WOLBACHIA-BASED INTERVENTIONS FOR MALARIA VECTOR CONTROL

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1004796
• Project Start Date: Dec 1, 2014
• Project End Date: Nov 30, 2019
• Program Code: [(N/A)]- (N/A)

Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824

Performing Department
Microbiology & Molecular Genetics

Non Technical Summary
Wolbachia are maternally-transmitted, Gram-negative, endosymbiotic bacteria that are estimated to infect more than 65% of all the insect species and approximately 28% of surveyed mosquito species, but surprisingly neither Anopheles malaria mosquitoes nor the major dengue mosquito vector, Aedes aegypti. A unique feature of Wolbachia biology in many arthropods is their ability to spread through host populations by means of a reproduction-interfering mechanism referred to as sperm-egg cytoplasmic incompatibility (CI). Lack of naturally occurring Wolbachia in those important disease vectors provides both a challenge, and a tantalizing opportunity to spread these bacteria into wild populations without the need for a complex driver system. As the first that introduced stable Wolbachia infection into Ae. aegypti, we recently achieved another milestone by successfully transferring Wolbachia from Ae. albopictus into the important Asian malaria vector, Anopheles stephensi, by embryonic microinjection. Wolbachia maintains a stable infection in An. stephensi and induces almost complete embryos death when infected males mate with uninfected females. Our long-term goal is to develop a Wolbachia-based control strategy as an eco-friendly, cost-effective and sustainable intervention to either suppress mosquito populations or block malaria transmission through population replacement. The objective of this proposal is to define the factors that enable infection with Wolbachia to reduce the Anopheles mosquitoes' vectorial capacity for malaria. Our central hypotheses are that a stable association formed between Wolbachia and Anopheles will alter the mosquitoes' physiological environment and result in a persistently tolerated Wolbachia infection as well as immune-mediated resistance to malaria parasites. Our project has the following four specific aims: 1). Determine the impact of Wolbachia on vector competence of An. stephensi for malaria parasites. 2). Characterize changes in the internal physiological environment of An. stephensi due to Wolbachia infection. 3). Evaluate the mosquito fitness costs associated with Wolbachia infections. 4). Introduce Wolbachia into two additional Anopheles malaria vectors, An. gambiae and An. arabiensis. In addition to addressing fundamental interactions among Wolbachia, malaria parasite, and mosquito host, this work will lead to development of Wolbachia-based control strategies for malaria control. This novel intervention is promising because it can target vectors that do not rest or feed indoors (and are therefore not susceptible to current control methods) and also permanently reduce the high vectorial capacity of the dominant malaria vectors.

Animal Health Component

10%

Research Effort Categories

Basic

60%

Applied

10%

Developmental

30%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
722	4010	1080	70%
721	4050	1130	30%

Knowledge Area
722 - Zoonotic Diseases and Parasites Affecting Humans; 721 - Insects and Other Pests Affecting Humans;

Subject Of Investigation
4050 - Protozoa; 4010 - Bacteria;

Field Of Science
1130 - Entomology and acarology; 1080 - Genetics;

Keywords

wolbachia

malaria

mosquito

vector

bacterium

tropical diseases

Goals / Objectives
Our long-term goal is to develop a Wolbachia-based control strategy as an eco-friendly, cost-effective and sustainable intervention to either suppress mosquito populations or block malaria transmission through population replacement. The objective of this proposal is to define the factors that enable infection with Wolbachia to reduce the Anopheles mosquitoes' vectorial capacity for malaria.

