Host preference patterns in domestic and wild settings: Insights into Anopheles feeding behavior.

The adaptation of Anopheles malaria vectors to domestic settings is directly linked to their ability to feed on humans. The strength of this species-habitat association is unequal across the species within the genus, with the major vectors being particularly dependent on humans. However, our understanding of how blood-feeding behavior interacts with and adapts to environmental settings, including the presence of humans, remains limited. Using a field-based approach, we first investigated Anopheles community structure and feeding behavior patterns in domestic and sylvatic settings in La Lopé National Park in Gabon, Central Africa. We characterized the preference indices using a dual-host choice sampling approach across mosquito species, habitats, and seasons. We then quantified the plastic biting behavior of mosquito species in each habitat. We collected individuals from 16 Anopheles species that exhibited significant differences in species composition and abundance between sylvatic and domestic settings. The host-seeking behavior also varied among the seven most abundant species. The general attractiveness to each host, human or animal, remained relatively constant for each species, but with significant variations between habitats across species. These variations, to more generalist and to more anthropophilic behavior, were related to seasonal changes and distance from the village, respectively. Finally, we pointed out that the host choice of major malaria vectors changed in the absence of humans, revealing a plastic feeding behavior of these species. This study highlights the effect of humans on Anopheles distribution and feeding evolution. The characterization of feeding behavior in wild and domestic settings provides opportunities to better understand the interplay between genetic determinants of host preference and ecological factors. Our findings suggest that protected areas may offer alternative thriving conditions to major malaria vectors.

A single cell atlas of sexual development in Plasmodium falciparum.

The developmental decision made by malaria parasites to become sexual underlies all malaria transmission. Here, we describe a rich atlas of short- and long-read single-cell transcriptomes of over 37,000 Plasmodium falciparum cells across intraerythrocytic asexual and sexual development. We used the atlas to explore transcriptional modules and exon usage along sexual development and expanded it to include malaria parasites collected from four Malian individuals naturally infected with multiple P. falciparum strains. We investigated genotypic and transcriptional heterogeneity within and among these wild strains at the single-cell level, finding differential expression between different strains even within the same host. These data are a key addition to the Malaria Cell Atlas interactive data resource, enabling a deeper understanding of the biology and diversity of transmission stages.

Polyploidy is widespread in Microsporidia.

Microsporidia are obligate intracellular eukaryotic parasites with an extremely broad host range. They have both economic and public health importance. Ploidy in microsporidia is variable, with a few species formally identified as diploid and one as polyploid. Given the increase in the number of studies sequencing microsporidian genomes, it is now possible to assess ploidy levels across all currently explored microsporidian diversity. We estimate ploidy for all microsporidian data sets available on the Sequence Read Archive using k-mer-based analyses, indicating that polyploidy is widespread in Microsporidia and that ploidy change is dynamic in the group. Using genome-wide heterozygosity estimates, we also show that polyploid microsporidian genomes are relatively homozygous, and we discuss the implications of these findings on the timing of polyploidization events and their origin.IMPORTANCEMicrosporidia are single-celled intracellular parasites, distantly related to fungi, that can infect a broad range of hosts, from humans all the way to protozoans. Exploiting the wealth of microsporidian genomic data available, we use k-mer-based analyses to assess ploidy status across the group. Understanding a genome's ploidy is crucial in order to assemble it effectively and may also be relevant for better understanding a parasite's behavior and life cycle. We show that tetraploidy is present in at least six species in Microsporidia and that these polyploidization events are likely to have occurred independently. We discuss why these findings may be paradoxical, given that Microsporidia, like other intracellular parasites, have extremely small, reduced genomes.

A chromosomal reference genome sequence for the malaria mosquito, Anopheles gambiae, Giles, 1902, Ifakara strain.

