Domako Bacterial microbiome in C hagas disease vectors 1 Presence of B acterial Microbiome and Incidence of Infection in Chagas Disease Vectors T riatoma dimidiata Amber Domako Department of Integrative Biology University of California, Berkeley EAP Tropical Biology and Conservation Program, Fall 2016 16 December 2016 ABSTRACT Chagas di sease is a condition caused by the parasite Trypanosoma cruzi In Costa Rica, it is spread by one species of kissing bug T riatoma d imidiata Recent studies have concluded that the microbi omes of insect disease vectors My study examine s the relationship between the number of individual bacteria m orphospecies found in the microbiome of each T dimidiata and whether that insect was a Chagas disease carrier With this data, I analyze d the correlation between the presence of the trypanosome, or not, and the microbiome of the insect I collected Chagas bugs from various locations across the San Lu is Valley and the Monteverde area o f Costa Rica and took fecal samples from the live insects. Through microscopic examination, I determined whether the T. dimidiata carried the parasite T. cruzi Subsequently, I surveyed the numbe r of bacteria in the microbiome of each Chagas bug using oil immersion There was a significant relationship between the number of bacteria and the presence or absence of the parasite T. cruzi T. dimidiata individuals carrying T. cruzi had a greater number of total bacteria t han individuals who did not have the infection. In both types of bacteria studied, cocci and bacilli, the presence of the parasite was related to a higher number of individual bacteria in each Chagas bug. In summary my research provided information toward understanding microbiomes and their relationship to Chagas disease. Presencia de microbiomas bacteriales e incidencia del parsito de la enfermedad de Chagas en sus vectores Triatoma dimidiata RESUMEN La enfermedad de Chagas es una afeccin causada por el parsito Trypanosoma cruzi En Costa Rica, se contagia a travs de individuos de Triatoma dimidiata una especie de chinche. Estudios recientes en Sur Amrica han concluido que los microbiomas en estas chinches vectoras de la enfermedad juegan un pa pel en el proceso de transmisin de la enfermedad. Este estudio examina la relacin entre el nmero de morfoespecies de bacterias que se encuentran en el microbioma de cada T. dimidiata y si ese insecto era un portador de la enfermedad de Chagas. Analic l a correlacin entre la presencia o ausencia del tripanosoma y el microbioma de cada chinche. Colect chinches de Chagas en varios lugares del Valle de San Luis y en Monteverde, Costa Rica y tom muestras fecales de los insectos vivos. A travs de un anlis is microscpico, determin si cada T. dimidiata portaba el parsito T. cruzi Posteriormente, cont el nmero de bacterias en el microbioma de cada individuo. Hubo una relacin significativa entre el nmero de
Domako Bacterial microbiome in C hagas disease vectors 2 bacterias y la presencia o ausencia del parsi to T. cruzi Individuos de T. dimidiata portadores de T. cruzi posean un mayor nmero total de bacterias que individuos no portadores. Adems, la presencia del parsito estaba relacionada con un mayor nmero de bacterias cocos y bacilos individualmente. E stos datos muestran que la actividad de la enfermedad est correlacionada con los microbios de los insectos de Chagas. Mi investigacin proporciona informacin para entender mejor la relacin entre el microbioma y la enfermedad de Chagas dentro de los chin ches. The complex world of disease is expanding with the advancement of identifying bacteria that aid in disease transmission. In the case of one diseas e American trypanosomiasis, the information available about bacterial symbionts is increasing as new research studies emerge Symbioses occur when two organisms live together, such as in a mutualism, commensalism, or parasitism. Evidence of American trypanosomiasis, otherwise known as Chagas disease, has been discovered in mummies across the Americas estimated to be 9,000 years old ( World Health Organization 2016 ). T he ancient disease is caused by a parasite that is transmitted among mammals and insect vectors The parasite for this disease is Trypanosoma cruzi a zooflagellate protozoan or a flagellated eukaryote that lacks photosynthetic pigment and feeds on organic matter ( Encyclopedia Britannica 2011 ) There are 25 million people at risk for Chagas disease across the Americas 8 million current cases, and 10,000 deaths a year from i t ( World Health Organization 2016 ). The is disease characterized by symptoms such as swelling, fever, and if untreated, heart abnormalities and digestive organ dilation that can lead to death. As a result, it is a significant parasitic infection, especial ly in rural parts of Latin America. In Costa Rica, the disease is spread through the feces of nocturnal triatomine bugs, Triatoma dimidiata which are commonly called assassin bu gs, Chagas bugs, or kissing bugs because they bite close to the lips ( Starr et al, 1991 ). These insects are members of the Triatominae