Exploring the molecular mechanisms of increased intensity of pyrethroid resistance in Central African population of a major malaria vector Anopheles coluzzii.

Molecular mechanisms driving the escalation of pyrethroid resistance in the major malaria mosquitoes of Central Africa remain largely uncharacterized, hindering effective management strategies. Here, resistance intensity and the molecular mechanisms driving it were investigated in a population of Anopheles coluzzii from northern Cameroon. High levels of pyrethroid and organochloride resistance were observed in An. coluzzii population, with no mortality for 1× permethrin; only 11% and 33% mortalities for 5× and 10× permethrin diagnostic concentrations, and <2% mortalities for deltamethrin and DDT, respectively. Moderate bendiocarb resistance (88% mortality) and full susceptibility to malathion were observed. Synergist bioassays with piperonyl butoxide recovered permethrin susceptibility, with mortalities increasing to 53.39%, and 87.30% for 5× and 10× permethrin, respectively, implicating P450 monooxygenases. Synergist bioassays with diethyl maleate (DEM) recovered permethrin and DDT susceptibilities (mortalities increasing to 34.75% and 14.88%, respectively), implicating glutathione S-transferases. RNA-seq-based genome-wide transcriptional analyses supported by quantitative PCR identified glutathione S-transferase, GSTe2 (RNA-seqFC = 2.93 and qRT-PCRFC = 8.4, p < 0.0043) and CYP450, CYP6Z2 (RNA-seqFC = 2.39 and qRT-PCRFC = 11.7, p < 0.0177) as the most overexpressed detoxification genes in the pyrethroid-resistant mosquitoes, compared to mosquitoes of the susceptible Ngousso colony. Other overexpressed genes include P450s, CYP6M2 (FC = 1.68, p < 0.0114), CYP4G16 (FC = 2.02, p < 0.0005), and CYP4G17 (FC = 1.86, p < 0.0276). While high frequency of the 1014F kdr mutation (50%) and low frequencies of 1014S (6.61%) and 1575Y (10.29%) were observed, no ace-1 mutation was detected in bendiocarb-resistant populations, suggesting the preeminent role of metabolic mechanism. Overexpression of metabolic resistance genes (including GSTe2 and CYP6Z2 known to confer resistance to multiple insecticides) in An. coluzzii from the Sudan Savannah of Cameroon highlights the need for alternative management strategies to reduce malaria burden in northern Cameroon.

A parasitic nematode induces dysbiosis in susceptible but not resistant gastropod hosts.

Animals' gut microbiomes affect a wide array of biological processes including immunity and protection from pathogens. However, how the microbiome changes due to infection by parasites is still largely unknown, as is how the microbiome changes in hosts that differ in their susceptibility to parasites. To investigate this, we exposed two slug species of differing susceptibility to the parasitic nematode Phasmarhabditis hermaphrodita (Deroceras reticulatum is highly susceptible and Ambigolimax valentianus resistant to the nematode) and profiled the gut microbiota after 7 and 14 days. Before infection, both slug species' microbiota was dominated by similar bacterial genera: Pseudomonas (by far the most abundant), Sphingobacterium, Pedobacter, Chryseobacterium, and Flavobacterium. In the resistant host A. valentianus, there was no significant change in the bacterial genera after infection, but in D. reticulatum, the bacterial profile changed, with a decrease in the abundance of Pseudomonadaceae and an increase in the abundance of Flavobacteriaceae and Sphingobacteriaceae after 7 days postinfection. This suggests nematode infection causes dysbiosis in hosts that are susceptible to infection, but the microbiome of resistant species remains unaltered. In summary, the regulation of the immune system is tightly linked with host survival, and nematode infection can alter the microbiome structure.

Molecular drivers of insecticide resistance in the Sahelo-Sudanian populations of a major malaria vector Anopheles coluzzii.

