The work, published in the current edition of Parasites & Vectors, a BioMed Central open-access medical journal, focuses on “determining how informative well-established genetic markers of resistance to pyrethroids are in predicting the resistance phenotype of individual mosquitoes of Aedes aegypti within a population,” said lead author Geoffrey Attardo, medical entomologist-geneticist in the UC Davis Department of Entomology and Nematology.
“Specifically, we generated mosquito colonies from invasive A. aegypti populations from four locations in the Central Valley (Dinuba, Clovis, Sanger and Kingsburg) and from collections in the Greater Los Angeles Area,” he said. “Mosquitoes from these populations have all demonstrated resistance to pyrethroid-type insecticides and we think this may be part of the reason why these mosquitoes have been so successful in spreading throughout California.”
A. aegypti transmits such viruses as dengue, Zika, chikungunya, and yellow fever. Despite California's aggressive surveillance and treatment efforts, this species presents a “significant challenge to local control agencies,” the nine-member team wrote in their research paper, “Frequency of Sodium Channel Genotypes and Association with Pyrethrum Knockdown Time in Populations of Californian Aedes aegypti.“
The paper is online and publicly accessible at https://bit.ly/3vmUxXR.
“What was interesting was that while all the mosquitoes from California show resistance to pyrethroids, there is a lot of variability from one individual to the next in terms of the level of resistance, even when they are carrying genetically identical resistance mutations,” Attardo said. “In particular, there seem to be two levels of resistance in these populations. The two levels seem to represent a resistant group and a super resistant group. However, the proportions of resistant/super-resistant differ in the sampled mosquitoes from population to population.”
Of particular interest was that mosquitoes carrying the resistance mutations at all five genetic locations were very resistant, he said. “However, there was also a large amount of unexplained variability in terms of the knockdown phenotypes demonstrated by mosquitoes of the same age and rearing conditions. We compared the knockdown times of mosquitoes positive for all five resistance mutations from different populations and found that these mutations account for only a proportion of the observed level of resistance. We believe that the unexplained variability is likely being mediated by the presence or absence of an undefined resistance mechanism.”
In launching the project, the researchers designed an assay “to test for the presence of mutations in the gene coding for the pyrethroid target protein, the voltage gated sodium channel (the para gene),” Attardo explained. “Detection of these mutations is used to monitor the level or resistance in populations. However, the actual link between the effect the genotype has on the phenotype of individual mosquitoes has not been looked at in detail. “
The scientists identified mutations from genetic sequences of Californian mosquitoes provided by co-author Yoosook Lee, a former UC Davis mosquito researcher now at the University of Florida-Florida Medical Entomology Laboratory, Vero Beach.
The authors also include research entomologist Anthony Cornel and staff research associate Katherine Brisco of the Mosquito Control Research Laboratory, Kearney Agriculture and Extension Center and UC Davis Department of Entomology and Nematology; and Lindsey Mack, Erin Taylor Kelly, Katherine Brisco, Kaiyuan Victoria Shen, Aamina Zahid, and Tess van Schoor, all with the UC Davis Department of Entomology and Nematology.
First, they tested the individual resistance phenotype of mosquitoes by placing them into bottles coated with the pyrethroid insecticide permethrin, and observed them to determine how long it takes for them to respond to the insecticide. Said Attardo: “This is a modified version of the assay used by the Center for Disease Control and Prevention to evaluate phenotypic resistance in groups of mosquitoes.”
Then they isolated the DNA from and performed a high-throughput genetic analysis on each individual to determine the composition of the five mutations in each individual. Next they looked at the resulting data to see how well knockdown time correlates with individual genotypes of mosquitoes.
Although A. aegypti was first detected in California in 2013, researchers believe that its arrival involved multiple introductions. Populations in Southern California are thought to have crossed the border from Mexico, while Central Valley populations may have been introduced, in part, from the southeastern United States.
“Upon detection in 2013, the Consolidated Mosquito Abatement District implemented an integrated vector control management strategy which involved extensive public education, thorough property inspections, sanitation, insecticide treatment at larval sources and residual barrier spraying with pyrethroids,” the authors wrote. Despite their efforts, the species successfully overwintered and continued to spread, implicating that it arrived in California with genetic mutations “conferring resistance to the type I pyrethroid insecticides applied for vector control in California.”
