bims-mosdis Biomed News
on Mosquito distribution and disease
Issue of 2020‒09‒06
thirty-one papers selected by
Richard Halfpenny
Staffordshire University


  1. Parasit Vectors. 2020 Sep 04. 13(1): 444
      BACKGROUND: Mosquitoes are vectors of many tropical diseases. Understanding the ecology of local mosquito vectors, such as species composition, distributions, population dynamics, and species diversity is important for designing the optimal strategy to control the mosquito-borne diseases.METHODS: Entomological surveillance of adult mosquitoes was conducted in five sites representing different ecological settings across Hainan Island from January to December of 2018 using BG Sentinel (BGS) traps and Centers for Disease Prevention and Control (CDC) light traps. In each site, we selected three areas representing urban, suburban and rural settings. Eighteen trap-days were sampled in each setting at each site, and CDC light traps and BGS traps were setup simultaneously. Mosquito species composition, distribution, population dynamics, and species diversity were analyzed. Mosquito densities were compared between different study sites and between different settings.
    RESULTS: Nine species of mosquitoes belonging to four genera were identified. Culex quinquefasciatus (80.8%), Armigeres subalbatus (13.0%) and Anopheles sinensis (3.1%) were the top three species collected by CDC light traps; Cx. quinquefasciatus (91.9%), Ae. albopictus (5.1%), and Ar. subalbatus (2.8%) were the top three species collected by BGS traps. Predominant species varied among study sites. The population dynamics of Ae. albopictus, An. sinensis and Cx. quinquefasciatus showed clear seasonal variation regardless of study sites with a varied peak season for different species. Mosquito abundance of all species showed significant differences among different study sites and among urban, suburban and rural areas. Danzhou had the highest mosquito biodiversity, with an α, β, and Gini-Simpson biodiversity index of 8, 1.13 and 0.42, respectively. BGS traps captured Aedes mosquito at a higher efficiency than CDC light traps, whereas CDC light traps captured significantly more Anopheles and Armigeres mosquitoes than BGS traps.
    CONCLUSIONS: Mosquitoes were abundant on Hainan Island with clear seasonality and spatial heterogeneity. Population density, species composition, distribution, and species diversity were strongly affected by the natural environment. Different tools are required for the surveillance of different mosquito species.
    Keywords:  BGS trap; CDC light trap; Hainan Island; Mosquito composition; Population dynamics; Species diversity
    DOI:  https://doi.org/10.1186/s13071-020-04326-5
  2. Am J Trop Med Hyg. 2020 Aug 31.
      Vector-borne diseases affect more than 1 billion people a year worldwide, causing more than 1 million deaths, and cost hundreds of billions of dollars in societal costs. Mosquitoes are the most common vectors responsible for transmitting a variety of arboviruses. Dengue fever (DENF) has been responsible for nearly 400 million infections annually. Dengue fever is primarily transmitted by female Aedes aegypti and Aedes albopictus mosquitoes. Because both Aedes species are peri-domestic and container-breeding mosquitoes, dengue surveillance should begin at the local level-where a variety of local factors may increase the risk of transmission. Dengue has been endemic in Colombia for decades and is notably hyperendemic in the city of Cali. For this study, we use weekly cases of DENF in Cali, Colombia, from 2015 to 2016 and develop space-time conditional autoregressive models to quantify how DENF risk is influenced by socioeconomic, environmental, and accessibility risk factors, and lagged weather variables. Our models identify high-risk neighborhoods for DENF throughout Cali. Statistical inference is drawn under Bayesian paradigm using Markov chain Monte Carlo techniques. The results provide detailed insight about the spatial heterogeneity of DENF risk and the associated risk factors (such as weather, proximity to Aedes habitats, and socioeconomic classification) at a fine level, informing public health officials to motivate at-risk neighborhoods to take an active role in vector surveillance and control, and improving educational and surveillance resources throughout the city of Cali.
    DOI:  https://doi.org/10.4269/ajtmh.20-0080
  3. Bull Soc Pathol Exot. 2019 ;112(5): 302-310
      Anopheles hancocki is one of the secondary vectors of malaria whose larval ecology in the South Cameroonian forest block is still unknown. This information is however fundamental for developing efficient and sustainable control strategies against this mosquito in localities where it acts as a disease vector. The present study describes the larval ecology of A. hancocki and its contribution in malaria transmission in a riverbank village of the Ayos Health District. Mosquito sampling took place in 2018 on a quarterly rate, combining the dipping method for larval collection with adult mosquito capture on volunteers. For each of the breeding sites, physicochemical characteristics were measured and larvae were collected and reared to adult. Molecular alongside morpho-taxonomic techniques were used for the identification of mosquito species. Physiological age was determined based on the appearance of their ovarian tracheoles and CSP Elisa test was used to assess infectivity. In total, 3,618 adult mosquitoes belonging to seven species were collected in the study area, namely A. hancocki, Anopheles gambiae, Anopheles moucheti, Mansonia africana, Culex pipiens quinquefasciatus, Aedes albopictus and Aedes aegypti. Breeding sites of A. hancocki were similar to those of A. gambiae. However, the total dissolved solids and conductivity values were significantly higher in A. gambiae breeding sites than in those of A. hancocki. A. hancocki was the most aggressive mosquito species and represented 45.6% of the local aggressive culicidofauna. Male species' nocturnal cycle of aggression showed maximum activity between 8 pm and 10 pm. Females of this species were significantly older than those of A. gambiae and contributed to 40% of malaria transmission in the locality with an average annual Entomological Inoculation Rate (EIR) of 2.92 ib/p/year lower than that of A. gambiae (3.65 ib/p/year). Except few differences, ecological requirements for the development of A. hancocki larvae are similar to that of A. gambiae. A. hancocki contributes to the perennial transmission of malaria in the Ayos area; implementation of vector control strategies is therefore needed.