BACKGROUND: Information on common markers of metabolic resistance in malaria vectors from countries sharing similar eco-climatic characteristics can facilitate coordination of malaria control. Here, we characterized populations of the major malaria vector Anopheles coluzzii from Sahel region, spanning four sub-Saharan African countries: Nigeria, Niger, Chad and Cameroon. RESULTS: Genome-wide transcriptional analysis identified major genes previously implicated in pyrethroid and/or cross-resistance to other insecticides, overexpressed across the Sahel, including CYP450s, glutathione S-transferases, carboxylesterases and cuticular proteins. Several, well-known markers of insecticide resistance were found in high frequencies-including in the voltage-gated sodium channel (V402L, I940T, L995F, I1527T and N1570Y), the acetylcholinesterase-1 gene (G280S) and the CYP4J5-L43F (which is fixed). High frequencies of the epidemiologically important chromosomal inversion polymorphisms, 2La, 2Rb and 2Rc, were observed (~80% for 2Rb and 2Rc). The 2La alternative arrangement is fixed across the Sahel. Low frequencies of these inversions (<10%) were observed in the fully insecticide susceptible laboratory colony of An. coluzzii (Ngoussou). Several of the most commonly overexpressed metabolic resistance genes sit in these three inversions. Two commonly overexpressed genes, GSTe2 and CYP6Z2, were functionally validated. Transgenic Drosophila melanogaster flies expressing GSTe2 exhibited extremely high DDT and permethrin resistance (mortalities <10% in 24h). Serial deletion of the 5' intergenic region, to identify putative nucleotide(s) associated with GSTe2 overexpression, revealed that simultaneous insertion of adenine nucleotide and a transition (T->C), between Forkhead box L1 and c-EST putative binding sites, were responsible for the high overexpression of GSTe2 in the resistant mosquitoes. Transgenic flies expressing CYP6Z2 exhibited marginal resistance towards 3-phenoxybenzylalcohol (a primary product of pyrethroid hydrolysis by carboxylesterases) and a type II pyrethroid, α-cypermethrin. However, significantly higher mortalities were observed in CYP6Z2 transgenic flies compared with controls, on exposure to the neonicotinoid, clothianidin. This suggests a possible bioactivation of clothianidin into a toxic intermediate, which may make it an ideal insecticide against populations of An. coluzzii overexpressing this P450. CONCLUSIONS: These findings will facilitate regional collaborations within the Sahel region and refine implementation strategies through re-focusing interventions, improving evidence-based, cross-border policies towards local and regional malaria pre-elimination.

The Colorectal Cancer Gut Environment Regulates Activity of the Microbiome and Promotes the Multidrug Resistant Phenotype of ESKAPE and Other Pathogens.

Taxonomic composition of the gut microbiota in colorectal cancer (CRC) patients is altered, a newly recognized driving force behind the disease, the activity of which has been overlooked. We conducted a pilot study on active microbial taxonomic composition in the CRC gut via metatranscriptome and 16S rRNA gene (rDNA) sequencing. We revealed sub-populations in CRC (n = 10) and control (n = 10) cohorts of over-active and dormant species, as changes in activity were often independent from abundance. Strikingly, the diseased gut significantly influenced transcription of butyrate producing bacteria, clinically relevant ESKAPE, oral, and Enterobacteriaceae pathogens. A focused analysis of antibiotic (AB) resistance genes showed that both CRC and control microbiota displayed a multidrug resistant phenotype, including ESKAPE species. However, a significant majority of AB resistance determinants of several AB families were upregulated in the CRC gut. We found that environmental gut factors regulated AB resistance gene expression in vitro of aerobic CRC microbiota, specifically acid, osmotic, and oxidative pressures in a predominantly health-dependent manner. This was consistent with metatranscriptome analysis of these cohorts, while osmotic and oxidative pressures induced differentially regulated responses. This work provides novel insights into the organization of active microbes in CRC, and reveals significant regulation of functionally related group activity, and unexpected microbiome-wide upregulation of AB resistance genes in response to environmental changes of the cancerous gut. IMPORTANCE The human gut microbiota in colorectal cancer patients have a distinct population compared to heathy counterparts. However, the activity (gene expression) of this community has not been investigated. Following quantification of both expressed genes and gene abundance, we established that a sub-population of microbes lies dormant in the cancerous gut, while other groups, namely, clinically relevant oral and multi-drug resistant pathogens, significantly increased in activity. Targeted analysis of community-wide antibiotic resistance determinants found that their expression occurs independently of antibiotic treatment, regardless of host health. However, its expression in aerobes, in vitro, can be regulated by specific environmental stresses of the gut, including organic and inorganic acid pressure in a health-dependent manner. This work advances the field of microbiology in the context of disease, showing, for the first time, that colorectal cancer regulates activity of gut microorganisms and that specific gut environmental pressures can modulate their antibiotic resistance determinants expression.