subfamily. They bite mammals for blood meals and then defecate onto the wound spreading the parasite as their feces enters the broken skin ( Centers for Disease Control 2016 ). Currently, prevention mechanisms in both residential and agricultural areas are cent ered on insecticide application in most of South and Central America, as well as in parts of the Southern United States like Texas These methods are not entirely effective in South America as seen by the number of new Chagas cases a year. This is due, in part, to species developing resistance to the insecticides ( Durvasula et al, 2014) Furthermore, insecticides used for Chagas bugs often harm organisms in the same habitat as the target organism For example, pyrethroids are the most potent type of insecticides used and these chemicals have a high toxicity to other insects besides Chagas bugs ( Thatheyus et al, 2013 ) Insecticides, although valuable disease prevention strategies in some cases, are not always effective and can cause problems for the surrounding environment. One understudied aspect of Chagas disease that has potential for disease prevention is the comp lex relationship between microbes and the vector. A specific microbiome is necessary for the survival of a Chagas bug although some Chagas bugs have additional bacterial symbioses which allow them to harbor and transmit T. cruzi (Weiss et al., 2011) A microbiome is the collective term for all of the microorganisms, and their genetic material, living inside of an organism. Insect microbe associations hav e shown to be
Domako Bacterial microbiome in C hagas disease vectors 3 immune system strength, and host pathogen tran smission process es (Weiss et al., 2011). Two illustration s of this are the mutualistic symbioses of both Tsetse flies and Triatominae insects, which involve bacteria that are indispensable for their hosts survival in unique or resource limited environments (Weiss et a, 2011). Bacteria in these instances aid in the digestion of blood meals and are necessary for the survival of the insects Microbiomes have other valuable purposes, as well. In a study on one type of Triatominae insect found in South America Triatoma infesta ns, a particular bacterium was identified in its feces which is necessary for the sexual maturation of the insect ( Durvasula et al ., 2008). In this study, when n ymphs, or young insects, we re grown without the bacterial symbionts, they arrested in their growth. Once the symbionts were re added, the triatomes would complete their development (Durvasula et al., 2008). In other words, when the specific bacteria body, it prevent ed the insect from maturing. bacterial microbiomes hav e a wide variety of roles and whose composition have the potential to affect their survival and ability to transmit T. cruzi Further studies on Chagas vectors have sho wn that the bacterial composition of the microbiota in these insects may play a role in the epidemiolog y of the disease ( Gumiel et al., 2015). For instance symbionts in the gut microbiome of South American Chagas bugs Rhodinus proxilus can be cultured, genetically modified, and subsequently reinserted into the population with expression of anti parasitic molecules (Weiss et al, 2011). This process is called paratransgenesis and includes isolating and genetically modifying microbiome bac teria to express an anti parasitic protein and then reintroducing the recombinant bacteria into their hosts (Weiss et al, 2011). Once the genetically modi fied bacteria are inserted, they produce antibodies that target antigens on the parasite All of the parasites living within the vector are eventually killed through this process Furthermore, once that insect has offspring, the recombinant genes are inherited, creating a self sustaining prevention method. Applying this data to wild populations could laun ch targeted treatment with genetic engineering. This technology enable s scientists to safely eliminate specific diseases The end result of the genetic modification is halt ing ability to transfer the parasite A limited number of studies have been completed on the microbiome of Rhodinus proxilus the Triatominae insect that is a vector for Chagas disease in South America. These research studies, which I previously mentioned, discovered relationships between bacterial symbionts and the inse At this time, n o microbiome studies have been done on the species T. dimidiata in Costa Rica. As a result, I was interested in studying the morphospecies of bacteria and their abundance in the Chagas bug species fou nd in Monteverde Costa Rica, which differ s from the South American species with previous research Because South American experiments on Triatominae found a corre lation between the types of T. cruzi I wondered if the same trend was present in the different species found in Costa Rica. This led me to ask the question: d oes the number and morphosp ecies of individual bacteria in the microbiome of the Chagas bug, T. dimidiata relate to whether it has a positive or negative diagnosis of the parasite T. cruzi ?
