Team:Tec-Chihuahua/prueba
Overview
"Human Practices is the study of how your work affects the world, and how the world affects your work."
— Peter Carr, Director of Judging
Beyond the technical work on the lab, we comprehend our project exists within a world that requires us exploring aspects that involve issues such as ethics, sustainability, safety, legal framework, security, between others. These issues are complex and approached only through our human practices. Being aware of this we engaged with the communities that might be impacted by our work to invite them to shape the course of our investigation.
These social considerations were upfront in our project and integrated with the design and execution of our project, impacting in our Entrepreneurship, Wet lab, and Mathematical model work. We aimed to develop a thoughtful project with inspiring Human Practices for other teams to build upon and come up with new and creative approaches for this section, striving not only to understand how the world affects our project but also aiming to contribute to how synthetic biology is understood and practiced around the world.
Social Science Research Description
Since our Human practices involved interacting with communities to understand the social concern we are addressing, it was essential for us to approach these communities correctly by getting informed on our institution and national guidelines. This way we were able to conduct our social science research respectfully and always looking after the well-being of our cooperators.
The Mexican National Guidelines for integration and operation of the Research Ethics Committee (REC) declare the inclusion of ethical aspects as a quality indicator in social science research. The development of a bioethic culture is favorable to guarantee individual and social well-being since the discipline promotes the concepts of equity, solidarity, quality, principles of benefit, no maleficence, as well as the protection of human rights.1
Human subjects research entails risks; this makes it necessary to guarantee ethically acceptable conditions in the production of knowledge, considering that the legal framework is not sufficient to elucidate or resolve conflicts of a moral or ethical nature that arise in scientific activity.1
To protect our contributor’s human rights, respect their dignity and privacy, avoid any form of discrimination as well as ensure their integrity and wellness, we took into consideration the universally recognized ethical standards which are principles of benefit, no maleficence, justice and respect for autonomy.
We also made sure our procedures where according to the Mexican Federal Law on Protection of Personal Data Held by Individuals (2). First of all, we sought a pre-approval letter from our institution for doing social science research in accordance to their ethics code and values and under the stipulated in the annexed letter, we also requested a consent letter filled up by our participants where they accept we share their information on our webpage and in the Giant Jamboree's presentation. Trough this letter they also recognize our behavior and procedures were done according to the iGEM values.
References
- Ruiz de Chávez, M. (2016). Guía Nacional para la integración y el funcionamiento de los Comités de Ética en investigación. 5th ed. México: Secretaría de Salud, Comisión Nacional de Bioética.http://www.diputados.gob.mx/LeyesBiblio/pdf/LFPDPPP.pdf
Silver Human Practices
From the first time you set foot outside the laboratory and leave scientific articles aside to interact with people related to your project, you begin to realize how crucial human practices are.
Human practices allowed us to investigate and validate the purpose, design, scope, and execution of our project. Is our product suitable for beekeepers to use? Will it disturb the innocuousness of honey? Is it capable of counteracting more diseases? Is it permissible before the law? These are some of the questions that were answered throughout our human practices chronology; we were driven to think, reflect, and act on it as our integrated human practices.
Beekeepers, beekeeping societies, international organisms, government representatives, and researchers were all essential for fortifying our research.
Beekeepers
The world of beekeeping is much more than what appears in texts; no research could have compared being in the field with a beekeeper sharing knowledge, getting to know his community and their needs. Our main reasons for approaching them were to be informed about Beekeeping in their region, their opinion on our project and validate technical aspects of it. We wanted to choose with their help, the optimal application method for our product into the beehives, investigate the presence of American foulbrood and European foulbrood in their regions or countries as well as current existing treatments, their benefits, and risks.
April 28th
Cuauhtémoc, Chihuahua
Meeting with Mr. Guillermo Rodríguez
June 15th
Santiago de Querétaro City
Meeting with Engineer Alejandro Bianchi
September 22nd
Province of Melipilla
Videocall with Bryan Saavedra
May 26th
Delicias, Chihuahua
Meeting with Ing. Manuel Ramírez Salcido
July 16th
Castellón, Valencia
Valencian beekeeper interview
April 28th
Meeting with Mr. Guillermo Rodríguez
Viana and Ana Laura had the opportunity to travel to Cuauhtémoc, a city specialized in fruit growing; production and exportation of apples, peaches, strawberries, walnuts, among many others.1 Thanks to Viana, who is native from this city, we were able to get to contact this beekeeper. Mr. Rodríguez is the president of the association of beekeepers in the city and we saw an enormous opportunity in talking to him. This was our very first direct approach with the beekeeper’s community! We were very excited to start getting to know them. What would they think about our ideas? Would they be
interested in our project? How many things would they have to tell us? Our project was about to begin its transformation path.
This enriching talk lasted approximately 1 hour 15 minutes and different topics were discussed. We learned many general characteristics of bees' lives thanks to the great experience of the beekeeper: The different varieties of bees that exist, specific tasks of each type of bee and the joint work bees and beekeepers do to ensure pollination.
Regarding the existing methods against Foulbrood, Mr. Rodríguez mentioned how SAGARPA (National Secretary of Agriculture, Livestock, Rural Development, Fishing, and Food) does not allow the usage of chemical contaminants in beehives because wastes may end up in honey flows. Mr. Rodríguez mentioned how beekeepers sometimes use extracts of rosemary or oregano against Foulbrood, but it is always difficult for them to know what dose to add and ensure it is adequate for fighting the diseases and at the same time does not destabilize the hive. On another hand, he mentioned that the method of burning beehives results very expensive for them. Currently, in Cuauhtémoc, there is almost no presence of American Foulbrood, since, in the past, many sick hives were exposed to this burning method, then the disease decreased. On the other hand, European foulbrood is most commonly found in Cuauhtemoc.