The Colorectal Cancer Microbiota Alter Their Transcriptome To Adapt to the Acidity, Reactive Oxygen Species, and Metabolite Availability of Gut Microenvironments.

The gut microbiome is implicated in the pathology of colorectal cancer (CRC). However, the mechanisms by which the microbiota actively contribute to disease onset and progression remain elusive. In this pilot study, we sequenced fecal metatranscriptomes of 10 non-CRC and 10 CRC patient gut microbiomes and conducted differential gene expression analyses to assess any changed functionality in disease. We report that oxidative stress responses were the dominant activity across cohorts, an overlooked protective housekeeping role of the human gut microbiome. However, expression of hydrogen peroxide and nitric oxide-scavenging genes was diminished and augmented, respectively, positing that these regulated microbial responses have implications for CRC pathology. CRC microbes enhanced expression of genes for host colonization, biofilm formation, genetic exchange, virulence determinants, antibiotic, and acid resistances. Moreover, microbes promoted transcription of genes involved in metabolism of several beneficial metabolites, suggesting their contribution to patient metabolite deficiencies previously solely attributed to tumor cells. We showed in vitro that expression of genes involved in amino acid-dependent acid resistance mechanisms of meta-gut Escherichia coli responded differently to acid, salt, and oxidative pressures under aerobic conditions. These responses were mostly dictated by the host health status of origin of the microbiota, suggesting their exposure to fundamentally different gut conditions. These findings for the first time highlight mechanisms by which the gut microbiota can either protect against or drive colorectal cancer and provide insights into the cancerous gut environment that drives functional characteristics of the microbiome. IMPORTANCE The human gut microbiota has the genetic potential to drive colorectal cancer onset and progression; however, the expression of this genetic potential during the disease has not been investigated. We found that microbial expression of genes that detoxify DNA-damaging reactive oxygen species, which drive colorectal cancer, is compromised in cancer. We observed a greater activation of expression of genes involved in virulence, host colonization, exchange of genetic material, metabolite utilization, defense against antibiotics, and environmental pressures. Culturing gut Escherichia coli of cancerous and noncancerous metamicrobiota revealed different regulatory responses of amino acid-dependent acid resistance mechanisms in a health-dependent manner under environmental acid, oxidative, and osmotic pressures. Here, for the first time, we demonstrate that the activity of microbial genomes is regulated by the health status of the gut in vivo and in vitro and provides new insights for shifts in microbial gene expression in colorectal cancer.

Case Report: Simple Nodular Cutaneous Leishmaniasis Caused by Autochthonous Leishmania (Mundinia) orientalis in an 18-Month-Old Girl: The First Pediatric Case in Thailand and Literature Review.

