(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>
Researchers from Harvard Medical School and the University of California, Davis, blocked the progression of cancer growth caused by environmental carcinogens and food contaminants by resolving an eicosanoid/cytokine storm triggered by cell debris.
The research, from the laboratories of physician-researcher Dipak Panigrahy of Harvard Medical School and UC Davis distinguished professor Bruce Hammock, is published in the current edition of the Proceedings of the National Academy of Sciences.
“We advanced the hypothesis that cell debris from chemotherapy, resection of tumors and even immunotherapy can make these therapies a double-edged sword stimulating cancer growth and metastasis while treating it,” said Hammock, who holds a joint appointment in the UC Davis Department of Entomology and Nematology and the UC Davis Comprehensive Cancer Center.
In their paper, “Resolution of Eicosanoid/Cytokine Storm Prevents Carcinogen and Inflammation-Initiated Hepatocellular Cancer Progression,” the scientists covered the potent environmental carcinogen and food contaminant aflatoxin. Aflatoxins are toxins produced by certain fungi that are found in such agricultural crops as corn, peanuts, cottonseed, and nuts.
“Not only is this fungal metabolite genotoxic but it is also a tumor promoter,” said Hammock, defining a genotoxic agent as “a chemical that damages cellular DNA, resulting in mutations or cancer.”
Lead authors Anna Fishbein of Harvard University, a recently enrolled medical student in the Georgetown University School of Medicine, and Weicang Wang, a postdoctoral scholar in the Hammock lab, said aflatoxin exerts some of its cancer-promoting effects by generating cell debris which activate a pathway leading to eicosanoid and cytokine storms. These two classes of natural chemical mediators, they explained, control many of our defenses against pathogens, but when out of control, these storms lead to growth and metastasis of liver cancer.
“We demonstrated that debris generated by aflatoxin B1accelerates tumor dormancy escape in liver cancer models by stimulating a macrophage-derived eicosanoid and cytokine storm of pro-inflammatory mediators,” said Fishbein. “Thus, targeting a single inflammatory mediator or eicosanoid pathway is unlikely to prevent carcinogen-induced tumor progression.”
The researchers showed that the inhibition of the soluble epoxide hydrolase (sEH) pathway or the combined inhibition sEH and cyclooxygenase-2 (COX-2) pathways prevented the carcinogen debris-induced storm of both cytokines and lipid mediators by macrophages--specialized detect-and-destroy cells.
In animal models, the dual COX-2/sEH inhibitor PTUPB prevented the onset of debris-stimulated liver cancer. The dual inhibition of COX-2/sEH pathways may be “a novel approach” to control cancer of the liver, the researchers said.
“We also showed that carcinogen-generated debris stimulates an endoplasmic reticulum (ER) stress response which may promote HCC progression. Importantly, PTUPB prevents the ER stress response,” Wang added. “We created a novel model of debris-stimulated liver cancer designed to study new strategies for the prevention and treatment of carcinogen-induced cancers with tremendous potential to translate to the clinic.”
From a nutritional standpoint, aflatoxin is a common food contaminant, Wang said. “But good agricultural practice and post-harvest technology keep the levels very low. However, in much of the world, aflatoxin levels are so high that many crops are discarded. In other cases, these contaminated grain and nut crops enter the human food chain, where they cause acute toxicity, severe anemia and of course later lead to cancer.”
UC Davis co-author and nutritional scientist Yuxin Wang (who is the wife of Weicang Wang) said that “finding a way to modulate the events that lead to the eicosanoid storm would have a major effect on children's health in many developing countries.”
Fishbein and Allison Gartung of the Panigrahy lab not only used the soluble epoxide hydrolase inhibitors from the Hammock lab but also used some prototype drugs synthesized by chemist Sung Hee Hwang of the UC Davis School of Veterinary Medicine “which proved to be even better,” Hammock said.
“These compounds are a synthetic combination of cyclooxygenase inhibitors like celebrex with epoxide hydrolase inhibitors,” Hammock said. “Since epoxide hydrolase inhibitors stabilize the endoplasmic reticulium stress response and transcriptionally down regulate inflammatory cyclooxygenase we expected them to synergize with cyclooxygenase inhibitors. We were surprised and pleased with the dramatic interaction of these inhibitors when combined in the same molecule in reducing the cytokine and eicosanoid production by in response to cell debris.”