Project Methods
Specific Aim 1. Determine the effect of Wolbachia infection on the vector competence of An. stephensi for malaria parasites.Our working hypothesis is that a stable Wolbachia wAlbB infection will induce resistance to malaria parasites in An. stephensi. We will follow parasite's development in mosquitoes by comparing ookinete loads in the midgut lumen, oocysts in the midgut, and sporozoites in the salivary glands of Wolbachia-infected and -uninfected mosquitoes. Six-day-old An. stephensi Liston strain, Wolbachia-infected LB1 strain, and aposymbiotic strain (derived from tetracycline treatment of the LB strain) mosquitoes will be fed with Plasmodium gametocytes using a membrane feeding apparatus as previously described (Xi et al. 2007, Isaacs et al. 2011). After 30 min of feeding, un-engorged mosquitoes will be removed, and engorged mosquitoes will be maintained in the insectary under standard conditions with daily access to a 10% sucrose solution. P. falciparum prevalence and mean intensity of infection in midguts (ookinete and oocyst) and in salivary glands (sporozoite) will be compared between the LB1, Liston, and aposymbiotic strains. Prevalence will be reported as the percentage of mosquitoes infected, and the mean intensity of infection as the mean number of parasites found in infected mosquitoes. To determine ookinete loads, midguts will be dissected at 28 hr post-bloodmeal and gently homogenized in PBS by pipetting, using low-retention hydrophobic tips. Homogenates will be spotted onto a slide, air-dried, fixed in ice-cold acetone, blocked with 10% goat serum, and stained with primary anti-Pfs25 (MR4) and secondary AlexaFluor 488-conjugated goat anti-mouse IgG (Invitrogen) antibodies. As an alternative approach, the air-dried slides will be fixed in methanol, followed by Giemsa staining. Ookinetes will be enumerated using a Nikon microscope. To determine oocyst loads, mosquito midguts will be dissected at 8 days post-infection, oocysts will be stained with 0.2% mercurochrome, and parasite numbers per midgut will be determined using a light contrast microscope (Olympus). Sporozoite numbers will be determined at 14 days postinfection by dissecting pairs of salivary glands from an individual mosquito and homogenizing in 120 μl PBS. The homogenates will be centrifuged and resuspended in a final volume of 30 μl. One-third of this volume will be placed in a Neubauer counting chamber and the organisms allowed to settle before sporozoites are enumerated using a Leica phase-contrast microscope at 400x total magnification. For all infection assays, P-values will be determined using Mann-Whitney statistical tests, comparing the LB1, Liston, and aposymbiotic strains.Specific Aim 2. Characterize the changes in the internal physiological environment of An. stephensi resulting from Wolbachia infection.Our working hypothesis is that: (1) Wolbachia infection will induce an increased level of ROS in An. stephensi, and these ROS species will act as signaling molecules to activate the Toll pathway; and (2) the activated Toll pathway can mediate the expression of antioxidants to counterbalance the oxidative stress, promoting a persistent Wolbachia infection in An. stephensi. We will compare the hydrogen peroxide (H2O2) levels and the genome-wide transcriptome of LB1 and the Wolbachia-uninfected strains. We will knock down NADPH oxidase (NOX) and dual oxidase (DUOX) or feed mosquitoes with the antioxidant vitamin C to reduce the production of ROS, and then measure the expression of two Toll pathway marker genes, Serpin-27 and Spaetzle 1 (SPZ1), in both LB1 and Liston mosquitoes. Another experiment will be conducted to turn off the Toll pathway by silencing MYD88 and measuring the expression of antioxidants in the two mosquito strains, as we have previously done in the Wolbachia-infected Ae. aegypti WB1 line (Pan et al. 2011. PNAS).Specific Aim 3. Evaluate the mosquito fitness costs associated with Wolbachia infections.Different from the Wolbachia wMelPop strain, wAlbB was seen to provide a fitness advantage to both its original host, Ae. Albopictus, and to transinfected Ae. aegypti (Dobson et al. 2002, Bian et al. 2010). Our working hypothesis here is that the An. stephensi LB1 strain will have a fitness advantage when compared to the Liston strain. We will measure the mosquitoes' longevity, fecundity, male mating performance, survivorship, and sex ratio and construct a life table for LB1 and Liston mosquitoes. These experiment will be conducted at both the individual and population levels. Previous studies have shown that transgenic mosquitoes refractory to Plasmodium gain a fitness advantage after feeding on mice infected with P. berghei, when compared to their sibling nontransgenic mosquitoes (Marrelli et al. 2007). We will also compare the fitness of the LB1 and Liston strains after feeding on malaria parasite-infected blood.Specific Aim 4. Introduce Wolbachia into two additional Anopheles malaria vectors, An. gambiae and An. arabiensis.We will use a technique similar to that developed for An. stephensi to transfer Wolbachia into the two most effective and efficient, predominant vectors of human malaria in Africa: An. gambiae and An. arabiensis. We selected these two species because of their importance in malaria transmission and reduced susceptibility to current vector control tools. An. gambiae is considered to be one of the most efficient vectors of malaria in the world. An. arabiensis is the sibling species of An. gambiae and is also of major importance as a malaria vector. Development of insecticide resistance in these two species has led to an urgent need for a novel control tool such as we have designed. Specifically, An. arabiensis exhibits outdoor feeding and resting behavior and can develop behavior avoidance from a sprayed surface (Sinka et al. 2010). This behavior severely reduces the efficicacy of the two main vector control tools that are currently available, indoor insecticide residue spray and insecticide-treated nets, with regard to thses species (Control 2011).We will first use Ae. albopictus as a donor to transfer Wolbachia into the three abovementioned Anopheles species. This choice is based on our successful experience in transferring wAlbB into new hosts, including An. stephensi. The wAlbB strain has the ability to maintain a stable infection, induce complete CI, and cause pathogen interference in its new mosquito hosts (Xi et al. 2005a, Bian et al. 2010). In addition, we will also transfer two additional Wolbachia types, wMel and wMelPop, from their original D. melanogaster host into An. gambiae . These two Wolbachia types have also been shown to be capable of forming a stable association with a new mosquito species and inducing strong resistance to various pathogens, including malaria (Moreira et al. 2009, Walker et al. 2011).