We present a genome assembly from an individual female Anopheles gambiae (the malaria mosquito; Arthropoda; Insecta; Diptera; Culicidae), Ifakara strain. The genome sequence is 264 megabases in span. Most of the assembly is scaffolded into three chromosomal pseudomolecules with the X sex chromosome assembled. The complete mitochondrial genome was also assembled and is 15.4 kilobases in length.

Future of DNA-based insect monitoring.

Insects are crucial for ecosystem health but climate change and pesticide use are driving massive insect decline. To mitigate this loss, we need new and effective monitoring techniques. Over the past decade there has been a shift to DNA-based techniques. We describe key emerging techniques for sample collection. We suggest that the selection of tools should be broadened, and that DNA-based insect monitoring data need to be integrated more rapidly into policymaking. We argue that there are four key areas for advancement, including the generation of more complete DNA barcode databases to interpret molecular data, standardisation of molecular methods, scaling up of monitoring efforts, and integrating molecular tools with other technologies that allow continuous, passive monitoring based on images and/or laser imaging, detection, and ranging (LIDAR).

Regulators of male and female sexual development are critical for the transmission of a malaria parasite.

Malaria transmission to mosquitoes requires a developmental switch in asexually dividing blood-stage parasites to sexual reproduction. In Plasmodium berghei, the transcription factor AP2-G is required and sufficient for this switch, but how a particular sex is determined in a haploid parasite remains unknown. Using a global screen of barcoded mutants, we here identify genes essential for the formation of either male or female sexual forms and validate their importance for transmission. High-resolution single-cell transcriptomics of ten mutant parasites portrays the developmental bifurcation and reveals a regulatory cascade of putative gene functions in the determination and subsequent differentiation of each sex. A male-determining gene with a LOTUS/OST-HTH domain as well as the protein interactors of a female-determining zinc-finger protein indicate that germ-granule-like ribonucleoprotein complexes complement transcriptional processes in the regulation of both male and female development of a malaria parasite.

The genome sequence of the dark-edged bee fly, Bombylius major (Linnaeus, 1758).

We present a genome assembly from an individual male Bombylius major (the dark-edged bee fly; Arthropoda; Insecta; Diptera; Bombyliidae). The genome sequence is 304.3 megabases in span. The whole assembly is scaffolded into 7 chromosomal pseudomolecules, including the X and Y sex chromosomes. The mitochondrial genome has also been assembled and is 17.8 kilobases in length. Gene annotation of this assembly on Ensembl identified 10,852 protein coding genes.

High-resolution species assignment of Anopheles mosquitoes using k-mer distances on targeted sequences.

The ANOSPP amplicon panel is a genus-wide targeted sequencing panel to facilitate large-scale monitoring of Anopheles species diversity. Combining information from the 62 nuclear amplicons present in the ANOSPP panel allows for a more senstive and specific species assignment than single gene (e.g. COI) barcoding, which is desirable in the light of permeable species boundaries. Here, we present NNoVAE, a method using Nearest Neighbours (NN) and Variational Autoencoders (VAE), which we apply to k-mers resulting from the ANOSPP amplicon sequences in order to hierarchically assign species identity. The NN step assigns a sample to a species-group by comparing the k-mers arising from each haplotype's amplicon sequence to a reference database. The VAE step is required to distinguish between closely related species, and also has sufficient resolution to reveal population structure within species. In tests on independent samples with over 80% amplicon coverage, NNoVAE correctly classifies to species level 98% of samples within the An. gambiae complex and 89% of samples outside the complex. We apply NNoVAE to over two thousand new samples from Burkina Faso and Gabon, identifying unexpected species in Gabon. NNoVAE presents an approach that may be of value to other targeted sequencing panels, and is a method that will be used to survey Anopheles species diversity and Plasmodium transmission patterns through space and time on a large scale, with plans to analyse half a million mosquitoes in the next five years.

Single-cell transcriptomics reveals expression profiles of Trypanosoma brucei sexual stages.