Domako Bacterial microbiome in C hagas disease vectors 4 MATERIALS AND METHODS Study Sites Chagas bugs are characteristically located in dark, dry wood piles or dusty, decaying sheds near vector animal s Weather conditions do not play a signific ant role in finding the insects because they are normally located inside covered areas with little light present. The properties where Chagas bugs are found are typically farms with horses, cows, dogs cats, pigs, rodents, or other animals located adjacent to older sheds or large wood p iles. Animals often slept in the sheds where the insects were located For my first method of collection I gathered Chagas bugs at private houses in the San Luis valley and Monteverde area El Jard n Finca Lindora, and Finca la Bella I spent one to fou r hour s at each of these site s searching through wood piles with assistance from professors and other students Finca Lindora had a plentiful wood pile within a horse tack room where we located ten T. dimidiata o rganisms within ten minutes. This location provided the most samples, most likely due to its large collection of stacked, dry wood situated close to a variety of farm animals. I collected individuals of T. dimidiata during daylight hours from 6 November 2016 to 26 November 2016 with instructors who are familiar with the private properties and farms in the Montverde and San Luis regions of the Puntarenas province of Costa Rica. I located them with the help of UCEAP staff members Eladio Cruz, Emilia Triana, and Eric McAdam. At each site, I used a headlamp to see the T. dimidiata and forceps and gloves to collect them. After I collected each insect with forceps, I immediately placed it into plastic containers labeled with the site and date. For my second m ethod of collection plastic containers were given to voluntarily cooperative property owners in the same region the San Luis Valley and Monteverde area who resided on land with a known presence of Chagas bugs. These owners had the ability to search thei r homes at night and collect live insects, which I would gather and sample the next day. The benefit see the nocturnal insects moving throughout their homes at night, thereby catching them during the period when they are most active. This is an advantageous method because it avoids the issue with the daytime collections where locating hidden insects is often challenging and time consuming. Lab Methods Wearing full personal protective equipment, I individually examine d each T. dimidiata under a pl astic bag. This method was used to prevent infected fecal material from contacting my skin. I determined the nymphal stage through the presence of red colored w ings to categorize them into either an adult or juvenile life stage. Adults are larger with wings and have red coloration, and juveniles are smaller with no wings and gray coloration. I placed the insects on their backs and use d forceps to press the feces out of each insect, beginning at the ventral portion of the abdomen and ending at the posterior portion. I would repeatedly apply pressure in this manner with the forceps until I collected a fecal sample. The feces were collecte d onto a glass
Domako Bacterial microbiome in C hagas disease vectors 5 slide within the plastic bag. The slides had a uniform amount of liquid waste collected from each insect. The slide was then examined within ten minutes of collection to prevent desiccation and death of the microbiome bacteria To examine each sample I place d a glass coverslip over the plated feces and viewed it underneath a compound microscope at 400x to determine the presence or absence of the parasite, T. cruzi This diagnosis was made if I saw the live parasite moving. The parasite is a long and narrow flagellated protozoan and it uses its flagella for locomotion through the feces After a diagnosis was made and recorded, I reposition ed the slide to view an open area ne ar the edge of the fecal sample. I then count ed the number of bacter ia present using immersion oil at 1000x. Once the immersion oil is applied the slide cannot be moved Thus, I counted bacteria inside of only one viewing area I used the same location on each slide, relative to each fecal sample to account for differenc es in the amount of feces present on different slide locations. I viewed each slide on the outer right edge of the fecal sample assuming that the total distribution