Biology subjects

Lawniczak, M. K. N.

Publications and source records attributed to Lawniczak, M. K. N..

15 recordsLinked to original sources

bioRxiv2026

Exploring the diversity and spatial distribution of Anopheles mosquitoes and their associated Plasmodium species in Tanzania using the ANOSPP amplicon panel

Species-level identification of malaria vectors and parasites is essential for targeted vector control, surveillance, and clinical case management. However, routine surveillance often relies on morphology, species group-restricted PCR assays, and parasite-detection workflows optimised for Plasmodium falciparum, potentially masking wider vector and parasite diversity. To address this gap, we applied the previously published ANOSPP amplicon sequencing panel to Anopheles mosquitoes collected across 25 districts in mainland Tanzania between 2020 and 2023, using it to characterise vector and parasite diversity, evaluate routine species assignments, and examine how primary-vector occurrence related to national malaria endemicity patterns. Of 71,146 mosquitoes collected, 10,020 were morphologically identified as Anopheles. ANOSPP-based identification resolved 15 Anopheles taxa, including the first Tanzanian record of Anopheles funestus-like mosquitoes (n = 10) and the first resolution of Anopheles longipalpis to Type C in the country (n = 13). Using ANOSPP as the reference, morphology misclassified 6.2% of ANOSPP-analysed specimens, with Anopheles rufipes and Anopheles maculipalpis frequently misclassified, and Anopheles pretoriensis and Anopheles marshallii sensu lato completely missed. Five Plasmodium species were detected across 37 parasite-positive mosquitoes: P. falciparum, P. vivax, P. ovale, P. malariae, and P. caprae. Plasmodium caprae was detected only in Anopheles arabiensis (n = 6), representing the first record of this parasite in Tanzania and its first detection in An. arabiensis globally. Co-occurrence of An. arabiensis, An. funestus s.s., and An. gambiae s.s. was more common in higher malaria transmission strata, whereas occurrence of An. arabiensis alone predominated in very-low and low strata. These findings show that integrating ANOSPP into surveillance can substantially improve vector and parasite resolution, uncover overlooked diversity, and strengthen species-informed malaria surveillance. Author summaryEffective malaria control requires accurate data on mosquito species distributions and the parasites they carry. However, in many settings surveillance still relies mainly on morphological identification or molecular tests targeting limited vector groups, while parasite detection often focuses on Plasmodium falciparum. This limits understanding of mosquito and parasite diversity, obscuring transmission dynamics. We used the previously published ANOSPP amplicon panel to analyse Anopheles mosquitoes across Tanzania. This revealed hidden biological complexity with direct implications for malaria transmission, resolving 15 mosquito and five Plasmodium species. Notably, for the first time in Tanzania, we detected the goat-associated parasite Plasmodium caprae in Anopheles arabiensis and identified An. funestus-like, whose vectorial capacity remains unclear, and An. longipalpis Type C, implicated in malaria transmission in dryland Kenya. Interestingly, co-occurrence of all three major Tanzanian vectors aligned with higher transmission settings, whereas areas where An. arabiensis was the sole vector were mainly in lower transmission settings. These findings show that species-resolved surveillance can reveal hidden biological complexity and provide a stronger basis for locally targeted malaria control.

genomics↗

bioRxiv2026

High quality chromosomal genome assemblies of three human Plasmodium species directly from natural infections

Malaria, caused by mosquito-transmitted Plasmodium parasites, remains a major global health challenge. While P. falciparum accounts for most malaria deaths, the lesser-studied P. ovale spp. and P. malariae also contribute to disease burden across many endemic regions, typically causing milder but chronic infections. Here, we generated high-quality, chromosome-level reference genomes for P. ovale wallikeri, P. malariae, and P. falciparum directly from blood samples taken from natural infections. Using PacBio Ultra-Low-Input HiFi long-read sequencing and Hi-C chromatin conformation capture, we overcome limitations posed by low parasite biomass and the absence of in vitro culture for P. ovale wallikeri and P. malariae. These high quality reference genomes resolve previously inaccessible subtelomeric regions that include expanded gene families likely involved in host-parasite interactions, and they enhance our understanding of parasite persistence, transmission, immune evasion and comparative evolutionary studies. This work provides a critical foundation for including these neglected species in global malaria elimination efforts.