Early diverging lineages such as trypanosomes can provide clues to the evolution of sexual reproduction in eukaryotes. In Trypanosoma brucei, the pathogen that causes Human African Trypanosomiasis, sexual reproduction occurs in the salivary glands of the insect host, but analysis of the molecular signatures that define these sexual forms is complicated because they mingle with more numerous, mitotically-dividing developmental stages. We used single-cell RNA-sequencing (scRNAseq) to profile 388 individual trypanosomes from midgut, proventriculus, and salivary glands of infected tsetse flies allowing us to identify tissue-specific cell types. Further investigation of salivary gland parasite transcriptomes revealed fine-scale changes in gene expression over a developmental progression from putative sexual forms through metacyclics expressing variant surface glycoprotein genes. The cluster of cells potentially containing sexual forms was characterized by high level transcription of the gamete fusion protein HAP2, together with an array of surface proteins and several genes of unknown function. We linked these expression patterns to distinct morphological forms using immunofluorescence assays and reporter gene expression to demonstrate that the kinetoplastid-conserved gene Tb927.10.12080 is exclusively expressed at high levels by meiotic intermediates and gametes. Further experiments are required to establish whether this protein, currently of unknown function, plays a role in gamete formation and/or fusion.

Why sequence all eukaryotes?

Life on Earth has evolved from initial simplicity to the astounding complexity we experience today. Bacteria and archaea have largely excelled in metabolic diversification, but eukaryotes additionally display abundant morphological innovation. How have these innovations come about and what constraints are there on the origins of novelty and the continuing maintenance of biodiversity on Earth? The history of life and the code for the working parts of cells and systems are written in the genome. The Earth BioGenome Project has proposed that the genomes of all extant, named eukaryotes-about 2 million species-should be sequenced to high quality to produce a digital library of life on Earth, beginning with strategic phylogenetic, ecological, and high-impact priorities. Here we discuss why we should sequence all eukaryotic species, not just a representative few scattered across the many branches of the tree of life. We suggest that many questions of evolutionary and ecological significance will only be addressable when whole-genome data representing divergences at all of the branchings in the tree of life or all species in natural ecosystems are available. We envisage that a genomic tree of life will foster understanding of the ongoing processes of speciation, adaptation, and organismal dependencies within entire ecosystems. These explorations will resolve long-standing problems in phylogenetics, evolution, ecology, conservation, agriculture, bioindustry, and medicine.

Standards recommendations for the Earth BioGenome Project.

A global international initiative, such as the Earth BioGenome Project (EBP), requires both agreement and coordination on standards to ensure that the collective effort generates rapid progress toward its goals. To this end, the EBP initiated five technical standards committees comprising volunteer members from the global genomics scientific community: Sample Collection and Processing, Sequencing and Assembly, Annotation, Analysis, and IT and Informatics. The current versions of the resulting standards documents are available on the EBP website, with the recognition that opportunities, technologies, and challenges may improve or change in the future, requiring flexibility for the EBP to meet its goals. Here, we describe some highlights from the proposed standards, and areas where additional challenges will need to be met.

The genome sequence of the blue-rayed limpet, Patella pellucida Linnaeus, 1758.

We present a genome assembly from an individual Patella pellucida (the blue-rayed limpet; Mollusca; Gastropoda; Patellidae). The genome sequence is 712 megabases in span. The majority of the assembly (99.85%) is scaffolded into 9 chromosomal pseudomolecules. The mitochondrial genome was assembled and is 14.9 kilobases in length.

A targeted amplicon sequencing panel to simultaneously identify mosquito species and Plasmodium presence across the entire Anopheles genus.