The beekeeper emphasized how a natural product against Foulbrood is needed, so there are not left residues in the honey and he also highlighted how beekeeping varies around the world and sometimes there are specific necessities for each region.
Thanks to the sharing we made with the beekeeper of our project, he was able to have a new panorama of beekeeping, and how emerging technologies, in this case, synthetic biology, can accurately respond to specific problematics and can relegate past methods that imply many setbacks.
Mr. Rodríguez kindly invited us to present our project with the Cuauhtémoc beekeepers association and commented us about M.C. Alejandro Romo who has done different investigations on the diseases and who we would talk with later in this story.
Getting to know real experiences of the treatment methods used against our interest diseases helped us to better measure the severity of the problem and the obstacles with which beekeepers have to fight to keep their bees’ welfare.
References
- Soto, A. (2017). Cuauhtémoc, región en crecimiento económico. Retrieved from http://referente.mx/@Almer/cuauhtemoc-region-en-crecimiento-economico
- Image retrieved from: Visita Chihuahua. (2018). Chihuahua y sus alrededores. http://www.visita-chihuahua.com/index.php/2015-05-06-09-42-18
May 26th
Meeting with Ing. Manuel Ramírez Salcido
Key team members related to the development of the mathematical model, human practices, legal management, and wet lab traveled southeast from our city to visit Salcido apiaries in Delicias; a municipality that bases its economy fundamentally on the food industry. Since we arrived with Mr. Ramírez, it was amazing how the discussion flowed and flowed, getting a meeting that end-up lasting approximately 3 hours; all were valuable minutes that taught us many relevant aspects for the development of our project.
First, Mr. Ramírez covered the topic of the importance of bees, emphasizing how they have always been considered an essential factor for environmental health and he even showed us a funny cucumber that had not been correctly pollinated; it was very small and thin.
Bee diseases were our next topic. He commented how Foulbrood
presence isn’t that strong in Mexico, however, in Europe and in the USA, this is not the case. Mr. Ramírez took advantage and included implications of other frequent bee diseases like Nosema and Varroa in the conversation. He mentioned a very important point that we had previously started to notice: Most of the serious problems in bees caused by viruses or bacteria, are not correctly detected or even known. As Mr. Rodríguez had previously commented, Mr. Ramírez mentioned how different extracts of plants are used against diseases in bees, in this case, mint and grapefruit were the examples. On another hand it was mentioned how antibiotics are not an option at all for treatment, since the European Union does not accept them, neither does accept the presence of transgenics in the honey; there are rigorous studies exercised and if there is something wrong the EU may return an entire shipment of honey back to the remitter. We discussed the issue of innocuity of honey by highlighting that when there is a new product added to the hive, it should not generate any residue at all or change the expected properties from the consumer.
After talking about our project, a question arose in Mr. Ramírez, an issue that would be asked many times since that day and that we would do everything possible to solve and validate: Our product would actually pass from the food that the nurse bee ingest all through its system until reaching the larva by means of the royal jelly? And regarding the method of application, (initially consisting in applying the final product in the bee bread), Mr. Ramírez explained to us how this bee bread is created, and the fermentation process it goes through in the bee cell.
We also had the great opportunity of listening to some of the concerns Mr. Ramírez has as a beekeeper: He thinks the beekeeping area could be greatly impulsed by receiving more support from governmental institutions and he perceives that the validation of honey innocuousness here in Mexico results complicated as there are not enough tools to endorse the required diagnostics. On another hand, he mentioned how there are no young people able to continue with the job of the current farmers, and this is an alarming situation that requires a prompt resolution.
Mr. Ramírez gave us a lot of priceless information that was essential for the development of our whole project. He was the first to comment us about the beekeeping congress that would take place in the city of Delicias in the coming months, and which we would have the great privilege to attend, he told us that by means of the different sites where he frequently researches information he had noticed another iGEM team that was working with bees, for the month of September we could establish a collaboration with this team (consult collaborations for more information) and on this same point of the media, Mr. Ramírez explained to us how he always tried to receive literature to focus and inform himself, he mentioned the magazine “Bee Culture”, where we successfully managed to make a publication about our project and synthetic biology regarding beekeeping (consult public engagement for more information).
He also shared with us the contacts of highly recommended specialists with whom we could consult about our product and with which we would have contact later: Juan Diego Pérez de la Rosa, Efraín Rocha, Felipe Esquivel Díaz.
We were able to share a new perspective on beekeeping in relation to synthetic biology with Mr. Ramírez by explaining him our project, the beekeeper mentioned how he was more than willing to help us, and he even shared some honey and bee bread with us! This meeting was an invaluable experience for us!
Bee Bread
References
- Image retrieved from: Administración. (2018). ¡Conócenos! http://municipiodelicias.com/
June 15th
Meeting with Engineer Alejandro Bianchi
Engineer Alejandro Bianchi studied Agronomy at the Monterrey Institute of Technology and Higher Education and has been dedicated to beekeeping since 30 years ago. Currently, he has a company called Kolmena Yothë that offers bee products for health and beauty purposes, his company also provides apitherapy treatments, complementing them with Bach flowers, Schuessler salts and homeopathy.
As we were speaking with him about our project, he mentioned
the importance of the Varroa mite, a parasite that sucks the hemolymph of bees, the equivalent to blood in this arthropods, weakening their immune system and making them susceptible to other diseases. He ensured that enhancing the natural defenses of bees would have a positive response since healthy bees are a lot less susceptible to diseases.
He mentioned the Varroa mite as part of the named “colony collapse disorder”, baptized by this name in the US, and described it to be a phenomenon where bees leave their beehives. Perhaps he announced foulbrood are not a current problem in his state, Varroa is, so he recommends us to expand our horizons to consider our product as a product that can also be applied when there is the presence of Varroa.