The project drew financial support from the Pacific Southwest Regional Center of Excellence for Vector-Borne Diseases, funded by the U.S. Centers for Disease Control and Prevention.
"Tsetse flies house an assortment of endosymbiotic bacteria and serve as the prominent vectors of pathogenic African trypanosomes," Weiss says in his abstract. "Tsetse and insect stage trypanosomes are metabolically dependent on the fly's endosymbiotic bacteria in order to maintain their physiological homeostasis. I will describe these interdependencies and
how they can be exploited to decrease tsetse's vector competency."
Weiss received his master's degree from the University of Queensland, Brisbane, in 1997 and his doctorate from the University of Alberta, Canada (2003).
"My research focuses on acquiring a better understanding of the relationship between insect disease vectors and their associated micro-organisms," he writes on his website. "To this end, I currently use the tsetse fly (Glossina morsitans morsitans) as a model system. These insects are the sole vectors of pathogenic African trypanosomes, which are the causative agent of Human African trypanosomiasis. Tsetse flies also harbor indigenous endosymbiotic bacteria that are intimately associated with their host's physiological well-being. I am interested in learning more about (1) the evolution adaptations that permit host tolerance of bacterial endosymbionts, (2) how symbiotic bacteria impact host physiology, with specific emphasis on nutritional supplementation and host immunity, and (3) how to use microbial symbionts to reduce disease vector competence."
Cooperative Extension specialist Ian Grettenberger coordinates the winter seminars. For technical issues, contact him at email@example.com.
He and other members of the research team are exploring the intact organs and tissues of tsetse flies using a powerful 3D X-ray imaging technique. The study, “Unraveling Intersexual Interactions in Tsetse”), is funded by the National Institute of Allergy and Infectious Diseases (NAIAD) of the National Institutes of Health.
“We started this project in 2019 and the work is ongoing,” said Attardo, an assistant professor in the UC Davis Department of Entomology and Nematology and chair of the Designated Emphasis in the Biology of Vector Borne Diseases. “We actually have scans of flies through the entire reproductive cycle, however, the segmentation is ongoing. We are working on developing ways to train artificial intelligence based software to assist us with the tissue segmentations.”
The tsetse fly transmits the parasite that causes the deadly human and animal trypanosomiasis, better known as African sleeping sickness, says Attardo, who is featured in a recently posted article, "A Detailed Look Inside Tsetse Flies," on the Berkeley Lab website. (See YouTube)
“The imaging technique provided new insights into how the flies' specialized biology governs mating and reproductive processes, including female flies' unique lactation and their delivery of a single fully developed larvae per birthing cycle – whereas most other insect species lay eggs,” according to the article. “The ALS (National Laboratory Advanced Light Source) produces X-rays and other forms of light for a broad range of simultaneous scientific experiments.”
Attardo, who specializes in medical entomology, reproductive physiology, molecular biology and genetics, says that tsetse flies resemble house flies, but are distinguished from other Diptera by their unique adaptations, including lactation and the birthing of live young. They carry only one offspring in their uterus at one time.
The parasite invades the central nervous system and disrupts the sleep cycle, says Attardo. “If not treated, the disease can result in progressive mental deterioration, coma, systemic organ failure and death.” An estimated 65 million people in 36 countries in sub-Saharan Africa are at risk for the deadly disease, according to the World Health Organization.
Attardo led a study, published in September 2020 in the journal Insects, detailing the ALS imaging work. The article, “Interpreting Morphological Adaptations Associated with Viviparity in the Tsetse Fly Glossina morsitans (Westwood) by Three-Dimensional Analysis,” received widespread attention. ALS experiments allow the researchers to create a detailed 3D visualizatiaon of the reproductive tissues without dissection and staining processes that introduce damage to the delicate samples.
“We want to understand what changes are happening during this process, how the process is being mediated, and if it can be manipulated to artificially repress females in the wild from mating,” Attardo told the Berkeley Lab News Center.