Progress 12/01/14 to 11/30/19

Outputs
Target Audience:I have given seven presentations during this report period to introduce Wolbachia-based strategies for vector-borne disease control. This includes the 2nd International Conference for Vector-borne Diseases, 2018 World Life Science Conference, the 4th international workshop on dengue/Zika and vector mosquitoes. We also hosted "High-level Scientific Visit for Key Stakeholders on the Requirements to Develop an SIT Pilot Project against Disease Transmitting Mosquitoes" for the International Atomic Energy Agency. In order to promote development of Wolbachia for biological control of malaria in India, we hosted the International Workshop on Wolbachia-based Dengue and malaria control from October 20 to 22, 2018 in Guangzhou, China, with the particpants from India's National Institute of Malaria Research, Indian Council of Medical Research (ICMR), VIT University, ICMR's Regional Medical Research Centre, and Energy and Resources Institute and Johns Hopkins Malaria Reseacrh Institue. In addtion, we also visited National Institute of Allergy and Infectious Diseases (NIAID) of NIH to give program officers a presentation on developing Wolbachia as a novel tool for malaria control. Changes/Problems:Except that it takes longer time than we expect to get fund supports for field trial, no major changes/problems during this reporting period. What opportunities for training and professional development has the project provided?One scientist from India's National Malaria Research Insititue has visited us to get training on Wolbachia-based vetcor control. I have given many classes, presentation and workshop to the people interested in developing Wolbachia for vector control. Through IAEA, we have provided trainings to a number of scientists from disease endemic countries on mosquito mass rearing, quality control and release which are esstential steps to develop our technology for disease control. How have the results been disseminated to communities of interest?Our results have been disseminated to communities through presentation in conference, scientific journal publication, internet, social media, public media, and interview through TV program. What do you plan to do during the next reporting period to accomplish the goals?We will continue seeking fund support to our proposed research, including from NIH, WHO and Bill & Melinda Gates Foundation. Our team is ready, in term of experence, skill and knowledge, to develop and deploy Wolbachia technology for malaria control as long as we receive the fund support. In additon to India, we will explore the opportunities to develop collabartions with Pakistan and Africa countries where our technology are in need with new funding possiblity emerging.

