publications

2025

1. Ancient origin of an urban underground mosquito

   Yuki Haba, Matthew L. Aardema, Maria O. Afonso, Natasha M. Agramonte, John Albright, Ana Margarida Alho, Antonio P.G. Almeida, Haoues Alout, Bulent Alten, Mine Altinli, and 207 more authors

   Jan 2025

   Abs DOI

   Abstract Understanding how life is adapting to urban environments represents an important challenge in evolutionary biology. Here we investigate a widely cited example of urban adaptation, Culex pipiens form molestus , also known as the London Underground Mosquito. Population genomic analysis of ∼350 contemporary and historical samples counter the popular hypothesis that molestus originated belowground in London less than 200 years ago. Instead, we show that molestus first adapted to human environments aboveground in the Middle East over the course of \textgreater1000 years, likely in concert with the rise of agricultural civilizations. Our results highlight the role of early human society in priming taxa for contemporary urban evolution and have important implications for understanding arbovirus transmission.

2. Detection and Characterization of Antiviral Proteins Against Bunyaviruses in Mosquito-Derived Cells

   P. Böhmer, M. Reuter, M. Altinli, and E. Schnettler

   In Bunyaviruses, Jan 2025

   Series Title: Methods in Molecular Biology

   DOI

2024

1. Are Leibniz PostDocs alright? Insights into the careers, working lives, and mental health of Leibniz PostDocs

   Harry M. Williams, Regina Becker, Lena Roemer, Mine Altinli, Sabrina Sobieraj, Thomas Lösch, and Daniela Fiedler

   Jan 2024

   Abs

   Postdoctoral researchers play a critical role in scientific research. This is the case not only for the Leibniz Association but for research worldwide. However, postdoctoral researchers are often overlooked or misrepresented as a homogeneous group of researchers. The principal aim of the Leibniz PostDoc Survey was to understand who Leibniz PostDocs are and to provide some insight into their experiences, interests, and needs. This report summarises the results of the second Leibniz PostDoc Survey, which was open for submission from November 2022 to January 2023. Overall, we had questions covering the following sections: (1) demographics, (2) contract and payments, (3) working hours and tasks, (4) career goals, (5) career development – experiences, (6) career development – supervisor and institutional support, (7) satisfaction and working situation, (8) good scientific practice, workplace conflict, and discrimination, (9) mental health, as well as (10) the experience of international Leibniz PostDocs. The report includes a summary of the key findings and recommendations as well as a comprehensive description of all results from each survey question.

2023

1. Antiviral RNAi Response in Culex quinquefasciatus-Derived HSU Cells

   Mine Altinli, Mayke Leggewie, Jonny Schulze, Rashwita Gyanwali, Marlis Badusche, Vattipally B. Sreenu, Janina Fuss, and Esther Schnettler

   Viruses, Feb 2023

   Abs DOI

   Culex spp. mosquitoes are important vectors of viruses, such as West Nile virus, Eastern equine encephalitis virus and Rift valley fever virus. However, their interactions with innate antiviral immunity, especially RNA interference (RNAi), are not well known. Most research on RNAi pathways in mosquitoes is focused on the tropical vector mosquito Aedes aegypti. Here, we investigated the production of arbovirus-specific small RNAs in Cx. quinquefasciatus-derived HSU cells. Furthermore, by silencing RNAi-related proteins, we investigated the antiviral role of these proteins for two different arboviruses: Semliki Forest virus (SFV) and Bunyamwera orthobunyavirus (BUNV). Our results showed an expansion of Ago2 and Piwi6 in Cx. quinquefasciatus compared to Ae. aegypti. While silencing Ago2a and Ago2b increased BUNV replication, only Ago2b showed antiviral activity against SFV. Our results suggest differences in the function of Cx. quinquefasciatus and Ae. aegypti RNAi proteins and highlight the virus-specific function of these proteins in Cx. quinquefasciatus.

