Search bioRxiv⌕ Search

Biology subjects

Kristan, M.

Publications and source records attributed to Kristan, M..

14 recordsLinked to original sources

Large deletion variants in a Plasmodium falciparum ookinete protein gene and associations with different endemic populations and mosquito vectors

The Plasmodium falciparum PIMMS43 gene encodes an ookinete protein that is important for mosquito infection. Here, large indel variants within the coding sequence are identified, and frequencies in natural infections of humans and mosquitoes investigated. Comparing long-read genome sequences in a small panel of P. falciparum strains and related species revealed a 150 bp deletion in the central part of the gene in several strains including the standard 3D7 reference genome. Mapping of short-read genome sequence data from 524 P. falciparum infections from nine different countries to full-length PIMMS43 showed the 150 bp deletion and an alternative 90 bp deletion to be common structural variants. Across African populations, the 150 bp deletion had a mean allele frequency of 32%, and the 90 bp deletion a mean frequency of 12%, with significant geographical variation. Targeted genotyping of 30 oocysts from naturally infected mosquitoes in Tanzania by nested PCR showed overall deletion variant frequencies similar to those seen in human infections in the same country. Although sample size was limited, a difference in the variant frequencies in oocysts from Anopheles gambiae and Anopheles funestus suggests potential vector-specific selection. The findings highlight the importance of surveying structural genomic variation and its potential role in parasite adaptation. Data summaryThe long-read genome sequence data sources are listed in the Supplementary Information (Supplementary Table 1). The short-read genome sequence data from human infections were extracted as a selected subset of those in the Pf7 release of global data from the MalariaGEN Consortium as described in the Methods. The SNP and indel genotypes derived from each sample of parasites cultured in the laboratory and from natural mosquito infections are given in the Supplementary Information (Supplementary Tables 1 and 3, Supplementary Figure 1). Impact statementThis study identifies large indel polymorphisms in the malaria parasite transmission-stage gene PIMMS43, reflecting substantial structural variation previously overlooked by genome-wide SNP-focused analyses. Using long- and short-read genomic data across diverse samples followed by targeted genotyping, we show that deletion variants are frequent and geographically structured, with initial evidence consistent with potential vector-specific selection. This highlights the importance of incorporating structural variation into population genomic surveillance and studies of parasite adaptation.

genomics↗

The translatome of quiescent Plasmodium falciparum gametocytes reveals parasite pyridoxal 5'-phosphate (PLP) biosynthesis is essential for efficient mosquito stage development

The ability of Plasmodium falciparum gametocytes to remain quiescent within the vertebrate host but poised for rapid onward development in the mosquito is an adaptation essential to maximise the onward spread of malaria. In this dormant state, mature infectious stage V gametocytes are largely unaffected by most antimalarial drugs and our limited understanding of how gametocytes prepare for mosquito transmission has hindered the identification of new molecular targets for transmission-blocking therapeutics. In this study, we move beyond the total proteome of gametocytes and define the translatome of mature stage V gametocytes using L-azidohomoalanine incorporation into nascent proteins, click chemistry purification and proteomic analysis. We identify the proteins and pathways that gametocytes sustain in preparation for transmission during this dormant period and through genetic disruption, we validate this approach by demonstrating the importance of parasite pyridoxal 5-phosphate biosynthesis for mosquito transmission.

microbiology↗

The First Chromosome-Scale Genome Assembly for Dermacentor reticulatus: A Key Vector of Tick-Borne Pathogens of Public and Veterinary Health Importance in Europe

BackgroundDermacentor reticulatus is a key tick species across Europe and an established vector of multiple pathogens that affect both human and animal health. Despite its significant role in disease transmission, genomic data for this species remain limited. Here, we present the first chromosome-scale genome assembly of D. reticulatus, constructed using Oxford Nanopore long-read sequencing data. MethodsHigh molecular weight DNA was extracted from a female Dermacentor reticulatus collected in Devon, UK, and sequenced using Oxford Nanopore long-read technology. Genome assemblies were generated using both guided and unguided approaches, followed by repeat masking, gene prediction, and functional annotation. Genome completeness was assessed using BUSCO, and comparative, phylogenetic, and functional analyses were performed against other Dermacentor and tick species. ResultsThis chromosome-scale genome assembly revealed a repeat-rich genome, with approximately 61.9% of the total sequence consisting of repetitive elements. Benchmarking universal single-copy ortholog (BUSCO) analysis demonstrated strong genome completeness, with guided assembly (chromosome) achieving a score of 97.1%, closely matching those of related Dermacentor species. For comparison, unguided assembly (scaffold) yielded a BUSCO score of 96.7%. Gene annotation following repeat masking resulted in BUSCO completeness scores of 95.1% (guided) and 95.0% (unguided). Functional characterisation included Pfam domain assignment and Gene Ontology analysis. Additionally, we assembled the mitochondrial genome (15,103 bp), comprising 38 genes, providing further insight into D. reticulatus phylogenetic placement. ConclusionsThis genomic resource establishes a foundation for functional genomics, evolutionary studies and supports future research in vector biology and the control of tick-borne diseases.