Impacts
What was accomplished under these goals? We have successfully established collaborations with Indian government to develop Wolbachia-based field trial for malaria control. Now India government has sponsored a scientist to visit my lab for training in order to enhance this collaboration. In additon, we have successfully completed a fiedl trial to demonstarte the potential of area-wide application of our technology for mosquoito vector control. This has laid a foundation to develop a similar effort for malaria control. This summer we submitted a mutiple-million grant to NIH, by collaborating with Johns Hopkins Malaria Research Institue, to understand Wolbachia-malaria interactions in mosquito vectors for further technology development. We alos started to work with Pennsylvania State University to understand Wolbachia-mediated plasmodial interference in Anopheles vectors.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Zheng, X., D. Zhang, Y. Li, C. Yang, Y. Wu, X. Liang, Y. Liang, X. Pan, L. Hu, Q. Sun, X. Wang, Y. Wei, J. Zhu, W. Qian, Z. Yan, A. G. Parker, J. R. L. Gilles, K. Bourtzis, J. Bouyer, M. Tang, B. Zheng, J. Yu, J. Liu, J. Zhuang, Z. Hu, M. Zhang, J. T. Gong, X. Y. Hong, Z. Zhang, L. Lin, Q. Liu, Z. Hu, Z. Wu, L. A. Baton, A. A. Hoffmann, and Z. Xi. (2019). Incompatible and sterile insect techniques combined eliminate mosquitoes. Nature 572: 56-61.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Zhang, M., Zhang, D., Li, Y., Sun, Q., Li, Q., Fan, Y., Wu, Y., Xi, Z., Zheng, X. (2019). Water-induced strong protection against acute exposure to low subzero temperature of adult Aedes albopictus. PLoS Negl Trop Dis. 13(2): e0007139.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Zheng, B., Yu, J., Xi, Z., and Tang, M. (2018). The annual abundance of dengue and Zika vector Aedes albopictus and its stubbornness to suppression. Ecological Modelling. 387:38-48.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:I have given fourteenpresentations during this report period to introduce Wolbachia-based strategies for vector-borne disease control. This includes talks in American Society for Microbiology, Pan American Health Organization/World Health Organization (PAHO/WHO), the 3rd International Congress on Biological Invasions, the 14th International Organization for Biological Control-Mass Rearing and Quality Assurance and the 66th Annual meeting of American Society of Tropical Medicine and Hygiene. In order to promote development of Wolbachia for biological control of malaria, I, together with MSU colleagues in a delegation, spent one-week in India to establish collaborations with Indian government, including Indian Council of Medical Research (ICMR) and its National Institute of Malaria Research (NIMR), National Institute for Research in Tribal Health, and Vector Control Research Centre. As a result from this trip, MSU has signed an agreement with ICMR, in partner with NIMR, to develop feasibility to prepare for a field trial in India. To support this effort, ICMR has agreed to fund NIMR's research actives at India's side. Changes/Problems:No major changes/problems during this reporting period. What opportunities for training and professional development has the project provided?I have given a number of presentation and workshop to the people interested in developing Wolbachia for vector control. This has greatly helped them to develop their own program to work on Wolbachia-based population suppression or replacement for disease control. This project has also provided training opportunitiesto postdoc and graduate students from India and Mexico for technique transfer and capacity building. Additional training opportunities are also provided to those interested in mosquito embryonic microinjection and development of genetic sex sorting system. How have the results been disseminated to communities of interest?Our results have been disseminated to communities through presentation in conference, scientific journal publication, public media, including seminar and newspaper, and interview through TV program. What do you plan to do during the next reporting period to accomplish the goals?Effort will continue to put into developing field trial in India. Toward that, we will invite our Indian collaborators to attend a workshop in China in October such that they can better prepare for this project. In addition, research proposal on feasibility and preparation of field trial will be prepared to seek for funding support from NIH and other funding agency. In the meanwhile, we will work with our colleagues to start community education and regulation approval which are essential for field trial.

