Brief Summary of Minutes of Annual Meeting

The annual meeting was organized as an Organized Meeting during the 2020 Entomological Society of America (ESA) Annual Meeting. It consisted of an on-demand session with seven 12-minute seminars and a 1-hr video chat. 

The speakers and their seminar titles were as follows: 

Dr. Paul Leisnham, University of Maryland: New Insights into the Effects of Tire Leachate on Urban Mosquito Communities

Dr. Susan Paskewitz, University of Wisconsin: Assessments of Vector Control Strategies in the Upper Midwest.

Dr. Allison Gardner, University of Maine: Tick-borne Disease Ecology and Management in Maine.

Anna Rosalee Pasternak, University of Kentucky: Getting Ticky in Kentucky: State-wide Surveillance and Pathogen Testing of Ticks

James Occi, Rutgers University: First Record of Soft Ticks in New Jersey and their Public Health Risk

Dr. Adela S. Oliva Chavez, Texas A&M: Epigenetic Drivers in the Transmission of Tick-borne Diseases in the US.

Emily Reed, North Carolina State University - Population Genetics of an Invasive Mosquito Along an Urban-rural Landscape.

During the 1-hr video chat, the Business Meeting was held. Dr. Leisnham chairs the meeting, first allowing each speaker to introduce themselves and summarize their work. Other attendees then contribute, summarising additional work. Dr. Leisnham begins a discussion by reminding attendees that this project started in October 2019 and will extend to September 2024, building on two prior multistate projects of the same name, which have been very projective and successful. Dr. Leisnham mentions that the preceding multistate project (NE1443) had staged its annual meeting in March to coincide with the annual arbovirus surveillance and mosquito control workshop at the Anastasia Mosquito Control District in St. Augustine, FL. Through email correspondence, project participants decided it was too soon after the establishment of this project to hold this annual meeting in March again; hence, the decision to stage the meeting at the Entomological Society of America's annual meeting. There was then a short discussion on when the next annual meeting should be held, with a recognition that it is always a difficult decision given the conflicting time schedules of project participants. Three society conferences: ESA, the American Society of Tropical Medicine & Hygiene, and the Society of Vector Ecology were identified as arguably the best venues to stage future annual meetings and maximize participant attendance, but that attendance in 2021 would depend heavily on the management of the COVID-19 pandemic. Dr. Leisnham continued the meeting by emphasizing the diversity of the 30 official project participants and their institutions, that we need to contribute to an annual report, and that the project will need to decide on a new Chair. Dr. Leisnham explained that chairs of the preceding projects had served 2-yr appointments and that his 2-yr appointment straddled the end of NE1443 and this project (NE1943). Attendees agreed that 2-yr terms were suitable and there was a consensus to seek a new Chair over the coming months, before the next annual meeting. Last, USDA's representative on this project, Erica Kistner Thomas, introduced herself and briefly described her role. The Meeting adjourned on time after 1 hour.

Dr. Paul Leisnham and his student, Oswaldo Villena, continued work on tire leachate and mosquito ecology at the University of Maryland. Specifically, they tested the hypothesis that more degraded tires contain greater tire leachate and alter interspecific mosquito competition, producing a condition-specific advantage for the competitively inferior resident, Culex pipiens, by relaxing the effects of competition with the invasive Aedes albopictus. Varying densities of newly hatched Ae. albopictus and Cx. pipiens larvae were added to tires that had been exposed to different ultra-violet (U.V.)-B conditions that mimicked either full-sun, shade, or no UV-B conditions in the field. There were higher competitive effects of Cx. pipiens on the population performance and survival of Ae. albopictus in tires exposed to shade and full-sun U.V. conditions that had higher concentrations of zinc, a marker contaminant of tire leachate. Zinc concentration was also higher in Ae. albopictus than Cx. pipiens indicating greater exposure of the invasive species to tire leachate. These results indicate that tire leachate can affect the outcome of the competition between Ae. albopictus and Cx. pipiens, helping foster the regional persistence of the resident Cx. pipiens. Cx. pipiens is the main vector circulating West Nile virus (WNv) within avian populations in many cities in the northern United States. Ae. albopictus may play an important role in WNv transmission, bridging the virus from avian into human populations. Increased coexistence of these two species resulting from condition-specific competition in tire habitats may increase WNV transmission risk.