genomics↗

Towards environmental detection, quantification, and molecular characterization of Anopheles stephensi and Aedes aegypti from larval breeding sites

The invasion and establishment of An. stephensi mosquitoes in the Horn of Africa represents a significant regional threat, which may jeopardise malaria control, particularly in urban areas which were formally free from disease transmission. Novel vector surveillance methods are urgently needed, both agnostic to mosquito larval morphology, and simple to implement at the sampling stage. Using new multiplex TaqMan assays, specifically targeting An. stephensi and Ae. aegypti, we validated the use of environmental DNA (eDNA) for simultaneous vector detection in shared artificial breeding sites. Study findings demonstrated that An. stephensi and Ae. aegypti eDNA deposited by as few as one second instar larva in 1L of water was detectable. Characterization of molecular insecticide resistance mechanisms, using novel amplicon-sequencing panels for both vector species, was possible from eDNA shed by as few as 32 second instar larvae in 50ml of water. An. stephensi eDNA, derived from emergent pupae for 24 hours, was remarkably stable, and still detectable ~2 weeks later. eDNA surveillance has the potential to be implemented in local endemic communities and points of country entry, to monitor the spread of invasive vector species. Further studies are required to validate the feasibility of this technique under field conditions.

molecular biology↗

Overabundance of Asaia and Serratia bacteria is associated with deltamethrin insecticide susceptibiltiy in Anopheles coluzzii from Agboville, Cote d'Ivoire

BackgroundInsecticide resistance among mosquito species is now a pervasive phenomenon, which threatens to jeopardise global malaria vector control efforts. Evidence of links between the mosquito microbiota and insecticide resistance is emerging, with significant enrichment of insecticide degrading bacteria and enzymes in resistant populations. Using 16S rRNA amplicon sequencing, we characterised and compared the microbiota of Anopheles (An.) coluzzii in relation to their deltamethrin resistance and exposure profiles. ResultsComparisons between 2-3 day old deltamethrin resistant and susceptible mosquitoes, demonstrated significant differences in microbiota diversity (PERMANOVA, pseudo-F = 19.44, p=0.0015). Ochrobactrum, Lysinibacillus and Stenotrophomonas genera, each of which comprised insecticide degrading species, were significantly enriched in resistant mosquitoes. Susceptible mosquitoes had a significant reduction in alpha diversity compared to resistant individuals (Shannon index: H=13.91, q=0.0003, Faiths phylogenetic diversity: H=6.68, q=0.01), with Asaia and Serratia dominating microbial profiles. There was no significant difference in deltamethrin exposed and unexposed 5-6 day old individuals, suggesting that insecticide exposure had minimal impact on microbial composition. Serratia and Asaia were also dominant in 5-6 day old mosquitoes, regardless of exposure or phenotype, and had reduced microbial diversity compared with 2-3 day old mosquitoes. ConclusionsOur findings revealed significant alterations of An. coluzzii microbiota associated with deltamethrin resistance, highlighting the potential for identification of novel microbial markers for insecticide resistance surveillance. qPCR detection of Serratia and Asaia was consistent with 16S rRNA sequencing, suggesting that population level field screening of the bacterial microbiota may be feasibly integrated into wider resistance monitoring if reliable and reproducible markers associated with phenotype can be identified.

microbiology↗

Genomic and microscopic evidence of stable high density and maternally inherited Wolbachia infections in Anopheles mosquitoes

