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Debug Helps Build Improved Aedes aegypti Genome Assembly
Monday, November 19, 2018
We are pleased to announce the publication of a highly improved reference genome of the mosquito
Aedes aegypti
, details of which were
reported last week
in the journal Nature. Led by Dr. Leslie Vosshall from Rockefeller University, the project to build the genome of
Ae. aegypti
began more than 2 years ago and involved 72 different authors from 32 institutions, including four Debug researchers from Verily. Below we discuss why this genome assembly is so important, how the broader team addressed some of the research challenges, and what Verily contributed to the effort.
Why is a good genome assembly important?
Like humans and other animals, mosquito DNA is organized into long strands called chromosomes. Not all organisms have the same number of chromosomes, for example humans have twenty-three pairs of chromosomes, while
Ae. aegypti
has only three. Understanding the order and sequence of DNA along the different chromosomes enables scientists to answer a wide range of questions about the biology of an organism, which in the case of the mosquito, can help us control the spread of diseases they carry.
Indeed, this new assembly of the
Ae. aegypti
genome has already helped researchers better understand fundamental aspects of mosquito biology including how mosquitoes find blood meals using chemosensory receptors that guide them to their human hosts, why some mosquito populations are better at transmitting disease such as dengue than others, and the molecular basis of how mosquito sex is determined.
What were some challenges?
High-quality genome assemblies have been available for insects related to
Aedes
, such as the fruit fly (
Drosophila melanogaster
) and the malaria mosquito (
Anopheles gambiae
), for more than a decade. Despite having roughly the same number of genes as these other species, the genome of
Ae. aegypti
has more than five times as much DNA! So what is all that other DNA? It is mostly repeated regions of DNA sequence known as repetitive elements, which account for more than two-thirds of the
Aedes
genome (Figure 1). With so much repetitive DNA, it was challenging to determine the correct order of DNA along the chromosomes, and to correctly annotate different parts of genes (exons) as they are separated by vast stretches of repeats. A previous genome assembly contained over 36,204 DNA fragments for just three chromosomes! To overcome these challenges, our team of collaborators utilized a variety of cutting-edge technologies, including a number of long-read sequencing platforms, to correctly map the genome of
Ae. aegypti
.
Figure 1. As shown in the pie chart above, a majority of the
Aedes aegypti
genome is composed of DNA repeats as opposed to unique DNA that codes for genes.
Our contribution
After a genome is correctly assembled, the next step is identifying and annotating the genes encoded. Verily produced data that helped identify the locations and sequences of genes. We did this through a process called iso-seq, which unlike many commonly employed short read technologies, allows researchers to sequence genes in their entirety. The long-read sequencing platform we used, developed by Pacific Biosciences, can achieve reads as long as 20,000 DNA base-pairs, over 60 times longer than short read techniques! Our full length transcript data helped to ensure that when scientists have a gene of interest, they will find the correct sequence in the genome.
Verily scientists also generated data highlighting the utility of the new genome. To do this, we performed whole genome sequencing on four
Ae. aegypti
colonies collected from different parts of the world. The sequencing data provided insight into regions of the genome that are especially diverse or conserved across the globe, an inference that would not have been possible without the new genome assembly.
Figure 2. Results of whole genome sequencing performed by the Debug team. On the left, diversity (e.g. nucleotide variation) of
Aedes aegypti
is visualized along the three chromosomes of the mosquito. A pronounced decrease in diversity occurs around the centromeres (center) of each chromosome. Summary statistics for each sequenced colony are given on the right.
How will the new genome assembly benefit the field going forward?
Debug is also leading an effort to sequence the genomes of 1000 wild
Ae. aegypti
from > 40 populations around the globe. This collaborative effort with members of the mosquito research community, will enable the Debug team to understand genetic factors that could impede efforts for mosquito control on the ground. With the improved genome assembly, we will gain insights into when this mosquito migrated out of Africa, how it has been able to adapt to so many different environments around the world, and determine the genetic flow between different populations. Such data will be critical as we scale our
Wolbachia
-based SIT program and bring Debug to new locations.
Sara Mitchell, PhD, Senior Scientist, Verily and Brad White, PhD, Debug Lead Scientist, Verily
Debug Fresno 2018 results in 95% suppression!
Tuesday, November 13, 2018
As Debug Fresno’s second season comes to a close, we are excited to share our 2018 results as well as some of the key insights we have learned along the way. Around this time last year, we were wrapping up
Debug Fresno’s first season
and soon after, preparation for a second year began. Throughout the winter, we continued to develop our automated production and release systems and designed plans for the 2018 mosquito season with our partners,
Consolidated Mosquito Abatement District
(CMAD) and
MosquitoMate
. In April 2018, we hatched the first batch of eggs, fueled up the release vans, and embarked on a plan to conduct the world’s largest study of
Wolbachia
-based
Sterile Insect Technique
targeting
Aedes aegypti
.
