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Summary: Mosquitoes, particularly Aedes aegypti, are critical vectors for globally significant pathogenic viruses. This study examines the limitations of oral RNA interference (RNAi) as a strategy to disrupt viral transmission by Ae. aegypti. We hypothesized that double-stranded RNA (dsRNA) targeting the Zika virus (ZIKV) or chikungunya virus (CHIKV) genomes produced by engineered bacterial symbionts could trigger an antiviral response. Mosquitoes mono-colonized with Escherichia coli producing dsZIK or dsCHIK did not display reduced viral titers following exposure to virus-contaminated bloodmeals and failed to generate dsZIK- or dsCHIK-derived small interfering RNAs. To address potential limitations of bacterial dsRNA release, we explored dsRNA inoculation via feeding and injection. Although viral replication was impeded in mosquitoes injected with dsZIK or dsCHIK, no antiviral effect was observed in dsRNA-fed mosquitoes. These findings highlight complexities of implementing oral RNAi as an antiviral strategy in Ae. aegypti and warrant further exploration of local and systemic RNAi mechanisms.

Abstract Secure environmental contexts are crucial for hominin interpretation and comparison. The discovery of a Denisovan individual and associated fauna at Tam Ngu Hao 2 (Cobra) Cave, Laos, dating back to 164–131 ka, allows for environmental comparisons between this (sub)tropical site and the Palearctic Denisovan sites of Denisova Cave (Russia) and Baishiya Karst Cave (China). Denisovans from northern latitudes foraged in a mix of forested and open landscapes, including tundra and steppe. Using stable isotope values from the Cobra Cave assemblage, we demonstrate that, despite the presence of nearby canopy forests, the Denisovan individual from Cobra Cave primarily consumed plants and/or animals from open forests and savannah. Using faunal evidence and proxy indicators of climates, results herein highlight a local expansion of rainforest at ~ 130 ka, raising questions about how Denisovans responded to this local climate change. Comparing the diet and habitat of the archaic hominin from Cobra Cave with those of early Homo sapiens from Tam Pà Ling Cave (46–43 ka), Laos, it appears that only our species was able to exploit rainforest resources.

Abstract The timing of the first arrival of Homo sapiens in East Asia from Africa and the degree to which they interbred with or replaced local archaic populations is controversial. Previous discoveries from Tam Pà Ling cave (Laos) identified H. sapiens in Southeast Asia by at least 46 kyr. We report on a recently discovered frontal bone (TPL 6) and tibial fragment (TPL 7) found in the deepest layers of TPL. Bayesian modeling of luminescence dating of sediments and U-series and combined U-series-ESR dating of mammalian teeth reveals a depositional sequence spanning ~86 kyr. TPL 6 confirms the presence of H. sapiens by 70 ± 3 kyr, and TPL 7 extends this range to 77 ± 9 kyr, supporting an early dispersal of H. sapiens into Southeast Asia. Geometric morphometric analyses of TPL 6 suggest descent from a gracile immigrant population rather than evolution from or admixture with local archaic populations.

Summary: Arthropod-borne viruses (arboviruses) transmitted by Aedes aegypti mosquitoes are an increasing threat to global health. The small interfering RNA (siRNA) pathway is considered the main antiviral immune pathway of insects, but its effective impact on arbovirus transmission is surprisingly poorly understood. Here, we use CRISPR-Cas9-mediated gene editing in vivo to mutate Dicer2, a gene encoding the RNA sensor and key component of the siRNA pathway. The loss of Dicer2 enhances early viral replication and systemic viral dissemination of four medically significant arboviruses (chikungunya, Mayaro, dengue, and Zika viruses) representing two viral families. However, Dicer2 mutants and wild-type mosquitoes display overall similar levels of vector competence. In addition, Dicer2 mutants undergo significant virus-induced mortality during infection with chikungunya virus. Together, our results define a multifaceted role for Dicer2 in the transmission of arboviruses by Ae. aegypti mosquitoes and pave the way for further mechanistic investigations.

Evidence for the presence of Homo during the Middle Pleistocene is limited in continental Southeast Asia. Here, the authors report a hominin molar from Tam Ngu Hao 2 (Cobra Cave), dated to 164–131 kyr. They use morphological and paleoproteomic analysis to show that it likely belonged to a female Denisovan.