The Berkeley Lab is a multiprogram science lab in the national laboratory system supported by the U.S. Department of Energy through its Office of Science.
In his UC Davis lab, Attardo researches one of 35 tsetse fly species, Glossina morsitans morsitans, which prefers feeding on cattle to humans. Its strong mouthparts can easily puncture the tough cattle hide. In his lab, he feeds them warm cow blood.
Attardo holds a doctorate in genetics from Michigan State University, where he researched the molecular biology of mosquito reproduction in the lab of Alexander. Prior to joining the UC Davis faculty in 2017, Attardo worked for 13 years in the Department of Epidemiology of Microbial Diseases at the Yale School of Public Health, first as a postdoctoral associate and then as a research scientist studying the reproductive biology of tsetse flies.
The Zoom seminar, open to all interested persons, will take place from 4:10 to 5 p.m. Click here for the form to obtain the Zoom link to connect.
"In this talk, we are going to demonstrate the tripartite interactions between the microbiome, mosquitoes of the genus Aedes and Zika virus that they transmit," she says. Aedes albopictus is also known as the Asian tiger mosquito.
"My research focuses on the tripartite interactions between the microbiome, mosquitoes as vectors and the arboviruses they transmit," Onyango says. "In addition, I am interested in the role the vector-host- pathogen interface plays in enhancing disease severity in the vertebrate host. The goal of my research is to develop innovative control mechanisms both for the vector and pathogens they transmit."
Host is medical entomologist-geneticist Geoffrey Attardo, assistant professor, UC Davis Department of Entomology and Nematology. Cooperative Extension specialist and assistant professor Ian Grettenberger coordinates the fall seminars.
"Dr. Maria Onyango works on the biology underlying interactions between arboviruses (Zika virus), vector mosquitoes and the associated microbiome," Attardo said.
Along with seven other scientists, Attardo and Onyango co-authored a research article in the Oct. 2nd edition of Frontiers in Microbiology on"Zika Virus Infection Results in Biochemical Changes Associated With RNA Editing, Inflammatory and Antiviral Responses in Aedes albopictus."
"Rapid and significant range expansion of both the Zika virus (ZIKV) and its Aedes vector species has resulted in the declaration of ZIKV as a global health threat. Successful transmission of ZIKV by its vector requires a complex series of interactions between these entities including the establishment, replication and dissemination of the virus within the mosquito. The metabolic conditions within the mosquito tissues play a critical role in mediating the crucial processes of viral infection and replication and represent targets for prevention of virus transmission. In this study, we carried out a comprehensive metabolomic phenotyping of ZIKV infected and uninfected Ae. albopictus by untargeted analysis of primary metabolites, lipids and biogenic amines. We performed a comparative metabolomic study of infection state with the aim of understanding the biochemical changes resulting from the interaction between the ZIKV and its vector. We have demonstrated that ZIKV infection results in changes to the cellular metabolic environment including a significant enrichment of inosine and pseudo-uridine levels which may be associated with RNA editing activity. In addition, infected mosquitoes demonstrate a hypoglycemic phenotype and show significant increases in the abundance of metabolites such as prostaglandin H2, leukotriene D4 and protoporphyrinogen IX which are associated with antiviral activity. These provide a basis for understanding the biochemical response to ZIKV infection and pathology in the vector. Future mechanistic studies targeting these ZIKV infection responsive metabolites and their associated biosynthetic pathways can provide inroads to identification of mosquito antiviral responses with infection blocking potential."
Onyango holds two degrees from the University of Nairobi, Kenya: a bachelor of science degree in biochemistry and zoology and a master's degree in applied parasitology. She received her doctorate in veterinary entomology from Deakin University and Australian Animal Health Laboratory, Commonwealth Scientific and Industrial Research Organisation (CSIRO), and then completed postdoctoral training at the Yale School of Public Health, Department of Epidemiology of Microbial Diseases.
For any technical issues regarding the seminar, contact Grettenberger at firstname.lastname@example.org
(Editor's Note: Geoffrey Attardo, assistant professor, UC Davis Department of Entomology and Nematology, published this piece July 29, 2020 on The Conversation website. This article is republished from The Conversation under a Creative Commons license. Read the original article.)