Anopheles is a diverse genus of mosquitoes comprising over 500 described species, including all known human malaria vectors. While a limited number of key vector species have been studied in detail, the goal of malaria elimination calls for surveillance of all potential vector species. Here, we develop a multilocus amplicon sequencing approach that targets 62 highly variable loci in the Anopheles genome and two conserved loci in the Plasmodium mitochondrion, simultaneously revealing both the mosquito species and whether that mosquito carries malaria parasites. We also develop a cheap, nondestructive, and high-throughput DNA extraction workflow that provides template DNA from single mosquitoes for the multiplex PCR, which means specimens producing unexpected results can be returned to for morphological examination. Over 1000 individual mosquitoes can be sequenced in a single MiSeq run, and we demonstrate the panel's power to assign species identity using sequencing data for 40 species from Africa, Southeast Asia, and South America. We also show that the approach can be used to resolve geographic population structure within An. gambiae and An. coluzzii populations, as the population structure determined based on these 62 loci from over 1000 mosquitoes closely mirrors that revealed through whole genome sequencing. The end-to-end approach is quick, inexpensive, robust, and accurate, which makes it a promising technique for very large-scale mosquito genetic surveillance and vector control.

A Minimally Morphologically Destructive Approach for DNA Retrieval and Whole-Genome Shotgun Sequencing of Pinned Historic Dipteran Vector Species.

Museum collections contain enormous quantities of insect specimens collected over the past century, covering a period of increased and varied insecticide usage. These historic collections are therefore incredibly valuable as genomic snapshots of organisms before, during, and after exposure to novel selective pressures. However, these samples come with their own challenges compared with present-day collections, as they are fragile and retrievable DNA is low yield and fragmented. In this article, we tested several DNA extraction procedures across pinned historic Diptera specimens from four disease vector genera: Anopheles, Aedes, Culex, and Glossina. We identify an approach that minimizes morphological damage while maximizing DNA retrieval for Illumina library preparation and sequencing that can accommodate the fragmented and low yield nature of historic DNA. We identify several key points in retrieving sufficient DNA while keeping morphological damage to a minimum: an initial rehydration step, a short incubation without agitation in a modified low salt Proteinase K buffer (referred to as "lysis buffer C" throughout), and critical point drying of samples post-extraction to prevent tissue collapse caused by air drying. The suggested method presented here provides a solid foundation for exploring the genomes and morphology of historic Diptera collections.

A single-cell atlas of Plasmodium falciparum transmission through the mosquito.

Malaria parasites have a complex life cycle featuring diverse developmental strategies, each uniquely adapted to navigate specific host environments. Here we use single-cell transcriptomics to illuminate gene usage across the transmission cycle of the most virulent agent of human malaria - Plasmodium falciparum. We reveal developmental trajectories associated with the colonization of the mosquito midgut and salivary glands and elucidate the transcriptional signatures of each transmissible stage. Additionally, we identify both conserved and non-conserved gene usage between human and rodent parasites, which point to both essential mechanisms in malaria transmission and species-specific adaptations potentially linked to host tropism. Together, the data presented here, which are made freely available via an interactive website, provide a fine-grained atlas that enables intensive investigation of the P. falciparum transcriptional journey. As well as providing insights into gene function across the transmission cycle, the atlas opens the door for identification of drug and vaccine targets to stop malaria transmission and thereby prevent disease.

Genetic variation at the Cyp6m2 putative insecticide resistance locus in Anopheles gambiae and Anopheles coluzzii.