“The observations from Harvard show that by inhibiting soluble epoxide hydrolase, we can block the activation of these inflammatory cascades leading to tumor promotion, growth and metastasis,” Hammock said. “We have a compound in human clinical trials that inhibits sEH, which should be clinically available in a few years. In addition. we have found natural inhibitors of the epoxide hydrolase in a variety of plants, including crop plants. This may allow us to reduce the cancer risk and block the gastrointestional erosion and bleeding caused by dietary aflatoxin using natural means.”
Other members of the 15-member team are UC Davis researchers Jun Yang, Yuxin Wang and Sung Hee Hwang; Harvard researchers Haixia Yang, Victoria Hallisey, Jianjun Deng, Sanne Verheul, Allison Gartung, Diane Bielenberg and Mark Kiernan (now of Bristol-Myers Squibb); and Sui Huang, Institute for Systems Biology, Seattle. Hammock and Panigrahy are the corresponding authors.
The research drew strong financial support as the Panigrahy's laboratory is generously supported by the Credit Unions Kids at Heart Team, the CJ Buckley Pediatric Brain Tumor Fund, and the Joe Andruzzi Foundation; and Hammock's UC Davis grants from the National Institute of Environmental Health (NIEHS) Superfund Research Program, and the NIEHS RIVER Award (Revolutionizing Innovative, Visionary, Environmental Health Research).
Hammock, a member of the UC Davis faculty since 1980, has directed the UC Davis Superfund Research Program for nearly four decades. It supports scores of pre- and postdoctoral scholars in interdisciplinary research in five different colleges and graduate groups on campus. Last year Hammock received a $6 million, eight-year “Outstanding Investigator” federal grant for his innovative and visionary environmental health research: The award is part of the Revolutionizing Innovative, Visionary Environmental Health Research (RIVER) Program of NIEHS.
“Toxic environmental carcinogens promote cancer via genotoxic and nongenotoxic pathways, but nongenetic mechanisms remain poorly characterized. Carcinogen-induced apoptosis may trigger escape from dormancy of microtumors by interfering with inflammation resolution and triggering an endoplasmic reticulum (ER) stress response. While eicosanoid and cytokine storms are well-characterized in infection and inflammation, they are poorly characterized in cancer. Here, we demonstrate that carcinogens, such as aflatoxin B1 (AFB1), induce apoptotic cell death and the resulting cell debris stimulates hepatocellular carcinoma (HCC) tumor growth via an ‘eicosanoid and cytokine storm.' AFB1-generated debris up-regulates cyclooxygenase-2 (COX-2), soluble epoxide hydrolase (sEH), ER stress-response genes including BiP, CHOP, and PDI in macrophages. Thus, selective cytokine or eicosanoid blockade is unlikely to prevent carcinogen-induced cancer progression. Pharmacological abrogation of both the COX-2 and sEH pathways by PTUPB prevented the debris-stimulated eicosanoid and cyto- kine storm, down-regulated ER stress genes, and promoted macrophage phagocytosis of debris, resulting in suppression of HCC tumor growth. Thus, inflammation resolution via dual COX-2/sEH inhibition is an approach to prevent carcinogen-induced cancer.”
As of July 8, the commentary, “Inflammation Resolution: a Dual-Pronged Approach to Averting Cytokine Storms in COVID-19?”—the work of a nine-member team of Harvard University and UC Davis researchers—has been downloaded 11,444 times since its publication May 8, 2020. It is online at https://rdcu.be/b33IN.
In comparison, the most downloaded publication in CMR in 2019 received 5,712, statistics show.
Editor-in-Chief and Professor Kenneth Honn, who selected their commentary as the top paper of the month, said it drew more downloads the first week of publication than any other in the journal's history. The work is based on more than 40 years of eicosanoid research from the Hammock lab and more than 40 years of eicosanoid research from the Charles Serhan lab at Harvard Medical School.
“COVID-19 results in excessive inflammation and a cytokine storm caused by the human body's reaction to the SARS-CoV-2 virus,” said lead author Dipak Panigrahy, a Harvard University physician and researcher who collaborates with the Hammock laboratory.