The overall goal of our 2018 study was the same as in 2017: test whether releasing sterile male mosquitoes reared and released using Debug’s automated systems could achieve strong suppression of
Aedes aegypti
mosquitoes in our release areas compared to similar non-release neighborhoods. For Debug Fresno 2018, we aimed high by expanding our release areas to include three suburban Fresno County neighborhoods and increasing the variety of landscapes in the study. We also started at the beginning of the season before the wild mosquito population had the opportunity to reproduce in large numbers, in hopes that we could continue to have a large impact while releasing fewer male mosquitoes. As in 2017, we adjusted according to data and feedback from the field and compared the number of biting female
Aedes aegypti
in release and non-release areas at peak season to tally our final result.
The outcome was decisive. Our data indicates that we achieved a greater than 95% reduction in the number of biting, female
Aedes aegypti
mosquitoes compared to similar, non-release neighborhoods (confidence interval: 93.67 - 96.78).
Aedes aegypti
is a highly invasive mosquito that has proven difficult to combat with conventional mosquito control methods, so strong suppression across three different release areas is an important validation of a new approach to controlling these mosquitoes with a dangerous potential to transmit human disease.
Above, the trend of biting, female
Aedes aegypti
mosquitoes within our three primary release neighborhoods compared to similar non-release neighborhoods, starting in April 2018.
Our team worked closely with our Debug Fresno partners, CMAD and MosquitoMate, to gain insights on program performance and design. Critically, our data suggest that a suppression rate beyond 95% was limited in our release sites by incursions of female
Aedes aegypti
mosquitoes from neighboring non-release areas, indicating that integrated control strategies and larger release areas may make this approach even more effective. We also showed that starting early in the season is a highly efficient strategy to achieve maximum suppression. This information provides invaluable guidance for future Debug programs both in the US and abroad.
Verily Sr. Scientist Jacob Crawford shows a local community member our demo box of sterile, male mosquitoes who may land on your hand - but like all male mosquitoes - cannot bite.
As the mosquito season winds down in Fresno County and we wrap up this year’s field study, we want to sincerely thank the community residents who welcomed us in 2018.
Aedes aegypti
is not just a nuisance, but also a potential public health threat to the people of the Central Valley of California and actively dangerous to millions of others around the world. We are energized by what we achieved this year and look forward to bringing this technology to communities most burdened by
Aedes aegypti
throughout the world.
Jacob Crawford, PhD, Sr. Scientist, Verily and David Clarke, PhD, Operations Manager, Verily
Singapore welcomes the Debug Project!
Monday, September 17, 2018
Today, at the Fifth Singapore International Dengue Workshop (SIDW), the
National Environment Agency
(NEA) announced that we have become a partner in
Project
Wolbachia
– Singapore
, a multi-year research program founded and led by Singapore’s NEA. We’re excited to bring the
Debug Project
’s technology to the Phase 2 field study of Project
Wolbachia
– Singapore, which has already
shown indications
that a
Wolbachia
-based sterile insect technique may be successful in suppressing the
Aedes aegypti
population in Singapore’s urban environment.
Yanni Yoong (left) and Nigel Snoad (right) preparing to sort mosquitoes.
One of the unique challenges of deploying the sterile insect technique in Singapore is effectively distributing sterile, male mosquitoes across the many high-rise apartment complexes that fill its landscape. In Singapore, as in many other dense, urban environments,
Aedes aegypti
breed in clean, stagnant water found in planters, vases and other containers in the hallways and homes within multi-floor buildings. To tackle this challenge, we have designed a new automated release system, contained within a 1.3m x 1m cart, lightweight enough to be pushed by an individual. With this new release device, we can precisely control the distribution of sterile, male mosquitoes within the corridors of the structures. Additionally, Project
Wolbachia
– Singapore will employ our mosquito sex-sorting technology, which has been successfully used in
Debug Fresno
and
Debug Innisfail
to separate male and female mosquitoes using a computer vision algorithm and artificial intelligence. Our team of scientists and automation specialists look forward to collaborating with NEA to drive the science and technology of the sterile insect technique forward.
This is an exciting time for the Debug team as we enter into a new region of the world, which challenges us to consider new ecologies, mosquito behavior, and human environments. With our first field study, Debug Fresno, launching just a year and a half ago, we continue to move forward into new environments, representative of areas where dengue, Zika, and other mosquito-borne diseases commonly spread. We look forward to all we will learn in this new region and to taking another step in realizing Debug’s mission to reduce the devastating impact that disease-carrying mosquitoes inflict on people around the world.