“If the bees are strong they can’t get sick, but how will they be strong if there are not enough flowers?” He mentioned urbanization is a problem bees are facing directly, and they are dying because of it. As flowering and bee population lower, he went from collecting a bucket of honey per box to a bucket every 40 boxes.
He said the actual treatments for American foulbrood are formic acid, oxalic acid, thymol (thyme), among others treatments
that don’t pollute the environment. Previously, fluvalinates were used to treat this diseases, however, they stopped using them since they are carcinogenic and pollute the environment. The Secretariat of Agriculture, Livestock, Rural Development, Fisheries and Food (Spanish: SAGARPA) is the governmental institution responsible of inspecting their hives, it allows under certain requirements the use of antibiotics for treating foulbrood but has to avoid products that pollute the environment and honey. He also shared with us the treatment he uses to fight Varroa which consists in an essential oils mix and stressed it is important not to use the same treatment for a long time since the pathogen can develop a resistance towards it.
We asked him if he could share with us certain regulations or standards that had to be followed to ensure the honey’s quality, he mentioned the "Good livestock practices".Discussing other issues affecting beekeeping in his state, another serious problem in Queretaro is bee theft, as beekeepers visit their bees every 8 or 15 days, they are the target for many people that take advantage of this situation. Engineer Bianchi assists to the Beekeeper's society meetings, and he sees there are not young beekeepers to take the hard work of beekeeping, he sees that as another issue.
Finally, after approximately an hour of conversating, he shared us his book of memories from the 25th International Apicultural Update Congress which was held in Queretaro last year and invited us to visit his beehives as well as the facilities of his company.
July 16th
Valencian beekeeper interview
Thanks to our colleagues from Valencia_UPV, we received written feedback from a Valencian beekeeper and information about the current status of foulbrood in his province. Our Valencian colleagues interviewed the beekeeper for us, thank you Valencia_UPV team! (LINK collaborations) Given that our interest diseases have a global presence, it is essential for us to make contact with beekeepers from all around the world; knowing how they face illnesses, the used treatment methods, their effectiveness and consequences, and the beekeeper's concerns and opinions about our solution proposal in
order to also integrate them into the development of our project.
The interviewed beekeeper has spent approximately 70 years dedicating himself to the invaluable work of beekeeping and he shared with us some already experienced foulbrood implications that are lived in his province. In Valencia the treatment against foulbrood consist in the following:
First, the combs with the infected brood are replaced by healthy brood and then antibiotic with powdered sugar, is applied. The adult bees, when they realize the larvae are dead, clean the cell. The beekeeper ensures that with this treatment he has managed to heal all his hives, and has not suffered any loss from the disease, the drawback for this method is that it contaminates pollen, so it would no longer be suitable for human consumption, and when this treatment is not applied on time, beekeepers get to have very large losses of money and even the total loss of their hives (between 20 to 100 hives per beekeeper). Regarding this information, we could realize that antibiotics indeed are currently used and although they are effective in treating the disease, the innocuity of the honey is always altered resulting in market issues.
Once the beekeeper heard about our project and learned about biotechnological alternatives that seek to solve beekeeping problems, he considered it an adequate proposal which does not contaminate the bee products and he was expectant before the results of the project to evaluate if it is actually an effective method.
September 22nd
Videocall with Bryan Saavedra
Keeping on with our international validation on the presence of foulbrood, its implications, and our project insights, we were able to contact a Chilean beekeeper thanks to our colleagues from UChile (LINK collaborations). Bryan Saavedra dedicates to honey production, pollination, and queen bee breeding and exportation. He has dedicated more than a decade of his life to beekeeping and he counts with more than 1000 beehives.
“Approximately 13% of the Chilean working population is engaged in
agriculture, and agricultural products represent about 4% of the gross domestic product (GDP). The fruit sector is very important and includes crops such as grapes, melons, apples, and peaches.1”
After UChile team sent us the beekeeper’s contact we were able to get in touch with him and schedule a video-call. During the meeting, that lasted approximately 35 minutes, we were able to discuss the presence of bee diseases in Chile, its implications and some activities of Chilean agencies in charge of supporting the development of agriculture, forests, and livestock.
Foulbrood occur in Chile as foci of infection, once an infection is detected, which most likely originated
from contaminated equipment, the SAG (Agricultural and Livestock Service) proceeds to burn and then bury the infected hives; the decision of burning the beehives is quite drastic but it is the only effective way to take care of the rest of the Chilean apiculture. The last outbreak of foulbrood the beekeeper noticed about, was in last February; normally the infection comes from Argentina, which is where the disease most often occurs. In Chile, they mostly struggle with varroa and nosema and for purposes of the SAG and exportations, the varroa mite is treated with organic products, that result inefficient and expensive. Antibiotics are also used against Varroa, getting back to the resistance problem. Regarding this topic, the beekeeper wondered if our product would help against varroa, and we could tell him how one of the most important consequences of varroa is that the immune system is weakened; providing the bee with new immunity components (AMP’s) will greatly help it, so the answer is yes! Our product can help bees get fortified when they are suffering from varroa.
The beekeeper also shared with us the main routes through which the disease is transmitted from Argentina to Chile, how the bee's life duration varies depending on the production stage and also shared with us details about SAC normative.
Regarding the discussed topic on how bee’s longevity varies depending on different production stages, we explained the beekeeper another approach of our project: When the bee uses less energy in the production of antimicrobial peptides it is possible that its longevity increases.2
Bryan Saavedra did not know about synthetic biology and we gave him an approach to this area and how it had so many reaches, one of them being beekeeping. While explaining our project we absent-mindedly use a somewhat technical word that is usually complicated for the community in general. A peptide. The beekeeper, as in other times of the conversation, didn't hesitate to ask us and he attentively listened to us while we explained to him what we were referring to; he was always intrigued by our project.