The thalamus is organized into nuclei that have distinct input and output connectivities with the cortex. Whereas first-order (FO) nuclei - also called core nuclei - relay input from sensory organs on the body surface and project to primary cortical sensory areas, higherorder (HO) nuclei - matrix nuclei - instead receive their driver input from the cortex and project to secondary and associative areas within cortico-thalamo-cortical loops. Input-dependent processes have been shown to play a crucial role in the emergence of FO thalamic neuron identity from a ground-state HO neuron identity, yet how this identity emerges during development remains unknown. Here, using single-cell RNA sequencing of the developing mouse embryonic thalamus, we show that, although they are born together, HO neurons start differentiating earlier than FO neurons. Within the FO visual thalamus, postnatal peripheral input is crucial for the maturation of excitatory, but not inhibitory, neurons. Our findings reveal different differentiation tempos and input sensitivities of HO and FO neurons, and highlight neuron type-specific molecular differentiation programs in the developing thalamus. [ABSTRACT FROM AUTHOR]

Total RNA sequencing (RNA-seq) is an important tool in the study of mosquitoes and the RNA viruses they vector as it allows assessment of both host and viral RNA in specimens. However, there are two main constraints. First, as with many other species, abundant mosquito ribosomal RNA (rRNA) serves as the predominant template from which sequences are generated, meaning that the desired host and viral templates are sequenced far less. Second, mosquito specimens captured in the field must be correctly identified, in some cases to the sub-species level. Here, we generate mosquito rRNA datasets which will substantially mitigate both of these problems. We describe a strategy to assemble novel rRNA sequences from mosquito specimens and produce an unprecedented dataset of 234 full-length 28S and 18S rRNA sequences of 33 medically important species from countries with known histories of mosquito-borne virus circulation (Cambodia, the Central African Republic, Madagascar, and French Guiana). These sequences will allow both physical and computational removal of rRNA from specimens during RNA-seq protocols. We also assess the utility of rRNA sequences for molecular taxonomy and compare phylogenies constructed using rRNA sequences versus those created using the gold standard for molecular species identification of specimens—the mitochondrial cytochrome c oxidase I (COI) gene. We find that rRNA- and COI-derived phylogenetic trees are incongruent and that 28S and concatenated 28S+18S rRNA phylogenies reflect evolutionary relationships that are more aligned with contemporary mosquito systematics. This significant expansion to the current rRNA reference library for mosquitoes will improve mosquito RNA-seq metagenomics by permitting the optimization of species-specific rRNA depletion protocols for a broader range of species and streamlining species identification by rRNA sequence and phylogenetics.

Abstract The capability of Pleistocene hominins to successfully adapt to different types of tropical forested environments has long been debated. In order to investigate environmental changes in Southeast Asia during a critical period for the turnover of hominin species, we analysed palaeoenvironmental proxies from five late Middle to Late Pleistocene faunas. Human teeth discoveries have been reported at Duoi U’Oi, Vietnam (70–60 ka) and Nam Lot, Laos (86–72 ka). However, the use of palaeoproteomics allowed us to discard the latter, and, to date, no human remains older than ~ 70 ka are documented in the area. Our findings indicate that tropical rainforests were highly sensitive to climatic changes over that period, with significant fluctuations of the canopy forests. Locally, large-bodied faunas were resilient to these fluctuations until the cooling period of the Marine Isotope Stage 4 (MIS 4; 74–59 ka) that transformed the overall biotope. Then, under strong selective pressures, populations with new phenotypic characteristics emerged while some other species disappeared. We argue that this climate-driven shift offered new foraging opportunities for hominins in a novel rainforest environment and was most likely a key factor in the settlement and dispersal of our species during MIS 4 in SE Asia.

Summary: Transgenerational immune priming (TGIP) allows memory-like immune responses to be transmitted from parents to offspring in many invertebrates. Despite increasing evidence for TGIP in insects, the mechanisms involved in the transfer of information remain largely unknown. Here, we show that Drosophila melanogaster and Aedes aegypti transmit antiviral immunological memory to their progeny that lasts throughout generations. We observe that TGIP, which is virus and sequence specific but RNAi independent, is initiated by a single exposure to disparate RNA viruses and also by inoculation of a fragment of viral double-stranded RNA. The progeny, which inherit a viral DNA that is only a fragment of the viral RNA used to infect the parents, display enriched expression of genes related to chromatin and DNA binding. These findings represent a demonstration of TGIP for RNA viruses in invertebrates, broadly increasing our understanding of the immune response, host genome plasticity, and antiviral memory of the germline.