We report an autochthonous case of simple, localized cutaneous leishmaniasis in a healthy 18-month-old girl from southern Thailand. The patient presented with a solitary chronic cutaneous nodular lesion on her left cheek for approximately 1 year. Histopathological dissection of the cheek skin biopsy demonstrated remarkably nodular and interstitial infiltrates of lymphocytes and histiocytes full of intracellular oval-shaped amastigotes, consistent with cutaneous leishmaniasis. The Leishmania promastigotes were also cultured successfully from the lesion biopsy and were designated with the WHO code MHOM/TH/2021/CULE5. Using internal transcribed spacer 1-specific polymerase chain reaction, the parasite DNA was demonstrated in both saliva and lesion biopsy. Based on the BLASTn and phylogenetic analysis, the parasite was identified as Leishmania orientalis, clustered in the Mundinia subgenus. The patient responded well to a 6-week course of oral itraconazole, without recurrence. To our knowledge, this is the fourth case of autochthonous leishmaniasis resulting from L. orientalis and the youngest patient of leishmaniasis ever reported in Thailand. More importantly, we also demonstrate the clinical course of the lesion according to the timeline before and after treatment, which can help physicians better understand and provide an accurate diagnosis with appropriate treatment of this emerging parasitic disease.

Gene Conversion Explains Elevated Diversity in the Immunity Modulating APL1 Gene of the Malaria Vector Anopheles funestus.

Leucine-rich repeat proteins and antimicrobial peptides are the key components of the innate immune response to Plasmodium and other microbial pathogens in Anopheles mosquitoes. The APL1 gene of the malaria vector Anopheles funestus has exceptional levels of non-synonymous polymorphism across the range of An. funestus, with an average πn of 0.027 versus a genome-wide average of 0.002, and πn is consistently high in populations across Africa. Elevated APL1 diversity was consistent between the independent pooled-template and target-enrichment datasets, however no link between APL1 diversity and insecticide resistance was observed. Although lacking the diversity of APL1, two further mosquito innate-immunity genes of the gambicin anti-microbial peptide family had πn/πs ratios greater than one, possibly driven by either positive or balancing selection. The cecropin antimicrobial peptides were expressed much more highly than other anti-microbial peptide genes, a result discordant with current models of anti-microbial peptide activity. The observed APL1 diversity likely results from gene conversion between paralogues, as evidenced by shared polymorphisms, overlapping read mappings, and recombination events among paralogues. In conclusion, we hypothesize that higher gene expression of APL1 than its paralogues is correlated with a more open chromatin formation, which enhances gene conversion and elevated diversity at this locus.

Multi-omics analysis identifies a CYP9K1 haplotype conferring pyrethroid resistance in the malaria vector Anopheles funestus in East Africa.

Metabolic resistance to pyrethroids is a menace to the continued effectiveness of malaria vector controls. Its molecular basis is complex and varies geographically across Africa. Here, we used a multi-omics approach, followed-up with functional validation to show that a directionally selected haplotype of a cytochrome P450, CYP9K1 is a major driver of resistance in Anopheles funestus. A PoolSeq GWAS using mosquitoes alive and dead after permethrin exposure, from Malawi and Cameroon, detected candidate genomic regions, but lacked consistency across replicates. Targeted sequencing of candidate resistance genes detected several SNPs associated with known pyrethroid resistance QTLs. The most significant SNPs were in the cytochrome P450 CYP304B1 (Cameroon), CYP315A1 (Uganda) and the ABC transporter gene ABCG4 (Malawi). However, when comparing field resistant mosquitoes to laboratory susceptible, the pyrethroid resistance locus rp1 and SNPs around the ABC transporter ABCG4 were consistently significant, except for Uganda where SNPs in the P450 CYP9K1 was markedly significant. In vitro heterologous metabolism assays with recombinant CYP9K1 revealed that it metabolises type II pyrethroid (deltamethrin; 64% depletion) but not type I (permethrin; 0%), while moderately metabolising DDT (17%). CYP9K1 exhibited reduced genetic diversity in Uganda underlying an extensive selective sweep. Furthermore, a glycine to alanine (G454A) amino acid change in CYP9K1 was fixed in Ugandan mosquitoes but not in other An. funestus populations. This study sheds further light on the evolution of metabolic resistance in a major malaria vector by implicating more genes and variants that can be used to design field-applicable markers to better track resistance Africa-wide.