    Keywords:  Afrique subsaharienne; Anopheles gambiae; Anopheles hancocki; Arrondissement de Nyakokombo; Ayos; Cameroun; Kobdombo; Lutte antivectorielle; Transmission du paludisme; Écologie
    DOI:  https://doi.org/10.3166/bspe-2020-0109
  4. Pest Manag Sci. 2020 Sep 01.
      BACKGROUND: Aedes albopictus is the primary vector of mosquito-borne diseases, including dengue and chikungunya, in China. The management of vector mosquitoes is the primary strategy for the control of such infectious diseases. The gravid Ae. albopictus prefers to skip-oviposit its eggs into different small water containers, and the management of these breeding places is critical for mosquito control. Bacillus thuringiensis subspecies. israelensis (Bti) is a useful biological larvicide, but the effective period of the currently available commercial product is relatively short. This study aimed to develop a long-lasting formulation of Bti to control the dengue vector mosquito Ae. albopictus.RESULTS: Water-soluble polyethylene glycols and water-insoluble hexadecanol were mixed with Bti to develop the long-lasting formulation, Bti-BLOCK, based on the solid dispersion technique. The controlled release of Bti-BLOCK and its effect on Ae. albopictus were assayed in the laboratory and in the field. The results showed that Bti toxins were slowly released from Bti-BLOCK into the water and maintained at an effective dose for at least six months. Bti-BLOCK caused high mortality during the immature stage (>90%) and achieved full inhibition during pupation (100%), and the efficacy lasted at least twelve weeks in the laboratory and six weeks in the field. Furthermore, we confirmed an 89% reduction in Ae. albopictus density and reduction in the R0 of dengue to a low-risk level after six months of open-field interventions.
    CONCLUSIONS: We developed a long-lasting biological larvicide, Bti-BLOCK, which displayed very good efficacy in the control of the dengue vector mosquito Ae. albopictus. This article is protected by copyright. All rights reserved.
    Keywords:  Aedes albopictus; Bacillus thuringiensis subspecies. israelensis; Dengue; Larvicide
    DOI:  https://doi.org/10.1002/ps.6069
  5. Malar J. 2020 Sep 04. 19(1): 326
      BACKGROUND: Indoor residual spraying (IRS) is an effective method to control malaria-transmitting Anopheles mosquitoes and often complements insecticide-treated mosquito nets, the predominant malaria vector control intervention. With insufficient funds to cover every household, malaria control programs must balance the malaria risk to a particular human community against the financial cost of spraying that community. This study creates a framework for modelling the distance to households for targeting IRS implementation, and applies it to potential risk prioritization strategies in four provinces (Luapula, Muchinga, Eastern, and Northern) in Zambia.METHODS: Optimal network models were used to assess the travel distance of routes between operations bases and human communities identified through remote sensing. Network travel distances were compared to Euclidean distances, to demonstrate the importance of accounting for road routes. The distance to reaching communities for different risk prioritization strategies were then compared assuming sufficient funds to spray 50% of households, using four underlying malarial risk maps: (a) predicted Plasmodium falciparum parasite rate in 2-10 years olds (PfPR), or (b) predicted probability of the presence of each of three main malaria transmitting anopheline vectors (Anopheles arabiensis, Anopheles funestus, Anopheles gambiae).
    RESULTS: The estimated one-way network route distance to reach communities to deliver IRS ranged from 0.05 to 115.69 km. Euclidean distance over and under-estimated these routes by - 101.21 to 41.79 km per trip, as compared to the network route method. There was little overlap between risk map prioritization strategies, both at a district-by-district scale, and across all four provinces. At both scales, agreement for inclusion or exclusion from IRS across all four prioritization strategies occurred in less than 10% of houses. The distances to reaching prioritized communities were either lower, or not statistically different from non-prioritized communities, at both scales of strategy.
    CONCLUSION: Variation in distance to targeted communities differed depending on risk prioritization strategy used, and higher risk prioritization did not necessarily translate into greater distances in reaching a human community. These findings from Zambia suggest that areas with higher malaria burden may not necessarily be more remote than areas with lower malaria burden.
    Keywords:  Malaria; Network modeling; Optimal routes; Residual spraying; Risk mapping; Zambia
    DOI:  https://doi.org/10.1186/s12936-020-03398-z
  6. Sci Rep. 2020 Sep 03. 10(1): 14527
      Despite significant reductions in malaria transmission across Africa since 2000, progress is stalling. This has been attributed to the development of insecticide resistance and behavioural adaptations in malaria vectors. Whilst insecticide resistance has been widely investigated, there is poorer understanding of the emergence, dynamics and impact of mosquito behavioural adaptations. We conducted a longitudinal investigation of malaria vector host choice over 3 years and resting behaviour over 4 years following a mass long-lasting insecticidal nets (LLINs) distribution in Tanzania. By pairing observations of mosquito ecology with environmental monitoring, we quantified longitudinal shifts in host-choice and resting behaviour that are consistent with adaptation to evade LLINs. The density of An. funestus s.l., declined significantly through time. In tandem, An. arabiensis and An. funestus s.l. exhibited an increased rate of outdoor relative to indoor resting; with An. arabiensis reducing the proportion of blood meals taken from humans in favour of cattle. By accounting for environmental variation, this study detected clear evidence of intra-specific shifts in mosquito behaviour that could be obscured in shorter-term or temporally-coarse surveys. This highlights the importance of mosquito behavioural adaptations to vector control, and the value of longer-term behavioural studies.
    DOI:  https://doi.org/10.1038/s41598-020-71187-4
  7. PLoS One. 2020 ;15(9): e0237322
      A bite from a La Crosse virus (LACV) infected Aedes mosquito can cause La Crosse encephalitis (LACE), which is a neuro-invasive disease that disproportionately affects children under the age of 16 in Southern Appalachia. The three vectors for LACV are Aedes albopictus (Skuse), Ae. japonicus (Theobald), and Ae. triseriatus (Say). Localized maps of the geographic distribution of vectors are practical tools for mosquito management personnel to target areas with high mosquito abundance. This study hypothesized that LACV vectors have unique species-specific spatial and temporal clusters. To test this, 44 sites were identified in Knox County, Tennessee for their land use/type. At each site, host-seeking mosquitoes were collected approximately every other week from May-October 2018. Spatial clusters of host-seeking mosquito collections for each of the three mosquito species were investigated using Kulldorff's spatial scan statistic, specifying a retrospective space-time Bernoulli model. Most vector clusters were identified in south-central Knox County while the seasonality of clusters varied by mosquito species. Clusters of Ae. albopictus were observed throughout the entire study period while clusters of Ae. japonicus and Ae. triseriatus only occurred May-June. The findings indicate that the relative abundance of LACV vectors were more abundant in south-central Knox County compared to the rest of the county. Of interest, these clusters spatially overlapped with previous LACE diagnosed cases. These findings are useful in guiding decisions on targeted mosquito control in Knox County and may be applied to other counties within Southern Appalachia.