“Controlling the body's inflammatory response to COVID-19 will likely be as important as anti-viral therapies or a vaccine,” Panigraphy said. “Stimulation of inflammation resolutions via pro-resolution lipid mediators that are currently in clinical trials for other inflammatory diseases is a novel approach to turning off the inflammation and preventing the cytokine storm caused by COVID-19.”
The drug is an inhibitor to the soluble epoxide hydrolase (sEH) enzyme, a key regulatory enzyme involved in the metabolism of fatty acids.
Panigraphy and Hammock said they are receiving “tons of calls, media requests and emails” from all over the world, including Norway, Japan, United Kingdom, Germany, France, Mexico and Belgium.
Pioneering research from the Panigrahy and Hammock labs shows that cell debris from surgery, chemotherapy, toxin exposure and other causes lead to production of high levels of pro-inflammatory mediators commonly called cytokines as well as eicosanoids.
“A rapid immune response is critical to controlling this virus,” Panigrahy emphasized.
“We believe it holds promise to combat the inflammation involved with this disease,” said co-author Hammock, a UC Davis distinguished professor who holds a joint appointment with the Department of Entomology and Nematology and the UC Davis Comprehensive Cancer Center. “It hit me in March that what we really need to do is not so much block cytokines as to move upstream to modulate them and resolve them rather than block inflammation.”
“We can increase the concentration of natural pro-resolving mediators termed EETs which act on a biological system to produce other pro-resolution mediators which modulate inflammation and actively resolve the process,” explained Hammock, who founded the Davis-based company EicOsis Human Health LLC, to bring the inhibitor to human clinical trials, which are underway in Texas.
The co-authors include two physician-researchers: Patricia Sime of the Division of Pulmonary and Critical Care Medicine, Virginia Commonwealth University, Richmond, and Irene Cortés-Puch of the Division of Pulmonary, Critical Care and Sleep Medicine, UC Davis Medical Center, and an EicOsis project scientist.
“It is this resolution of inflammation and the subsequent repair that is critical to restore patient health,” said Serhan, whose studies with collaborator Sime show that immune resolution and repair are active processes in the lungs and other tissues. What drives the process, Serhan said, is the production of specific pro-resolving agents (SPMs).
Other co-authors of the paper are Molly Gilligan and Allison Gartung of the Panigrahy lab; Sui Huang of the Institute for Systems Biology, Seattle; and Richard Phipps, independent scholar, Richmond, Va.
National Institutes of Health (NIH) grants, including a National Institute of Environmental Health Science (River Award) to Hammock, helped fund the research. The Panigrahy laboratory is generously supported by the Credit Unions Kids at Heart Team; the C.J. Buckley Pediatric Brain Tumor Fund; and the Joe Andruzzi Foundation.
Macaluso received $1000, and her research paper, “The Biological Basis for Alzheimer's Disease," will be published in eScholarship, an open-access scholarly publishing service affiliated with the University of California.
This is the first time a student enrolled in a UC Davis Department of Entomology and Nematology class has won the universitywide competition, now in its fourth year. The award memorializes Norma J. Lang (1931-2015), professor emerita of botany.
Macaluso, who is majoring in psychology with a biological emphasis, and minoring in aging and adult development, anticipates receiving her bachelor of science degree in the fall of 2020.
Carey, an internationally recognized teacher, instructs undergraduates in his classes--which usually exceed 200 students--how to research topics, use style sheets, and structure their papers. He has produced 13 videos on how to research and write a research paper, along with a new video on the use of style sheets.
The Lang Prize recognizes undergraduate students whose research projects make extensive use of library resources, services and expertise. First, second and third-place prizes are awarded each year in two categories: science, engineering and mathematics; and arts, humanities and social sciences. Second place in the science, engineering and mathematics category went to Vincent Pan, a student doing research in the lab of ecologist Rick Karban, UC Davis Department of Entomology and Nematology, for several years. His paper: "Recent Advances in Elucidating the Function of Zebra Stripes: Parasite Avoidance and Thermoregulation Do Not Resolve the Mystery." (See recipients at https://bit.ly/3cPPsNt.)
“Macaluso's term paper gives an in-depth synopsis of the biology of Alzheimer's disease, a prevalent form of dementia that impairs memory and cognition,” wrote the Norma J. Lang Prize judges. “Utilizing the library's databases and subject guides, Macaluso identified 20 sources from top scientific journals across multiple disciplines, including Nature and the Annual Reviews of Medicine, Neuroscience, Psychology and Public Health, to provide a comprehensive overview of the current state of the science on Alzheimer's.”