Summary: Diseases caused by mosquito-borne viruses have been on the rise for the last decades, and novel methods aiming to use laboratory-engineered mosquitoes that are incapable of carrying viruses have been developed to reduce pathogen transmission. This has stimulated efforts to identify optimal target genes that are naturally involved in mosquito antiviral defenses or required for viral replication. Here, we investigated the role of a member of the Tudor protein family, Tudor-SN, upon dengue virus infection in the mosquito Aedes aegypti. Tudor-SN knockdown reduced dengue virus replication in the midgut of Ae. aegypti females. In immunofluorescence assays, Tudor-SN localized to the nucleolus in both Ae. aegypti and Aedes albopictus cells. A reporter assay and small RNA profiling demonstrated that Tudor-SN was not required for RNA interference function in vivo. Collectively, these results defined a novel proviral role for Tudor-SN upon early dengue virus infection of the Ae. aegypti midgut. : Genetics; Virology Subject Areas: Genetics, Virology

Sensory input and neuronal activity are crucial for proper morphological development of neurons. Here, Frangeul and colleagues show that membrane excitability is a critical component of dendritic development in mouse somatosensory thalamocortical neurons.

Recent sequencing and assembly of the genome for the fungal pathogen Candida albicans used simple automated procedures for the identification of putative genes. We have reviewed the entire assembly, both by hand and with additional bioinformatic resources, to accurately map and describe 6,354 genes and to identify 246 genes whose original database entries contained sequencing errors (or possibly mutations) that affect their reading frame. Comparison with other fungal genomes permitted the identification of numerous fungus-specific genes that might be targeted for antifungal therapy. We also observed that, compared to other fungi, the protein-coding sequences in the C. albicans genome are especially rich in short sequence repeats. Finally, our improved annotation permitted a detailed analysis of several multigene families, and comparative genomic studies showed that C. albicans has a far greater catabolic range, encoding respiratory Complex 1, several novel oxidoreductases and ketone body degrading enzymes, malonyl-CoA and enoyl-CoA carriers, several novel amino acid degrading enzymes, a variety of secreted catabolic lipases and proteases, and numerous transporters to assimilate the resulting nutrients. The results of these efforts will ensure that the Candida research community has uniform and comprehensive genomic information for medical research as well as for future diagnostic and therapeutic applications.

Palm Creek virus (PCV) is an insect-specific flavivirus that can interfere with the replication of mosquito-borne flaviviruses in Culex mosquitoes, thereby potentially reducing disease transmission. We examined whether PCV could interfere with arbovirus replication in Aedes (Ae.) aegypti and Ae. albopictus mosquitoes, major vectors for many prominent mosquito-borne viral diseases. We infected laboratory colonies of Ae. aegypti and Ae. albopictus with PCV to evaluate infection dynamics. PCV infection was found to persist to at least 21 days post-infection and could be detected in the midguts and ovaries. We then assayed for PCV–arbovirus interference by orally challenging PCV-infected mosquitoes with Zika and chikungunya viruses. For both arboviruses, PCV infection had no effect on infection and transmission rates, indicating limited potential as a method of intervention for Aedes-transmitted arboviruses. We also explored the hypothesis that PCV–arbovirus interference is mediated by the small interfering RNA pathway in silico. Our findings indicate that RNA interference is unlikely to underlie the mechanism of arbovirus inhibition and emphasise the need for empirical examination of individual pairs of insect-specific viruses and arboviruses to fully understand their impact on arbovirus transmission.

Mosquitoes tolerate long-lasting viral infections, which makes them efficient vectors for human viral diseases. Here, the authors show that the tolerance of Aedesmosquitoes to infection with dengue or chikungunya viruses depends on the production of viral-derived DNA by cellular reverse transcriptases.