of bacteria in the fecal sample was similar to this single visual field I chose to examine I then note d the morphology and presence of each bacterium in the sample I defined a live bacterium as having change s in direction or visible locomotion not caused by the liquid on the slide A majority of the fecal samples were dry enough to appear stationary when I examined them on the slide, due to the higher proportion of feces to liquid in the samples To control for unaccounted movement, I did not include individuals whose samples had feces with liquids continuously flowing through them In the se samples, bacteria and fecal material would be constantly moving, making a standardized or accurate count unreasonable. This criterion removed two individuals from the study. Data analysis I utilized chi squared tests to find the significance between t he number of bacteria in each individual and whether that individual carried the trypanosome. I used a logistical regression to present the data because it was comprised of a continuous category and a binomial category The regression analyzed the relation ship between the variables infection with T. cruzi and the number of bacteria per individual and created probabilities of infection based on the data. RESULTS Number of Bacteria Present In my sample size of 25, the presence of the trypanosome in the Triatoma dimidiata indicated an elevated number of total bacteria, whereas the absence of the trypanosome in the T. dimidiata sampled indicated a reduced number of total bacteria ( Figure 1). T he number of total bacteria present in T. dimidiata is correlated to the presence or absence of the trypanosome, T. cruzi ( X 2 = 15.59 df = 24, p < 0.0001). The number of cocci present in each sample is also correlated to the presence or absence of T. cruzi ( X 2 = 14.566 df = 24 p < 0.001 ). The number of bacilli present in each T. dimidiata is correlated to the presence of the trypanosome (X 2 = 11 713 d f =24 p < 0.0001 ) With both of these bacterial morphospecies, the presence of T. cruzi was correlated wit h a higher number of bacteria present.
Domako Bacterial microbiome in C hagas disease vectors 6 Figure 1 Number of total b acteria present per i ndividual T. dimidiata with and without the p resence of T. c ruzi Each total number of bacteria for individuals without T. cruzi lies below ten. The total numbers of bacteria for individuals with T. cruzi are, on average higher than the individuals without the parasite. The logistic regression graph shows the fitted model, which is the probability of being infected with T. cruzi, as a function of Total Bacteria Found (Figure 2). It demonstrates that the number of individual bacteria found in each T. dimidiata has a significant effect on whether an insect carries T. cruzi For each respective bacteria morphotypes, higher numb ers of individual bacteria predict a higher chance of carrying Chagas disease. As the probability of infection with T. cruzi increases to 1, there is a higher chance that the overall microbiome of the Chagas bug will contain more live bacteria for total c ounts, bacilli counts, and cocci counts (Figure 2). Figure 2. Nominal Logistic Fit for Trypanosoma cruzi Present. There was a significant difference in the total number of bacteria found between infected and non infected individuals ( p < 0.0001, X 2 = 193.871, df=24). 0 5 10 15 20 25 30 Number Of Individual Bacteria per T. dimidiata With T. cruzi Without T. cruzi
Domako Bacterial microbiome in C hagas disease vectors 7 Prevalence of Chagas Disease The percentage of insects carrying the trypanosome now resides at 22 percent. Six of the individuals sampled, from the total sample size of twenty five, were infected. These individuals were concentrated at two sites, Finca Lindora, and a private residence in San Luis. DISCUSSION The number of bacteria found in T. dimidiata individuals was correlated to whether or not the insect was a current vector for T. cruzi Infected individuals have more bacteria in their microbiome than non infected individuals For cocci and bacilli, respectively, there was a significant ly higher amount of each morphospecies when T. cruzi was present (Figure 2 ) These results indicate a clear distinction between the microbiomes of insects carrying Chagas disease and those without the parasite. The misclassification rate for the logistical regre ssion showed a