18S rDNA gene metabarcoding of microeukaryotes and epi-endophytes in the holobiome of seven species of large brown algae

Brown algae (Phaeophyceae) are habitat-forming species in coastal ecosystems and include kelp forests and seaweed beds that support a wide diversity of marine life. Host-associated microbial communities are an integral part of phaeophyte biology, and whereas the bacterial microbial partners have received considerable attention, the microbial eukaryotes associated with brown algae have hardly been studied. Here, we used broadly targeted “pan-eukaryotic” primers (metabarcoding) to investigate brown algal-associated eukaryotes (the eukaryome). Using this approach, we aimed to investigate the eukaryome of seven large brown algae that are important and common species in coastal ecosystems. We also aimed to assess whether these macroalgae harbor novel eukaryotic diversity and to ascribe putative functional roles to the host-associated eukaryome based on taxonomic affiliation and phylogenetic placement. We detected a significant diversity of microeukaryotic and algal lineages associated with the brown algal species investigated. The operational taxonomic units (OTUs) were taxonomically assigned to 10 of the eukaryotic major supergroups, including taxonomic groups known to be associated with seaweeds as epibionts, endobionts, parasites, and commensals. Additionally, we revealed previously unrecorded sequence types, including novel phaeophyte OTUs, particularly in the Fucus spp. samples, that may represent fucoid genomic variants, sequencing artifacts, or undescribed epi-/endophytes. Our results provide baseline data and technical insights that will be useful for more comprehensive seaweed eukaryome studies investigating the evidently lineage-rich and functionally diverse symbionts of brown algae.publishedVersio

Antiviral RNAi response in Culex quinquefasciatus-derived HSU cells

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

Agua Salud alphavirus infection, dissemination and transmission in Aedes aegypti mosquitoes

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

An Aedes aegypti-derived Ago2 knockout cell line to investigate arbovirus infections

Mosquitoes are known as important vectors of many arthropod-borne (arbo)viruses causing disease in humans. These include dengue (DENV) and Zika (ZIKV) viruses. The exogenous small interfering (si)RNA (exo-siRNA) pathway is believed to be the main antiviral defense in arthropods, including mosquitoes. During infection, double-stranded RNAs that form during viral replication and infection are cleaved by the enzyme Dicer 2 (Dcr2) into virus-specific 21 nt vsiRNAs, which are subsequently loaded into Argonaute 2 (Ago2). Ago2 then targets and subsequently cleaves complementary RNA sequences, resulting in degradation of the target viral RNA. Although various studies using silencing approaches have supported the antiviral activity of the exo-siRNA pathway in mosquitoes, and despite strong similarities between the siRNA pathway in the Drosophila melanogaster model and mosquitoes, important questions remain unanswered. The antiviral activity of Ago2 against different arboviruses has been previously demonstrated. However, silencing of Ago2 had no effect on ZIKV replication, whereas Dcr2 knockout enhanced its replication. These findings raise the question as to the role of Ago2 and Dcr2 in the control of arboviruses from different viral families in mosquitoes. Using a newly established Ago2 knockout cell line, alongside the previously reported Dcr2 knockout cell line, we investigated the impact these proteins have on the modulation of different arboviral infections. Infection of Ago2 knockout cell line with alpha- and bunyaviruses resulted in an increase of viral replication, but not in the case of ZIKV. Analysis of small RNA sequencing data in the Ago2 knockout cells revealed a lack of methylated siRNAs from different sources, such as acute and persistently infecting viruses-, TE- and transcriptome-derived RNAs. The results confirmed the importance of the exo-siRNA pathway in the defense against arboviruses, but highlights variability in its response to different viruses and the impact the siRNA pathway proteins have in controlling viral replication. Moreover, this established Ago2 knockout cell line can be used for functional Ago2 studies, as well as research on the interplay between the RNAi pathways

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

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 co-infection 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

Plant regulatory networks : RNA binding proteins as mediators of communication between DNA containing compartments by dual targeting

The endosymbiosis-derived organelles within a plant cell, plastids and mitochondria, have to be equipped with a certain set of proteins to be fully functional. This set of proteins is encoded by different genomes: the organellar genomes and the nuclear genomes. This setup poses some interesting challenges for the regulation of gene expression and protein transport. On the one hand, the targeting signals that transport proteins to the organelles have to be highly specific and on the other hand, the communication between the DNA containing compartments to coordinate their gene expression has to be transmitted somehow, not only from the nucleus but also from the organelles back to the nucleus. In this thesis, two prediction programs are introduced. One of them can predict dual targeted proteins to both organelles (ATP, ambiguous targeting predictor) and the other one is species- specific for Physcomitrella patens (GTP_Pp; green targeting predictor – P. patens-specific). The first predictor can help to gain a more complete picture of the proteins potentially present in the organelles. With the help of that predictor, we predicted that the amount of proteins with dual targeting signals is higher than anticipated and that we to date know only a minor part of actually dual targeted proteins. The second predictor can help to answer the question on the evolutionary consistency of targeting signals within the plant kingdom and the importance of having species-specific approaches in analyzing protein targeting. We actually observed a surprisingly big difference in composition and recognition of mitochondrial and dual-targeting protein signals, which led to the conclusion, that species-specific approaches always should be considered as the optimal option for both, in silico and in vivo experiments. The second part of this thesis focuses the mechanisms of communication between nucleus and the organelles, especially the plastid possibly mediated by dual targeting. We chose several plastid RNA-/DNA-binding proteins to analyze their sub-plastidic localization and their potential additional nuclear localization. Those candidates were AtWHY1 (Arabidopsis thaliana Whirly1), four members of the AtcpRNP (chloroplast ribonucleoprotein) family and AtEF-Tu (elongation factor thermo-unstable). The analyzed members of the AtcpRNP family reflect their described multiple functions within the plastid also in a multiple localization pattern. Furthermore, we were able to show interactions of different members of the AtcpRNP family by yeast-two-hybrid interaction assays. The localization pattern of AtEF-Tu was very similar to the one observed for the AtcpRNPs, which indicated, together with a confirmed localization within the transcriptionally active chromosome, a multiple function for AtEF-Tu. The sub-plastidic localization data suggest overlapping networks of activity for the proteins by observed co-localizations. This was also shown with respect to several marker proteins for plastid functions. For AtEF-Tu and the AtcpRNPs, we also showed experimentally that a second localization in the nucleus is possible for the mature protein, which makes them interesting candidates for a possible mediation of plastid signals to the nucleus next to AtWHY1. For AtWHY1, we were able to show an effect of the DNA binding domain on the known localization pattern which seemed to reflect an aberration in transport processes through the envelope. This offers a potential regulatory mechanism that needs to be explored in detail in the future