Yanni Yoong, Program Manager, Verily and Nigel Snoad, Product Manager, Verily
Debug Innisfail achieves strong suppression
Friday, July 20, 2018
During this past wet season in Far North Queensland,
Debug Innisfail
, a collaboration between Australia’s
Commonwealth Scientific and Industrial Research Organisation
(CSIRO),
James Cook University
(JCU) and Verily, released sterile, male
Aedes aegypti
mosquitoes with
Wolbachia
into central Innisfail and several nearby communities to research whether we could suppress the population of these dangerous invasive mosquitoes.
We came to Innisfail with CSIRO and JCU to see how
Debug’s approach
to reduce mosquito populations with SIT would work in a tropical environment where these mosquitoes thrive, and to learn what it was like to operate our technology with research collaborators a long way from our home base. For more than six months, our partners at CSIRO and JCU reared millions of
Aedes aegypti
, used our tools to remove the biting females and released more than 3 million males into three neighbourhoods around Innisfail. The CSIRO field team monitored mosquito traps and performed releases three times a week through a drought,
a cyclone
, and flooding in a week that brought nearly 30 inches of rain.
With the Debug Innisfail releases now finished for this season, we’re extremely happy to see the program achieve strong suppression of
Aedes aegypti
mosquitoes. The trap and monitoring data show that the occurrence of biting, female
Aedes aegypti
mosquitoes decreased by more than 80% in the three release areas relative to similar, untreated sites in the Cassowary Coast. This is a result we’re proud of and it reflects the hard work and collaboration across our partnership.
We are particularly thankful to the people of Mourilyan, South Johnstone, Goondi Bend and the rest of the Cassowary Coast region for their strong support. The community has been incredibly welcoming, hosting surveillance traps in their homes, coming to events, and supporting the team as they conducted releases.
Residents of Mourilyan wave at the Debug Innisfail van. Photo credit: CSIRO.
Beyond the field results, Debug Innisfail represents an important step in the development of Verily’s Debug project: we’ve worked with and supported our partners at CSIRO and JCU as they used our technologies on the other side of the world to make and release mosquitoes. We’ve all learnt a huge amount about how to design and operate a successful Sterile Insect Technique program, and this experience is already being applied to our ongoing work.
Nigel Snoad, Product Manager, Debug
Debug Fresno continues!
Monday, April 30, 2018
Mosquitoes in Fresno take the winter off and wait for spring. Not so for the Debug team. We spent the winter months carefully reviewing the data from our
2017 field study in Fresno
, brainstorming on scientific and operational improvements, and updating our technologies and techniques. We think these updates put us in a great place to make an even bigger impact on the population of invasive
Aedes aegypti
in Fresno - and so on April 16th, Verily, in collaboration with
Consolidated Mosquito Abatement District
and
MosquitoMate
, began our first releases of sterile male
Aedes aegypti
mosquitoes of the 2018 season in Fresno County.
Like last year, the primary goal is to see a steep decline in the number of biting female
Aedes aegypti
mosquitoes in the areas where we release sterile male mosquitoes, but there are a few differences in the program this year. Last year, we began releasing mosquitoes in mid-July when the mosquito populations in Fresno were already approaching peak density. This year, we began releasing our sterile male mosquitoes in mid-April when mosquito reproduction is just beginning for the season, in order to outnumber and outcompete the wild male mosquito population with the release of fewer sterile male mosquitoes. In addition, we are conducting releases in more neighborhoods in Fresno County to increase the number and variety of environments included in the study.
We are pleased with what we achieved so far in Fresno County and are looking forward to continue towards the goal of reducing the number of biting, female
Aedes aegypti
mosquitoes during the 2018 mosquito season.
You can read more about Debug Fresno at
debugfresno.com
Jacob Crawford, Sr. Scientist
Targeting Mosquitoes on their Home Turf: Debug heads to Australia
Tuesday, December 5, 2017
This November, the Debug team at Verily took an exciting next step with the launch of our first international field study,
Debug Innisfail
, as part of the collaboration we
formed
in October 2016 with the Australian
Commonwealth Scientific and Industrial Research Organisation
(CSIRO) and
James Cook University
(JCU).
For the Debug team at Verily, Innisfail and the greater Cassowary Coast region of Queensland, Australia is an appealing place to undertake our research to reduce the invasive
Aedes aegypti
mosquito. As a tropical climate, Far North Queensland (FNQ) is more representative of the areas where
Aedes aegypti
mosquitoes usually thrive. In fact, FNQ not only has an established population of
Aedes aegypti
but also has experienced outbreaks of dengue, and as we noted in our blog post last year, JCU and CSIRO have a great deal of mosquito research experience in this region. Additionally, Debug Innisfail represents an essential next step and challenge for our technology and research design: the ability to support partners at a distance as they use our technologies to help make and release mosquitoes.