BACKGROUND: The emergence of insecticide resistance is a major threat to malaria control programmes in Africa, with many different factors contributing to insecticide resistance in its vectors, Anopheles mosquitoes. CYP6M2 has previously been recognized as an important candidate in cytochrome P450-mediated detoxification in Anopheles. As it has been implicated in resistance against pyrethroids, organochlorines and carbamates, its broad metabolic activity makes it a potential agent in insecticide cross-resistance. Currently, allelic variation within the Cyp6m2 gene remains unknown. METHODS: Here, Illumina whole-genome sequence data from Phase 2 of the Anopheles gambiae 1000 Genomes Project (Ag1000G) was used to examine genetic variation in the Cyp6m2 gene across 16 populations in 13 countries comprising Anopheles gambiae and Anopheles coluzzii mosquitoes. To identify whether these alleles show evidence of selection either through potentially modified enzymatic function or by being linked to variants that change the transcriptional profile of the gene, hierarchical clustering of haplotypes, linkage disequilibrium, median joining networks and extended haplotype homozygosity analyses were performed. RESULTS: Fifteen missense biallelic substitutions at high frequency (defined as > 5% frequency in one or more populations) are found, which fall into five distinct haplotype groups that carry the main high frequency variants: A13T, D65A, E328Q, Y347F, I359V and A468S. Despite consistent reports of Cyp6m2 upregulation and metabolic activity in insecticide resistant Anophelines, no evidence of directional selection is found occurring on these variants or on the haplotype clusters in which they are found. CONCLUSION: These results imply that emerging resistance associated with Cyp6m2 is potentially driven by distant regulatory loci such as transcriptional factors rather than by its missense variants, or that other genes are playing a more significant role in conferring metabolic resistance.

Using scRNA-seq to Identify Transcriptional Variation in the Malaria Parasite Ookinete Stage.

The crossing of the mosquito midgut epithelium by the malaria parasite motile ookinete form represents the most extreme population bottleneck in the parasite life cycle and is a prime target for transmission blocking strategies. However, we have little understanding of the clonal variation that exists in a population of ookinetes in the vector, partially because the parasites are difficult to access and are found in low numbers. Within a vector, variation may result as a response to specific environmental cues or may exist independent of those cues as a potential bet-hedging strategy. Here we use single-cell RNA-seq to profile transcriptional variation in Plasmodium berghei ookinetes across different vector species, and between and within individual midguts. We then compare our results to low-input transcriptomes from individual Anopheles coluzzii midguts infected with the human malaria parasite Plasmodium falciparum. Although the vast majority of transcriptional changes in ookinetes are driven by development, we have identified candidate genes that may be responding to environmental cues or are clonally variant within a population. Our results illustrate the value of single-cell and low-input technologies in understanding clonal variation of parasite populations.

The genome sequence of the spiny starfish, Marthasterias glacialis (Linnaeus, 1758).

We present a genome assembly from an individual Marthasterias glacialis (the spiny starfish; Echinodermata; Asteroidea; Forcipulatida; Asteriidae). The genome sequence is 521 megabases in span. The majority of the assembly, 99.44%, is scaffolded into 22 chromosomal pseudomolecules. The mitochondrial genome has also been assembled, and is 16 kb in span.

Transmission of Artemisinin-Resistant Malaria Parasites to Mosquitoes under Antimalarial Drug Pressure.

Resistance to artemisinin-based combination therapy (ACT) in the Plasmodium falciparum parasite is threatening to reverse recent gains in reducing global deaths from malaria. While resistance manifests as delayed parasite clearance in patients, the phenotype can only spread geographically via the sexual stages and mosquito transmission. In addition to their asexual killing properties, artemisinin and its derivatives sterilize sexual male gametocytes. Whether resistant parasites overcome this sterilizing effect has not, however, been fully tested. Here, we analyzed P. falciparum clinical isolates from the Greater Mekong Subregion, each demonstrating delayed clinical clearance and known resistance-associated polymorphisms in the Kelch13 (PfK13var) gene. As well as demonstrating reduced asexual sensitivity to drug, certain PfK13var isolates demonstrated a marked reduction in sensitivity to artemisinin in an in vitro male gamete formation assay. Importantly, this same reduction in sensitivity was observed when the most resistant isolate was tested directly in mosquito feeds. These results indicate that, under artemisinin drug pressure, while sensitive parasites are blocked, resistant parasites continue transmission. This selective advantage for resistance transmission could favor acquisition of additional host-specificity or polymorphisms affecting partner drug sensitivity in mixed infections. Favored resistance transmission under ACT coverage could have profound implications for the spread of multidrug-resistant malaria beyond Southeast Asia.