Microbiome Analysis of Malacopathogenic Nematodes Suggests No Evidence of a Single Bacterial Symbiont Responsible for Gastropod Mortality.

Nematodes and bacteria are prevalent in soil ecosystems, and some have evolved symbiotic relationships. In some cases, symbionts carry out highly specialized functions: a prime example being entomopathogenic nematodes (EPNs), which vector bacteria (Xenorhabdus or Photorhabdus) into insect hosts, killing them to provide a food source for the nematodes. It is thought that the commercially available malacopathogenic (kills slugs and snails) biocontrol nematode Phasmarhabditis hermaphrodita vectors a bacterium (Moraxella osloensis) into slugs to kill them. To investigate this further we used a metagenomic approach to profile the bacteria present in the commercial strain of P. hermaphrodita, a wild strain of P. hermaphrodita and two other Phasmarhabditis species (P. californica and P. neopapillosa), after they had killed their slug host (Deroceras invadens). We show that these nematodes do not exclusively associate with one bacterium but a range of species, with members of the phyla Pseudomonadota, Bacillota, Actinobacteriota and Bacteroidota the most prevalent. The commercial strain of P. hermaphrodita had the least diverse bacterial community. Furthermore, we found that the bacterium P. hermaphrodita has been cultured on for 25 years is not the expected species M. osloensis but is Psychrobacter spp. and the only strain of the Phasmarhabditis species to associate with Psychrobacter spp. was the commercial strain of P. hermaphrodita. In summary, we found no evidence to show that P. hermaphrodita rely exclusively on one bacterium to cause host mortality but found variable and diverse bacterial communities associated with these nematodes in their slug hosts.

Characterization of the glutathione S-transferase genes in the sand flies Phlebotomus papatasi and Lutzomyia longipalpis shows expansion of the novel glutathione S-transferase xi (X) class.

Leishmaniasis control often relies upon insecticidal control of phlebotomine sandfly vector populations. Such methods are vulnerable to the evolution of insecticide resistance via a range of molecular mechanisms. There is evidence that two major resistance mechanisms, target site insensitivity and metabolic resistance, have evolved in some sandfly populations and further genetic characterization of resistance would be useful to understand and combat it. To facilitate the study of the mechanisms of metabolic resistance, here we improved the annotation and characterized a major detoxification gene family, the glutathione-s-transferases (GST), in the genomes of two sand fly species: Phlebotomus papatasi and Lutzomyia longipalpis. The compositions of the GST gene family differ markedly from those of Aedes and Anopheles mosquitoes. Most strikingly, the xi (X) class of GSTs appears to have expanded in both sand fly genomes. Our results provide a basis for further studies of metabolic resistance mechanisms in these important disease vector species.

Malaria in the 'Omics Era'.

Genomics has revolutionised the study of the biology of parasitic diseases. The first Eukaryotic parasite to have its genome sequenced was the malaria parasite Plasmodium falciparum. Since then, Plasmodium genomics has continued to lead the way in the study of the genome biology of parasites, both in breadth-the number of Plasmodium species' genomes sequenced-and in depth-massive-scale genome re-sequencing of several key species. Here, we review some of the insights into the biology, evolution and population genetics of Plasmodium gained from genome sequencing, and look at potential new avenues in the future genome-scale study of its biology.

Taxon-Specific Proteins of the Pathogenic Entamoeba Species E. histolytica and E. nuttalli.