2. Agua Salud Alphavirus Infection, Dissemination and Transmission in Aedes aegypti Mosquitoes

   Swati V. Jagtap, Jorn Brink, Svea C. Frank, Marlis Badusche, Mayke Leggewie, Vattipally B. Sreenu, Janina Fuss, Esther Schnettler, and Mine Altinli

   Viruses, May 2023

   Abs DOI

   Mosquitoes are competent vectors for many important arthropod-borne viruses (arboviruses). In addition to arboviruses, insect-specific viruses (ISV) have also been discovered in mosquitoes. ISVs are viruses that replicate in insect hosts but are unable to infect and replicate in vertebrates. They have been shown to interfere with arbovirus replication in some cases. Despite the increase in studies on ISV–arbovirus interactions, ISV interactions with their hosts and how they are maintained in nature are still not well understood. In the present study, we investigated the infection and dissemination of the Agua Salud alphavirus (ASALV) in the important mosquito vector Aedes aegypti through different infection routes (per oral infection, intrathoracic injection) and its transmission. We show here that ASALV infects the female Ae. aegypti and replicates when mosquitoes are infected intrathoracically or orally. ASALV disseminated to different tissues, including the midgut, salivary glands and ovaries. However, we observed a higher virus load in the brain than in the salivary glands and carcasses, suggesting a tropism towards brain tissues. Our results show that ASALV is transmitted horizontally during adult and larval stages, although we did not observe vertical transmission. Understanding ISV infection and dissemination dynamics in Ae. aegypti and their transmission routes could help the use of ISVs as an arbovirus control strategy in the future.

3. The Aedes aegypti RNA interference response against Zika virus in the context of co-infection with dengue and chikungunya viruses

   Mayke Leggewie, Christina Scherer, Mine Altinli, Rommel J. Gestuveo, Vattipally B. Sreenu, Janina Fuss, Marie Vazeille, Laurence Mousson, Marlis Badusche, Alain Kohl, and 2 more authors

   PLOS Neglected Tropical Diseases, Jul 2023

   Abs DOI

   Since its detection in 2015 in Brazil, Zika virus (ZIKV) has remained in the spotlight of international public health and research as an emerging arboviral pathogen. In addition to single infection, ZIKV may occur in co-infection with dengue (DENV) and chikungunya (CHIKV) viruses, with whom ZIKV shares geographic distribution and the mosquito Aedes aegypti as a vector. The main mosquito immune response against arboviruses is RNA interference (RNAi). It is unknown whether or not the dynamics of the RNAi response differ between single arboviral infections and co-infections. In this study, we investigated the interaction of ZIKV and DENV, as well as ZIKV and CHIKV co-infections with the RNAi response in Ae. aegypti. Using small RNA sequencing, we found that the efficiency of small RNA production against ZIKV -a hallmark of antiviral RNAi—was mostly similar when comparing single and co-infections with either DENV or CHIKV. Silencing of key antiviral RNAi proteins, showed no change in effect on ZIKV replication when the cell is co-infected with ZIKV and DENV or CHIKV. Interestingly, we observed a negative effect on ZIKV replication during CHIKV coinfection in the context of Ago2-knockout cells, though his effect was absent during DENV co-infection. Overall, this study provides evidence that ZIKV single or co-infections with CHIKV or DENV are equally controlled by RNAi responses. Thus, Ae. aegypti mosquitoes and derived cells support co-infections of ZIKV with either CHIKV or DENV to a similar level than single infections, as long as the RNAi response is functional.

4. Interaction of Mesonivirus and Negevirus with arboviruses and the RNAi response in Culex tarsalis-derived cells

   Eric Agboli, Jonny Schulze, Stephanie Jansen, Daniel Cadar, Vattipally B. Sreenu, Mayke Leggewie, Mine Altinli, Marlis Badusche, Hanna Jöst, Jessica Börstler, and 2 more authors