Impacts
What was accomplished under these goals? We have successfully established collaborations with Indian government to develop Wolbachia-based field trial for malaria control. With the approval by Indian Council of Medical Research (ICMR) and in partner with National Institute of Malaria Research (NIMR), now we are negotiating with NIH and other fund agency for their funding support. In addition, our collaboration with Hawaii government to develop Wolbachia to protect birds from avian malaria has recently resulted in an award from US Fish and Wildlife Service (USFWS). Our mosquito factory in Merida Mexico, sponsored by USAID, has recently opened and started mosquito mass rearing for field trial for Zika control, providing us additional experience on how to collaborate with foreign governments for field trials.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Pan, X., Parker, A., Joshi, D., Bian, G., McFadden, M. J., Lu, P., Liang, X., Zhang, F., Raikhel, S.A. and Xi, Z. (2018). The bacterium Wolbachia exploits host innate immunity to establish a symbiotic relationship with the dengue vector mosquito Aedes aegypti. ISME Journal. 12:277-288
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Zhang, D., Li, Y., Sun, Q., Zheng, X., Gilles, J. R. L., Yamada, H., Wu, Z., Xi, Z. and Wu, Y. (2018). Establishment of a medium-scale mosquito facility: tests on mass production cages for Aedes albopictus (Diptera: Culicidae). Parasites & Vectors 11: 189.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Zheng, B., Yu, J., Xi, Z., and Tang, M. (2018). The annual abundance of dengue and Zika vector Aedes albopictus and its stubbornness to suppression. Ecological Modelling. 387:38-48.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:I have given presentation in Third FAO-IAEA International Conference on Area-wide Management of Insect Pests in Vienna with participants from 168 countries. Both my postdoc and I have given talks in International Congress of Entomology to introduce our work on Wolbachia-based malaria control. To help control bird-malaria in Hawaii, I have been invited to attend a workshop there and share our experience in field trial, resulting in initiating a project to develop Wolbachia to protect bird in Hawaii with support by Google (Verily). Our mosquito factory has been well reported by the top media, including CNN. In addition, I have been invited by Google (Verily) to visit their mosquito factory in California and introduce the potential of Wolbachia technology for vector-borne disease control. Changes/Problems:No major changes/problems during this reporting period. What opportunities for training and professional development has the project provided?I have given a number of presentation and workshop to the people working on vector control in Hawaii, Taiwan, Grand Cayman and Jamaica. This has greatly helped them to develop their own program to work on Wolbachia-based population suppression for disease control. This project has also provided training opportunity to postdoc and graduate students for studying mosquito embryonic microinjection and developing genetic sex sorting system. How have the results been disseminated to communities of interest?Our results have been disseminated to communities through presentation in conference, scientific journal publication, public media, including seminar andnewspaper, and interview through TV program. What do you plan to do during the next reporting period to accomplish the goals?Effort will be mainly put to develop field trial in India. Toward that, we will have a workshop in India and form partnership with India scientists. A five-year research plan will then be prepared to seek for funding support. In the meanwhile, we will work with our colleagues to start community education and regulation approval which are essential for field trial.

Impacts
What was accomplished under these goals? We have further characterized the Wolbachia-infected Anopheles stephensi line in preparation for field trial, including fitness and anti-plasmodium spectrum, in the labortory. Inorder to movefrom lab benchinto field trial, we have actively seeked support from Michigan State University (MSU) in order to use MSU's existing strong connection with India to promote the work. This has ended in a recentsuccessful visit to Indiaby MSU team led by Professor Maredia to build paretnership and investigate the regulatory process. It has sigificantly facilitated the effort to develop a field trialfor testing Wolbachia-based malaria control in India.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Joshi, D., Pan, X., McFadden, M.J., Bevins, D., Liang, X., Lu, P., Thiem, S., and Xi, Z. (2017). The maternally inheritable Wolbachia wAlbB confers resistance to Plasmodium berghei in Anopheles stephensi. Front Microbiol. 8:366. doi: 10.3389/fmicb.2017.00366.
• Type: Book Chapters Status: Published Year Published: 2017 Citation: Pan, X., Thiem, S. and Xi. Z. Wolbachia-mediated immunity induction in mosquito vectors. In the book Arthropod Vector: Controller of Disease Transmission. Edited by Aksoy, S., Wikel, S., and Dimopoulos, G. 2017.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Joshi, D. and Xi. Z. The maternally inheritable Wolbachia wAlbB confers resistance to Plasmodium berghei in Anopheles stephensi. International Congress of Entomology. Sept 27th, Orlando, Florida