Research at the Midwest Center of Excellence for Vector-Borne Disease, headed by Dr. Susan Paskewitz of the University of Wisconsin, explored control efforts for the blacklegged tick, Ixodes scapularis, and the northern house mosquito, Cx. pipiens. These two species are responsible for most cases of vector-borne human disease in the upper Midwest and are a focus for this project. Tick control is most often performed at the individual household level.  Using the Tick App and field sampling, researchers confirmed that backyard environments can be important sources of blacklegged ticks in Wisconsin.  Researchers evaluated the efficacy of several low cost, do-it-yourself strategies for homeowners.  Perimeter treatments with a granular formulation of cyahalothrin were highly effective in reducing tick encounters.  Deployment of tick tubes, containing host-targeted nest materials, was less effective although reduction in the density of infected nymphs could still be detected.  Combining tick tubes with removal of invasive vegetation did not increase the effectiveness of treatment.  By comparison, mosquito control for reduction of West Nile virus risk is often done by professional, tax-funded mosquito control operations.  Control of Cx. pipiens focuses on larval control through catch basement treatments and on application of adulticides.  Researchers tested the impacts of these strategies on adult mosquito populations in Chicago and Milwaukee.  While larval control in the basins was good, there was little evidence that this impacted adult populations.  The main impact of application of adulticides was a change in age structure of the population that favored younger mosquitoes.

Dr. Allison Gardner and the Vector Biology Lab of the University of Maine completed a study that investigated the impacts of active forest management on the risk of exposure to ticks and tick-borne pathogens across spatial scales, as well as the casual ecological mechanisms underlying observed patterns. Dr. Gardner also introduced an active surveillance citizen science project that seeks to understand the economic, environmental, and production factors that influence private forest landowners’ forest management decision-making processes and the implications for tick-borne disease exposure risk in the landscape.

Graduate student Anna Pasternak and her advisor Dr. Subba Reddy Palli of the University of Kentucky conducted Kentucky-wide surveillance of tick distributions at the county level and pathogen testing for detection of Rickettsia rickettsii, Ehrlichia chaffeensis and Borrelia burgdorferi. Preliminary data supports the presence of established lone star tick, Amblyomma americanum, American dog tick, Dermacentor variabilis, and Ixodes scapularis populations infected with Ehrlichia chaffeensis, Rickettsia rickettsia and Borrelia burgdorferi, respectively. These results indicate that Kentuckians are at risk for these bacterial diseases and calls for the implementation of the largescale surveillance program that this study provides.

Under the supervision of his advisor Dr. Dina Fonseca from Rutgers University, graduate student James Occi has described the first occurrence of the soft tick Carios kelleyi (Cooley and Kohls) from New Jersey, based on larvae collected from big brown bats, Eptesicus fuscus. Although C. kelleyi is known to occur on bats in at least 29 of the 48 conterminous U.S. states, its ecology of is not well understood, despite reports of this species feeding on humans and its consequent potential as a disease vector. The association of C. kelleyi with bat species that regularly roost in human-made structures, such as attics and barns, and recent isolations from this tick of pathogens capable of infecting humans, companion animals, and livestock underscore the need for further studies of these bat ectoparasites.

Dr. Adela Oliva Chavex and her colleagues of Texas A&M University conducted research that sought to determine the role of epigenetics in the vectorial capacity of different tick populations. Researchers investigated the variation in methylation of I. scapularis collected in Minnesota (high-Lyme disease area) and Texas (low-Lyme disease area). Global methylation was assessed by ELISA and bisulfite genome sequencing. The expression of DNA methyltransferases was evaluated by Reverse-Transcriptase (R.T.)-PCR. Preliminary results indicated that the genomes of ticks collected from MN and TX are differentially methylated. Further, bisulfite methylation analysis of TX ticks has identified differential methylation between sexes. These results suggest that similar to bees, DNA methylation plays a role in the life cycle of I. scapularis, one that may influence Lyme disease transmission.