Wolbachia, a widespread bacterium that can reduce pathogen transmission in mosquitoes, has been detected within populations of Anopheles (An.) malaria vectors. In the An. gambiae complex, the primary vectors in Sub-Saharan Africa, Wolbachia strains are at low density and infection frequencies in wild populations. PCR-independent evidence is required to determine whether Wolbachia strains are true endosymbionts in Anopheles given most studies to date have used nested-PCR to identify strains. Here we report high-density strains found in geographically diverse populations of An. moucheti and An. demeilloni. Fluorescent in situ hybridization localized a heavy infection in the ovaries of An. moucheti and maternal transmission was observed. Genome sequencing of both strains obtained genome depths and coverages comparable to other known infections. Notably, homologs of cytoplasmic incompatibility factor (cif) genes were present indicating these strains possess the capacity to induce the phenotype cytoplasmic incompatibility which allows Wolbachia to spread through populations. The characteristics of these two strains suggest they are ideal candidates for Wolbachia biocontrol strategies in Anopheles.

microbiology↗

Reduced long-lasting insecticidal net efficacy and pyrethroid insecticide resistance are associated with over-expression of CYP6P4, CYP6P3 and CYP6Z1 in populations of Anopheles coluzzii from South-East Cote d Ivoire

BackgroundResistance to major public health insecticides in Cote dIvoire has intensified and now threatens the long-term effectiveness of malaria vector control interventions. MethodsThis study evaluated the bioefficacy of conventional and next-generation long-lasting insecticidal nets (LLINs), determined resistance profiles, and characterized molecular and metabolic mechanisms in wild Anopheles coluzzii from South-East Cote dIvoire in 2019. ResultsPhenotypic resistance was intense: more than 25% of mosquitoes survived exposure to ten times the doses of pyrethroids required to kill susceptible populations. Similarly, 24-hour mortality to deltamethrin-only LLINs was very low and not significantly different to an untreated net. Sub-lethal pyrethroid exposure did not induce significant delayed vector mortality 72 hours later. In contrast, LLINs containing the synergist piperonyl butoxide (PBO), or new insecticides, clothianidin and chlorfenapyr, were highly toxic to An. coluzzii. Pyrethroid-susceptible An. coluzzii were significantly more likely to be infected with malaria, compared to those that survived insecticidal exposure. Pyrethroid resistance was associated with significant over-expression of CYP6P4, CPY6Z1 and CYP6P3. ConclusionsStudy findings raise concerns regarding the operational failure of standard LLINs and support the urgent deployment of vector control interventions incorporating PBO, chlorfenapyr or clothianidin in areas of high resistance intensity in Cote dIvoire.

genetics↗

Determination of the amount of insecticide picked up by mosquitoes from treated nets

BackgroundInsecticides used in vector control mostly rely on vectors being exposed through contact with treated surfaces, yet little is known about the amount picked up by the insect. Measuring this amount is relevant not only for determining the actual doses that are lethal to the mosquito, but also for understanding effects on the physiology and vector competence of mosquitoes. Insecticides at sub-lethal doses can affect both parasites developing inside mosquitoes and mosquito microbiota, hence it is important to understand the processes by which parasites are exposed to insecticide inside the insect. These doses will inevitably depend on the amount of insecticide that mosquitoes pick up when they come into contact with treated nets. MethodsThree to five days old non-blood fed female Anopheles coluzzii mosquitoes were exposed to a long-lasting insecticidal net (PermaNet 2.0 containing 55 mg/m2 deltamethrin), using a wire ball frame, for 0.5-5.0 minutes. Our in-house developed colorimetric test was used to visually detect the amount of deltamethrin on different parts of the mosquito (legs, heads, thoraxes, abdomens) following exposure to the net. The amount of insecticide picked up by mosquitoes from the net over a range of exposure times was measured using a high-performance liquid chromatography with diode array detection (HPLC-DAD). ResultsThe colorimetric test, designed to only detect the type 2 pyrethroids (i.e deltamethrin, -cypermethrin and {lambda}-cyhalothrin) on fabrics (e.g. ITNs) and sprayed walls, was successfully used for the first time to detect deltamethrin on mosquitoes following exposure to the net. The confirmatory HPLC-DAD analysis determined that after 2 min exposure up to 12 ng of deltamethrin adhered to mosquitoes following exposure to PermaNet 2.0 (mean = 5.2 ng/mosquito, SE = 1.9) and that the final dose depends on the length of exposure time. ConclusionsThis study demonstrated the potential of a screening (type 2 pyrethroid colorimetric test) and a confirmatory test (HPLC-DAD) to determine the amount of insecticide that adheres to mosquitoes on contact with treated surfaces. This has implications for a precise lethal dose determination and detection of specific insecticide that causes the greatest mosquito mortality in circumstances where mixtures of insecticides may be used to maximise effectiveness of interventions.

biochemistry↗

An assessment of adult mosquito collection techniques for studying species abundance and diversity in Maferinyah, Guinea

