1. Discover the Microbes Within! The Wolbachia Project Real-world Research in the Classroom

2. Discover the Microbes Within! The Wolbachia Project  1,000,000,000,000,000,000 insects on the planet  Insects present on earth for ~500 MY  85% of all animal species are insects (1-30 million species)  20% of all insect species harbor heritable symbionts called Wolbachia  Scientists cant discover them all on their own.  YOU ARE OUR BIGGEST ASSET TO SCIENTIFIC DISCOVERY AND REAL RESEARCH!

3. What is symbiosis? The living together of dissimilar organisms (de Bary 1859) Often for mutual benefit, but also parasitism and commensalism Endosymbiosis is when one organism lives and replicates inside another one

4. Vertically-transmitted (i.e., Inherited) Symbionts

5. Insect egg Insect egg with symbiotic bacteria Credit: Michael Clark & Seth Bordenstein

6. Ways that vertically transmitted microbes can increase in frequency Increase host survival & reproduction (mutualism) –Very common Why might vertical transmission be associated with mutualistic effects on hosts? 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

7. Must heritable symbionts always be beneficial?

8. Reproductive Parasites

9. Asymmetric (Uniparental) Inheritance Dead end Distortion of the sex-ratio

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

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

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

13. x x x x = = = = Wolbachia- infected offspring Uninfected offspring Wolbachia- infected offspring X CI Late prophasePrometaphase MetaphaseTelophase Courtesy of U. Tram Cytoplasmic Incompatibility (CI) Testes Embryo Wolbachia Host

14. Wolbachia-to-host lateral gene transfer in Drosophila ananassae NATURE REVIEWS | microbiology Vol OCT 2008, p741

15. …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?" Science fiction? Definitely. For many insect species and other arthropods, however, the truth can be as strange as fiction when bacteria known as Wolbachia are around. By JOHN TRAVIS Undesirable Sex Partners Bacteria manipulate reproduction ofinsects and other species Imagine…

16. The Wolbachia Pandemic

17. Arthropods Nematodes Insects (at least 20% !) The Wolbachia Pandemic Crustaceans Chelicerates (family Onchocercidae) 2-6 million insect species are infected with Wolbachia!! Filarial nematodes

18. Number of scientific papers with Wolbachia in title Nobel prize awarded for PCR PCR detection of Wolbachia in ~20% of all insects

19. How Important Are Wolbachia?

20. 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

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

22. Population Replacement Transmits Disease Does Not Transmit Disease

23. Human Health: Bill and Melinda Gates Foundation

24. 24

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

26. 26 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 Coinfection with Wolbachia bacteria causes river blindness Treatment: tetracycline and ivermectin

27. Overview of Onchocerciasis River blindness is the symptomatic stage of a filarial infection It is a painful and debilitating disease. 18 million people in Africa are infected

28. Overview of Onchocerciasis Symptoms include: visual impairment, rashes, lesions, intense itching, depigmentation, inflammation of lymph nodes, and general debilitation.

29. Overview of Onchocerciasis Disease is the result of infection by the parasitic worm, Onchocerca volvulus. Females produce millions of microfilariae that migrate through the body and cause a manifestation of symptoms

30. Overview of Onchocerciasis Simulans spp. (black flies) are vectors Female black flies require a blood meal prior to egg laying Microfilariae are taken in with infected blood Transfer takes place when the fly bites uninfected person

31. CDC/DPD Summary Report 2001 Black flies are infected with Wolbachia bacteria Worldwide distribution Infection rates have been found to be up to 76% of insects in some regions Also found in millipedes, crustaceans, and mites Wolbachia is passed horizontally and vertically

32. Lancet 2005; 365: 2116–21

33. 33 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

34. 34 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

35. 35 Tissue Nematode (Roundworm): Elephantitis

36. 36 Elephantiasis: Wucheria bancrofti

37. How do YOU discover the Wolbachia within?

38. 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)

39. 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

40. 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

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

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

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

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

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

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

48. MBL High School ATGCGCATGCGC Primers, Insect controls,

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