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:I have given presentation to vector control program in South America, Asia and Africa in the meetings organized by International Atomic energy Agency. In addition, I also submitted proposal to NIH in order to get funding support. Lastly, we have introduced the potential of Wolbachia technology in use for vector-borne disease control to different media and industry partner such as google. Changes/Problems:No major changes/problems during this reportingperiod. What opportunities for training and professional development has the project provided?I have given a number of presentation and workshop to the people working on vector control in many disease endemic countries to train them how to develop Wolbachia-based population suppression for vector-borne disease control through field trial. This project also provides training opportunity to postdoc for studying and developing an improved genetic sex sorting system for population suppression of malaria vectors. How have the results been disseminated to communities of interest?As the previous year, we have continued to use Wolbachia-based dengue control as a concept to deliver the knowledge of this technology to the community through a number of public media, including seminar, newspaper, and interview through TV program. What do you plan to do during the next reporting period to accomplish the goals?We will continue determining the spectrum of Wolbachia-mediated anti-malaria effect in Anopheles stephensi. In addition, we will evaluate the role of host immunity in Plasmodium interference and symbiosis formation between Wolbachia and mosquitoes. To improve the efficiency of this technology in implementation, we will develop a novel sex sorting system which can be used in Wolbachia-based population suppression.

Impacts
What was accomplished under these goals? We have showed that Wolbachia wAlbB can inhibit Plasmodium berghei and found that anti-plasmodium immune genes were induced by wAlbB in different mosquito body tissues. These findings suggest that immune priming is a potential cause of wAlbB-mediated antimalarial response in Anopheles stephensi. More importantly, no evidence was found for any enhancement of Plasmodium infection in An. stephensi stably infected with wAlbB, supporting the safety in development of Wolbachia-based approach for malaria control.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Deepak Joshi, Xiaoling Pan, Michael J McFadden, David Bevins, Xiao Liang, Peng Lu, Suzanne Thiem, and Zhiyong Xi. The maternally inheritable Wolbachia wAlbB confers resistance to Plasmodium berghei in Anopheles stephensi. Frontiers in Microbiology. Submitted.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Chen, S., Zhao, J., Joshi, D., Xi, Z., Norman, B., Walker, E., 2016. Persistent infection by Wolbachia wAlbB has no effect on composition of the gut microbiota in adult female Anopheles stephensi. Frontiers in Microbiology 7, doi: 10.3389/fmicb.2016.01485
• Type: Book Chapters Status: Published Year Published: 2015 Citation: Xi, Z. and Joshi, D. Genetic control of malaria and dengue using Wolbachia. In the book Genetic Control of Arboviruses and Malaria. Edited by Adelman, Z. N. 2015.
• Type: Book Chapters Status: Submitted Year Published: 2017 Citation: Xiaoling Pan, Suzanne Thiem, Zhiyong Xi. Wolbachia-mediated immunity inductions in mosquito vectors.