The mosquito antiviral response has mainly been studied in the context of arthropod-borne virus (arbovirus) infection in female mosquitoes. However, in nature, both female and male mosquitoes are frequently infected with insect-specific viruses (ISVs). ISVs are capable of infecting the reproductive organs of both sexes and are primarily maintained by vertical transmission. Since the RNA interference (RNAi)-mediated antiviral response plays an important antiviral role in mosquitoes, ISVs constitute a relevant model to study sex-dependent antiviral responses. Using a naturally generated viral stock containing three distinct ISVs, Aedes flavivirus (AEFV), Menghai rhabdovirus (MERV), and Shinobi tetra virus (SHTV), we infected adult Aedes albopictus females and males and generated small RNA libraries from ovaries, testes, and the remainder of the body. Overall, both female and male mosquitoes showed unique small RNA profiles to each co-infecting ISV regardless of the sex or tissue tested. While all three ISVs generated virus-derived siRNAs, only MERV generated virus-derived piRNAs. We also studied the expression of PIWI genes in reproductive tissues and carcasses. In contrast to Piwi5-9, Piwi1-4 were abundantly expressed in ovaries and testes, suggesting that Piwi5-9 are involved in exogenous viral piRNA production. Together, our results show that ISV-infected Aedes albopictus produce viral small RNAs in a virus-specific manner and that male mosquitoes mount a similar small RNA-mediated antiviral response to that of females.

Dengue virus (DENV) causes more human infections than any other mosquito-borne virus. The current lack of antiviral strategies has prompted genome-wide screens for host genes that are required for DENV infectivity. Earlier transcriptomic studies that identified DENV host factors in the primary vector Aedes aegypti used inbred laboratory colonies and/or pools of mosquitoes that erase individual variation. Here, we performed transcriptome sequencing on individual midguts in a field-derived Ae. aegypti population to identify new candidate host factors modulating DENV replication. We analyzed the transcriptomic data using an approach that accounts for individual co-variation between viral RNA load and gene expression. This approach generates a prediction about the agonist or antagonist effect of candidate genes on DENV replication based on the sign of the correlation between gene expression and viral RNA load. Using this method, we identified 39 candidate genes that went undetected by conventional pairwise comparison of gene expression levels between DENV-infected midguts and uninfected controls. Only four candidate genes were detected by both methods, emphasizing their complementarity. We demonstrated the value of our approach by functional validation of a candidate agonist gene encoding a sterol regulatory element-binding protein (SREBP), which was identified by correlation analysis but not by pairwise comparison. We confirmed that SREBP promotes DENV infection in the midgut by RNAi-mediated gene knockdown in vivo. We suggest that our approach for transcriptomic analysis can empower genome-wide screens for potential agonist or antagonist factors by leveraging inter-individual variation in gene expression. More generally, this method is applicable to a wide range of phenotypic traits displaying inter-individual variation.

During cortical development, the identity of major classes of long-distance projection neurons is established by the expression of molecular determinants, which become gradually restricted and mutually exclusive. However, the mechanisms by which projection neurons acquire their final properties during postnatal stages are still poorly understood. In this study, we show that the number of neurons co-expressing Ctip2 and Satb2, respectively involved in the early specification of subcerebral and callosal projection neurons, progressively increases after birth in the somatosensory cortex. Ctip2/Satb2 postnatal co-localization defines two distinct neuronal subclasses projecting either to the contralateral cortex or to the brainstem suggesting that Ctip2/Satb2 co-expression may refine their properties rather than determine their identity. Gain- and loss-of-function approaches reveal that the transcriptional adaptor Lmo4 drives this maturation program through modulation of epigenetic mechanisms in a time- and area-specific manner, thereby indicating that a previously unknown genetic program postnatally promotes the acquisition of final subtype-specific features.

Recent sequencing and assembly of the genome for the fungal pathogen Candida albicans used simple automated procedures for the identification of putative genes. We have reviewed the entire assembly, both by hand and with additional bioinformatic resources, to accurately map and describe 6,354 genes and to identify 246 genes whose original database entries contained sequencing errors (or possibly mutations) that affect their reading frame. Comparison with other fungal genomes permitted the identification of numerous fungus-specific genes that might be targeted for antifungal therapy. We also observed that, compared to other fungi, the protein-coding sequences in the C. albicans genome are especially rich in short sequence repeats. Finally, our improved annotation permitted a detailed analysis of several multigene families, and comparative genomic studies showed that C. albicans has a far greater catabolic range, encoding respiratory Complex 1, several novel oxidoreductases and ketone body degrading enzymes, malonyl-CoA and enoyl-CoA carriers, several novel amino acid degrading enzymes, a variety of secreted catabolic lipases and proteases, and numerous transporters to assimilate the resulting nutrients. The results of these efforts will ensure that the Candida research community has uniform and comprehensive genomic information for medical research as well as for future diagnostic and therapeutic applications.