Whirly proteins as communicators between plant organelles and the nucleus?

Whirly proteins belong to a small family of proteins with a characteristic secondary structure and a conserved DNA binding domain that is found mainly in angiosperms. At least one member of the Whirly protein family, Whirly1, is dually targeted to the nucleus and to the chloroplasts and it was shown that apart from its initially described function as a transcriptional regulator of nuclear disease resistance genes, this protein comprises many more functions. It seems to fulfil roles in nuclear telomere homeostasis and possibly chloroplast rRNA metabolism as well as chloroplast intron splicing. A homologous protein with a mitochondrial presequence, Whirly2, in contrast, is presumably involved in the replication of the mitochondrial genome and in mitochondrial gene expression. In addition, it seems to affect the expression of a small subset of nuclear genes. Both Whirly proteins show an antagonistic effect on leaf senescence. Although direct evidence for a nuclear localisation of Whirly2 has yet to be obtained, we hypothesise that all members of the Whirly family are intriguing candidates for organelle-to-nucleus crosstalk with an intricate interaction between each other

Draft genome assembly and transcriptome sequencing of the golden algae Hydrurus foetidus (Chrysophyceae)

Hydrurus foetidus is a freshwater alga belonging to the phylum Heterokonta. It thrives in cold rivers in polar and high alpine regions. It has several morphological traits reminiscent of single-celled eukaryotes, but can also form macroscopic thalli. Despite its ability to produce polyunsaturated fatty acids, its life under cold conditions and its variable morphology, very little is known about its genome and transcriptome. Here, we present an extensive set of next-generation sequencing data, including genomic short reads from Illumina sequencing and long reads from Nanopore sequencing, as well as full length cDNAs from PacBio IsoSeq sequencing and a small RNA dataset (smaller than 200 bp) sequenced with Illumina. We combined this data with, to our knowledge, the first draft genome assembly of a chrysophyte algae. The assembly consists of 5069 contigs to a total assembly size of 171 Mb and a 77% BUSCO completeness. The new data generated here may contribute to a better understanding of the evolution and ecological roles of chrysophyte algae, as well as to resolve the branching patterns within the Heterokonta

Meta-transcriptomic comparison of two sponge holobionts feeding on coral- and macroalgal-dissolved organic matter

[Background] Sponge holobionts (i.e., the host and its associated microbiota) play a key role in the cycling of dissolved organic matter (DOM) in marine ecosystems. On coral reefs, an ecological shift from coral-dominated to algal-dominated ecosystems is currently occurring. Given that benthic corals and macroalgae release different types of DOM, in different abundances and with different bioavailability to sponge holobionts, it is important to understand how the metabolic activity of the host and associated microbiota change in response to the exposure to both DOM sources. Here, we look at the differential gene expression of two sponge holobionts 6 hours after feeding on naturally sourced coral- and macroalgal-DOM using RNA sequencing and meta-transcriptomic analysis.[Results] We found a slight, but significant differential gene expression in the comparison between the coral- and macroalgal-DOM treatments in both the high microbial abundance sponge Plakortis angulospiculatus and the low microbial abundance sponge Haliclona vansoesti. In the hosts, processes that regulate immune response, signal transduction, and metabolic pathways related to cell proliferation were elicited. In the associated microbiota carbohydrate metabolism was upregulated in both treatments, but coral-DOM induced further lipid and amino acids biosynthesis, while macroalgal-DOM caused a stress response. These differences could be driven by the presence of distinct organic macronutrients in the two DOM sources and of small pathogens or bacterial virulence factors in the macroalgal-DOM.[Conclusions] This work provides two new sponge meta-transcriptomes and a database of putative genes and genetic pathways that are involved in the differential processing of coral- versus macroalgal-DOM as food source to sponges with high and low abundances of associated microbes. These pathways include carbohydrate metabolism, signaling pathways, and immune responses. However, the differences in the meta-transcriptomic responses of the sponge holobionts after 6 hours of feeding on the two DOM sources were small. Longer-term responses to both DOM sources should be assessed to evaluate how the metabolism and the ecological function of sponges will be affected when reefs shift from coral towards algal dominance.This work has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant number 715513; personal grant to JM de Goeij). AR was funded by the Spanish Ministry of Science and Innovation (RYC2018-024247-I and PID2019-105769GB-I00). EJ and GM were funded by the Research Priority Area Systems Biology of the University of Amsterdam.Peer reviewe

Antiviral RNAi Response in Culex quinquefasciatus-Derived HSU Cells

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