genomics↗

bioRxiv2025

An atlas of positive selection in the genomes of major malaria vectors

Insecticide resistance poses a major threat to malaria control efforts in Africa, yet our understanding of the genomic basis of adaptation in malaria vectors remains incomplete. Here we present a comprehensive atlas of recent positive selection in natural populations of the major African malaria vectors; the An. gambiae species complex and An. funestus. By analyzing whole-genome sequence data from 4,306 mosquitoes collected across twenty-one countries, we identify and characterize both known and novel genomic regions under strong positive selection. We develop an innovative approach for identifying and localising selection signals, and find extensive evidence for parallel evolution between species, with all important loci in An. funestus also under selection in An. gambiae s.l. We confirm intense selection at established resistance loci including known target site proteins (Vgsc, Rdl, Ace1) and metabolic enzymes (Cyp6p, Cyp9k1, Gste, Coeaexf) and describe novel signals at a diacylglycerol kinase on the X chromosome, and in An. gambiae s.l, at the metabolic regulator Keap1. To support real-time monitoring of emerging variants threatening vector control, we present an open-source web resource containing detailed selection scan results for all cohorts. The resource is driven by an automated computational workflow, which enables continuous updates as new whole-genome data are generated. The selection atlas provides a foundation for understanding vector adaptation to insecticide-based interventions and we hope will inform evidence-based resistance management strategies that are critical for sustaining malaria control.

genomics↗

bioRxiv2025

Breaking the cold chain: solutions for room temperature preservation of mosquitoes leading to high quality reference genomes

The Earth BioGenome Project (EBP) is a global endeavour to produce reference genomes for all described eukaryotic species. The majority of described species are arthropods, which tend to be small and require taxonomic expertise to identify to species level. Therefore, the ability to collect and preserve specimens in a suitable way for long read and Hi-C data generation using very simple approaches with minimal infrastructure is certain to be important in scaling up reference genome generation. Using Anopheles mosquitoes as an insect representative we evaluate how well different preservation liquids protect high molecular weight DNA, RNA, and nuclei for Hi-C when mosquitoes are held intact versus slightly squished. We find that squished samples stored in 100% ethanol and Allprotect held at room temperature for one week result in excellent preservation of both high molecular weight DNA and nuclei for Hi-C. Other tested buffers, including RNAlater, EDTA at several pHs, and DMSO Salt Solution (DESS) performed satisfactorily for long read data generation and RNA retrieval, but less ideally for Hi-C, which may have bigger negative impacts when aiming to generate data for organisms with larger genomes. Field collections requiring dry ice or dry shippers can be logistically challenging to arrange, are notoriously expensive, and DNA degrades rapidly if ultra-cold temperature is not maintained, which is devastating given how expensive and time consuming field work can be. Here we present multiple viable options for room temperature collection and/or shipment for arthropod samples. Further exploration across a broader range of species will hopefully enable cheaper and more widely available reference genome generation globally.

genomics↗

bioRxiv2025

Signatures of selection and mechanisms of insecticide resistance in Ugandan Anopheles funestus: Insights from embedding translational genomics into the LLINEUP cluster randomised trial.