    DOI:  https://doi.org/10.1371/journal.pone.0237322
  8. Ann Glob Health. 2020 Aug 06. 86(1): 94
      Background: Integrated management strategies for dengue prevention and control have been the main way to decrease the transmission of arboviruses transmitted by A. aegypti in Colombia. However, the increase of chikungunya (CHIKV), Zika, and dengue (DENV) fever cases suggests deficiencies in vector control strategies in some regions from this country.Objective: This work aimed to establish a baseline susceptibility profile of A. aegypti to insecticides, determine the presence of kdr mutations associated with resistance to pyrethroids, and detect natural arbovirus infection in this vector from Moniquirá - Boyacá, one of the most endemic cities in Colombia.
    Methods: Mosquitos were collected in six neighborhoods, and colonies established in the laboratory. Susceptibility to malathion and lambda-cyhalothrin insecticides was evaluated, and we examined the point mutations present in portions of domains I, II, III, and IV of the sodium channel gene using a simple allele-specific PCR-based assay (AS-PCR).
    Findings: A. aegypti from Moniquirá showed decreased susceptibility to pyrethroid insecticides, and kdr mutations 419L, 1016I, and 1558C with allelic frequencies of 0.39, 0.40 and 0.95, respectively, were observed. The minimal infection rate (MIR) to DENV-1 was 44.1, while to CHIKV was 14.7.
    Conclusions: We establish a baseline insecticide resistance, kdr mutations, and arbovirus circulation, which contain the elements necessary for the consolidation of a local surveillance strategy with an early warning system and rational selection and rotation of insecticides.
    DOI:  https://doi.org/10.5334/aogh.2805
  9. PLoS Negl Trop Dis. 2020 Sep;14(9): e0008561
      Combined incompatible and sterile insect technique (IIT-SIT) has been considered to be an effective and safe approach to control mosquito populations. Immobilization of male adults by chilling is a crucial process required for the packing, transportation and release of the mosquitoes during the implementation of IIT-SIT for mosquito control. In this study, effects of chilling on the Aedes albopictus males with triple Wolbachia infections (HC line), a powerful weapon to fight against the wild type Ae. albopictus population via IIT-SIT, were evaluated under both laboratory and field conditions. Irradiated HC (IHC) males were exposed to 1, 5 and 10°C for 1, 2, 3, 6 and 24 h. The survival rate of the post-chilled IHC males was then monitored. Longevity of post-chilled IHC males was compared to non-chilled males under laboratory and semi-field conditions. Mating competitiveness of IHC/HC males after exposure to 5 or 10°C for 0, 3 and 24 h was then evaluated. Effects of compaction and transportation under chilled conditions on the survival rate of IHC males were also monitored. The optimal chilling conditions for handling IHC males were temperatures between 5 and 10°C for a duration of less than 3 h with no negative impacts on survival rate, longevity and mating competitiveness when compared to non-chilled males. However, the overall quality of post-chilled IHC/HC males decreased when exposed to low temperatures for 24 h. Reduced survival was observed when IHC males were stored at 5°C under a compaction height of 8 cm. Transportation with chilling temperatures fluctuating from 8 to 12°C has no negative impact on the survival of IHC males. This study identified the optimal chilling temperature and duration for the handling and transportation of Ae. albopictus IHC male adults without any detrimental effect on their survival, longevity and mating competitiveness. Further studies are required to develop drone release systems specific for chilled mosquitoes to improve release efficiency, as well as to compare the population suppression efficiency between release of post-chilled and non-chilled males in the field.
    DOI:  https://doi.org/10.1371/journal.pntd.0008561
  10. Malar J. 2020 Aug 31. 19(1): 314
      BACKGROUND: Selection pressure from continued exposure to insecticides drives development of insecticide resistance and changes in resting behaviour of malaria vectors. There is need to understand how resistance drives changes in resting behaviour within vector species. The association between insecticide resistance and resting behaviour of Anopheles gambiae sensu lato (s.l.) in Northern Ghana was examined.METHODS: F1 progenies from adult mosquitoes collected indoors and outdoors were exposed to DDT, deltamethrin, malathion and bendiocarb using WHO insecticide susceptibility tests. Insecticide resistance markers including voltage-gated sodium channel (Vgsc)-1014F, Vgsc-1014S, Vgsc-1575Y, glutathione-S-transferase epsilon 2 (GSTe2)-114T and acetylcholinesterase (Ace1)-119S, as well as blood meal sources were investigated using PCR methods. Activities of metabolic enzymes, acetylcholine esterase (AChE), non-specific β-esterases, glutathione-S-transferase (GST) and monooxygenases were measured from unexposed F1 progenies using microplate assays.
    RESULTS: Susceptibility of Anopheles coluzzii to deltamethrin 24 h post-exposure was significantly higher in indoor (mortality = 5%) than outdoor (mortality = 2.5%) populations (P = 0.02). Mosquitoes were fully susceptible to malathion (mortality: indoor = 98%, outdoor = 100%). Susceptibility to DDT was significantly higher in outdoor (mortality = 9%) than indoor (mortality = 0%) mosquitoes (P = 0.006). Mosquitoes were also found with suspected resistance to bendiocarb but mortality was not statistically different (mortality: indoor = 90%, outdoor = 95%. P = 0.30). Frequencies of all resistance alleles were higher in F1 outdoor (0.11-0.85) than indoor (0.04-0.65) mosquito populations, while Vgsc-1014F in F0 An. gambiae sensu stricto (s.s) was significantly associated with outdoor-resting behaviour (P = 0.01). Activities of non-specific β-esterase enzymes were significantly higher in outdoor than indoor mosquitoes (Mean enzyme activity: Outdoor = : 1.70/mg protein; Indoor = 1.35/mg protein. P < 0.0001). AChE activity was also more elevated in outdoor (0.62/mg protein) than indoor (0.57/mg protein) mosquitoes but this was not significant (P = 0.08). Human blood index (HBI) was predominantly detected in indoor (18%) than outdoor mosquito populations (3%).
    CONCLUSIONS: The overall results did not establish that there was a significant preference of resistant malaria vectors to solely rest indoors or outdoors, but varied depending on the resistant alleles present. Phenotypic resistance was higher in indoor than outdoor-resting mosquitoes, but genotypic and metabolic resistance levels were higher in outdoor than the indoor populations. Continued monitoring of changes in resting behaviour within An. gambiae s.l. populations is recommended.