References
- Economía de Chile: agricultura y pesca de Chile. (2018). Retrieved from http://www.voyagesphotosmanu.com/economia_chile.html
- Turcatto, A. P., Lourenço, A. P., & De Jong, D. (2017). Propolis consumption ramps up the immune response in honey bees infected with bacteria. Apidologie, 49(3), 287–296. doi:10.1007/s13592-017-0553-z
- Image retrieved from: Melipilla Chile: Ubicación, mapa y todo lo que desconoce. (n.d.). Retrieved from http://conociendochile.com/c-region-metropolitana-de-santiago/melipilla-chile/
Specialists and Investigators
Our project considers particular parts as the immune system of the bee, as well as bigger picture aspects like how beekeepers provide the liquid food to their hives; the required panorama is vast, and we sought to make fit each small component of the project in the life of bees and beekeepers. Researchers who have been surrounded by the beekeeping world for years were the indicated to guide us in external aspects regarding the beekeepers' field of expertise.
June 7th
Mexico, City
Video call with MVZ Ernesto Tanús Sánchez
August 25th
São Paulo, Brazil
Video call with Ph.D. David De Jong
June 15th
Cuauhtémoc, Chihuahua
Center for Research in Food and Development A.C. (CIAD) visit
October 2nd
Guelph, Canada
Video conference with Dr. Ernesto Guzmán Novoa
June 7th
Video call with MVZ Ernesto Tanús Sánchez
- The current burning of beehives threatens the environment and the loss of its resources, which is why a viable alternative to avoid such burning is necessary.
- He clarified that the word of a beekeeper is not in doubt; however, it might be hard for us to obtain real data regarding the presence of American foulbrood as it is a mandatory complaint issue, so there is distrust of the producer.
- Dr. Tanus suggested liquid application (in the form of syrup) which beekeepers could feed their hives. This syrup (sweetened water) could contain the peptides in it and be fed directly to the box. This way, nurse bees would feed on here. Since Africanized bees appeared, he claims, it is better to have interior feeding to avoid foragers. This method would also avoid product loss from environmental conditions such as the wind or sun.
- We mentioned the possibility of adding the solution cell by cell, but he said we must think of the beekeeper. This just isn’t scalable for 3,000 or 4,000 boxes. We realized here that the easier the application, the more attractive the product.
- We should consider the effect our product would have on the bee's usual microbiome.
- He put us in contact with MSc Alejandro Romo, MSc Juan Diego Perez de la Rosa, and Dr. Alfonso Arroyo.
- We received an invitation to the XXVI International Congress for Beekeeping Updates in 2019.
Detailed Description
After receiving feedback from beekeepers, we looked into bees’ anatomy and behavior in a healthy environment. We were looking to enrich our investigation with the point of view of a veterinary expert. Dr. Tanus provided us with an hour and a half of his time to listen to our work. He began by introducing the arrival of foulbrood in Mexico and mentioning the National Program for Honey Harmlessness and Quality, which appeals to European standards (since the EU is Mexican honey’s largest purchaser). Among these standards, Mexico asks honey producers to create antibiotic-free products.
On American Foulbrood, he called the larvae’s first 24 hours “crucial”, since it is the time their defenses are at their lowest. He remarked that there aren´t effective remedies to these diseases, only preventive methods.
He said that bee colony arson was frowned upon and claimed that wood should not be used indiscriminately. When one suspects spores are present, it is imperative to change hives and flame the boxes by blowtorch, this is sterilization.
On our own account, we found NOM--SEMARNAT/SAGARPA-2007 which sets parameters under which it is legal to start fires on forestry, agricultural, and cattle land. It allows fires for the control of bacterial diseases, after meeting several requirements.
He mentioned foulbrood incidence has declined since 2008 thanks to the Good Honey Production Practices manual which recommends changing boxes and preventive strategies for beekeepers. He mentioned MSc Juan Diego Perez de la Rosa, Head of the Department of Genomic Sequencing at the National Center for Animal Health Verification Services, who has cultured Paenibacillus larvae for identification, from samples taken in 17 different states of the Mexican Republic.
Tanus stated that it is impossible to say that there is no presence of foulbrood in Mexico without first conducting an epizootiology study in the country. He clarified that the word of a beekeeper is not in doubt; however, it might be hard to obtain real data as it is a mandatory complaint issue, so there is distrust of the producer. He shared some characteristics that differentiate both foulbrood. He also commented on the incidence of these diseases throughout the seasons.
Dr. Tanus suggested liquid application (in the form of syrup) which beekeepers could feed their hives. This syrup (sweetened water) could contain the peptides in it and be fed directly to the box. This way, nurse bees would feed on here. Since Africanized bees appeared, he claims, it is better to have interior feeding to avoid foragers. This method would also avoid product loss from environmental conditions such as the wind or sun.
He reassured us that peptides would get to the brood, regardless of the application and only the time it takes might change. Peptides cannot arrive directly since larvae do not feed on their own: on their first 3 days, larvae are fed jelly, and with bee bread the following three. He also gave us a first draft of the path the peptides follow: from the digestive system to the hemolymph and finally to the hypopharyngeal glands in the head where they produce royal jelly.
Adult bees are not affected by P. larvae. In fact, in their digestive system, there is an anatomical structure called proventriculus and can close when the time comes to purify the food. He shared with us a new fact about hygienic behavior. As brood infected with AFB begin metamorphosis, proteolytic enzymes from P. larvae degrade fibers and reduce them to pure viscose biomass. Bees organs in their antennas can sense this decomposition; cleaner bees then break through the capped cell, remove the corpse, and clean the cell. All colonies of beehives exhibit this behavior being in some more marked than in others.
