboviruses and a malaria parasite from establishing productive infections in the mosquito ◦ Shows that the pathogen interference effect is general and fundamental. Mechanism unclear although data suggest that immune effector genes are up-regulated in the mosquito in the presence of the Wolbachia wMelPop-CLA strain and are likely to play a role.

54. Bottom Line Wolbachia-infected mosquitoes display priming of immune system genes, possibly underpinning viral inhibition. Arboviral infection in mosquitoes and some mosquito cell lines induces antimicrobial immune pathways such as Toll, JAK/STAT and Imd/JNK as well as RNAi based defenses.

55. How do YOU discover the Wolbachia within?

56. Integrated Set of Lab Exercises From Organisms to Molecules and Back! Lab 1 - Insect Identification (Biodiversity) Lab 2 - Isolation of Insect and Wolbachia DNA (Molecular Biology) Lab 3 - PCR of Wolbachia 16S rDNA (Molecular Biology) Lab 4 - Presence/Absence of Amplicon (Molecular Biology) Lab 5 - Evolutionary Sequence Analysis (Bioinformatics)

57. Features of These Labs Original Research in Lab Exercises Integrates across Science Biodiversity -> Molecular -> Evolution YOU can make new discoveries Covers Emerging Fields Microbial diversity, Bioinformatics, Molecular Phylogeny Integrates Science and Technology

58. Dictypotera cockroaches and mantids; 6,000 species Discover the Microbes Within: The Wolbachia Project Lab 1: Insect Identification Odonata dragonflies, damselflies; 5,000 species Dermaptera earwigs; 1,200 species Coleoptera beetles; 370,000 species Collembola spring tails; 6,000 species Diptera flies; 120,000 species Ephemeroptera mayflies; 2,000 species Hemiptera aphids; 67,500 species Hymenoptera bees, wasps, ants; 108,000 species Isoptera termites; 1,900 species Lepidoptera butterflies, moths, skippers; 140,000 species Orthoptera grasshoppers, crickets, katydids; 17,000 species

59. Discover the Microbes Within: The Wolbachia Project Lab 2: DNA Extraction EXTRACT DNA FROM YOUR SAMPLES WHY? Infected sample (+): Uninfected Sample (-) Insect Mitochondria WOLBACHIA -

60. Discover the Microbes Within: The Wolbachia Project Lab 3: Polymerase Chain Reaction

61. Discover the Microbes Within: The Wolbachia Project Lab 4: Gel Electrophoresis

62. +++++ + +++ +++++ ++++++ + Discover the Microbes Within: The Wolbachia Project Lab 4: Gel Electrophoresis

64. Discover the Microbes Within: The Wolbachia Project Lab 5: Sequence Analysis NCBI BLAST No programming skills required!

65. When It all comes together, it looks like this!

66. MBL High School ATGCGCATGCGC Primers, Insect controls,

67. Where Does Your Data Go? To… To… Class reports Peers, family, teachers, research scientists Online database (website repository for your data) Consequences Consequences New discoveries Collaborations between research scientists and high schools Summer “envisionships” Professional meetings Professional societies Journal publications