“This currently incurable disease is caused by significant neuronal death in the brain due to of the accumulation of two neurodegenerative proteins: intercellular amyloid-beta plaques and intracellular tau tangles,” she wrote. “The interaction of these two proteins creates a feedback loop that facilitates the continual destruction of nerve cells in the brain. Because the destruction of nerve cells disrupts the neuronal connections in the brain, Alzheimer's disease results in significant memory deficits as well as impaired cognition. Moreover, with the use of human models and transgenic mouse models, researchers have been able to analyze the role of biology, genetics, and physiology in Alzheimer's disease. For example, mutations in the presenilin 1 (PSEN1) gene or the amyloid precursor protein (APP) gene predispose an individual to acquire early-onset Alzheimer's disease.”
“Likewise, an individual can have an increased likelihood of developing late-onset Alzheimer's disease if they carry the ApoE4 variant of the apolipoprotein E (ApoE) gene. In summary, researchers are amply investigating Alzheimer's disease from a variety of biological faucets in an effort to treat or even cure this form of dementia.”
Macaluso went on to discuss three major risk factors affiliated with Alzheimer's disease: age, gender, and genetics.
Macaluso penned “The Biological Basis for Alzheimer's Disease” as her term paper for Human Development-Aging 117 (Longevity) in the fall of 2019. “The purpose of this assignment was to utilize the library databases for research, improve both my writing and editing skills, and broaden my understanding of longevity with a topic of my choice," she wrote in her Norma J. Lang Prize application. "Moreover, this research paper served to expand my communication skills and bolster my intellectual confidence. A key requirement for this paper was to use at least ten sources, seven of which needed to be primary sources such as a research article or a review paper. Initially, I was quite daunted by the prospect of this assignment because I had only modest experience reading research papers or using the online library databases. I distinctly recall reading about this assignment on the syllabus and questioning if I was capable of such an onerous task. To my surprise, by the end of this quarter and after countless hours exploring the online library reserves, I completed my assignment and felt confident in my ability to utilize the UC Davis library resources.”
A 2019-2020 McNair Scholar, Macaluso has worked as an undergraduate research assistant for the Dynamic Memory Lab (Charan Ranganath Lab) since 2017. She serves on the Animal Care Staff at Young Hall; as a genetics tutor for the Academic Assistance and Tutoring Centers; and as president of the America Red Cross Club at UC Davis.
The UC Davis student, a native of Santa Barbara but raised in nearby Buellton, plans to enroll in graduate school in the fall of 2021 to study cognitive neuroscience or cognitive psychology. Her career plans? "I'm thinking academia right now," she said. "I hope to finish my PhD, work as a postdoctoral fellow for a few years, and then pursue a professorship position."
Carey, a member of the UC Davis entomology faculty since 1980, is considered the preeminent global authority on arthropod demography. He directed the multidisciplinary, 11-institution, 20-scientist program, “Biodemographic Determinants of Lifespan,” which garnered more than $10 million in funding from the National Institute on Aging from 2003 to 2013.
Highly honored by his peers for his teaching expertise, Carey received the Entomological Society of America's 2015 Distinguished Teaching Award; a 2018 Robert Foster Cherry Award from Baylor University, which presents international teaching awards; and the UC Davis Academic Senate's 2014 Distinguished Teaching Award, an honor given to internationally recognized professors who excel at teaching.
(Undergraduate students can apply for the annual Norma J. Lang Prize here.)
In a newly published article on “The School of Food” in Futurum, Nansen advocates that all school curricula be “rooted in a single dominator: food.”
Biology, ecology and environmental science should be “taught based on subjects related to the growth of plants and animals,” Nansen writes. Literature, history, sociology and humanities should focus on “the importance of food concepts, like ‘breaking bread,' feasts and banquets.”
“What I am proposing here is already being done, in part, as individual initiatives and projects,” he writes. “For example, many schools have a butterfly garden, biology labs keep colonies of insects, students grow some vegetables and have a few livestock animals. In some schools, students learn how to eat and cook healthy food.”
Futurum is a website geared toward students and teachers to become inspired and interested in science. It focuses on research, analysis and insights.