   Parasites & Vectors, Oct 2023

   Abs DOI

   Background  Mosquito-specific viruses (MSVs) comprise a variety of different virus families, some of which are known to interfere with infections of medically important arboviruses. Viruses belonging to the family Mesoniviridae or taxon Negevirus harbor several insect-specific viruses, including MSVs, which are known for their wide geographical distri‑bution and extensive host ranges. Although these viruses are regularly identified in mosquitoes all over the world, their presence in mosquitoes in Germany had not yet been reported. Methods  A mix of three MSVs (Yichang virus [Mesoniviridae] and two negeviruses [Daeseongdong virus and Dezi‑dougou virus]) in a sample that contained a pool of Coquillettidia richiardii mosquitoes collected in Germany was used to investigate the interaction of these viruses with different arboviruses in Culex-derived cells. In addition, small RNA sequencing and analysis of different mosquito-derived cells infected with this MSV mix were performed. Results  A strain of Yichang virus (Mesoniviridae) and two negeviruses (Daeseongdong virus and Dezidougou virus) were identified in the Cq. richiardii mosquitoes sampled in Germany, expanding current knowledge of their circula‑tion in central Europe. Infection of mosquito-derived cells with these three viruses revealed that they are targeted by the small interfering RNA (siRNA) pathway. In Culex-derived cells, co-infection by these three viruses had vary‑ing effects on the representative arboviruses from different virus families (Togaviridae: Semliki forest virus [SFV]; Bunyavirales: Bunyamwera orthobunyavirus [BUNV]; or Flaviviridae: Usutu virus [USUV]). Specifically, persistent MSV co-infection inhibited BUNV infection, as well as USUV infection (but the latter only at specific time points). However, the impact on SFV infection was only noticeable at low multiplicity of infection (MOI 0.1) and at specific time points in combination with the infection status. Conclusions  Taken together, these results are important findings that will lead to a better understanding of the com‑plex interactions of MSVs, mosquitoes and arboviruses.

2022

1. Antiviral RNAi Response against the Insect-Specific Agua Salud Alphavirus

   Mine Altinli, Mayke Leggewie, Marlis Badusche, Rashwita Gyanwali, Christina Scherer, Jonny Schulze, Vattipally B Sreenu, Marvin Fegebank, Bernhard Zibrat, Janina Fuss, and 2 more authors

   mSphere, Oct 2022

   Abs DOI

   Arboviruses transmitted by mosquitoes are responsible for the death of millions of people each year. In addition to arboviruses, many insect-specific viruses (ISVs) have been discovered in mosquitoes in the last decade. ISVs, in contrast to arboviruses transmitted by mosquitoes to vertebrates, cannot replicate in vertebrate cells even when they are evolutionarily closely related to arboviruses. The alphavirus genus includes many arboviruses, although only a few ISVs have been discovered from this genus so far. Here, we investigate the interactions of a recently isolated insect-specific alphavirus, Agua Salud alphavirus (ASALV), with its mosquito host. RNA interference (RNAi) is one of the essential antiviral responses against arboviruses, although there is little knowledge on the interactions of RNAi with ISVs. Through the knockdown of transcripts of the different key RNAi pathway (small interfering RNA [siRNA], microRNA [miRNA], and P-element-induced wimpy testis [PIWI]-interacting RNA [piRNA]) proteins, we show the antiviral role of Ago2 (siRNA), Ago1 (miRNA), and Piwi4 proteins against ASALV in Aedes aegypti-derived cells. ASALV replication was increased in Dicer2 and Ago2 knockout cells, confirming the antiviral role of the siRNA pathway. In infected cells, mainly ASALV-specific siRNAs are produced, while piRNA-like small RNAs, with the characteristic nucleotide bias resulting from ping-pong amplification, are produced only in Dicer2 knockout cells. Taken together, ASALV interactions with the mosquito RNAi response differ from those of arthropod-borne alphaviruses in some aspects, although they also share some commonalities. Further research is needed to understand whether the identified differences can be generalized to other insect-specific alphaviruses.

2021

1. Symbiotic Interactions Between Mosquitoes and Mosquito Viruses

   Mine Altinli, Esther Schnettler, and Mathieu Sicard

   Frontiers in Cellular and Infection Microbiology, Aug 2021

   Abs DOI

   Mosquitoes not only transmit human and veterinary pathogens called arboviruses (arthropod-borne viruses) but also harbor mosquito-associated insect-specific viruses (mosquito viruses) that cannot infect vertebrates. In the past, studies investigating mosquito viruses mainly focused on highly pathogenic interactions that were easier to detect than those without visible symptoms. However, the recent advances in viral metagenomics have highlighted the abundance and diversity of viruses which do not generate mass mortality in host populations. Over the last decade, this has facilitated the rapid growth of virus discovery in mosquitoes. The circumstances around the discovery of mosquito viruses greatly affected how they have been studied so far. While earlier research mainly focused on the pathogenesis caused by DNA and some doublestranded RNA viruses during larval stages, more recently discovered single-stranded RNA mosquito viruses were heavily studied for their putative interference with arboviruses in female adults. Thus, many aspects of mosquito virus interactions with their hosts and host-microbiota are still unknown. In this context, considering mosquito viruses as endosymbionts can help to identify novel research areas, in particular in relation to their long-term interactions with their hosts (e.g. relationships during all life stages, the stability of the associations at evolutionary scales, transmission routes and virulence evolution) and the possible context-dependent range of interactions (i.e. beneficial to antagonistic). Here, we review the symbiotic interactions of mosquito viruses considering different aspects of their ecology, such as transmission, host specificity, host immune system and interactions with other symbionts within the host cellular arena. Finally, we highlight related research gaps in mosquito virus research.

