Biology subjects

Makunin, A.

Publications and source records attributed to Makunin, A..

11 recordsLinked to original sources

bioRxiv2026

Convergent evolutionary loss of chemosensory and blood-feeding pathways in non-blood-feeding mosquitoes

Complex traits that span multiple tissues and systems often integrate large numbers of genes across development, physiology, and behavior, making it challenging to identify their essential components. Blood feeding in mosquitoes is one such trait. It is ancestral to the mosquito family, maintained in most species for ~200 million years, and was independently lost in three lineages. These convergent losses offer a natural experiment to discover the genetic, physiological, and neural features required for blood feeding. We assembled high-quality, chromosome-level genomes for seven mosquito species, along with whole-brain tomographic reconstructions. Our study spanned the three known non-blood-feeding lineages (Toxorhynchites rutilus, Topomyia yanbarensis, and Malaya genurostris), blood-feeding relatives, and the variable blood feeder Wyeomyia smithii. Comparing orthologous gene clades, we detected convergent gene loss specific to the three lineages that had lost blood feeding. The losses include the salivary platelet-aggregation inhibitor Aegyptin, blood-activated serine proteases such as Chymotrypsin-1 and 2, and a carboxylesterase expressed in the female fat body and brain glia. The loss of blood feeding also extended to chemosensation. Non-blood feeders lack two odorant-binding protein clades, two ionotropic receptor clades associated with blood-component taste detection, and odorant receptor clades expressed in a discrete, strongly female-biased population of antennal neurons. Female-biased head gene expression was reduced in non-blood feeders. Finally, examination of whole-brain tomographic reconstructions across the species revealed smaller antennal lobes in non-blood-feeding females, consistent with reduced olfactory input. Together, these findings identify a compact set of genes, expression patterns, and brain regions associated with blood feeding, offering an evolutionary entry point for functional dissection of how this complex and dangerous trait is built and dismantled.

genomics↗

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

De novo assembly of complete Plasmodium falciparum isolate genomes using PacBio HiFi sequencing technology

Plasmodium falciparum possesses a highly structured genome with extensive sequence diversity concentrated in Variant Surface Antigen (VSA) families. These genes--var, rif, and stevor--play key roles in immune evasion and pathogenesis and are difficult to assemble using short-read sequencing technologies. Here, we applied PacBio HiFi long-read sequencing to generate high-quality de novo genome assemblies from 43 P. falciparum parasite cultures originating from community cases in The Gambia. Parasites were culture-adapted, cloned by limiting dilution where possible, and sequenced using high molecular weight DNA extracts. Assemblies from single-genotype lineages were constructed using hifiasm, producing complete chromosomal-length scaffolds with high base accuracy without requiring short-read polishing. We recovered full repertoires of var, rif, and stevor genes and classified them into known subgroups. Together, our results demonstrate that PacBio HiFi sequencing enables accurate assembly of complex P. falciparum genomes from natural infections. This work provides a valuable genomic resource for future studies of parasite evolution, transmission dynamics, and antigenic diversity, and suggests that VSA repertoires can serve as reliable proxies of genetic relatedness across infections.

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

Assessing the population genetic structure and demographic history of Anopheles gambiae and Anopheles arabiensis at island and mainland populations in Uganda: Implications for testing novel malaria vector control approaches.

Despite substantial investments in malaria control, the disease remains a major burden in sub-Saharan Africa, particularly Uganda. Novel tools such as gene drive systems are being developed to suppress malaria vector populations, but their deployment requires detailed knowledge of mosquito population genetics. We assessed the genetic structure, diversity, and demographic history of Anopheles gambiae and Anopheles arabiensis from six sites in Uganda: three islands in Lake Victoria and three mainland sites. A total of 2918 Anopheles gambiae and 173 Anopheles arabiensis were genotyped using targeted amplicon sequencing of 62 loci across coding and non-coding regions of the genome. Population structure analyses revealed clear separation between the two species but little differentiation within each species across sites. Pairwise FST values among An. gambiae populations were low (0.00054-0.028) but often significant, with mainland populations showing higher connectivity and island populations exhibiting greater isolation. Anopheles arabiensis mainland populations showed no statistically significant differentiation, suggesting panmixia. Principal Component Analysis and Bayesian clustering similarly distinguished species-level structure but no obvious substructure within sites. Mainland An. gambiae populations displayed higher nucleotide diversity than island populations, while An. arabiensis showed the lowest diversity overall. Tajimas D values were negative across sites, consistent with recent population expansions. Effective population size estimates indicated small populations at the islands (146 to 249) compared to large mainland populations (4,054 to 8,190). These findings demonstrate strong genetic differentiation between Anopheles gambiae and Anopheles arabiensis, and subtle but meaningful structure between island and mainland Anopheles gambiae populations. The reduced diversity and small effective population sizes at island sites suggest stronger genetic drift and limited gene flow, in contrast to the highly connected mainland populations. For malaria control, this contrast has direct implications. High connectivity among mainland populations may facilitate the spread of insecticide resistance alleles, while island populations, with their relative isolation and smaller sizes, may serve as suitable sites for contained field trials of gene drive strategies. This study highlights how geographic and ecological factors shape mosquito population structure and provides critical evidence for the design and monitoring of genetic-based vector control interventions.

evolutionary biology↗

bioRxiv2025

Forty New Genomes Shed Light on Sexual Reproduction and the Origin of Tetraploidy in Microsporidia

Microsporidia are single-celled, obligately intracellular parasites with growing public health, agricultural, and economic importance. Despite this, Microsporidia remain relatively enigmatic, with many aspects of their biology and evolution unexplored. Key questions include whether Microsporidia undergo sexual reproduction, and the nature of the relationship between tetraploid and diploid lineages. While few high-quality microsporidian genomes currently exist to help answer such questions, large-scale biodiversity genomics initiatives, such as the Darwin Tree of Life project, can generate high-quality genome assemblies for microsporidian parasites when sequencing infected host species. Here, we present 40 new microsporidian genome assemblies from infected arthropod hosts that were sequenced to create reference genomes. Out of the 40, 32 are complete genomes, eight of which are chromosome-level, and eight are partial microsporidian genomes. We characterised 14 of these as polyploid and five as diploid. We found that tetraploid genome haplotypes are consistent with autopolyploidy, in that they coalesce more recently than species, and that they likely recombine. Within some genomes, we found large-scale rearrangements between the homeologous genomes. We also observed a high rate of rearrangement between genomes from different microsporidian groups, and a striking tolerance for segmental duplications. Analysis of chromatin conformation capture (Hi-C) data indicated that tetraploid genomes are likely organised into two diploid compartments, similar to dikaryotic cells in fungi, with evidence of recombination within and between compartments. Together, our results provide evidence for the existence of a sexual cycle in Microsporidia, and suggest a model for the microsporidian lifecycle that mirrors fungal reproduction.

genomics↗

bioRxiv2024

Genomic diversity of the African malaria vector Anopheles funestus

Anopheles funestus s.s. is a formidable human malaria vector across sub-Saharan Africa. To understand how the species is evolving, especially in response to malaria vector control, we sequenced 656 modern specimens (collected 2014-2018) and 45 historic specimens (collected 1927-1967) from 16 African countries. We find high levels of genetic variation with clear and stable continental patterns. Six segregating inversions might be involved in adaptation of local ecotypes. Strong recent signals of selection centred on canonical insecticide resistance genes are shared by multiple populations. A promising gene drive target in An. gambiae is highly conserved in An. funestus. This work represents a significant advance in our understanding of the genetic diversity and population structure of An. funestus and will enable smarter targeted malaria control.

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

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↗