Nansen, whose research interests include insect ecology, integrated pest management, and remote sensing, says that such a focus on food “would strengthen, not weaken, the academic rigor that could be delivered to students of all age groups.”
“That is, ‘food' as an educational denominator can be taught and approached with multiple goals in mind, and these would be similar to the current distinctions between practical and more theoretical classes. By engaging with students through the prism of food, we can make math, physics, history, biology, literature--all these topics more relevant to students and make the teaching more interactive and challenge-based.”
Nansen acknowledges that it is crucial that schools teach students about traditional subjects and provide them with essential skill sets regarding problem solving, critical thinking and basic knowledge, but that that students “can all be taught very effectively through an underlying emphasis on food.”
For example, he mentions that students of all ages can grow crop plants in small pots inside a classroom or outside (small plots and roof gardens), “and study growth as a function of time and growing conditions.” More advanced practical tasks could include developing irrigation systems, and plant and animal breeding programs.
Another example: for engineering, computer sciences and food production, students could delve into solar panels, rainwater catchment systems and water recycling methods. At the more advanced level, they could integrate robotics and machine learning system.
Nansen, linking chemistry with cooking, comments: “Cooking is nothing more and nothing less than applied chemistry. How does the pickling of vegetables work? What is happening when cream is whipped? What happens to food during heating and/or frying? Salting olives, fish and other types of meat has been practiced for thousands of years—how does this means of preserving food actually work?”
In his article, Nansen also explains how food can be incorporated in such subjects as humanities, human history, social studies and math.
Eating has changed over time, the professor acknowledges, “and it varies among countries and cultures, meaning that not all students view food in the same way.” But teachers can capitalize on diversity in the classroom, he relates. They can also address “societal challenges, such as obesity” and elevate levels of empowerment related to stresses, such as fear.
In the article, Nansen shared a project he assigned to his 11-year daughter, Molly, during the sheltering-in requirements: “How much cabbage would be needed to meet the Vitamin K requirements for her entire class for a whole year?”
In addition to learning about the metric system, using Excel spread sheets, regression analyses and calculus, Molly investigated websites and came to several conclusions:
- A person can harvest about 3 kg per m2 (kilograms per square meter)
- A student her age has to have 105 grams of cabbage to meet daily vitamin K requirements
In addition, she created a cabbage-muffin recipe and calculated she would need to eat four muffins per day to meet the daily Vitamin K requirements. She also calculated she would need 2,291 m2 to grow enough cabbage to meet the daily vitamin K requirement for her entire school. It is age-dependent, so that was a bit tricky to figure out.
And lastly, using Google Earth, Molly suggested where to place the cabbage field next to her school. (Her entire project is online as a sidebar.)
Virtual Youth Summit on Food and Education 2021
Nansen said he seeks contact with teachers and headmasters "interested in pursuing this approach at some level at their school."
"The idea is now to take this several steps further, through collaboration with teachers and their students, and set up a web-based platform to host an annual virtual youth summit on food and education!" he said. "That is, groups of students, in collaboration with their teachers and as part of course curricula, produce a 3-5 minute video describing a particular project they have executed. These videos would then be shown at the virtual youth summit, and we will organize review panels of students, teachers, and scientists to comment on the videos. These video projects would be divided into age groups and topics – still to be determined."
"I am hoping that we will be able to create a very special category of video projects describing two schools (have to be on separate continents) doing a project together," Nansen said. "As a start, we are pursuing the potential of a school in California working with a school in Uganda… which would be awesome!"
"We may be able to obtain corporate sponsorships and therefore be able to offer prizes/awards to participating schools, teachers and student groups. With corporate sponsorships, we may also be able to offer logistical support to schools – computers, software licenses (to create videos), basic lab supplies and equipment to conduct experiments.'"
"Just imagine a school being able to put on its website that a group of students competed in the Virtual Youth Summit on Food and Education 2021 and was selected as one of the winners! Students can put this experience on their resume when they later apply to university or jobs. Teachers can include this in their evaluation dossiers."
"Initially, we need to identify teachers interested in joining this effort--ideally teachers from multiple countries," Nansen related. "Once we have 5-10 teachers committed, then we can start putting together the virtual platform and invite schools and teachers more broadly." School teachers and others potentially interested in getting involved can contact him at email@example.com.