In response to the emerging threat of insecticide resistance in malaria vectors, insecticides are being repurposed for vector control or developed de novo. Good stewardship of these finite new resources is essential if disease control programmes are to remain effective. This is dependent on timely data to help guide evidence-based decision-making for National Malaria Control programmes (NMCPs). By embedding genomics into cluster randomized control trials (cRCTs), we can perform surveillance and early detection of insecticide resistance variants to new and repurposed chemicals in natural field conditions, supporting effective stewardship. The LLIN Evaluation Uganda Project (LLINEUP) trial evaluated the efficacy of pyrethroid-piperonyl butoxide (PBO) and pyrethroids-only long-lasting insecticidal nets (LLINs). It was conducted in Uganda between 2017-2020 and was the largest cRCT to date, covering 40% of the country in 104 health sub-districts. We embedded genomic surveillance within LLINEUP to detect and track insecticide resistance variants. At baseline and throughout the trial, we sampled Anopheles mosquitoes with Prokopack aspirators and performed Illumina whole-genome sequencing. We show that An. funestus populations were relatively unaffected by the interventions, compared to An. gambiae s.l., which were markedly reduced six months following LLIN deployment. Standard approaches for describing genetic diversity and population structure e.g. fixation index (FST), Principal Component Analysis (PCA) and Neighbour-Joining (NJ) trees, were consistent with the density observations and suggestive of a single large An. funestus population in Uganda with little genetic differentiation. Genome-wide selection scans revealed strong signals of selection at the Resistance to pyrethroid-1 (RP1) locus and Cyp9k1, both loci previously implicated in pyrethroid resistance. We report two additional loci, eye diacylglycerol kinase (Dgk) ({cong}13.5Mb on the X chromosome) and O-mannosyl-transferase (TMC-like) ({cong}67.9Mb on 3RL) that showed signals of selection. Known DDT and permethrin resistance-associated variants at the Gste2 locus, L119F and L119V, were also identified. Over the trial period, changes in haplotype frequencies were observed in regions under selection, with more pronounced shifts in the PBO arm. Notably, there were significant reductions in the frequencies of swept haplotypes (measured by delta ({Delta}) H12) in the Dgk and Cyp6p9a regions, while significant increases in haplotype frequency were observed at Gste2 and Cyp9k1 loci. Our findings reveal the differential impact of the trial on An. gambiae s.l. and An. funestus densities and the differing responses of An. funestus populations to pyrethroid and pyrethroid-PBO selection pressure. These insights underscore the potential value of tailored, species- and region-specific vector control strategies, supported by regional genetic surveillance, to better control insecticide resistance evolution and spread. By embedding genomic surveillance in cRCTs we can facilitate the discovery of putative resistance variants and can provide evidence of their impact on vector control tool efficacy; both of crucial importance to evidence-based deployment of vector control tools by NMCPs.

genomics↗

bioRxiv2025

The 229 chromosomes of the Atlas blue butterfly reveal rules constraining chromosome evolution in Lepidoptera

Chromosomal arrangements are important for processes including genetic recombination, adaptation, and speciation. Related taxa often possess similar numbers of chromosomes, but some groups show remarkable variation in chromosome numbers. Most Lepidoptera, the butterflies and moths, have 31 or 32 chromosomes, but some species deviate from this norm. We present a chromosome-level genome assembly of a heterogametic female Atlas blue butterfly (Polyommatus atlantica; Lycaenidae), and find it has 227 autosomes and four sex chromosomes, the highest recorded chromosome number in non-polyploid Metazoa. We show that the 227 autosomes, exceptionally small even for Lepidoptera, are derived from extensive fragmentation of the 24 ancestral lycaenid autosomes. We show that autosomal fissions likely largely occurred in euchromatic, lightly-packed regions of chromosomes. We assemble two large Z chromosomes, one of which comprises the ancestral Z fused with an autosome and retains its ancestral length, while the other is a neo-Z, formed from the fusion of an intact ancestral autosome with a fragment of a second. We find two large W chromosomes, derived from copies of the Z-linked, ancestrally autosomal sequences. In contrast to the autosomes, the sex chromosomes have not experienced fission. We observe frequent presence of chromosome-internal arrays of the telomeric repeat motif in P. atlantica. Such arrays are not observed in the genomes of close relatives that have not undergone fission and suggest a possible mechanism for rapid, viable fragmentation. Altogether, our findings in P. atlantica make evident several constraints that govern karyotypic change, a key component of eukaryotic genome evolution.

evolutionary biology↗

bioRxiv2024

Discovery of knock-down resistance in the major malaria vector Anopheles funestus reveals the legacy of persistent DDT pollution.