    Keywords:  Anopheles gambiae; Indoor and outdoor behavior; Insecticide resistance; Northern Ghana
    DOI:  https://doi.org/10.1186/s12936-020-03388-1
  11. BMC Infect Dis. 2020 Sep 03. 20(1): 649
      BACKGROUND: More than 80,000 dengue cases including 215 deaths were reported nationally in less than 7 months between 2016 and 2017, a fourfold increase in the number of reported cases compared to the average number over 2010-2016. The region of Negombo, located in the Western province, experienced the greatest number of dengue cases in the country and is the focus area of our study, where we aim to capture the spatial-temporal dynamics of dengue transmission.METHODS: We present a statistical modeling framework to evaluate the spatial-temporal dynamics of the 2016-2017 dengue outbreak in the Negombo region of Sri Lanka as a function of human mobility, land-use, and climate patterns. The analysis was conducted at a 1 km × 1 km spatial resolution and a weekly temporal resolution.
    RESULTS: Our results indicate human mobility to be a stronger indicator for local outbreak clusters than land-use or climate variables. The minimum daily temperature was identified as the most influential climate variable on dengue cases in the region; while among the set of land-use patterns considered, urban areas were found to be most prone to dengue outbreak, followed by areas with stagnant water and then coastal areas. The results are shown to be robust across spatial resolutions.
    CONCLUSIONS: Our study highlights the potential value of using travel data to target vector control within a region. In addition to illustrating the relative relationship between various potential risk factors for dengue outbreaks, the results of our study can be used to inform where and when new cases of dengue are likely to occur within a region, and thus help more effectively and innovatively, plan for disease surveillance and vector control.
    Keywords:  Climate; Dengue; Human mobility; Land-use; Outbreaks; Risk factors; Spatial-temporal dynamics; Statistical modeling, Sri Lanka
    DOI:  https://doi.org/10.1186/s12879-020-05369-w
  12. Acta Trop. 2020 Aug 28. pii: S0001-706X(20)30352-1. [Epub ahead of print] 105680
      In this study, insecticide resistance and the mechanisms responsible were characterized in Ae. aegypti of Boyeros municipality from Havana, Cuba. Boyeros represents a high epidemiological risk because it is located near the Havana International Airport, it is highly urbanized, and it has a large influx of people from endemic countries so that it qualifies as a sentinel site for surveillance. The larvae collected from five areas of this municipality showed resistance to temephos associated with metabolic enzymes. The adult mosquitoes displayed a deltamethrin resistance and less distinctly to other pyrethroids associated with a high frequency of sodium channel gene mutations (F1534C and V1016I), detected for the first time in a field population from Cuba. The presence in the field populations of two insecticide resistance mechanisms represents a limiting factor in the success of the control operations of this vector, so other strategies should be considered to preserve the effectiveness of the insecticides available in public health for vector control in Cuba.
    Keywords:  Aedes aegypti; Insecticide resistance; Mechanisms of resistance; Sodium channel gene mutations
    DOI:  https://doi.org/10.1016/j.actatropica.2020.105680
  13. PLoS Negl Trop Dis. 2020 Aug 31. 14(8): e0008669
      Exposure of adult mosquitoes to pyriproxyfen (PPF), an analog of insect juvenile hormone (JH), has shown promise to effectively sterilize female mosquitoes. However, the underlying mechanisms of the PPF-induced decrease in mosquito fecundity are largely unknown. We performed a comprehensive study to dissect the mode of PPF action in Aedes aegypti mosquitoes. Exposure to PPF prompted the overgrowth of primary follicles in sugar-fed Ae. aegypti females but blocked the development of primary follicles at Christopher's Stage III after blood feeding. Secondary follicles were precociously activated in PPF-treated mosquitoes. Moreover, PPF substantially altered the expression of many genes that are essential for mosquito physiology and oocyte development in the fat body and ovary. In particular, many metabolic genes were differentially expressed in response to PPF treatment, thereby affecting the mobilization and utilization of energy reserves. Furthermore, PPF treatment on the previtellogenic female adults considerably modified mosquito responses to JH and 20-hydroxyecdysone (20E), two major hormones that govern mosquito reproduction. Krüppel homolog 1, a JH-inducible transcriptional regulator, showed consistently elevated expression after PPF exposure. Conversely, PPF upregulated the expression of several key players of the 20E regulatory cascades, including HR3 and E75A, in the previtellogenic stage. After blood-feeding, the expression of these 20E response genes was significantly weaker in PPF-treated mosquitoes than the solvent-treated control groups. RNAi-mediated knockdown of the Methoprene-tolerant (Met) protein, the JH receptor, partially rescued the impaired follicular development after PPF exposure and substantially increased the hatching of the eggs produced by PPF-treated female mosquitoes. Thus, the results suggested that PPF relied on Met to exert its sterilizing effects on female mosquitoes. In summary, this study finds that PPF exposure disturbs normal hormonal responses and metabolism in Ae. aegypti, shedding light on the molecular targets and the downstream signaling pathways activated by PPF.
    DOI:  https://doi.org/10.1371/journal.pntd.0008669
  14. J Med Entomol. 2020 Sep 02. pii: tjaa172. [Epub ahead of print]
      Control strategies exploiting the innate response of mosquitoes to chemicals are urgently required to complement existing traditional approaches. We therefore examined the behavioral responses of 16 field strains of Aedes aegypti (L.) from two countries, to deltamethrin and permethrin by using an excito-repellency (ER) test system. The result demonstrated that the escape percentage of Ae. aegypti exposed to pyrethroids did not vary significantly between the two countries in both contact and noncontact treatment despite the differing epidemiological patterns. Deltamethrin (contact: 3.57 ± 2.06% to 31.20 ± 10.71%; noncontact: 1.67 ± 1.67% to 17.31 ± 14.85%) elicited relatively lower responses to field mosquitoes when compared with permethrin (contact: 16.15 ± 4.07% to 74.19 ± 4.69%; noncontact: 3.45 ± 2.00% to 41.59 ± 6.98%) in contact and noncontact treatments. Compared with field strains, the mean percentage of escaping laboratory susceptible strain individuals were significantly high after treatments (deltamethrin contact: 72.26 ± 6.95%, noncontact: 61.10 ± 12.31%; permethrin contact: 78.67 ± 9.67%, noncontact: 67.07 ± 7.02%) and the escaped individuals spent significantly shorter time escaping from the contact and noncontact chamber. The results indicated a significant effect of resistance ratio on mean escape percentage, but some strains varied idiosyncratically compared to the increase in insecticide resistance. The results also illustrated that the resistance ratio had a significant effect on the mortality in treatments. However, the mortality in field mosquitoes that prematurely escaped from the treated contact chamber or in mosquitoes that stayed up to the 30-min experimental period showed no significant difference.