We mentioned the possibility of adding the solution cell by cell, but he said we must think of the beekeeper. This just isn’t scalable for 3,000 or 4,000 boxes. We realized here that the easier the application, the more attractive the product. This made us think of secondary factors that influence our products viability, other than its antimicrobial ability. This is further discussed in our business model. He noted that if the product is applied to cure it is susceptible to apply directly in the cells as mentioned above since in an apiary of 20 to 30 hives, this disease can be initiated in 2 or 3 of them, however, it should be applied as a preventive measure throughout the apiary.
He mentioned that we should consider the effect our product would have on the bee's usual microbiome. He asked us whether our product was a remedy or a preventive measure. We answered that the design wasn't enough to pick one or the other, and it might be both.
Finally, he recommended articles and contacts for further information. He put us in contact with MSc Alejandro Romo, MSc Juan Diego Perez de la Rosa, and Dr. Alfonso Arroyo.
He congratulated us for our work; he found our efforts and interest fascinating, especially when we put it to work to help the beekeeper community up close. He encouraged us to keep at it and invited us to the XXVI International Congress for Beekeeping Updates in 2019.
The chat was enlightening, and gave us valuable information for our research, hard to find in books or articles. He also recommended a website to check for information (OIE) which had epidemiological information on both foulbroods. We are still awaiting the 2018 update of foulbrood presence across the world.
We greatly thank Ernesto Tanus for his time and disposition, especially by giving us faith and encouragement to keep going and make this a reality.
June 15th
Center for Research in Food and Development A.C. (CIAD) visit
The main objective of the realization of this visit was to get to know and evaluate the ideal conditions with which M. plutonius and P. larvae grow and how are they handled in vitro. On another hand, we looked forward to knowing the practices of in vitro bee breeding.
Four members of the team had the opportunity to travel to Cuauhtémoc and attend this visit where we invested approximately four hours, first in the laboratory and then in the field. M. S. Alejandro Romo Chacón (who has
participated in more than 20 scientific dissemination articles related to bees or agriculture) welcomed us to the facilities and once we entered the laboratory he began to explain us about the bees’ life cycle and about some of the products and instruments beekeepers use to handle hives. Later, we were able to meet M.S. Adrián Ponce de León, who is
researching about foulbrood bacteria, and along with M.S. Romo, he explained us about procedures and safety considerations in order to manage the in vitro growth of P. larvae and M. plutonius.
The day of our visit, M.S. Romo and M.S. Ponce de León planned to go to the bee field and carry out bee larvae transferences, for its following in vitro breeding. This activity is one of the parts that implies our project once we reach the experimentation stage with the insects and M.S. Romo and M.S. Ponce de León attentively invited us to accompany them to the field, they even lent us suits and veils to interact directly with the bees! Previously
we had consulted a protocol that showed us all the details about the in vitro breeding of bees and by observing the techniques used by Ponce de León for the transference, we were able to validate and somehow, to begin with, the practice of this activity.
Interacting with the bees and being able to observe the composition of the bee boxes, the way in which the different foods were applied and the way in which beekeepers approached the bees were all key elements that allowed us to define the optimum application method of our product and helped us ensure its appropriate integration regarding each characteristic of the hive organization and the work done by beekeepers.
When returning to the CIAD facilities we had the opportunity to discuss the use of bees in the laboratory, how many bees were used? What was this number based on? Were the three R’s
considered? The reality is that in Mexico there does not exist such regulations and the bioethical part of working with bees in the laboratory is quite unattended. We found this situation somewhat unsettling and it led us to perform a more extensive analysis of this topic. (consult the bioethical essay in)
M.S. Romo and M.S. Ponce de León could broaden their panorama on the research that is currently being done with bees and their diseases thanks to the sharing we made with them of our project and the allusion that synthetic biology is its fundamental part.
Thanks to this visit we were able to have our first real approach with bees, we knew the reality of working with them and working with the two strains according to a specialized laboratory that has already experienced the proper biosafety considerations. This visit gave us different data that is very useful for the whole experimental plan of our project, and besides that, we lived a little of what beekeepers live every day, like traveling inside the truck with many bees that stayed inside from the field and even experiencing some bee-stings. We were also able to capture amazing photos of the hives!
References
- Image retrieved from: Grupo Caresner S. de R.L de C.V. (2018). Museo Menonita. https://www.mexicodestinos.com/chihuahua/tours/museo-menonita.html
August 25th
Video call with Ph.D. David De Jong
P.hD. David de Jong is a professor of genetics and entomology at the University of São Paulo, Ribeirão Preto School of Medicine (specialist in pathology, biology, and management of Africanised honey bees); he is also technical Editor of an online scientific journal: Genetics and Molecular Research (GMR).
Our team leader, human practices leader, and our ethics, legal and mathematical model representatives were present in this 40-minute call to
approach issues regarding their respective divisions. David the Jong is a researcher that has written in many papers we had previously found and read on bees; we thank MVZ Ernesto Tanús for contacting us with him. First off, he was curious if we were planning to test our peptides on the bee larvae or in the colony, so far, the idea was to test them against a bacterium within a culture in the lab, we were working on the time to see if we could test them in vivo on the larvae but we were not quite sure yet. He said that was a good first step and recommend us to emphasize that on our plan.
He was curious whether we know if it is feasible for beekeepers to eventually use our product, if it could be a costly production or if we have an idea of how much these peptides were going to cost in a final presentation. We mentioned him experimental procedures were still missing to solve these issues, like determining how much a bacterium can produce within a particular time and with given resources to give a better approximation of the cost.
He mentioned using AMPs in food for bees was newly and thinks it's feasible for us to test in culture and then determine which combination of AMPs works best to inhibit the bacteria. Before our phone call, we sent him our detailed project description to which he recommended us to add more details on our justification as well as mention the importance of honey, money
involved in its exportation and beekeeping in Mexico. He said we should also emphasize the impact of this diseases on the whole colony and beekeeping, “the larvae are interesting, but not so important concerning the application".