A major mechanism of insecticide resistance in insect pests is knock-down resistance (kdr) caused by mutations in the voltage-gated sodium channel (Vgsc) gene. Despite being common in most malaria Anopheles vector species, kdr mutations have never been observed in Anopheles funestus, the principal malaria vector in Eastern and Southern Africa. While monitoring 10 populations of An. funestus in Tanzania, we unexpectedly found resistance to DDT, a banned insecticide, in one location. Through whole-genome sequencing of 333 An. funestus samples from these populations, we found 8 novel amino acid substitutions in the Vgsc gene, including the kdr variant, L976F (L1014F in An. gambiae), in tight linkage disequilibrium with another (P1842S). The mutants were found only at high frequency in one region, with a significant decline between 2017 and 2023. Notably, kdr L976F was strongly associated with survivorship to the exposure to DDT insecticide, while no clear association was noted with a pyrethroid insecticide (deltamethrin). Further study is necessary to identify the origin and spread of kdr in An. funestus, and the potential threat to current insecticide-based vector control in Africa. SignificanceKnock-down resistance (kdr) mutations confer resistance to malaria vector control insecticides and pose a grave threat to malaria control. Here, we report the first discovery of kdr in An. funestus, the principal malaria vector in East and Southern Africa. Kdr in An. funestus conferred resistance to DDT but not deltamethrin. Based on extensive DDT contamination and unofficial usage in Tanzania, it is possible that kdr emerged because of widespread organic pollution as opposed to through public health efforts. Regardless of origin, the discovery of kdr in An. funestus is an alarming development that warrants immediate, extensive follow-up and close surveillance to establish the origin, and extent to which it may threaten malaria control in An. funestus.

genetics↗

bioRxiv2023

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 a Malian individual naturally infected with multiple P. falciparum strains. We investigated genotypic and transcriptional heterogeneity within and among these wild strains at a single-cell level for the first time, finding considerable differential expression between different strains even within the same host. This work is a key addition to the Malaria Cell Atlas, enabling a deeper understanding of the biology and diversity of transmission stages. One sentence summaryThis addition to the Malaria Cell Atlas presents an analysis of sexual development and uses it to explore a natural infection.

genomics↗

bioRxiv2022

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 nuanced 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 haplotypes 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.

evolutionary biology↗

bioRxiv2021

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

The transmission of malaria parasites from vertebrate host to mosquito vector 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 ten genes essential for the formation of either male or female sexual forms and validate their importance for transmission. High-resolution single-cell transcriptomics of wild-type and mutant parasites portrays the developmental bifurcation and reveals a regulatory cascade of putative gene functions in determination and subsequent differentiation of each sex. A male-determining gene with a LOTUS/OST-HTH domain points towards unexpected conservation of molecular mechanisms of gametogenesis in animals and a distantly related eukaryotic parasite.

genomics↗

bioRxiv2021

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 to present-day collections, as they are fragile and retrievable DNA is low yield and fragmented. In this paper 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 low salt Proteinase K buffer, 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. Significance statementLarge museum collections of pinned insects could provide important snapshots of genomes through time, but unfortunately DNA retrieval from such fragile samples often leads to severe morphological damage, especially in delicate species such as disease transmitting Diptera. In this study we have worked on a combined method that minimizes morphological damage while maximizing the retrieval of DNA from dry pinned Diptera species. We identified the importance of tissue rehydration, gentle DNA lysis buffer incubation, and critical point drying to restore collapsed tissues. We hope this approach will make it possible for more historic insect specimens to become available for genomic research while ensuring they remain intact for morphological studies.