    Keywords:   kdr resistance; avoidance behavior; field strain mosquito; indoor residual spraying; resistance ratio
    DOI:  https://doi.org/10.1093/jme/tjaa172
  15. Acta Trop. 2020 Sep 01. pii: S0001-706X(20)30748-8. [Epub ahead of print] 105683
      Understanding the diversity and dynamics of the microbiota within the mosquito holobiome is of great importance to apprehend how the microbiota modulates various complex processes and interactions. This study examined the bacterial composition of Aedes albopictus across land use type and mosquito sex in the state of Selangor, Malaysia using 16S rRNA sequencing. The bacterial community structure in mosquitoes was found to be influenced by land use type and mosquito sex, with the environment and mosquito diet respectively identified to be the most likely sources of microbes. We found that approximately 70% of the microbiota samples were dominated by Wolbachia and removing Wolbachia from analyses revealed the relatively even composition of the remaining bacterial microbiota. Furthermore, microbial interaction network analysis highlighted the prevalence of co-exclusionary patterns in all networks regardless of land use and mosquito sex, with Wolbachia exhibiting co-exclusionary interactions with other residential bacteria such as Xanthomonas, Xenophilus and Zymobacter.
    Keywords:  16S rRNA sequencing; Microbial interaction network; Microbiota; Wolbachia
    DOI:  https://doi.org/10.1016/j.actatropica.2020.105683
  16. Molecules. 2020 Aug 31. pii: E3978. [Epub ahead of print]25(17):
      The number of documented dengue cases has increased dramatically in recent years due to transmission through the Aedes aegypti mosquito bite. Vector control remains the most effective measure to protect against this and other arboviral diseases including Zika, chikungunya and (urban) yellow fever, with an established vaccine only available for yellow fever. Although the quinone class shows potential as leading compounds for larvicide development, limited information restricts the development of optimized structures and/or formulations. Thus, in this contribution we investigated the larvicidal and pupicidal activity of three quinone compounds isolated from a Connarus suberosus root wood ethyl acetate extract together with 28 quinones from other sources. Eight quinones demonstrated larvicidal activity, of which tectoquinone (4) proved to be the most active (LC50 1.1 µg/mL). The essential residual effect parameter of four of these quinones was evaluated in laboratory trials, with tectoquinone (4) and 2-ethylanthraquinone (7) presenting the most prolonged activity. In small-scale field residual tests, tectoquinone (4) caused 100% larvae mortality over 5 days, supporting its selection for formulation trials to develop a prototype larvicide to control Ae. aegypti.
    Keywords:  Aedes aegypti; Connarus suberosus; embelin; quinones; residual larvicidal activity; tectoquinone
    DOI:  https://doi.org/10.3390/molecules25173978
  17. J Vis Exp. 2020 Aug 13.
      Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of disease cases each year. E. aedis causes mortality and reduced reproductive fitness in the mosquito vector and has been explored for its potential as a biocontrol agent. The protocol we present for culturing E. aedis is based on its natural infection cycle, which involves both horizontal and vertical transmission at different life stages of the mosquito host. Ae. aegypti mosquitoes are exposed to spores in the larval stage. These infected larvae then mature into adults and transmit the parasite vertically to their offspring. Infected offspring are then used as a source of spores for future horizontal transmission. Culturing E. aedis can be challenging to the uninitiated given the complexities of the parasite's life cycle, and this protocol provides detailed guidance and visual aids for clarification.
    DOI:  https://doi.org/10.3791/61574
  18. Sci Rep. 2020 Aug 31. 10(1): 14316
      Plasmodium invasion of mosquito midguts is a mandatory step for malaria transmission. The roles of mosquito midgut proteins and parasite interaction during malaria transmission are not clear. This study aims to identify mosquito midgut proteins that interact with and affect P. falciparum invasion. Based on gene expression profiles and protein sequences, 76 mosquito secretory proteins that are highly expressed in midguts and up-regulated by blood meals were chosen for analysis. About 61 candidate genes were successfully cloned from Anopheles gambiae and expressed in insect cells. ELISA analysis showed that 25 of the insect cell-expressed recombinant mosquito proteins interacted with the P. falciparum-infected cell lysates. Indirect immunofluorescence assays confirmed 17 of them interacted with sexual stage parasites significantly stronger than asexual stage parasites. Knockdown assays found that seven candidate genes significantly changed mosquitoes' susceptibility to P. falciparum. Four of them (AGAP006268, AGAP002848, AGAP006972, and AGAP002851) played a protective function against parasite invasion, and the other three (AGAP008138, FREP1, and HPX15) facilitated P. falciparum transmission to mosquitoes. Notably, AGAP008138 is a unique gene that only exists in Anopheline mosquitoes. These gene products are ideal targets to block malaria transmission.
    DOI:  https://doi.org/10.1038/s41598-020-71186-5
  19. Saudi J Biol Sci. 2020 Sep;27(9): 2358-2365
      Mosquito borne diseases have remained a grave threat to human health and are posing a significant burden on health authorities around the globe. The understanding and insight of mosquito breeding habitats features is crucial for their effective management. Comprehensive larval surveys were carried out at 14 sites in Qatar. A total of 443 aquatic habitats were examined, among these 130 were found positive with Culex pipiens, Cx. quinquefasciatus, Cx. mattinglyi, Ochlerotatus dorsalis, Oc. caspius and Anopheles stephensi. The majority of positive breeding habitats were recorded in urban areas (67.6%), followed by livestock (13.8%), and least were in agriculture (10.7%). An. stephensi larvae were positively correlated with Cx. pipien, Cx. quinquefasciatus, and negatively with water salinity. Large and shaded habitats were the most preferred by An. stephensi. In addition, Cx. pipiens was positively associated with the turbidity and pH, and was negatively associated with vegetation and habitat size. A negative association of Cx. quinquefasciatus with dissolved oxygen, water temperature, and salinity, while positive with habitat surface area was observed. Oc. dorsalis was negatively correlated with pH, water temperature, depth, and habitat surface area, whereas salinity water was more preferable site for females to lay their eggs. These results demonstrate that environmental factors play a significant role in preference of both anopheline and culicine for oviposition, while their effective management must be developed as the most viable tool to minimize mosquito borne diseases.
