Preventing the spread of infectious diseases through the endosymbiotic relationships with Wolbachia By Lois Crozier BAMA Teacher Johnsonburg Area School District, Johnsonburg, PA

Doudoumis, V., Alma, U., Askoy, E., Abd-Alla, A. M. M., Tsiamis, G., Brelsfoard, C., Aksoy, S., & Bourtzis, K. (2013). Tsetse-wolbachia symbiosis: Comes of age and has great potential for pest and disease control. Journal of Invertebrate Pathology, 112, S94-S103. doi: PMID: Article:

Vector in the transmission of Trypanosomiasis the cause of African Sleeping Sickness Vector in the transmission of Trypanosomiasis the cause of African Sleeping Sickness Tsetse fly

 thread-like parasitic nematodes (roundworms) transmitted by arthropod vectors  microfilariae infect vector arthropods, in which they mature to larvae  adult worms inhabit specific tissues where they mate and produce microfilariae, thread-like larvae.

 60 million people live in regions where the parasite is located.  African Animal Trypanosomiasis is the biggest problem in preventing the increase of livestock populations  African Animal Trypanosomiasis is the leading cause of hunger and poverty

 Current vector control is not sustainable or environmentally friendly.  Urgent need for pesticide free, environmentally friendly method of vector control

Filarial Nematodes and Wolbachia Mutualistic relationship with Filarial nematodes Plays a role in nematode survival and reproduction. parasite: Trypanosoma brucei gambiens parasite: Trypanosoma brucei rhodesiense African Sleeping Sickness Parasite: Trypanosoma cruzi Chagas Disease Reproductive parasitic relationship with Tsetse flies Vector: Tsetse fly is the host for each of these parasites

1. Wigglesworth glossinidia Found in 2 distinct populations in the insect: Intracellular population in the specialized epithelial cells in the end gut Plays a role in providing essential vitamins and minerals absent from a blood diet Extracellular population in female milk secretion Method of transmission to the intrauterine larvae through mothers milk. An absence of Wigglesworth g. results in female sterility Wigglesworth is heterozygous in the natural population influences host juvenile larvae development of immune system 2.Sodalis glossinidius Present in all lab populations and heterozygous in natural populations Distributed in many tissues including haemolymph, salivary gland, milk gland, an midgut where it lives close to where trypanosomes develop. Passed to intrauterine larva through mothers milk 1. Wigglesworth glossinidia Found in 2 distinct populations in the insect: Intracellular population in the specialized epithelial cells in the end gut Plays a role in providing essential vitamins and minerals absent from a blood diet Extracellular population in female milk secretion Method of transmission to the intrauterine larvae through mothers milk. An absence of Wigglesworth g. results in female sterility Wigglesworth is heterozygous in the natural population influences host juvenile larvae development of immune system 2.Sodalis glossinidius Present in all lab populations and heterozygous in natural populations Distributed in many tissues including haemolymph, salivary gland, milk gland, an midgut where it lives close to where trypanosomes develop. Passed to intrauterine larva through mothers milk

3. Wolbachia pipiens  Obligatory endosymbiont  (lives within the organism and either the endosymbiont or the host cannot survive without the other)  maternally transmitted  Found in reproductive tissue of insect 3. Wolbachia pipiens  Obligatory endosymbiont  (lives within the organism and either the endosymbiont or the host cannot survive without the other)  maternally transmitted  Found in reproductive tissue of insect

 Parthenogenesis ( female reproduction without fertilization )  Feminization ( male killing, male zygotes don’t survive )  Cytoplasmic Incompatibility (CI)  Most common  Can be unidirectional or bidirectional  Results in a reduction in the population of the host

 I ncompatible I nsect T echnique ( IIT )  IIT causes sterility due to the presence of a Wolbachia strain genetically capable of inducing cytoplasmic incompatibility.  IIT Has been done successfully in the lab and in the field  I ncompatible I nsect T echnique ( IIT )  IIT causes sterility due to the presence of a Wolbachia strain genetically capable of inducing cytoplasmic incompatibility.  IIT Has been done successfully in the lab and in the field

 Wolbachia-induced CI can control populations of agricultural pests and disease vectors  might someday play a role in spreading or replacing desired phenotypes ( because infected females have a reproductive advantage of passing their DNA onto the offspring )  Methods for inducing the transfer of desired genes have yet to be worked out fully.

Higher frequency in the Population Proportional to the number of infected males No Yes No Yes

 vector genus of tsetse flies is Glossinia   Using 16S rRNA based PCR analysis Using 16S rRNA based PCR  3,750 flies from 12 species from 10 African countries were tested  Found multiple cytoplasmic strains and Wolbachia gene insertions on the Glossinia chromosomes ( host DNA)

3 Wolbachia genes were found on the host DNA of Glossinia m. morsitans in natural and lab populations 1. 16S rRNA (used in classifying and identifying bacterial species) 2. fbpA (ferric iron binding protein-encoding gene or fibronetin binding protein) 3. wsp (Wolbachia surface protein) 1. 16S rRNA (used in classifying and identifying bacterial species) 2. fbpA (ferric iron binding protein-encoding gene or fibronetin binding protein) 3. wsp (Wolbachia surface protein)

led to the identification of at least 2 horizontal gene transfer events 52% of Wolbachia genome transferred in one event and 47% of Wolbachia genome being transferred in the other event. Whole Genome Sequence of Glossinia m.morsitans

 The HGT of Wolbachia genome can provide clues into the evolution and transfer of endosymbiont genes into multicellular organisms  aiding in the development of Wolbachia as a transport device to get desired genes into vectors such as the tsetse fly to stop the spread of African Trypanosomiasis and other diseases spread by insects.

 disease produced by a filarial worm depends on the tissue locations preferred by adults and type of microfilariae  lymphatic filariasis  disease is caused by the presence of worms in the regional lymphatic vessels  and by the host response to the worms and worm products

 Elephantiasis of leg caused by chronic infection with the filarial nematode Wuchereria bancrofti spread by a mosquito vector  Onchocerca volvulus is a filarial worm that is transmitted to humans by blackflies.  Mature worms live in the subcutaneous tissues and produce microfilariae that migrate through the skin and connective tissues.  do not move through the circulatory system  3-4 months after infection, form nodules on outer layer of connective tissue which turns into calcifications

 HAT and AAT are serious problems in Africa and in North America someday, as climates change.  Insecticides are not environmentally responsible or sustainable, neither are baiting or releasing sterilized male tsetse flies.  Wolbachia has the potential to rid the world of this sickness and others, one fly at a time, one generation at a time. Wouldn’t that be nice.

 16S ribosomal RNA is a component of prokaryotic ribosomes having 1,542 nucleotides  used in reconstructing phylogenies  multiple sequences of 16S rRNA can exist within a single bacterium What is 16s rRNA ?

 Carl R.Woese, an American microbiologist and biophysicist along with George E. Fox, a researcher at the University of Houston, were well known for their defining work in identifying a new domain in classifying organisms, Archaea.  by phylogenetic taxonomy of 16S rRNA stemming from their introduction of the lowest common ancestor (a progenote).  comparative analysis of ribosomal RNA  which they believed was the earliest genetic resemblance to life on Earth today.

PCR applications of 16S rRNA  highly conservative primer binding sites making PCR easier ( very few differences in primer sites for the gene)  Has hyper variable regions in its gene sequence making gene sequences making it a very species specific tool used to classify bacteria  Used in medical microbiology due to it being fast and inexpensive, compared to using phenotypes to identify bacteria  Originally used to classify bacteria now used to identify new species of bacteria BACK