evolutionary biology↗

bioRxiv2020

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

BackgroundThe 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 mosquitoes. 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. ResultsHere, we use Illumina whole-genome sequence data from Phase 2 of the Anopheles gambiae 1000 Genomes Project (Ag1000G) to examine genetic variation in the Cyp6m2 gene across 16 populations in 13 countries comprising Anopheles gambiae and Anopheles coluzzii mosquitoes. We find 15 missense biallelic substitutions at high frequency (defined as >5% frequency in one or more populations), that fall into five distinct haplotype groups that carry the main high frequency variants: A13T, D65A, E328Q, Y347F, I359V and A468S. We examine 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. Despite consistent reports of Cyp6m2 upregulation and metabolic activity in insecticide resistant Anophelines, we find no evidence of directional selection occurring on these variants or on the haplotype clusters in which they are found. ConclusionOur 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.

genetics↗

bioRxiv2018

Whole genome sequencing reveals high complexity of copy number variation at insecticide resistance loci in malaria mosquitoes

BackgroundPolymorphisms in the copy number of a genetic region can influence gene expression, coding sequence and zygosity, making them powerful actors in the evolutionary process. Copy number variants (CNVs) are however understudied, being more difficult to detect than single nucleotide polymorphisms. We take advantage of the intense selective pressures on the major malaria vector Anopheles gambiae, caused by the widespread use of insecticides for malaria control, to investigate the role of CNVs in the evolution of insecticide resistance.\n\nResultsUsing the whole-genome sequencing data from 1142 samples in the An. gambiae 1000 genomes project, we identified 1557 independent increases in copy number, encompassing a total of 267 genes, which were enriched for gene families linked to metabolic insecticide resistance. The five major candidate genes for metabolic resistance were all found in at least one CNV, and were often the target of multiple independent CNVs, reaching as many as 16 CNVs in Cyp9k1. These CNVs have furthermore been spreading due to positive selection, indicated by high local CNV frequencies and extended haplotype homozygosity.\n\nConclusionsOur results demonstrate the importance of CNVs in the response to selection, with CNVs being closely associated with genes involved in the evolution of resistance to insecticides, highlighting the urgent need to identify their relative contributions to resistance and to track their spread as the application of insecticide in malaria endemic countries intensifies. Our detailed descriptions of CNVs found across the species range provides the tools to do so.

evolutionary biology↗

bioRxiv2017

Single-cell transcriptomics of malaria parasites

Single-cell RNA-sequencing is revolutionising our understanding of seemingly homogeneous cell populations, but has not yet been applied to single cell organisms. Here, we established a method to successfully investigate transcriptional variation across individual malaria parasites. We discover an unexpected, discontinuous program of transcription during asexual growth previously masked by bulk analyses, and uncover novel variation among sexual stage parasites in their expression of gene families important in host-parasite interactions.

genomics↗

bioRxiv2016

Rapid evolution of female-biased genes among four species of Anopheles malaria mosquitoes

Understanding how phenotypic differences between males and females arise from the sex-biased expression of nearly identical genomes can often reveal important insights into the biology and evolution of a species. Among Anopheles mosquito species, these phenotypic differences include vectorial capacity, as it is only females that blood feed and thus transmit human malaria. Here, we use RNA-seq data from multiple tissues of four vectors spanning the Anopheles phylogeny to explore the genomic and evolutionary properties of sex-biased genes. We find that in these mosquitoes, in contrast to what has been found in many other organisms, female-biased genes are more rapidly evolving in sequence, expression, and genic turnover, than male-biased genes. Our results suggests that this atypical pattern may be due to the combination of sex-specific life history challenges encountered by females, such as blood feeding. Furthermore, female propensity to only mate once in nature in male swarms likely diminishes sexual selection of post-reproductive traits related to sperm competition among males. We also develop a comparative framework to systematically explore tissue- and sex-specific splicing, to document its conservation throughout the genus and identify a set of candidate genes for future functional analyses of sex-specific isoform usage. Finally, our data reveals that the deficit of male-biased genes on the X chromosomes in Anopheles is a conserved feature in this genus and can be directly attributed to chromosome-wide transcriptional regulation that demasculinizes the X in male reproductive tissues.

genomics↗