BackgroundGuinea is a West African country with a high prevalence of vector-borne diseases where few entomological studies have been undertaken. Although several mosquito collection methods are routinely used for surveillance in vector control programmes, they target different behaviours causing bias in species diversity and abundance. Given the paucity of mosquito trap data in West Africa, we compared the performance of five trap-lure combinations and Human Landing Catches (HLCs) in Guinea.\n\nMethodsFive mosquito traps were compared in a 5x5 Latin Square design for 15 days in three villages in Guinea between June and July 2018. CDC light traps, BG sentinel 2 traps (with BG and MB5 lures), gravid traps and Stealth traps were deployed for 24-hour intervals with mosquitoes collected every 12 hours (day and night collections). HLCs were also performed for 15 nights. A Generalised Linear Mixed Model was applied to compare the effect of the traps, sites and collection times on the mosquito abundance. Species identification was confirmed using PCR-based analysis and Sanger sequencing.\n\nResultsIn total, 10,610 mosquitoes were captured across all five traps. Significantly more mosquitoes (P<0.005) were collected by Stealth traps (7,096) compared to the rest of the traps. Stealth traps and BG sentinel 2 traps were the best at capturing An. gambiae and Ae. aegypti mosquitoes respectively. HLCs captured predominantly An. coluzzii (41%) and hybrids of An. gambiae s.s. / An. coluzzii (36%) in contrast to the five adult traps, which captured predominantly An. melas (83%). Senguelen (rural) presented the highest abundance of mosquitoes and overall diversity in comparison with Fandie (semi-rural) and Maferinyah Centre One (semi-urban). To our knowledge, four species are reported for the first time in Guinea.\n\nConclusionsStealth traps presented the best performance overall, suggesting that this trap may play an important role for mosquito surveillance in Guinea and similar sites in West Africa. We recommend the incorporation of molecular tools in entomological studies since it has helped to reveal, together with morphological identification, the presence of 25 mosquito species in this area.

ecology↗

Characterizing the molecular and metabolic mechanisms of insecticide resistance in Anopheles gambiae s.l. in Faranah, Guinea

BackgroundIn recent years, the scale-up of long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) has greatly reduced malaria transmission. However, malaria remains a global public health concern with the majority of disease burden in sub-Saharan Africa. Insecticide resistance is a growing problem among Anopheles vector populations, with potential implications for the continued effectiveness of available control interventions. Improved understanding of current resistance levels and underlying mechanisms is essential to design appropriate management strategies and to mitigate future selection for resistance.\n\nMethodsAnopheles gambiae s.l. mosquitoes were collected from three villages in Faranah Prefecture, Guinea and their levels of susceptibility to seven insecticides were measured using CDC resistance intensity bioassays. Synergist assays with piperonyl butoxide (PBO) were also undertaken to assess the role of elevated mixed-function oxidases in resistance. RNA was extracted from 563 individuals and PCR was performed on cDNA to determine vector species, presence of target site mutations (L1014F kdr, N1575Y and G119S Ace-1), Plasmodium falciparum infection, and relative expression of three metabolic genes (CYP6M2, CYP6P3 and GSTD3).\n\nResultsIn Faranah, resistance to permethrin and deltamethrin was observed, as well as possible resistance to bendiocarb. All assayed vector populations were fully susceptible to alpha-cypermethrin, pirimiphos-methyl, clothianidin and chlorfenapyr. Plasmodium falciparum infection was detected in 7.3% (37/508) mosquitoes tested. The L1014F kdr mutation was found in 100% of a sub-sample of 60 mosquitoes, supporting its fixation in the region. The N1575Y mutation was identified in 20% (113/561) of individuals, with ongoing selection evidenced by significant deviations from Hardy-Weinberg equilibrium. The G119S Ace-1 mutation was detected in 62.1% (18/29) of mosquitoes tested and was highly predictive of bendiocarb bioassay survival. The metabolic resistance genes, CYP6M2, CYP6P3 and GSTD3, were found to be overexpressed in wild resistant and susceptible An. gambiae s.s. populations, compared to a susceptible G3 colony. Furthermore, CYP6P3 was significantly overexpressed in bendiocarb survivors, implicating its potential role in carbamate resistance in Faranah.\n\nConclusionsIdentification of intense resistance to permethrin and deltamethrin in Faranah, is of concern, as the Guinea National Malaria Control Program (NMCP) relies exclusively on the distribution of pyrethroid-treated LLINs for vector control. Study findings will be used to guide current and future control strategies in the region.