Progress 12/01/14 to 09/30/15

Outputs
Target Audience:We have given classes to undergraduate students on how Wolbachia can be used as a tool for malaria control, what the current progress and challenges are in this field, and how the knowledge and experience learnt from dengue control can be adapted for malaria. I have also spent efforts to introduce this concept to Gates foundation and welcome trust and seek their funding support. In addition, I have also visited international atomic energy agency (IAEA) to introduce how Wolbachia technique can be combined with traditional sterile insect technique (SIT)for vector control. Lastly, I have communicated with scientists in malaria endemic areas in India in order to initiate a field trial in Chennai where Anopheles stephensi is a primary malaria vector. Changes/Problems:One of the major challenges in this project is to enhance the ability of Wolbachia-mediated pathogen resistance in transinfected Anopheles mosquitoes. The parasite that escapes from the resistance will be the major risk if a population replacement strategy will be used. This requires us to select the right Wolbachia strain for transinfection. However, a strong anti-pathogen resistance is always associated with the fitness cost to the host. The symbiosis system will be no longer maintained if such fitness has reached to certain level. Another challenge is to develop collaboration with disease endemic country. The current mosquito species we have developed Wolbachia infection is An. stephensi, which is the primary malaria vector in India, Pakistan and Iran. After putting significant effort, we found it is a very difficult job due to difference in regulation of research and international collaboration in these countries. What opportunities for training and professional development has the project provided?This project has provided a opportunity for scientists from malaria endemic countries to learn how to develop Wolbachia for malaria control. We have a visiting student from Pakistan to get training on the aspect in my lab. It also provides opportunities for postdoc to get further training in this direction. How have the results been disseminated to communities of interest?Although research has not moved to implementation in developing Wolbachia for malaria control, a field trial has initiated to develop a similar strategy for dengue control in China. The concept thus has been delivered to the community through a number of public media, including seminar,newspaper, and interview through TV program. Our field trial program also has a team working on community education and engagement, through which we have regular meetings, door-to-door visits and surveys and questionnaire in order to get the community support. What do you plan to do during the next reporting period to accomplish the goals?We will continue validating Wolbachia-mediated anti-malaria effect in Anopheles stephensi and understanding its underlying mechanism. In addition, we will transfer WolbachiaintoAfrican malaria mosquito vectors such as Anopheles arabiensis, and test whether Wolbachia induces cytoplasmic incompatibility, pathogen interference, fitness costs in the transinfected lines. To increase the chance of success, we will used the Wolbachia strain, which has adapted to Anopheles host for many years, as a donor for the transfer. We will further enhance our collaboration with IAEA in develop integration of Wolbachia and SIT for malaria vector control in African malaria endemic areas.

Impacts
What was accomplished under these goals? We have completed the assay to test the impact of Wolbachia on mosquito vector competence for plasmodium berghei. Our results exclude the possiblity, as reported previously, that Wolbachia wAlbB will enhance p. berghei in mosquitoes. Moreover, we show that wAlbB can confer mosquito resistent to this parasite species. In addition, we have performed the study to understand the role of mosquito immunity underlying the anti-malaria resistence. Lastly, we study how the mosquito innate immunity plays roles in enhancing Wolbachia symbiosis with Anopheles mosquitoes.

Publications

• Type: Book Chapters Status: Awaiting Publication Year Published: 2015 Citation: Xi, Z. and Joshi, D. Genetic control of malaria and dengue using Wolbachia. In the book Genetic Control of Arboviruses and Malaria. Edited by Adelman, Z. N. In press
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Zhang, D., Lees, R. S., Xi, Z., Gilles, J. R., and Bourtzis, K. (2015). Combining the Sterile Insect Technique with Wolbachia-based approaches: II- A safer approach to Aedes albopictus population suppression programmes, designed to minimize the consequences of inadvertent female release. PLoS One. 10(8):e0135194. doi: 10.1371.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Rossi P, Ricci I, Cappelli A, Damiani C, Ulissi U, Mancini MV, Valzano M, Capone A, Epis S, Crotti E, Chouaia B, Scuppa P, Joshi D, Xi Z, Mandrioli M, Sacchi L, O'Neill SL, and Favia G. (2015). Mutual exclusion of Asaia and Wolbachia in the reproductive organs of mosquito vectors. Parasites & Vectors. 17; 8:278.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Zhang, D., Zheng, X., Xi, Z., Bourtzis, K., and Gilles, J. R. (2015). Combining the Sterile Insect Technique with the Incompatible Insect Technique: I-impact of Wolbachia infection on the fitness of triple- and double-infected strains of Aedes albopictus. PLoS One, 10(4):e0121126.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Li, Y., Liu, Q., and Xi, Z. (2015). Blocking the transmission of mosquito-borne diseases through Wolbachia-based population replacement. Chinese Journal of Vector Biology and Control. 26 (1): 11-15.