    Keywords:  Anopheles; Culex; Environmental variables; Larval habitat; Ochlerotatus; Qatar
    DOI:  https://doi.org/10.1016/j.sjbs.2020.07.006
  20. Geohealth. 2020 Aug;4(8): e2020GH000253
      The 2018 outbreak of dengue in the French overseas department of Réunion was unprecedented in size and spread across the island. This research focuses on the cause of the outbreak, asserting that climate played a large role in the proliferation of the Aedes albopictus mosquitoes, which transmitted the disease, and led to the dengue outbreak in early 2018. A stage-structured model was run using observed temperature and rainfall data to simulate the life cycle and abundance of the Ae. albopictus mosquito. Further, the model was forced with bias-corrected subseasonal forecasts to determine if the event could have been forecast up to 4 weeks in advance. With unseasonably warm temperatures remaining above 25°C, along with large tropical-cyclone-related rainfall events accumulating 10-15 mm per event, the modeled Ae. albopictus mosquito abundance did not decrease during the second half of 2017, contrary to the normal behavior, likely contributing to the large dengue outbreak in early 2018. Although subseasonal forecasts of rainfall for the December-January period in Réunion are skillful up to 4 weeks in advance, the outbreak could only have been forecast 2 weeks in advance, which along with seasonal forecast information could have provided enough time to enhance preparedness measures. Our research demonstrates the potential of using state-of-the-art subseasonal climate forecasts to produce actionable subseasonal dengue predictions. To the best of the authors' knowledge, this is the first time subseasonal forecasts have been used this way.
    Keywords:  Aedes albopictus; Réunion; dengue; outbreak; subseasonal forecast
    DOI:  https://doi.org/10.1029/2020GH000253
  21. J Med Entomol. 2020 Aug 31. pii: tjaa183. [Epub ahead of print]
      Recent studies report extensive reductions in the abundance of the North American rock pool mosquito, Aedes atropalpus (Diptera: Culicidae), following the invasion of Ae. japonicus japonicus in the United States. Although developmental temperature is recognized as an important component of the invasion biology of Ae. j. japonicus, its impacts on the population growth and fitness of Ae. atropalpus remain largely undefined. In this study we reared Ae. atropalpus larvae at three temperature ranges reflecting ecologically important temperatures in natural rock pools: a low temperature range (mean: 19°C) where Ae. j. japonicus is common and Ae. atropalpus is often rare, a middle temperature range (mean: 25°C) where both species are naturally found in similar relative abundances, and a higher temperature range (mean: 31°C) where Ae. atropalpus is the dominant species. We measured survival, development time, wing length, and fecundity to calculate a finite population growth rate at each temperature. Our results indicate that Ae. atropalpus population growth suffers in colder rock pools, which informs the perceived displacement of the species in temperate habitats. The population growth rate was highest in the middle temperature range, but not significantly higher than in the highest temperature range used in this study. The developmental success of Ae. atropalpus at the intermediate temperature range suggests that competition with Ae. j. japonicus in rock pools within that range may significantly impact natural Ae. atropalpus populations.
    Keywords:   Aedes atropalpus ; Aedes japonicus ; Ochlerotatus ; rate of increase
    DOI:  https://doi.org/10.1093/jme/tjaa183
  22. Rev Prat. 2020 Mar;70(3): 333-335
      West nile virus infection: an emerging arbovirosis in france and europe. West Nile virus is a mosquito-borne flavivirus, transmitted to humans by mosquitoes of the genus Culex, from an avian reservoir. Humans are accidental hosts and there is no report of human-to-human transmission, except via blood transfusion or organ transplantation. In 2018, Europe experienced the largest outbreak of West Nile virus infection ever. In France, 27 cases were identified including 7 neuro invasive forms. This infection is asymptomatic in most cases but may also manifest as an isolated fever or flu-like syndrome. In about 1% of cases, neuro-invasive forms with meningitis, meningoencephalitis or flaccid paralysis can be observed. There is no specific treatment for this viral infection. Prevention is based on the prevention of mosquito bites, but also on enhanced epidemiological surveillance during the period of circulation of the virus in Europe (from spring to autumn).
    Keywords:  Arbovirus Infections; Communicable Diseases; Emerging; West Nile virus
  23. Insects. 2020 Sep 01. pii: E584. [Epub ahead of print]11(9):
      Insecticide resistance in Aedes aegypti populations is a problem that hinders vector control and dengue prevention programs. In this study, we determined the susceptibility of Ae. aegypti populations from six Colombian regions to the pyrethroid lambda-cyhalothrin and evaluated the presence of the V1016I mutation in the sodium channel gene, which has been broadly involved in the resistance to this insecticide. The diversity of the gut microbiota of these mosquito populations was also analyzed. Only mosquitoes from Bello were susceptible to lambda-cyhalothrin and presented a lower allelic frequency of the V1016I mutation. Remarkably, there was not an important change in allelic frequencies among populations with different resistance ratios, indicating that other factors or mechanisms contributed to the resistant phenotype. Treatment of mosquitoes with antibiotics led us to hypothesize that the intestinal microbiota could contribute to the resistance to lambda-cyhalothrin. Beta diversity analysis showed significant differences in the species of bacteria present between susceptible and resistant populations. We identified 14 OTUs of bacteria that were unique in resistant mosquitoes. We propose that kdr mutations are important in the development of resistance to lambda-cyhalothrin at low insecticide concentrations but insect symbionts could play an essential role in the metabolization of pyrethroid insecticides at higher concentrations, contributing to the resistant phenotype in Ae. aegypti.