Bloodthirsty tsetse flies nurse their young, one live birth at a time – understanding this unusual strategy could help fight the disease they spread
Tsetse flies are bloodthirsty. Natives of sub-Saharan Africa, tsetse flies can transmit the microbe Trypanosoma when they take a blood meal. That's the protozoan that causes African sleeping sickness in people; without treatment, it's fatal, and millions of people are at risk due to the bite of a tsetse fly.
My entomology research focuses on insects that feed on the blood of people and animals. From a human health standpoint, understanding what makes all these bugs tick is key to developing ways to control them and prevent transmission of the diseases they carry, such as malaria, dengue, Lyme disease, West Nile virus and many others.
Tsetse flies stand out from their blood-feeding cousins the mosquitoes and ticks because of their unique reproductive biology. They give birth to live young and, even more unusual, the mother lactates and provides milk for her offspring. Here's how it all works – and why their unusual reproduction strategy might be a key to controlling tsetse flies and the parasite they carry once and for all.
From egg to larva
Scientists know of other flies that hold onto their eggs in their reproductive tract until they hatch into young larvae, with each brood consisting of dozens of offspring. The mother then tries to find a suitable source of nutrition in the environment, deposits the larvae and leaves them to survive on their own. The mother does not provide any nutrition for her young.
That's the standard fly way of life. Tsetse flies take a different approach.
Female tsetse flies develop just one single egg at a time. When the egg is complete, the mother moves it from her ovaries into her uterus in a process called ovulation. Once in the uterus, the egg is fertilized with sperm the female has stored in an organ called the spermatheca. While females can mate multiple times, they obtain all the sperm they need for their lifetime from a male fly during a single mating event.
After fertilization, the female keeps the egg in her uterus for five days while an embryo develops within the egg. When the embryo is ready, the egg hatches in the uterus of the female and the tsetse fly larva begins its life living inside its mother's uterus.
Milk meals for baby
Here's where tsetse flies dramatically diverge from most other insects.
Attached to the mother's uterus is a specialized gland that makes a milk-like substance. The organ is called the milk gland, and it produces a rich mixture of fats and particular proteins that provide the larva with all the nutrition it needs to develop into an adult.
Just like in mammals, the milk also transfers beneficial bacteria from the mother to the offspring. These bacteria are essential for tsetse flies, and without them adult female flies are unable to reproduce.
After five or six days of developing and feeding on milk, the larva is fully grown and ready to enter the world. The mother finds a safe spot and gives birth. The larva immediately burrows underground to avoid predators and parasites.
Once buried, the outer surface of the larva's skin hardens and turns black, forming a protective shell. This is called the pupal stage and it lasts for around three weeks. During this time, the pupa transforms into an adult fly.
It then emerges from the pupa, climbs out of the ground, and begins its life as an adult tsetse fly looking for hosts to blood-feed on and other tsetse flies to mate with.
Why live birth?
Why would an insect evolve this slow and resource-intensive way to reproduce?
One idea is that this method provides a defensive advantage relative to free-living larvae against parasites and predation. Larvae on their own have few (if any) ways to defend against these threats. But keeping larvae in the mother's uterus provides shelter and a guaranteed food source. While this strategy is much slower, scientists think the extra maternal care results in higher larval survival rates. It's a matter of quality over quantity.
A result of this reproductive strategy is that tsetse fly populations are small and slow to recover from control efforts, relative to more prolific insects like mosquitoes.
My colleagues and I hope that we can parlay our understanding of the molecular processes that regulate tsetses' milk production and mating behavior into new environmentally friendly, cost-effective and tsetse-specific control strategies for these insects.
The sleeping sickness tsetse flies spread is a potential issue for millions of people in 36 sub-Saharan countries, though the number of annual cases has decreased drastically thanks to major control efforts – including trapping flies, applying insecticides and releasing sterile males to the environment where they mate with wild females but don't produce offspring. Ultimately, we'd like to contribute to the World Health Organization's goal of eliminating African sleeping sickness by 2030 with a new way to prevent the transmission of disease-causing trypanosomes to people and animals./h2>/h2>/h2>/figcaption>/h1>