Regarding the application method of our product, we were considering putting nanocapsules containing our AMPs within the bee’s liquid food substitute. He said it’s feasible, but we would want to make sure bees could digest them. An excellent measure he suggested us was to eventually plan to extract the hypopharyngeal glands in bees and determine if our AMPs are entering these glands.
He believes okay for us to supply our peptides and then try to see if they help bees resist foulbrood, as for now, he thinks it's an excellent first step if we can produce some material and prove it to work on the laboratory, he mentioned that's a paper already. He emphasized is not easy to have real larvae on the laboratory.
We were wondering his opinion whether our AMPs could affect the larvae negatively in their development or worker bees in general. He compared or product to a human antibiotic, it kills the disease, but it also eliminates part of its microbiota, he thinks we could mention it, but we would have to test these possibilities experimentally and see what happens. He stated antibiotic treatments available now for foulbrood not always work, and they could contaminate the honey. Regarding the possibility of our AMPs getting into the honey, he thinks there are going to be low levels of our peptides, and they are going to be on the larvae, he doesn't think it's going to be a problem our AMPs getting to the honey.
If we did get to test within larvae, we asked him which would be an optimum method to find out if our peptides are getting to the developing brood.
- Consider two scenarios, feeding the larvae in vitro, where we won't be making use of a nurse bee or the second scenario where the nurse bee feeds the larvae.
- See if our peptides get into the worker jelly; we can look at the hypopharyngeal glands and take a worker jelly sample from the cells where larvae are swirling.
- Finally, compare to a control
If we want to see if our AMPs are getting into the food nurse bees are feeding the larvae in the colony, we would have to use a colony or a mini colony, which is more complicated, he said.
"Many people have been working on ways to protect the bees, they have different systems they are using, but none of these experiments involve all these different steps you are needing because it's very ambitious and requires much time". He thinks if we can get the peptides off our bacterium and then show that they inhibit P. larvae and M. plutonius, we can talk about the next steps on a plan.
We were wondering if our peptides could activate some biochemical signal or have the larvae reject them. He said if an animal is given something it produces, the tendency is this animal will produce less. He recommended us to see if naturally, bee AMPs get into their food, it could be that maybe if bees have available more AMPs, they disactivate the bacteria, he wasn't sure about it.
When nanoencapsulated medicine is done, there is the risk that it can get accumulated somewhere within the organism since honey bees live within a month, he doesn't deem that is something we should worry about, yet he would say the fat body, that's the place bees tend to accumulate.
Regarding the incidence of American or European foulbrood, he mentioned he has worked in the US for control of these two diseases, the percentage was always around one percent, sometimes two. In Brazil, it's not established, they have few European foulbrood cases and there is no presence of American foulbrood, by the other hand, in Argentina, it has been a big problem. He was there to diagnose the first official finding of it, they had many thousands of colines dying, since then, some of their people went to Brazil to learn bee genetics and the incidence was very much reduced.
In Mexico, we have the presence of Varroa, he mentioned that's a concern because is probably affecting the bee's immune system, another reason to be concerned about the AMPs levels. If we get to see if nurse bee AMPs get to the food of larvae, that is an interesting result because it helps researches understand how bees can resist diseases. Beyond the work, we are proposing, or as part of it, he thinks is interesting to have that information.
As we continue talking about foulbrood, we asked him if it's legal to burn beehives on Brazil, he answered it’s not for someone who is not a beekeeper, the fireman is only supposed to do it if it’s a matter of environmental protection. He has burned a few beehives in a demonstration of how they control the disease when it is found, normally the government would burn beehives is there's a new problem or disease, but they are supposed to pay the beekeeper. He told us about when he was a US bee inspector, what they would do is when they find the disease, they would burn the colonies, quarantine the apiary, and tell the beekeeper he had five days to challenge, after that period, colonies that had symptoms would be burned.
What he would do is he would burn the frames and the columns on the bees and then crush the boxes so the beekeeper couldn't put bees in it anymore, nowadays, gasoline is used. The reason he did that is he wanted to make sure all the honey was destroyed because the honey stores were contaminated with the spores of American foulbrood and if the honey wasn't destroyed, all the apiaries around would get infected. Another way the disease is spread between beekeepers is through the places where honey is processed, they bring honey from different beekeepers or different countries and process it, if it is contaminated and bees get access to it, the disease is spread in the surroundings. Another way of spreading is when a beekeeper wants to buy or sell beehives or equipment that is contaminated because they don't see the symptoms and the person who is selling or buying, doesn't know there is a contamination.
We were curious if there were any biotechnical regulations that apply to bees in Brazil and the US regarding experimental work on them for example, for eradicating diseases. In Brazil they have no regulation affecting research with honey bees, the only regulation is involving bees in the wild. For experimental work, for instance, he is on the ethics committee of the hospital, he mentioned for human research a permission is needed as for animal research, but there are no insects’ permissions.
He offered to contact us to speak with Erica Weinstein Teixeira, she was a student of him, she set up a laboratory and it’s a good bee pathologist. Erika has worked with viruses and diagnosis and said she probably knows about AMPs. He also offered to contact us with more researchers in his lab in case we needed to.
Ultimately, he mentioned American foulbrood was not a problem in Brazil, but in Argentina is. He sent us some papers that could be useful to our research and said it was a good project. We are thankful for his help and for his willingness to keep supporting us.
October 2nd
Video conference with Dr. Ernesto Guzmán Novoa
Dr. Ernesto Guzmán is a professor and head of the Honey Bee Research Centre at the University of Guelph; he received a DVM degree from the National Autonomous University of Mexico and a MSc and Ph.D. in entomology from the University of California, Davis. Dr. Guzmán has advised or co-advised the theses of more than 50 DVM, MSc, and Ph.D. students. Research in his lab focuses on three main areas: genetics, behavior and parasitic mites of honey bees, identifying factors that affect honey bee health.