Graduate student Emily Reed and her advisor Dr. Michael Reiskind of North Carolina State University completed work exploring the impact of urban landscapes on genetic connectivity of Ae. albopictus. Researchers collected adult mosquitoes across Wake County, North Carolina from 60 locations and built genomic libraries of 336 Ae. albopictus individuals using double digest restriction-site associated DNA sequencing (ddRADseq). Researchers processed sequence data with the STACKS de novo pipeline and analyzed genetic structure, differentiation, and inbreeding between populations with STRUCTURE, DAPC, and popgraph. Using spatial data, researchers then examined how local landscape features correlate with genetic signatures at several spatial scales. This project contributes to a growing body of genetic research that demonstrates how environmental processes affect the genetic structure and evolution of invasive species at fine scales. These studies can improve existing programs for controlling invasive species and will help fill knowledge gaps needed for effective management.

Led by Dr. Goudarz Molaei, researchers at the Connecticut Agricultural Experimental Station have worked on four studies. First, they conducted work exploring vector-host interactions of Ae. albopictus in Virginia. Engorged Ae. albopictus were collected from a variety of habitat types using the Centers for Disease Control and Prevention light traps, Biogents Sentinel 2 traps, and modified Reiter gravid traps in southeast Virginia. Sources of blood meals were determined by the analysis of mitochondrial cytochrome b gene sequences amplified in PCR assays. Degrees of Ae. albopictus interactions with vertebrate hosts were quantified, the influence of key socioecological conditions on spatial variability in Ae. albopictus blood feeding was assessed, and temporal differences in blood feeding by season were investigated. Analysis of 961 engorged specimens of Ae. albopictus sampled between 2017-2019 indicated that 96%, 4%, and less than 1% obtained blood meals from mammalian, reptilian, and avian hosts, respectively. Domestic cats were the most frequently identified (50%) hosts for Ae. albopictus followed by Virginia opossums (17%), white-tailed deer (12%), and humans (7%), together representing 86% of all identified blood hosts. A small proportion of blood meals acquired from avian hosts in mixed blood meals suggests that this species may rarely mediate epidemic/epizootic transmission of arboviruses as a bridge vector. Screening of the head and thorax of engorged Ae. albopictus mosquitoes by cell culture and RT-PCR resulted in a single isolate of Potosi virus. Spatial patterns in blood feeding were found to be linked to socioecological conditions and seasonal shifts in Ae. albopictus blood feeding with implications for understanding human biting and disease risk.  In Suffolk Virginia in areas of lower human development, the likelihood of human blood feeding increased as median household income increased and human blood feeding was more likely early in the season (May-June) compared to later (July-October). Understanding of the mosquito-host interactions in nature is vital for evaluating vectorial capacity in mosquitoes and reservoir competency in vertebrate hosts in transmission, maintenance, and amplification of zoonotic agents of human diseases. Results of our study in conjunction with abundance in urban/suburban settings, virus isolations from field-collected mosquitoes, and vector competence of Ae. albopictus, highlight the potential of this species in the transmission of a number of arboviruses such as dengue, chikungunya, and Zika to humans. A manuscript has been submitted to PLoS NTD and is currently under review.

Second, Dr. Molaei and his colleagues studied climatic and environmental determinants of the spatial distribution and abundance of I. scapularis (blacklegged tick) and A. americanum (lone star tick). Spatially explicit statistical models were used to link extensive passive tick surveillance data to mean annual temperature and the Wildland-Urban Interface. Blacklegged ticks, endemic in Connecticut, were not associated with mean annual temperature while lone star ticks, recently reported to be established in Connecticut, were positively associated with mean annual temperature. Blacklegged tick submission rates were higher in towns with a higher proportion of land classified as intermix, areas where houses and undeveloped wildland vegetation mix. Lone star tick submission rates were higher in towns with a lower proportion of very low housing density. These findings have implications for tick-borne disease dynamics in the northeastern U.S. as the climate continues to warm and for land use planning to mitigate tick-borne disease risk. A manuscript has been submitted to the CDC Emerging Infections Diseases (EID) and is currently under review.

Dr. Molaei identified the first reported established population of Gulf Coast ticks (Amblyomma maculatum) infected with Rickettsia parkeri in Connecticut, representing the northernmost range limit of this medically relevant tick species. This finding highlights the importance of tick surveillance and public health challenges posed by geographic expansion of tick vectors and their pathogens. A manuscript has been submitted to the Journal of Medical Entomology and is currently under review. 