    Keywords:  Aedes aegypti; insecticide resistance; lambda-cyhalothrin; microbiome
    DOI:  https://doi.org/10.3390/insects11090584
  24. Vector Borne Zoonotic Dis. 2020 Sep 01.
      Background: The mosquito species Culex pipiens is a known vector of several pathogens and occurs in two distinct bioforms, pipiens and molestus. The bioform molestus thrives in urban environments where there are below-ground habitats; it can mate in confined spaces and feed on mammals as well as birds. In contrast, the bioform pipiens is found above ground, is thought to require more space for mating, and mainly feeds on birds. The pipiens bioform is present in large parts of Sweden but the molestus bioform has previously only been found in major cities. Materials and Methods: People experiencing mosquito nuisance in southern Sweden submitted mosquito samples as part of a citizen science project, and these samples were analyzed to determine the geographical distribution of the molestus bioform of Cx. pipiens. Mosquito specimens were identified to the species level by DNA barcoding of the cytochrome C oxidase subunit I (COI) gene, and the bioforms were determined through the CQ11 microsatellite marker. Results: Culex pipiens f molestus was observed to be spread across large parts of Gothenburg as well as in the suburbs. This bioform was found both in urban and rural areas at several sites across southern Sweden. In one site, hybrids between the two bioforms were found. Conclusions: The detection of Cx. pipiens f molestus in several rural areas was surprising, indicating that it may be more widely spread than urban areas alone, where it has been previously reported.
    Keywords:  CQ11; Culex pipiens f molestus; Culex pipiens f pipiens; DNA barcoding
    DOI:  https://doi.org/10.1089/vbz.2020.2631
  25. mSphere. 2020 Sep 02. pii: e00692-20. [Epub ahead of print]5(5):
      The mosquito midgut is a critical barrier that Plasmodium parasites must overcome to complete their developmental cycle and be transmitted to a new vertebrate host. Previous confocal studies with fixed infected midguts showed that ookinetes traverse midgut epithelial cells and cause irreversible tissue damage. Here, we investigated the spatiotemporal dynamics of ookinete midgut traversal and the response of midgut cells to invasion. A novel mounting strategy was established, suitable fluorescent dye combinations were identified and protocols optimized to label mosquito tissues in vivo, and live imaging protocols using confocal microscopy were developed. Tracking data showed that ookinetes gliding on the midgut surface travel faster and farther than those that remain in the lumen or those that have invaded the epithelium. Image analysis confirmed that parasite invasion and cell traversal occur within a couple of minutes, while caspase activity in damaged cells, indicative of cellular apoptosis, and F-actin cytoskeletal rearrangements in cells extruded into the gut lumen persist for several hours. This temporal difference highlights the importance of hemocyte-mediated cellular immunity and the mosquito complement system to mount a coordinated and effective antiplasmodial response. This novel in vivo imaging protocol allowed us to continuously observe individual ookinetes in live mosquitoes within the gut lumen and during cell traversal and to capture the subsequent cellular responses to invasion in real time for several hours, without loss of tissue integrity.IMPORTANCE Malaria is one of the most devastating parasitic diseases in humans and is transmitted by anopheline mosquitoes. The mosquito midgut is a critical barrier that Plasmodium parasites must overcome to complete their developmental cycle and be transmitted to a new host. Here, we developed a new strategy to visualize Plasmodium ookinetes as they traverse the mosquito midgut and to follow the response of damaged epithelial cells by imaging live mosquitoes. Understanding the spatial and temporal aspects of these interactions is critical when developing novel strategies to disrupt disease transmission.
    Keywords:  F-actin; Plasmodium ; anopheles; apoptosis; caspases; live imaging; midgut invasion; mosquito; ookinete
    DOI:  https://doi.org/10.1128/mSphere.00692-20
  26. Infect Dis Poverty. 2020 Sep 03. 9(1): 126
      BACKGROUND: The social-ecological systems theory, with its unique conception of resilience (social-ecological systems & resilience, SESR), provides an operational framework that currently best meets the need for integration and adaptive governance as encouraged by the Sustainable Development Goals. SESR accounts for the complex dynamics of social-ecological systems and operationalizes transdisciplinarity by focusing on community engagement, value co-creation, decentralized leadership and social innovation. Targeting Social Innovation (SI) in the context of implementation research for vector-borne diseases (VBD) control offers a low-cost strategy to contribute to lasting and contextualized community engagement in disease control and health development in low and middle income countries of the global south. In this article we describe the processes of community engagement and transdisciplinary collaboration underpinning community-based dengue management in rural primary schools and households in two districts in Cambodia.METHODS: Multiple student-led and community-based interventions have been implemented focusing on empowering education, communication for behavioral change and participatory epidemiology mapping in order to engage Cambodian communities in dengue control. We describe in particular the significance of the participatory processes that have contributed to the design of SI products that emerged following iterative consultations with community stakeholders to address the dengue problem.
    RESULTS: The SI products that emerged following our interaction with community members are 1) adult mosquito traps made locally from solid waste collections, 2) revised dengue curriculum with hands-on activities for transformative learning, 3) guppy distribution systems led by community members, 4) co-design of dengue prevention communication material by students and community members, 5) community mapping.
    CONCLUSIONS: The initiative described in this article put in motion processes of community engagement towards creating ownership of dengue control interventions tools by community stakeholders, including school children. While the project is ongoing, the project's interventions so far implemented have contributed to the emergence of culturally relevant SI products and provided initial clues regarding 1) the conditions allowing SI to emerge, 2) specific mechanisms by which it happens and 3) how external parties can facilitate SI emergence. Overall there seems to be a strong argument to be made in supporting SI as a desirable outcome of project implementation towards building adaptive capacity and resilience and to use the protocol supporting this project implementation as an operational guiding document for other VBD adaptive management in the region.
    Keywords:  Community engagement; Health development; Integrated vector management; Social innovation in health; Social-ecological system; Sustainability; Transdisciplinarity
    DOI:  https://doi.org/10.1186/s40249-020-00734-y
  27. EFSA J. 2020 Apr;18(4): e06093
      Rift Valley fever (RVF) is a vector-borne disease transmitted by different mosquito species, especially Aedes and Culex genus, to animals and humans. In November 2018, RVF re-emerged in Mayotte (France) after 11 years. Up to the end of October 2019, 126 outbreaks in animals and 143 human cases were reported. RVF mortality was 0.01%, and the number of abortions reported in polymerase chain reaction (PCR)-positive ruminants was fivefold greater than the previous 7 years. Milk loss production in 2019 compared to 2015-2018 was estimated to be 18%, corresponding to an economic loss of around €191,000 in all of Mayotte. The tropical climate in Mayotte provides conditions for the presence of mosquitoes during the whole year, and illegal introductions of animals represent a continuous risk of (re)introduction of RVF. The probability of RVF virus (RVFV) persisting in Mayotte for 5 or more years was estimated to be < 10% but could be much lower if vertical transmission in vectors does not occur. Persistence of RVF by vertical transmission in Mayotte and Réunion appears to be of minor relevance compared to other pathways of re-introduction (i.e. animal movement). However, there is a high uncertainty since there is limited information about the vertical transmission of some of the major species of vectors of RVFV in Mayotte and Réunion. The only identified pathways for the risk of spread of RVF from Mayotte to other countries were by infected vectors transported in airplanes or by wind currents. For the former, the risk of introduction of RVF to continental France was estimated to 4 × 10-6 epidemic per year (median value; 95% CI: 2 × 10-8; 0.0007), and 0.001 epidemic per year to Réunion (95% CI: 4 × 10-6; 0.16). For the latter pathway, mosquitoes dispersing on the wind from Mayotte between January and April 2019 could have reached the Comoros Islands, Madagascar, Mozambique and, possibly, Tanzania. However, these countries are already endemic for RVF, and an incursion of RVFV-infected mosquitoes would have negligible impact.