genetics↗

MOSQUITO TRANSMISSION AND HUMAN HEPATOCYTE INFECTIONS WITH Plasmodium ovale curtisi AND P. ovale wallikeri

Human ovale malaria is caused by the two closely related species Plasmodium ovale curtisi and P. ovale wallikeri. Both species are known to relapse from quiescent hepatic forms months or years after the primary infection occurred. Although some studies have succeeded in establishing mosquito transmission for ovale malaria, none have specifically described transmission and human hepatocyte infection of both sibling species. Here we describe a simplified protocol for successful transmission of both P. ovale curtisi and P. ovale wallikeri to Anopheles coluzzii mosquitoes, and streamlined monitoring of infection using sensitive parasite DNA detection, by loop-activated amplification, in blood-fed mosquitoes. In one experimental infection with P. ovale curtisi and one with P. ovale wallikeri, viable sporozoites were isolated from mosquito salivary glands, and used to successfully infect cultured human hepatocytes. This protocol provides a method for the utilisation of pre-treatment clinical blood samples from ovale malaria patients, collected in EDTA, for mosquito infection studies and generation of the hepatic life cycle stages of P. ovale curtisi and P. ovale wallikeri. We also demonstrate the utility of LAMP as a rapid and sensitive alternative to dissection for estimating the prevalence of infection in Anopheles mosquitoes fed with Plasmodium-infected blood.

microbiology↗

Assessing the blood-host plasticity and dispersal rate of the malaria vector Anopheles coluzzii

Difficulties with observing the dispersal of insect vectors in the field have hampered understanding of several aspects of their behaviour linked to disease transmission. Here, a novel method based on detection of blood-meal sources is introduced to inform two critical and understudied mosquito behaviours: plasticity in the malaria vectors blood-host choice and vector dispersal. Strategically located collections of Anopheles coluzzii from a malaria-endemic village of southern Ghana showed statistically significant variation in host species composition of mosquito blood-meals. Trialling a new sampling approach gave the first estimates for the remarkably local spatial scale across which host choice is plastic. Using quantitative PCR, the blood-meal digestion was then quantified for field-caught mosquitoes and calibrated according to timed blood digestion in colony mosquitoes. We demonstrate how this new molecular Sella score approach can be used to estimate the dispersal rate of blood-feeding vectors caught in the field.

ecology↗

A systematic review and meta-analysis of the human blood index of the major African malaria vectors

BACKGROUNDThe proportion of mosquito blood-meals that are of human origin, referred to as the human blood index or HBI, is a key determinant of malaria transmission. We conducted a systematic review of the HBI for the major African malaria vectors.\n\nRESULTSEvidence is presented for higher HBI among Anopheles gambiae (M/S forms and An. coluzzii/An. gambiae s.s. are not distinguished for most studies and therefore combined) as well as An. funestus when compared with An. arabiensis (prevalence odds ratio adjusted for collection location [i.e. indoor or outdoor]: 1.62; 95%CI 1.09-2.42; 1.84; 95%CI 1.35-2.52, respectively). This finding is keeping with the entomological literature which describes An. arabiensis to be more zoophagic than the other major African vectors. However, analysis also revealed that HBI was more associated with location of mosquito captures (R2=0.29) than with mosquito (sibling) species (R2=0.11).\n\nCONCLUSIONSOur findings call into question the appropriateness of current methods of assessing host preferences among disease vectors and have important implications for strategizing vector control.

ecology↗

Novel Wolbachia strains in Anopheles malaria vectors from Sub-Saharan Africa

Anopheles (An.) mosquitoes contain bacteria that can influence Plasmodium parasites. Wolbachia, a common insect endosymbiont, has historically been considered absent from Anopheles but has recently been found in An. gambiae populations. Here, we assessed a range of Anopheles species from five malaria-endemic countries for Wolbachia and Plasmodium infection. Strikingly, we found Wolbachia infections in An. coluzzii, An. gambiae s.s, An. arabiensis, An. moucheti and An. species A increasing the number of Anopheles species known to be naturally infected by this endosymbiont. Molecular analysis suggests the presence of phylogenetically diverse novel strains, while qPCR and 16S rRNA sequencing indicates that Wolbachia is the dominant member of the microbiota in An. moucheti and An. species A. We found no evidence of Wolbachia/Asaia co-infections, and presence of these endosymbionts did not have significant effects on malaria prevalence. We discuss the importance of novel Wolbachia strains in Anopheles and potential implications for disease control.

microbiology↗