2. Cytoplasmic Incompatibility Variations in Relation with Wolbachia cid Genes Divergence in Culex pipiens

   Mathieu Sicard, Alice Namias, Marco Perriat-Sanguinet, Eric Carron, Sandra Unal, Mine Altinli, Frederic Landmann, and Mylène Weill

   mBio, Feb 2021

   Abs DOI

   In arthropods, Wolbachia endosymbionts induce conditional sterility, called cytoplasmic incompatibility (CI), resulting from embryonic lethality. CI penetrance (i.e., embryonic death rate) varies depending on host species and Wolbachia strains involved. All Culex pipiens mosquitoes are infected by the endosymbiotic alphaproteobacteria Wolbachia wPip. CI in Culex, characterized as a binary “compatible/incompatible” phenomenon, revealed an unparalleled diversity of patterns linked to the amplification-diversification of cidA and cidB genes. Here, we accurately studied CI penetrance variations in the light of cid genes divergence by generating a C. pipiens compatibility matrix between 11 lines hosting different phylogenetic wPip groups and exhibiting distinct cid gene repertoires. We showed, as expected, that crosses involving wPip from the same group were mostly compatible. In contrast, only 22% of the crosses involving different wPip groups were compatible, while 54% were fully incompatible. For the remaining 24% of the crosses, “intermediate” compatibilities were reported, and a cytological observation of the first zygotic division confirmed the occurrence of “canonical” CI phenotypes in a fraction of the eggs. Backcross experiments demonstrated that intermediate compatibilities were not linked to host genetic background but to the Wolbachia strains involved. This previously unstudied intermediate penetrance CI was more severe and frequent in crosses involving wPip-IV strains exhibiting cid variants markedly divergent from other wPip groups. Our data demonstrate that CI is not always a binary compatible/incompatible phenomenon in C. pipiens but that intermediate compatibilities putatively resulting from partial mismatch due to Cid proteins divergence exist in this species complex.

2020

1. Wolbachia modulates prevalence and viral load of Culex pipiens densoviruses in natural populations

   Mine Altinli, Sebastian Lequime, Celestine Atyame, Fabienne Justy, Mylene Weill, and Mathieu Sicard

   Molecular Ecology, Oct 2020

   Abs DOI

   The inadequacy of standard mosquito control strategies calls for ecologically safe novel approaches, for example the use of biological agents such as the endosymbiotic α-proteobacteria Wolbachia or insect-specific viruses (ISVs). Understanding the ecological interactions between these “biocontrol endosymbionts” is thus a fundamental step. Wolbachia are transmitted vertically from mother to offspring and modify their hosts’ phenotypes, including reproduction (e.g., cytoplasmic incompatibility) and survival (e.g., viral interference). In nature, Culex pipiens (sensu lato) mosquitoes are always found infected with genetically diverse Wolbachia called wPip that belong to five phylogenetic groups. In recent years, ISVs have also been discovered in these mosquito species, although their interactions with Wolbachia in nature are unknown. Here, we studied the interactions between a widely prevalent ISV, the Culex pipiens densovirus (CpDV, Densovirinae), and Wolbachia in northern Tunisian C. pipiens populations. We showed an influence of different Wolbachia groups on CpDV prevalence and a general positive correlation between Wolbachia and CpDV loads. By investigating the putative relationship between CpDV diversification and wPip groups in the different sites, we detected a signal linked to wPip groups in CpDV phylogeny in sites where all larvae were infected by the same wPip group. However, no such signal was detected where the wPip groups coexisted, suggesting CpDV horizontal transfer between hosts. Overall, our results provide good evidence for an ecological influence of Wolbachia on an ISV, CpDV, in natural populations and highlight the importance of integrating Wolbachia in our understanding of ISV ecology in nature.