The human protozoan parasite Entamoeba histolytica can live in the human intestine for months or years without generating any symptoms in the host. For unknown reasons, amoebae can suddenly destroy the intestinal mucosa and become invasive. This can lead to amoebic colitis or extraintestinal amoebiasis whereby the amoebae spread to other organs via the blood vessels, most commonly the liver where abscesses develop. Entamoeba nuttalli is the closest genetic relative of E. histolytica and is found in wild macaques. Another close relative is E. dispar, which asyptomatically infects the human intestine. Although all three species are closely related, only E. histolytica and E. nuttalli are able to penetrate their host's intestinal epithelium. Lineage-specific genes and gene families may hold the key to understanding differences in virulence among species. Here we discuss those genes found in E. histolytica that have relatives in only one or neither of its sister species, with particular focus on the peptidase, AIG, Ariel, and BspA families.

A differential expression of pyrethroid resistance genes in the malaria vector Anopheles funestus across Uganda is associated with patterns of gene flow.

BACKGROUND: Insecticide resistance is challenging the effectiveness of insecticide-based control interventions to reduce malaria burden in Africa. Understanding the molecular basis of insecticides resistance and patterns of gene flow in major malaria vectors such as Anopheles funestus are important steps for designing effective resistance management strategies. Here, we investigated the association between patterns of genetic structure and expression profiles of genes involved in the pyrethroid resistance in An. funestus across Uganda and neighboring Kenya. METHODS: Blood-fed mosquitoes An. funestus were collected across the four localities in Uganda and neighboring Kenya. A Microarray-based genome-wide transcription analysis was performed to identify the set of genes associated with permethrin resistance. 17 microsatellites markers were genotyped and used to establish patterns of genetic differentiation. RESULTS: Microarray-based genome-wide transcription profiling of pyrethroid resistance in four locations across Uganda (Arua, Bulambuli, Lira, and Tororo) and Kenya (Kisumu) revealed that resistance was mainly driven by metabolic resistance. The most commonly up-regulated genes in pyrethroid resistance mosquitoes include cytochrome P450s (CYP9K1, CYP6M7, CYP4H18, CYP4H17, CYP4C36). However, expression levels of key genes vary geographically such as the P450 CYP6M7 [Fold-change (FC) = 115.8 (Arua) vs 24.05 (Tororo) and 16.9 (Kisumu)]. In addition, several genes from other families were also over-expressed including Glutathione S-transferases (GSTs), carboxylesterases, trypsin, glycogenin, and nucleotide binding protein which probably contribute to insecticide resistance across Uganda and Kenya. Genotyping of 17 microsatellite loci in the five locations provided evidence that a geographical shift in the resistance mechanisms could be associated with patterns of population structure throughout East Africa. Genetic and population structure analyses indicated significant genetic differentiation between Arua and other localities (FST>0.03) and revealed a barrier to gene flow between Arua and other areas, possibly associated with Rift Valley. CONCLUSION: The correlation between patterns of genetic structure and variation in gene expression could be used to inform future interventions especially as new insecticides are gradually introduced.

A 6.5-kb intergenic structural variation enhances P450-mediated resistance to pyrethroids in malaria vectors lowering bed net efficacy.

Elucidating the complex evolutionary armory that mosquitoes deploy against insecticides is crucial to maintain the effectiveness of insecticide-based interventions. Here, we deciphered the role of a 6.5-kb structural variation (SV) in driving cytochrome P450-mediated pyrethroid resistance in the malaria vector, Anopheles funestus. Whole-genome pooled sequencing detected an intergenic 6.5-kb SV between duplicated CYP6P9a/b P450s in pyrethroid-resistant mosquitoes through a translocation event. Promoter analysis revealed a 17.5-fold higher activity (p < .0001) for the SV- carrying fragment than the SV- free one. Quantitative real-time PCR expression profiling of CYP6P9a/b for each SV genotype supported its role as an enhancer because SV+/SV+ homozygote mosquitoes had a significantly greater expression for both genes than heterozygotes SV+/SV- (1.7- to 2-fold) and homozygotes SV-/SV- (4-to 5-fold). Designing a PCR assay revealed a strong association between this SV and pyrethroid resistance (SV+/SV+ vs. SV-/SV-; odds ratio [OR] = 2,079.4, p < .001). The 6.5-kb SV is present at high frequency in southern Africa (80%-100%) but absent in East/Central/West Africa. Experimental hut trials revealed that homozygote SV mosquitoes had a significantly greater chance to survive exposure to pyrethroid-treated nets (OR 27.7; p < .0001) and to blood feed than susceptible mosquitoes. Furthermore, mosquitoes homozygote-resistant at the three loci (SV+/CYP6P9a_R/CYP6P9b_R) exhibited a higher resistance level, leading to a far superior ability to survive exposure to nets than those homozygotes susceptible at the three loci, revealing a strong additive effect. This study highlights the important role of structural variations in the development of insecticide resistance in malaria vectors and their detrimental impact on the effectiveness of pyrethroid-based nets.