    Keywords:  Mayotte; Rift Valley Fever; impact; ruminants; spread; vector‐borne
    DOI:  https://doi.org/10.2903/j.efsa.2020.6093
  28. BMC Genomics. 2020 Aug 31. 21(1): 604
      BACKGROUND: Aedes aegypti is the principle vector of many arboviruses, including dengue virus and Zika virus, which are transmitted when an infected female mosquito takes a blood meal in order to initiate vitellogenesis. During blood digestion, ~ 10 mM heme-iron is ingested into the midgut lumen. While heme acts as both a nutrient and signaling molecule during blood digestion, it can also be highly toxic if left unchaperoned. Both signaling by, and degradation of, heme are intracellular processes, occurring in the nucleus and cytoplasm, respectively. However, the precise mechanism of heme uptake into the midgut epithelium is not currently known.RESULTS: We used next generation RNA sequencing with the goal to identify genes that code for membrane bound heme import protein(s) responsible for heme uptake into the midgut epithelium. Heme deprivation increased uptake of a heme fluorescent analog in cultured cells, while treatment of midguts with an excess of heme decreased uptake, confirming physiological changes were occurring in these heme-sensitive cells/tissues prior to sequencing. A list of candidate genes was assembled for each of the experimental sample sets, which included Aag2 and A20 cultured cells as well as midgut tissue, based on the results of a differential expression analysis, soft cluster analysis and number of predicted transmembrane domains. Lastly, the functions related to heme transport were examined through RNAi knockdown.
    CONCLUSIONS: Despite a large number of transmembrane domain containing genes differentially expressed in response to heme, very few were highly differentially expressed in any of the datasets examined. RNAi knockdown of a subset of candidates resulted in subtle changes in heme uptake, but minimal overall disruption to blood digestion/egg production. These results could indicate that heme import in Ae. aegypti may be controlled by a redundant system of multiple distinct transport proteins. Alternatively, heme membrane bound transport in Ae. aegypti could be regulated post-translationally.
    Keywords:  Aedes aegypti; Heme; Mosquito midgut; RNAi; Transcriptomes; Transporter
    DOI:  https://doi.org/10.1186/s12864-020-06981-5
  29. PLoS Negl Trop Dis. 2020 Sep 04. 14(9): e0008617
      The zoonotic malaria parasite, Plasmodium knowlesi, is now a substantial public health problem in Malaysian Borneo. Current understanding of P. knowlesi vector bionomics and ecology in Sabah comes from a few studies near the epicentre of human cases in one district, Kudat. These have incriminated Anopheles balabacensis as the primary vector, and suggest that human exposure to vector biting is peri-domestic as well as in forest environments. To address the limited understanding of vector ecology and human exposure risk outside of Kudat, we performed wider scale surveillance across four districts in Sabah with confirmed transmission to investigate spatial heterogeneity in vector abundance, diversity and infection rate. Entomological surveillance was carried out six months after a cross-sectional survey of P. knowlesi prevalence in humans throughout the study area; providing an opportunity to investigate associations between entomological indicators and infection. Human-landing catches were performed in peri-domestic, farm and forest sites in 11 villages (3-4 per district) and paired with estimates of human P. knowlesi exposure based on sero-prevalence. Anopheles balabacensis was present in all districts but only 6/11 villages. The mean density of An. balabacensis was relatively low, but significantly higher in farm (0.094/night) and forest (0.082/night) than peri-domestic areas (0.007/night). Only one An. balabacensis (n = 32) was infected with P. knowlesi. Plasmodium knowlesi sero-positivity in people was not associated with An. balabacensis density at the village-level however post hoc analyses indicated the study had limited power to detect a statistical association due low vector density. Wider scale sampling revealed substantial heterogeneity in vector density and distribution between villages and districts. Vector-habitat associations predicted from this larger-scale surveillance differed from those inferred from smaller-scale studies in Kudat; highlighting the importance of local ecological context. Findings highlight potential trade-offs between maximizing temporal versus spatial breadth when designing entomological surveillance; and provide baseline entomological and epidemiological data to inform future studies of entomological risk factors for human P. knowlesi infection.
    DOI:  https://doi.org/10.1371/journal.pntd.0008617
  30. Microb Biotechnol. 2020 Aug 30.
      Aedes albopictus transmits several arboviral infections. In the absence of vaccines, control of mosquito populations is the only strategy to prevent vector-borne diseases. As part of the search for novel, biological and environmentally friendly strategies for vector control, the isolation of new bacterial species with mosquitocidal activity represents a promising approach. However, new bacterial isolates may be difficult to grow and genetically manipulate. To overcome these limits, here we set up a system allowing the expression of mosquitocidal bacterial toxins in the well-known genetic background of Bacillus subtilis. As a proof of this concept, the ability of B. subtilis to express individual or combinations of toxins of Bacillus thuringiensis israelensis (Bti) was studied. Different expression systems in which toxin gene expression was driven by IPTG-inducible, auto-inducible or toxin gene-specific promoters were developed. The larvicidal activity of the resulting B. subtilis strains against second-instar Ae. albopictus larvae allowed studying the activity of individual toxins or the synergistic interaction among Cry and Cyt toxins. The expression systems here presented lay the foundation for a better improved system to be used in the future to characterize the larvicidal activity of toxin genes from new environmental isolates.
    DOI:  https://doi.org/10.1111/1751-7915.13648