2019

1. Mosquito-Specific Viruses—Transmission and Interaction

   Agboli,  Leggewie,  Altinli, and  Schnettler

   Viruses, Sep 2019

   Abs DOI

   Mosquito-specific viruses (MSVs) are a subset of insect-specific viruses that are found to infect mosquitoes or mosquito derived cells. There has been an increase in discoveries of novel MSVs in recent years. This has expanded our understanding of viral diversity and evolution but has also sparked questions concerning the transmission of these viruses and interactions with their hosts and its microbiome. In fact, there is already evidence that MSVs interact with the immune system of their host. This is especially interesting, since mosquitoes can be infected with both MSVs and arthropod-borne (arbo) viruses of public health concern. In this review, we give an update on the different MSVs discovered so far and describe current data on their transmission and interaction with the mosquito immune system as well as the effect MSVs could have on an arboviruses-coinfection. Lastly, we discuss potential uses of these viruses, including vector and transmission control.

2. Evolution and phylogeography of Culex pipiens densovirus

   Mine Altinli, Sebastian Lequime, Maxime Courcelle, Sarah François, Fabienne Justy, Anne-Sophie Gosselin-Grenet, Mylene Ogliastro, Mylene Weill, and Mathieu Sicard

   Virus Evolution, Jul 2019

   Abs DOI

   Viruses of the Parvoviridae family infect a wide range of animals including vertebrates and invertebrates. So far, our understanding of parvovirus diversity is biased towards medically or economically important viruses mainly infecting vertebrate hosts, while invertebrate infecting parvoviruses—namely densoviruses—have been largely neglected. Here, we investigated the prevalence and the evolution of the only mosquito-infecting ambidensovirus, Culex pipiens densovirus (CpDV), from laboratory mosquito lines and natural populations collected worldwide. CpDV diversity generally grouped in two clades, here named CpDV-1 and -2. The incongruence of the different gene trees for some samples suggested the possibility of recombination events between strains from different clades. We further investigated the role of selection on the evolution of CpDV genome and detected many individual sites under purifying selection both in non-structural and structural genes. However, some sites in structural genes were under diversifying selection, especially during the divergence of CpDV-1 and -2 clades. These substitutions between CpDV-1 and -2 clades were mostly located in the capsid protein encoding region and might cause changes in host specificity or pathogenicity of CpDV strains from the two clades. However, additional functional and experimental studies are necessary to fully understand the protein conformations and the resulting phenotype of these substitutions between clades of CpDV.

3. Sharing cells with Wolbachia : the transovarian vertical transmission of Culex pipiens densovirus

   Mine Altinli, Julien Soms, Marc Ravallec, Fabienne Justy, Manon Bonneau, Mylene Weill, Anne‐Sophie Gosselin‐Grenet, and Mathieu Sicard

   Environmental Microbiology, Sep 2019

   Abs DOI

   Culex pipiens densovirus (CpDV), a single stranded DNA virus, has been isolated from Culex pipiens mosquitoes but differs from other mosquito densoviruses in terms of genome structure and sequence identity. Its transmission from host to host, the nature of its interactions with both its host and host’s endosymbiotic bacteria Wolbachia are not known. Here, we report the presence of CpDV in the ovaries and eggs of Cx. pipiens mosquitoes in close encounters with Wolbachia. In the ovaries, CpDV amount significantly differed between mosquito lines harbouring different strains of Wolbachia and these differences were not linked to variations in Wolbachia densities. CpDV was vertically transmitted in all laboratory lines to 17%–20% of the offspring. For some females, however, the vertical transmission reached 90%. Antibiotic treatment that cured the host from Wolbachia significantly decreased both CpDV quantity and vertical transmission suggesting an impact of host microbiota, including Wolbachia, on CpDV transmission. Overall our results show that CpDV is transmitted vertically via transovarian path along with Wolbachia with which it shares the same cells. Our results are primordial to understand the dynamics of densovirus infection, their persistence and spread in populations considering their potential use in the regulation of mosquito vector populations.