On October 2, we met with Dr. Guzmán on a video conference to speak about our project; he referred to it as something new since to the best of his knowledge, no one has worked with bee’s antimicrobial peptide genes to treat diseases in these arthropods. He mentioned there might be some questions we won’t be able to answer until we get into bee experimentation, but we should consider possible risks such as the addition of external AMPs to the colony atrophying the natural production of AMPs in bees, as well as the possibility of weakening their microbiota.
He emphasized that it is not an easy project to develop as working with American and European foulbrood of honey bees is extremely complicated because of the
specialized infrastructure, techniques and studies required to work with honey bee brood diseases. It can take months or even years to standardize the protocols needed, so he was curious if we were planning to continue working on this project after the competition.
Finally, we were curious whether there were any regulations regarding experimentation on bees in his country, to which he answered there are none in Canada concerning experimentation on them, yet, he doesn't consider there should be since regulating it would hinder and inhibit research with them. Ultimately, he wished us luck and referred to our project as good and novel.
Beekeeping Societies
We had the great privilege of being able to get closer to complete and organized associations that show how beekeepers gather, collaborate and strengthen beekeeping. Dialogue is fundamental in a society and what a better way to join this dialogue than by discussing in these reunions, listening to the concerns or contributions expressed by the societies and building-up new group insights.
May 5th
Cuauhtémoc, Chihuahua
Monthly beekeepers’ association meeting
July 27th, 28th and 29th
Delicias City, Chihuahua
IV State Beekeeping Forum
July 19th
Rome, Italy
Video call with FAI presidential staff member Lorenzo Della Morte
May 5th
Monthly beekeepers’ association meeting
We thank Mr. Guillermo Rodríguez for inviting us to the monthly meeting of the Cuauhtemoc's beekeeping society where we had our first approach with the beekeeping community. The society consisted of approximately 33 beekeepers and researchers who gave us a warm welcome, listened to our project, and gave us their opinion on particular details that we had to consider or improve. They were happy that young students of other disciplines were interested in their work and bees, we explained to them the reason it was
important for us to talk with them and why we had chosen to work with bees in our project, something that made them curious.
Until that meeting, our project consisted of the production of antimicrobial peptides in Pichia pastoris for exclusively the inhibition of the pathogen Paenibacillus larvae. We planned on using yeasts due to their high capacity to carry out post-translational modifications, similar to the protein folding mechanisms of bees and their ability to secrete heterologous proteins to the environment, in this case, our antimicrobial peptides. The intention was to apply these yeasts in a solution with hexane extract of the plant Achyrocline satureioides which its efficiency against P. larvae has already been shown previously 2, in presence also of surfactins, the surfactin would have a synergistic effect with the hexane extract. Previous studies on B. subtilis have shown that surfactin, a lipopeptide produced by the bacteria, inhibits the pathogens of honey bees, including P. larvae, A. apis and Nosema ceranae.1 This solution would be applied to bee bread, which we knew, the larvae ingested in their diet.
Through their feedback, we evaluated the risks of using yeasts in our product and the risks of introducing GMOs in what is known as "harmless honey". They emphasized that an overdose of yeasts would cause a fermentation of the honey and an alteration in the microbiota of the bees which could be counterproductive since this would cause diseases such as American foulbrood to arrive, they recommended us to be very careful with the dose and consider the addition of a fermentation inhibitor in our formula. From this meeting, we set out to investigate the export requirements of honey, which is the main product exported by beekeepers, and how our genetically modified yeasts would affect or play a role in these requirements.
One issue we were asked to review was whether the hexane extract of A.satureioides oxidized because if so, it loses its qualities. Currently, a treatment that they use to fight diseases in bees is the oregano essential oil which they have already found to be oxidized. After further investigation, we verified that certainly, the extract that we wanted to add is oxidized by exposure to air. Another treatment that beekeepers use to fight diseases, is the sodium bicarbonate which, when it is spread in the hive, causes the bees to begin a cleaning protocol with which they take that bicarbonate out of their hive taking away the dirt present.
Thanks to this meeting, we set out to investigate the export requirements of honey, which is the main product exported by beekeepers, and how our genetically modified yeasts would affect or play a role in these requirements. They mentioned that they do not usually have much time to try different combinations of treatments and find out the optimal combination. They said there is not much research in the beekeeping sector, so it seems excellent that we intend to carry out scientific research in this field and try different combinations of peptides thus ensuring the best combination.
They also told us about other bee pathogens, Nosema apis and Varroa destructor. They commented that, if effective, our product is excellent, however, they also mentioned that in general there are many good products and at a reasonable price, however, they are challenging to work with, occasional applications are less complicated than regular ones. They look for a practical alternative, cheap and that doesn't consume time. Concerning the final product being a dilution, they commented that this was difficult to apply since, if our product was to be applied frequently, it would be laborious to have to spray every three days, for example, 2,000 boxes of bees.
Subsequently, they mentioned that, in their city, the foulbrood that represents a problem is the European, which occurs when bees have little accessibility to pollen because of the little flowering throughout the year. Previously we had read about this disease; however, it was from this meeting that we investigated more about it and after validating the theoretical efficacy of our antimicrobial peptides of interest against this disease, we decided to address it as part of the pathogens to combat with our product.
Finally, they congratulated us for our research and stressed the importance of feedback between researcher and producer since in this way we can collaborate in a more effective way, guaranteeing a bidirectional communication. They invited us to continue attending their and MSc Alejandro Romo, a researcher at the Center for Food Research and Development (For its acronym in Spanish: CIAD), offered to do the experimentation with bees from our research in his laboratory in Cuauhtémoc, the same laboratory that we visited one month later.