Last, Dr. Molaei and his colleagues continued the Connecticut Agricultural Experiment Station (CAES)-Passive Tick Surveillance Program (PTSP) that was established in 1990. This program monitors: 1) tick vector populations for distribution, abundance and range expansion of existing species, 2) introduction of exotic/invasive tick vectors and tick-borne diseases, 3) spatiotemporal dynamics of activity of existing pathogens transmitted by tick vectors to determine the risk of human infection, and 4) conducts investigations on tick biology, ecology, and their roles in disease transmission.  Each year, an average of 3,000 ticks are submitted by the state residents, health departments, and physicians’ offices; however, in recent years the number of submissions has substantially increased, and in some years, it has reached to nearly 6,000. From 10/1/2019 to 9/30/2020, the CAES-PTSP received a total of 3,711 tick submissions. Of these, 3,175 (85.6%) were identified as I. scapularis (blacklegged tick), 369 (9.9%) as D. variabilis (American dog tick), 156 (4.2%) A. americanum (lone star tick), and 11 (0.3%) a few other species. Of the 3,711 total tick submissions, 3,011 (94.8%) engorged nymph and adult female I. scapularis were tested for evidence of infection. Of these, 28.8% (n=867) tested positive for B. burgdorferi, 4.9% (n=149) for Anaplasma phagocytophilum, and 5.4% (n=162) for Babesia microti. Coinfections with B. burgdorferi and A. phagocytophilum were identified in 2.1% (n=63); B. burgdorferi and B. microti in 2.2% (n=66); A. phagocytophilum and B. microti 0.3% (n=9); and with all three pathogens/parasites in 0.2% (n=5).

Further research at CAES, under the leadership of Dr. Philip Armstrong, has explored the ecology and control of disease vectors through three projects. The first project continues longstanding work understanding environmental determinants of Ae. albopictus abundance at a northern range limit. Dr. Armstrong ans colleagues constructed linear models to evaluate how trapping methodology, land cover, as well as temperature and precipitation influenced A. albopictus abundance at the northern limits of its range in New York and Connecticut. BGS traps were 2.78 times as efficient as gravid traps and 1.49 times as efficient as CO2-baited CDC light traps. Low and medium-intensity development and low proportions of deciduous cover around the trap site were positively associated with increased abundance, as were minimum winter temperature and March precipitation. The cumulative precipitation within a 28-day time window before the date of collection had a nonlinear relationship with abundance, such that as precipitation increased beyond 70 mm, there was a decrease in abundance. This study shows the impact of landscape and climate variables on A. albopictus distribution at the northern limit of its current range. The second study led by Dr. Armstrong found that successive bloodmeals enhance virus dissemination within mosquitoes and increase transmission potential. Vector competence and the extrinsic incubation period (EIP) are two key entomological parameters used to assess the public health risk posed by arboviruses. These are typically measured by offering mosquitoes an infectious bloodmeal and temporally sampling mosquitoes to determine infection and transmission status. However, this approach does not accurately capture the biology and behavior of many mosquito vectors which refeed frequently (every 2-3 days). The work showed that a second non-infectious bloodmeal significantly shortens the extrinsic incubation period (EIP) of Zika virus (ZIKV) in adult Ae. aegypti by enhancing virus escape from the mosquito midgut. Similarly, a second bloodmeal increased the competence of this species for dengue and chikungunya viruses as well. This effect was also observed for ZIKV in A. albopictus. Bloodmeals induced fissures in the virus-impenetrable basal lamina surrounding the midgut providing a mechanism for enhanced virus escape. Modeling of these findings revealed that a shortened EIP would result in a significant increase in the basic reproductive number, R0. This study helps explain how A. aegypti can sustain an explosive epidemic like ZIKV despite its relatively poor vector competence in single-feed laboratory trials. Last, Dr. Armstrong and colleagues published research that evaluated of novel trapping lures for monitoring exotic and native container-breeding Aedes mosquitoes. During 2018, researchers tested new scent lures, TrapTech Lure-A and Lure-H (Bedoukian Research, Inc.), using BG-Sentinel traps with CO2 in two regions of Connecticut, Stamford and Hamden, against the BG-Lure. Pooled mosquitoes were additionally screened for arbovirus infection.  A total of 47,734 mosquitoes representing 8 genera and 32 species were captured during the study, with the Stamford site deriving on average three times as many mosquitoes per trap, adjusting for effort.  Lure-A and Lure-H outperformed the BG-Lure in terms of overall numbers, diversity evenness, and the proportion of both Ae. japonicus and Aedes triseriatus. There were no significant differences among lures in capturing Ae. albopictus, and in terms of species richness.  Fifty-seven isolates of virus (West Nile, Jamestown Canyon, and La Crosse viruses) were obtained during the study, with no significant difference between trap-lure.  Both novel lures were highlighted as effective potential attractants for use in combination with CO2 in mosquito surveillance. 