2018

1. Wolbachia diversity and cytoplasmic incompatibility patterns in Culex pipiens populations in Turkey

   Mine Altinli, Filiz Gunay, Bulent Alten, Mylene Weill, and Mathieu Sicard

   Parasites & Vectors, Dec 2018

   Abs DOI

   Background: Wolbachia are maternally transmitted bacteria that can manipulate their hosts’ reproduction causing cytoplasmic incompatibility (CI). CI is a sperm-egg incompatibility resulting in embryonic death. Due to this sterilising effect on mosquitoes, Wolbachia are considered for vector control strategies. Important vectors for arboviruses, filarial nematodes and avian malaria, mosquitoes of Culex pipiens complex are suitable for Wolbachia-based vector control. They are infected with Wolbachia wPip strains belonging to five genetically distinct groups (wPip-I to V) within the Wolbachia B supergroup. CI properties of wPip strongly correlate with this genetic diversity: mosquitoes infected with wPip strains from a different wPip group are more likely to be incompatible with each other. Turkey is a critical spot for vector-borne diseases due to its unique geographical position as a natural bridge between Asia, Europe and Africa. However, general wPip diversity, distribution and CI patterns in natural Cx. pipiens (s.l.) populations in the region are unknown. In this study, we first identified wPip diversity in Turkish Cx. pipiens (s.l.) populations, by assigning them to one of the five groups within wPip (wPip-Ito V). We further investigated CI properties between different wPip strains from this region. Results: We showed a wPip fixation in Cx. pipiens (s.l.) populations in Turkey by analysing 753 samples from 59 sampling sites. Three wPip groups were detected in the region: wPip-I, wPip-II and wPip-IV. The most dominant group was wPip-II. While wPip-IV was restricted to only two locations, wPip-I and wPip-II had wider distributions. Individuals infected with wPip-II were found co-existing with individuals infected with wPip-I or wPip-IV in some sampling sites. Two mosquito isofemale lines harbouring either a wPip-I or a wPip-II strain were established from a population in northwestern Turkey. Reciprocal crosses between these lines showed that they were fully compatible with each other but bidirectionally incompatible with wPip-IV Istanbul infected line. Conclusion: Our findings reveal a high diversity of wPip and CI properties in Cx. pipiens (s.l.) populations in Turkey. Knowledge on naturally occurring CI patterns caused by wPip diversity in Turkey might be useful for Cx. pipiens (s.l.) control in the region.

2015

1. The Mutualistic Side of Wolbachia–Isopod Interactions: Wolbachia Mediated Protection Against Pathogenic Intracellular Bacteria

   Christine Braquart-Varnier, Mine Altinli, Romain Pigeault, Frédéric D. Chevalier, Pierre Grève, Didier Bouchon, and Mathieu Sicard

   Frontiers in Microbiology, Dec 2015

   Abs DOI

   Wolbachia is a vertically transmitted endosymbiont whose radiative success is mainly related to various host reproductive manipulations that led to consider this symbiont as a conflictual reproductive parasite. However, lately, some Wolbachia have been shown to act as beneficial symbionts by protecting hosts against a broad range of parasites. Still, this protection has been mostly demonstrated in artificial Wolbachia-host associations between partners that did not co-evolved together. Here, we tested in two terrestrial isopod species Armadillidium vulgare and Porcellio dilatatus whether resident Wolbachia (native or non-native) could confer protection during infections with Listeria ivanovii and Salmonella typhimurium and also during a transinfection with a Wolbachia strain that kills the recipient host (i.e., wVulC in P. dilatatus). Survival analyses showed that (i) A. vulgare lines hosting their native Wolbachia (wVulC) always exhibited higher survival than asymbiotic ones when infected with pathogenic bacteria (ii) P. dilatatus lines hosting their native wDil Wolbachia strain survived the S. typhimurium infection better, while lines hosting non-native wCon Wolbachia strain survived the L. ivanovii and also the transinfection with wVulC from A. vulgare better. By studying L. ivanovii and S. typhimurium loads in the hemolymph of the different host-Wolbachia systems, we showed that (i) the difference in survival between lines after L. ivanovii infections were not linked to the difference between their pathogenic bacterial loads, and (ii) the difference in survival after S. typhimurium infections corresponds to lower loads of pathogenic bacteria. Overall, our results demonstrate a beneficial effect of Wolbachia on survival of terrestrial isopods when infected with pathogenic intracellular bacteria. This protective effect may rely on different mechanisms depending on the resident symbiont and the invasive bacteria interacting together within the hosts.