An Africa-wide genomic evolution of insecticide resistance in the malaria vector Anopheles funestus involves selective sweeps, copy number variations, gene conversion and transposons.

Insecticide resistance in malaria vectors threatens to reverse recent gains in malaria control. Deciphering patterns of gene flow and resistance evolution in malaria vectors is crucial to improving control strategies and preventing malaria resurgence. A genome-wide survey of Anopheles funestus genetic diversity Africa-wide revealed evidences of a major division between southern Africa and elsewhere, associated with different population histories. Three genomic regions exhibited strong signatures of selective sweeps, each spanning major resistance loci (CYP6P9a/b, GSTe2 and CYP9K1). However, a sharp regional contrast was observed between populations correlating with gene flow barriers. Signatures of complex molecular evolution of resistance were detected with evidence of copy number variation, transposon insertion and a gene conversion between CYP6P9a/b paralog genes. Temporal analyses of samples before and after bed net scale up suggest that these genomic changes are driven by this control intervention. Multiple independent selective sweeps at the same locus in different parts of Africa suggests that local evolution of resistance in malaria vectors may be a greater threat than trans-regional spread of resistance haplotypes.

The Entamoeba lysine and glutamic acid rich protein (KERP1) virulence factor gene is present in the genomes of Entamoeba nuttalli, Entamoeba dispar and Entamoeba moshkovskii.

The lysine and glutamic acid rich protein KERP1 is a cell surface-expressed virulence factor in the human pathogen Entamoeba histolytica. It was originally suggested that the gene was absent from the related, avirulent human commensal Entamoeba dispar, an absence which would be relevant to the differential virulence of these species. Here, the gene is shown to be present in E. dispar, and its sequence is presented, as well as in a virulent parasite of macaques, Entamoeba nuttalli, and the primarily free living, opportunistically parasitic Entamoeba moshkovskii.

The genome of the sea anemone Actinia equina (L.): Meiotic toolkit genes and the question of sexual reproduction.

The beadlet anemone Actinia equina (L.) (Cnidaria: Anthozoa: Actiniaria: Actiniidae) is one of the most familiar organisms of the North European intertidal zone. Once considered a single, morphologically variable species across northern Europe, it is now recognised as one member of a variable species complex. Previous studies of distribution, aggression, allozymes and mitochondrial DNA suggest that the diversity in form and colour within A. equina may hide still unrecognised species diversity. To empower further study of A. equina population genetics and systematics, we sequenced (PacBio Sequel) the genome of a single A. equina individual to produce a high-quality genome assembly (contig N50 = 492,607 bp, 1485 contigs, number of protein coding genes = 47,671, 97% BUSCO completeness). There is debate as to whether A. equina reproduces solely asexually, since no reliable, consistent evidence of sexual reproduction has been found. To gain further insight, we examined the genome for evidence of a 'meiotic toolkit' - genes believed to be found consistently in sexually reproducing organisms - and demonstrate that the A. equina genome appears not to have this full complement. Additionally, Smudgeplot analysis, coupled with high haplotype diversity, indicates this genome assembly to be of ambiguous ploidy, suggesting that A. equina may not be diploid. The suggested polyploid nature of this species coupled with the deficiency in meiotic toolkit genes, indicates that further field and laboratory studies of this species is warranted to understand how this species reproduces and what role ploidy may play in speciation within this speciose genus.