For this field study, CSIRO is releasing sterile, male
Aedes aegypti
mosquitoes bred with
Wolbachia
into central Innisfail and several nearby communities through the region’s wet season. We will measure our success by monitoring changes in the number of biting, female mosquitoes in these release areas relative to similar neighborhoods nearby that will serve as controls. Though only minutes drive apart, the behavior of
Aedes aegypti
coupled with the separation of townships by fields of sugar-cane and river tributaries makes this a perfect place to see how far we can suppress the population of biting, female
Aedes aegypti
using our technology. This ecological isolation is because
Aedes aegypti
hover around human activity and travel on average
less than 100 meters
in the search for a mate, effectively making each township an ecological island, thus allowing us to measure our impact with more precision.
On Wednesday, November 15, the Debug Innisfail van (aka the “Mobile Mosquito Unit”, see photo) began work in Mourilyan, with CSIRO staff releasing mosquitoes produced at JCU near Cairns. As production ramps up, our partners will continue to expand the study to neighboring communities. Over the next week, releases will hopefully begin in two additional neighborhoods, South Johnston and Goondi Bend. Once Debug Innisfail is running at scale releases will occur in all study sites three times a week for up to 24 weeks.
Members of the research and field operations team, including members of CSIRO and James Cook University, on the first day of releases.
This month’s first releases are the culmination of over a year’s work in scientific and operational preparation. During this time, CSIRO and JCU, as well as researchers from the University of Queensland and the QIMR Berghofer Research Institute, reared
Wolbachia
Aedes aegypti
mosquitoes, studied the size and behavior of the
Aedes aegypti
mosquito population in and around Innisfail, and built strong links with the community members and local government, including the Cassowary Coast regional council whose support has been a critical element of our launch. The research our teams have done together over the last 12 months has not only benefited our project design, but has also resulted in our first
peer reviewed publication
which we hope will benefit the field of mosquito research overall.
CSIRO, JCU and ourselves all still have a huge amount to learn: how these non-biting males disperse and survive in the landscape, how well they find females in and around people’s houses, and how to operate our equipment a long way from our home base in California. We’re eager to see the impact these males can make on the biting, female population in these towns and take the next step in testing our technology in the field.
Nigel Snoad, Product Manager
Rhodamine B: A New Tool for Monitoring Mosquito Mating
Tuesday, November 21, 2017
We are very excited to announce the Debug Project’s first
publication
, which explores tracking mosquito movement and mating in the wild, a collaborative effort between Verily’s Debug team, The Australian
Commonwealth Scientific and Industrial Research Organization
(CSIRO) and
James Cook University
(JCU) in Australia. The study demonstrated that rhodamine B can be utilized to successfully perform mark-release-recapture (MRR) experiments and observe mating in the field.
Rhodamine B is a red dye that fluoresces when exposed to certain wavelengths of light. When fed to mosquitoes, it binds to internal proteins and stains them bright red. Rhodamine B staining is observable in adult mosquitoes by eye or by a microscope when looking at small tissues, which can be useful when studying mosquitoes in the wild. Mosquitoes can be tracked in the wild using an approach known as mark-release-recapture (MRR) where insects are marked (in our research, with rhodamine B), released into the wild, and then recaptured for marking examination. MRRs provide information on how far mosquitoes move across the landscape, as well as their lifespan, and can help us estimate the wild population size by comparing the ratio of marked recaptured individuals to unmarked insects.
A male Aedes aegypti mosquito fed on a mixture of rhodamine B and honey is stained visibly red (A) compared to males fed on honey alone
(B).
Traditional methods for MRR labeling rely on dusting mosquitoes with fluorescent powders, however, these powders can be difficult to detect and can impair a mosquito's ability to fly. Rhodamine B, on the other hand, can be easily visualized and evidence suggests it does not impact a mosquito’s activity or health.
Rhodamine B labeled males will seek out wild females. During mating males transfer their labelled semen to females, which can be detected by its red fluorescent properties.
Rhodamine B has another useful property: it can stain the male mosquito’s semen, which females store in dedicated capsules called spermathecae. After mating with a rhodamine B fed male, stained semen can be observed in the female’s spermatheca using a microscope. So not only can rhodamine B be used to track males in the wild, it can also be used to measure how successfully they mate with wild females.
Rhodamine B labeled sperm within the female spermathcal capsules after mating.
In our paper, we demonstrate that rhodamine B is a very effective label when mixed into the honey normally fed to male mosquitoes, with the stain visible up to three days after males are released. We performed several small scale MRR experiments in Cairns, Australia and were able to successfully detect mating between our lab reared males and wild females through rhodamine B fluorescence.
For future sterile male releases rhodamine B labeling will be a useful tool allowing us to easily and efficiently assess the dispersal, fitness and mating ability of the males we rear in our Debug factory. We are excited to continue this research and further demonstrate rhodamine B’s utility.
Sara Mitchell, Senior Scientist
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