low error rate for the data representation indicating a well fitted line that matches the significance of the data collect ed In accordance with previous studies depicting the value of microbiome research in insects, this study provides further evidence that disease activity for insect vectors is correlated to the bacteria found within an insect. The logistic regression shows that with a higher probability of T. cruzi infection, an insect is more likely to have higher numbers of bacteria (Figure 2 ). Correspondingly, a lower probability of T. cruzi infection suggests that an insect will be more likely to have a smaller microbiome bacterial count (Figure 2 ). This provides significant evidence to support the idea that the number of bacteria in the microbiomes of T. dimidiata is correlated to disease transmission for Chagas disease. Additionally, the 22 percent pre sence of T. cruzi in the population of San Luis T. dimidiata has changed since the last sampling study, which reported a 45 percent infection rate across San Luis, Monteverde, and Peas Blancas (Morton, 2014). The majority of the data collection occurred i n primarily the same geographic locations between the two studies, and a variety of different residences or farms, indicating similar study sites. Due my sample sizes, this data may not be representative of the whole population. A larger sample size would aid in determining the true prevalence of T. cruzi in the Monteverde and San Luis areas. Although I did not have the ability to classify the bacteria I examined into species, the results of this study showed a significant correlation between the number of bacteria in the microbiomes of T. dimidiata and their status as a vector for T. cruzi As a general trend, a positive identification of the parasite in the insects I sampled contributed to a greater number of bacteria in their microbiomes. My data supports the premise that internal bacteria numbers correlate to the epidemiology of Chagas disease, showing that there is a correlation of the microbiome with disease prevalence. This study has implications which could be applied in future research to fur ther our amount of available information about the relationship to microbiomes and disease. Bacteria that are part of a microbiome are genetically inherited, and they have a variety of functions in Triatominae Past research done on Chagas disease in Sout h America has focused on
Domako Bacterial microbiome in C hagas disease vectors 8 identifying specific bacterial symbionts that relate either to development or disease transmission These investigations have found important relationships between insects, their microbiomes, and the diseases they have the potentia l to transmit (Gumiel et al, 2015) Specifically, former studies have shown that microbiomes of the R. prolixus species of Chagas bugs are related to the epidemiology of Chagas disease by successfully genetically modifying the bacteria to eliminate T. cruz i No bacterial studies have been conducted on the Chagas bug species located in the Monteverde region of Costa Rica T. dimidiata (Gumiel et al, 2015) Quantification of the bacteria amounts in the microbiomes of T. dimidiata has also not been previously undertaken. Additionally, because I found a difference between the number of bacteria in the microbiomes of disease positive T. dimidiata versus the insects who were not infected with T. cruzi my study shows that the microbiome is correlated with disease prevalence in the Chagas bug species of Costa Rica as it is in other species found in South America Scientists found a similar correlation in Rhodinus proxilus with the presence of Chagas disease (Durvasula et al., 1999). After discovering this correlation, further research showed that species of Triatoma found in South America contain As a result, the larger numbers of bacteria I examined in insects carrying T. cruzi may contain possi ble symbionts, similar to the South American varieties, that also help the Costa Rican T. dimidiata carry the Chagas parasite. Thus, the correlation I established relate s to previous preliminary microbiome studies done on different species of Triatoma in S outh America and has the potential for further studies. Using genetic techniques like sequencing, the correlation discovered between microbiomes and Chagas disease led to further studies in South America which identified the