Dr. Gabe Hamer of Texas A&M has made progress studying the ecology of Aedes aegypti and associated arboviruses in Texas and Mexico and has several related manuscripts published in 2020 or in preparation for submission (see publications). These include evaluating multiple control tools, including a Autocidal Gravid Ovitrap Intervention which has a manuscript in review.  The intervention using the Autodissemination Stations with pyriproxyfen was originally planned for the summer of 2020 but was postponed to 2021 due to COVID.  Another manuscript is in preparation looking at the unique human dimensions of community engagement in South Texas.

Dr. Stephen Dobson at the University of Kentucky has been focused on developing improved monitoring tools that combine internet-connected databases and quality control with the multiple trap types being used by mosquito abatement districts, e.g., B.G. traps, CDC traps, ovitraps, etc. The data is updated in real time as collections are made and then presented as a ‘Dashboard’ for managers and supervisors, who can then generate standard and customize report summaries for stakeholders. Furthermore, the sample collections can be tracked through subsequent assays and tests, e.g., PCR and virus assays, to avoid tracking errors.

Dr. Zhijian Jake Tu at Virginia Polytechnic Institute and State University have improved the characterization of the male-determining locus (M) in the dengue and Zika vector Ae. aegypti. The key improvement is the determination of the content of the ~160 Kilobase gap in the M-locus. The red-eye mutation within a 5 Mb region that is linked to the M locus was mapped. This is a naturally occurring mutation that gives predominantly wild-type males (only ~2% wild-type individuals are females while most of the females are red-eyed) in progeny. A few candidate genes within this region have been identified.

Dr. Bruce Noden from Oklahoma State University is conducting work describing the regional spread of Culex coronator, an invasive mosquito species which has spread throughout the southern U.S. in the last 20 years. So far, they have reported Cx. coronator in 16 of Oklahoma’s 77 counties with expansion throughout the state.

Armstrong PM, Ehrlich HY, Magalhaes T, Miller MR, Conway PJ, Bransfield A, Misencik MJ, Gloria-Soria A, Warren JL, Andreadis TG, Shepard JJ, Foy BD, Pitzer VE, Brackney DE (2020) Successive blood meals enhance virus dissemination within mosquitoes and increase transmission potential. Nat Microbiol 5(2):239-247.

Aryan, A., Anderson M., Biedler J.K., Qi, Y., Overcas J. M., Naumenko A., Sharakhova M.A., Mao C., Adelman Z.A. and Tu Z. 2020. Nix alone is sufficient to convert female Aedes aegypti into fertile males and myo-sex is needed for male flight. Proc Natl Acad Sci U S A. 117 (30), 17702-17709.

Compton A, Liang J, Chen C, Lukyanchikova V, Qi Y, Potters M, Settlage R, Miller D, Deschamps S, Mao C, Llaca V, Sharakhov IV, Tu Z. 2020. The beginning of the end: a chromosomal assembly of the New World malaria mosquito ends with a novel telomere. G3 Genes|Genomes|Genetics doi: 10.1534/g3.120.401654, 10, 3811-3819

Compton, A., Sharakhov, IV and Tu, Z. 2020. Recent Advances and Future Perspectives in Vector-omics. Current Opinion in Insect Science. https://doi.org/10.1016/j.cois.2020.05.006

Eastwood G, Donnellycolt AK, Shepard JJ, Misencik MJ, Bedoukian R, Cole L, Armstrong PM, Andreadis TG (2020) Evaluation of novel trapping lures for monitoring exotic and native container-inhabiting Aedes spp. (Diptera: Culicidae) mosquitoes. J Med Entomol 57(2):534-541.