Preventive antibiotic treatment of calves: emergence of dysbiosis causing propagation of obese state-associated and mobile multidrug resistance-carrying bacteria.

In agriculture, antibiotics are used for the treatment and prevention of livestock disease. Antibiotics perturb the bacterial gut composition but the extent of these changes and potential consequences for animal and human health is still debated. Six calves were housed in a controlled environment. Three animals received an injection of the antibiotic florfenicol (Nuflor), and three received no treatment. Faecal samples were collected at 0, 3 and 7 days, and bacterial communities were profiled to assess the impact of a therapy on the gut microbiota. Phylogenetic analysis (16S-rDNA) established that at day 7, antibiotic-treated microbiota showed a 10-fold increase in facultative anaerobic Escherichia spp, a signature of imbalanced microbiota, dysbiosis. The antibiotic resistome showed a high background of antibiotic resistance genes, which did not significantly change in response to florfenicol. However, the maintenance of Escherichia coli plasmid-encoded quinolone, oqxB and propagation of mcr-2, and colistin resistance genes were observed and confirmed by Sanger sequencing. The microbiota of treated animals was enriched with energy harvesting bacteria, common to obese microbial communities. We propose that antibiotic treatment of healthy animals leads to unbalanced, disease- and obese-related microbiota that promotes growth of E. coli carrying resistance genes on mobile elements, potentially increasing the risk of transmission of antibiotic resistant bacteria to humans.

Cis-regulatory CYP6P9b P450 variants associated with loss of insecticide-treated bed net efficacy against Anopheles funestus.

Elucidating the genetic basis of metabolic resistance to insecticides in malaria vectors is crucial to prolonging the effectiveness of insecticide-based control tools including long lasting insecticidal nets (LLINs). Here, we show that cis-regulatory variants of the cytochrome P450 gene, CYP6P9b, are associated with pyrethroid resistance in the African malaria vector Anopheles funestus. A DNA-based assay is designed to track this resistance that occurs near fixation in southern Africa but not in West/Central Africa. Applying this assay we demonstrate, using semi-field experimental huts, that CYP6P9b-mediated resistance associates with reduced effectiveness of LLINs. Furthermore, we establish that CYP6P9b combines with another P450, CYP6P9a, to additively exacerbate the reduced efficacy of insecticide-treated nets. Double homozygote resistant mosquitoes (RR/RR) significantly survive exposure to insecticide-treated nets and successfully blood feed more than other genotypes. This study provides tools to track and assess the impact of multi-gene driven metabolic resistance to pyrethroids, helping improve resistance management.

Old World cutaneous leishmaniasis treatment response varies depending on parasite species, geographical location and development of secondary infection.

BACKGROUND: In the Kingdom of Saudi Arabia (KSA), Leishmania major and L. tropica are the main causative agents of Old World cutaneous leishmaniasis (CL). The national CL treatment regimen consists of topical 1% clotrimazole/2% fusidic acid cream followed by 1-2 courses of intralesional sodium stibogluconate (SSG); however, treatment efficacy is highly variable and the reasons for this are not well understood. In this study, we present a complete epidemiological map of CL and determined the efficacy of the standard CL treatment regime in several endemic regions of KSA. RESULTS: Overall, three quarters of patients in all CL-endemic areas studied responded satisfactorily to the current treatment regime, with the remaining requiring only an extra course of SSG. The majority of unresponsive cases were infected with L. tropica. Furthermore, the development of secondary infections (SI) around or within the CL lesion significantly favoured the treatment response of L. major patients but had no effect on L. tropica cases. CONCLUSIONS: The response of CL patients to a national treatment protocol appears to depend on several factors, including Leishmania parasite species, geographical location and occurrences of SI. Our findings suggest there is a need to implement alternative CL treatment protocols based on these parameters.