After this meeting, our project changed to production of antimicrobial peptides in Escherichia coli for the inhibition of Paenibacillus larvae and Melissococcus plutonius
References
- Wu, M., Sugimura, Y., Iwata, K., Takaya, N., Takamatsu, D., Kobayashi, M., … Yoshiyama, M. (2014). Inhibitory effect of gut bacteria from the Japanese honey bee, Apis cerana japonica, against Melissococcus plutonius, the causal agent of European foulbrood disease. Journal of Insect Science, 14, 129. http://doi.org/10.1093/jis/14.1.129
- Chanchao, C., Stoić, M., Yoshiyama, M. and Kimura, K. (2018). Review on Apiary Management and Antimicrobial Compounds as Alternative Prevention or Treatment for Honeybee Diseases. OnLine Journal of Biological Sciences, 18(1), pp.46-61.
July 19th
Video call with FAI presidential staff member Lorenzo Della Morte
Looking forward to expanding our international reach, we encountered the possibility to contact the Italian Federation of Beekeepers (FAI). Successfully, after exchanging some emails we were able to schedule a virtual meeting with the FAI presidential staff member Lorenzo Della Morte. We were very excited to have the opportunity to share and also nurture our project with insights, concerns and regulations that came from the city that hosts the headquarters of many important multinational food companies and agencies,
such as the United Nations Food and Agriculture Organization (FAO), the International Fund for Agricultural Development (IFAD) and The World Food Programme (WFP). Eight members of the team could attend this important reunion that lasted approximately 40 minutes.
FAI President Raffaele Cirone and his staff member Lorenzo Della Morte shared with us very invaluable information about how foulbrood is experienced in Italy:
Foulbrood incidence rate in Italy usually has a seasonal trend with the largest number of registered infections during the second semester of the year (July-December). Regarding the impact and incidence of these two diseases on Italian beekeeping, FAI Italian Federation of Beekeepers has consulted the National Centre of Reference on bee diseases, who elaborated an official data reporting 51 complaints of colony infections from 7 on the 20 Italian Regions, for the year 2017. The number of infected and then destroyed colonies is a few tens of units compared to the 1.300.000 colonies, which constitute the Italian beekeeping heritage.
From the FAI point of view, the data is not to be considered statistically reliable both for the low number of registered cases and for the missing complaints. In fact, in order to avoid the Veterinarian Police controls, many beekeepers often don’t report the infection and directly destroy the colonies in secret. According to FAI estimates the incidence rate of foulbrood can’t be lower than the 10-20% of the Italian beekeeping heritage in total.
The treatment methods used in Italy to treat AFB and EFB are the following:
- For AFB:
Must be compulsorily notifiable to the Health Authority. It involves serious restrictive measures in accordance with the Veterinarian Police Regulation (DPR 8/2/1954, n.320). Apart from the Beekeeping Best Practices (BBP) there are no official and authorized control methodologies so that the infected colonies must be burned. Furthermore, the apiary is subject to health constraints until the recovery plan has been ascertained. All the beekeeper’s instruments must be disinfected and sterilized.
- For EFB:
Must be compulsorily notifiable to the Health Authority. It involves serious restrictive measures in accordance with the Veterinarian Police Regulation (DPR 8/2/1954, n.320). Differently, from the American foulbrood (AFB) regulation, the European foulbrood (EFB), has a less restricted regulation. In fact, the infected colonies must be distinguished in two different clusters related to their clinical picture:
- Severely compromised colonies: In this case, the approach is the same as the one for AFB and it lies in destroying both the colony and the hive and in disinfecting and sterilizing the beekeeper’s instruments.
- Not compromised colonies but with clinical signs: In this case, the “swarming technique” is carried out to overcome the disease. It consists of:
- Removing the infected combs from the hive.
- Replacing the queen bee.
- Adding adult bees from external healthy colonies.
- Feeding bees with an appropriate nutrition.
- Further practices suggested by the veterinarian in charge.
The apiary will be seized for at least 9 days and until the positive outcome of the treatment, in order to assess the efficacy of the “swarming technique”. Anyway, also, in this case, the beekeeper can ask the destruction of all the affected colonies to the veterinarian authority.
In both the cases AFB and EFB, the use of antibiotics is not allowed in Italy because of its ineffectiveness and to prevent the spread of antibiotic-resistant bacterial strains.
FAI emphasized a very important point that seems not only to apply in Italy but throughout the whole world: there are no different solutions to AFB and EFB diseases these days and the legislative tools are often perceived as repressive, so many beekeepers don’t notify the infection in their apiary, destroying the infected colonies and concealing the fact. This is one of the most relevant problems for the health and statistical assessment studies.
After Mr. Della Morte learned about our project and the scopes of synthetic biology regarding beekeeping, he considered our proposal as a possibility of prevention method against the diseases. Our product would help beekeepers do not have to lose all their beehives and save money.
After the video call, Mr. Raffaele Cirone and his staff reflected on our project information and kindly sent us their final insights:
The Italian Federation of Beekeepers believes that AMPABEE Project is a new and innovative approach to such incurable diseases as AFB and EFB. Preventing the spread of these bacteria and increasing the immune system of bee larvae is an important goal to achieve in the health bee risk prevent. In particular, nowadays the only preventing tools are the Beekeeping Best Practices (BBP) which depends on the personal application beekeepers carry out. In this respect, FAI considers essential to test this research on the pure Italian Apis mellifera ligustica, as it is the most widespread in the world. In this regard, FAI will collaborate and provide the necessary support, remaining at iGEM team Tec-Chihuahua disposal in case of need for further information for the research project and programme. FAI is totally sure that such a research could find a spreading interest in the beekeeping world at different scales. FAI can support the product commercialization thanks to the FAI’s Service Company and the FAI trade partnerships covering the entire national territory.
July 27th, 28th, and 29th
IV State Beekeeping Forum
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