Juarez, J. G., S. Garcia-Luna, L. F. Chaves, E. Carbajal, E. Valdez, C. Avila, W. Tang, E. Martin, R. Barrera, R. Hemme, J. P. Mutebi, N. Vuong, B. Roark, C. R. Maupin, I. E. Badillo-Vargas, G. L. Hamer. 2020. Dispersal of female and male Aedes aegypti from discarded container habitats using a stable isotope mark-capture study design in South Texas. Nature Scientific Reports. 10:6803.

Kache PA, Eastwood G, Collins-Palmer K, Katz M, Falco RC, Bajwa WI, Armstrong PM, Andreadis TG, Diuk-Wasser MA (2020) Environmental determinants of Aedes albopictus abundance at a northern limit of its range in the United States. Am J Trop Med Hyg 102(2):436-447.

Little EAH, Williams SC, Stafford KC 3rd, Linske MA, Molaei G. 2019. Evaluating the Effectiveness of An Integrated Tick Management Approach on Multiple Pathogen Infection in Ixodes scapularis Questing Nymphs and Larvae Parasitizing White-Footed Mice. Experimental and Applied Acarology. 80: 127–136. 

Martin E, W. Tang, C. Briggs, H. Hopson, J. G. Juarez, S. Garcia Luna, M. Wise de Valdez, I. Badillo-Vargas, M. Borucki, M. Frank, G. L. Hamer. In press. Cell fusing agent virus (Flavivirus) infection in Aedes aegypti in Texas: seasonality, comparison by trap type, and individual viral loads. Archives of Virology.

Molaei G, Little EAH. 2020. A Case of Morphological Anomalies in Amblyomma americanum(Acari: Ixodidae) Collected from Nature. Experimental and Applied Acarology 2020; 81: 279–285.

Molaei G, Little EAH, Williams SC, Stafford KC. 2019. Bracing for the Worst–Range Expansion of the Lone Star Tick in the Northeastern United States. New England Journal of Medicine 2019; 381: 2189–2192.

Molaei G, Mertins JW, Stafford III KC. 2020. Enduring Challenge of Invasive Ticks: Introduction of Amblyomma Oblongoguttatum(Acari: Ixodidae) into the United States on A Human Traveler Returning from Central America. Journal of Parasitology 2020; 106: 670–674.

Olson M. F., S. Garcia-Luna, J. G. Juarez, E. Martin, L. C. Harrington, M. D. Eubanks, I. E. Badillo-Vargas, G. L. Hamer. In press. Sugar feeding patterns for Aedes aegypti and Culex quinquefasciatus (Diptera: Culicidae) mosquitoes in South Texas. Journal of Medical Entomology.

Olson, M. F., M. L. Ndeffo-Mbah, J. G. Juarez, S. Garcia-Luna, E. Martin, M. K. Borucki, M. Frank, J. G. Estrada Franco, M. A. Rodriguez-Perez, N. A. Fernandez-Santos, G. J. Molina-Gamboa, S. D. Carmona Aguirre, B. L. Reyes-Berrones, L. J. Cortes-De la cruz, A. Garcia-Barrientos, R. E. Huidobro-Guevara, R. M. Brussolo-Ceballos, J. Ramirez, A. Salazar, L. F. Chaves, I. E. Badillo-Vargas, G. L. Hamer. 2020. High rate of non-human feeding by Aedes aegypti reduces Zika virus transmission in South Texas. Viruses. 12:453.

Pokutnaya D, Molaei G, Weinberger DM, Vossbrinck CR, Diaz AJ. 2020. Prevalence of Infection and Co-infection and Presence of Rickettsial Endosymbionts in Ixodes scapularis (Acari: Ixodidae) in Connecticut, USA. Journal of Parasitology; 106: 30–37.

Stafford III KC, Ridge GE, Molaei G, Zarb C, Bevilacqua P. 2020. Rabbit Bot Fly Furuncular, Tracheopulmonary, and Human Bot Fly Infestations in Connecticut (Oestridae: Cuterebrinae). Journal of Medical Entomology 58(1):114-120. doi: 10.1093/jme/tjaa181.

Stafford III KC, Williams SC, van Oosterwijk JG, Linske MA, Zatechka S, Richer LM, Molaei G, Przybyszewski C, Wikel SK. Field Evaluation of a Novel Oral Reservoir‐Targeted Vaccine Against Borrelia burgdorferiUtilizing an Inactivated Whole‐Cell Bacterial Antigen Expression Vehicle. Experimental and Applied Acarology. 2020; 80: 257–268.