species of the bacterial symbionts in individuals of Rhodinus proxilus carrying the parasite (Durvasula et al., 1999). This information has since been used in develop ing a targeted, promising way to prevent disease that does not involve pesticides but instead genetically alters t he bacteria to produce anti parasitic proteins Future Chagas disease prevention in Costa Rica could build off of the potential genetic difference s I found between bacterial microbiomes to act as a vector for and transmitter of T. cruzi with anti parasitic gene inserts (Weiss et al, 2011) This would be done through the process of paratransgenesi s, the process that involves inserting anti parasitic genes into the microbiome population. These new genes cause the production of antibodies that target the parasite, eventually eliminating its population inside the vector. What remains unclear, however, is whether the same bacterial symbionts are found in all of the vectors for Chagas disease acro ss the Americas Accordingly specific research needs to be conducted to discover the species of the bacteria in Costa Rican vectors T. dimidiata Once that is conducted, genetic modification strategies being developed in South America that affect the micr obiome bacteria can be attempted in Costa Rica, as well One topic for future research would be to introduce anti parasitic genes into microbiomes of Costa Rican vectors and analyze if this method would be a via ble option for disease control in the wild Investigating the effects of removing prevalent bacteria in vector individuals could also be a topic for additional studies These further studies would contribute to our understanding of
Domako Bacterial microbiome in C hagas disease vectors 9 the role of the micr obiome in disease transmission and would be the first step to ward developing a safer and more effect ive way to prevent Chagas disease in Costa Rica. ACKNOWLEDGEMENTS First, I would like to thank my incredible advisor s Emilia Triana and Frank Joyce for their wisdom, time, patience, knowledge, and guidance through the various stages of my study. I would also like to thank Eladio Cru z for his unbelievable skill in locating and catching Chagas bugs, along with Eric McAdam and Chirag Govardhan for their time and assistance with my insect collection and sampling process Additionally, I would like to acknowledge the landow ners of Finca La Bella, El Jard n, and other private residences in San Luis and Monteverde for allowing me to collect insects on their properties I would also like to thank the homeowners who graciously collected Chagas bugs overnight on their properties for me Lastly, I would like to thank the Monteverde Institute and the Estacion Biol gica for their respective lab spaces and equipment that allowed me to complete this study This study could not have been possible without the help of these individuals and all of the students of the EAP Fall 2016 Program. LITERATURE CITED Corynebacterial Symbiont fr om the Chagas Disease Vector Triatoma Infestans Experimental parasitology 119.1 (2008): 94 98. PMC Web. 7 Nov. 2016. "History of Chagas Disease." World Health Organization (2016) World Health Organization, n.d. Web. 06 Nov. 2016. Gumiel, Marcia, Fabio Faria Da Mota, Vanessa De Sousa Rizzo, Otlia Sarquis, Daniele Pereira De Castro, Marli Maria Lima, Eloi De Souza Garcia, Nicolas Carels, and Patricia Azambuja. "Characterization of the Microbiota in the Guts of Triatoma Brasiliensis and Triatoma Pseudomaculata Infected by Trypanosoma Cruzi in Natural Conditions Using Culture Independent Methods." Parasites & Vectors 8.1 (2015): n. pag. Web Morton, Amanda M. Adult sex ratio determination of Chagas disease vector Triatoma dimidiata and incidence of Trypanosoma cruzi in the Monteverde region of Costa Rica. UCEAP, Fall 2014. "Parasites American Trypanosomiasis." Centers for Disease Control Global Health, 24 May 2016 Web. 6 Nov. 2016. Starr, Mark D., Julio C. Rojas, Rodrigo Zeledn, David W. Hird, and Tim E. Carpenter. "Chagas' Disease: Risk Factors for House Infestation by Triatoma Dimidiata, the Major Vector of Trypanosoma Cruzi in Costa Rica." American Journal of Epidemiology 133.7 (1991): 740 47. Oxford Journals Web. 15 Dec. 2016. Vector Trends in parasitology 27.11 (2011): 514 522. PMC Web. 7 Nov. 2016. "Trypanosome." Encyclopedia Britannica Online Encyclopedia Britannica (2011) Web. 21 Nov. 2016.