Sand Fly (Phlebotomus papatasi) Embryo Microinjection for CRISPR/Cas9 Mutagenesis

Sand flies are the natural vectors for Leishmania species, protozoan parasites producing a broad spectrum of symptoms ranging from cutaneous lesions to ...

National Institutes of Health

Invertebrate stages of Leishmania are capable of genetic exchange during their extracellular growth and development in the sand fly vector. Here we explore two variables: the ability of diverse L. major strains from across its natural range to undergo mating in pairwise tests; and the timing of the appearance of hybrids and their developmental stage associations within both natural (Phlebotomus duboscqi) and unnatural (Lutzomyia longipalpis) sand fly vectors. Following co-infection of flies with parental lines bearing independent drug markers, doubly-drug resistant hybrid progeny were selected, from which 96 clonal lines were analyzed for DNA content and genotyped for parent alleles at 4-6 unlinked nuclear loci as well as the maxicircle DNA. As seen previously, the majority of hybrids showed '2n' DNA contents, but with a significant number of '3n' and one '4n' offspring. In the natural vector, 97% of the nuclear loci showed both parental alleles; however, 3% (4/150) showed only one parental allele. In the unnatural vector, the frequency of uniparental inheritance rose to 10% (27/275). We attribute this to loss of heterozygosity after mating, most likely arising from aneuploidy which is both common and temporally variable in Leishmania. As seen previously, only uniparental inheritance of maxicircle kDNA was observed. Hybrids were recovered at similar efficiencies in all pairwise crosses tested, suggesting that L. major lacks detectable 'mating types' that limit free genetic exchange. In the natural vector, comparisons of the timing of hybrid formation with the presence of developmental stages suggest nectomonads as the most likely sexually competent stage, with hybrids emerging well before the first appearance of metacyclic promastigotes. These studies provide an important perspective on the prevalence of genetic exchange in natural populations of L. major and a guide for experimental studies to understand the biology of mating.

The mating competence of geographically diverse Leishmania major strains in their natural and unnatural sand fly vectors.

Genetic exchange between Leishmania major strains during their development in the sand fly vector has been experimentally shown. To investigate the possibility of genetic exchange between different Leishmania species, a cutaneous strain of L. major and a visceral strain of Leishmania infantum, each bearing a different drug-resistant marker, were used to coinfect Lutzomyia longipalpis sand flies. Eleven double-drug-resistant progeny clones, each the product of an independent mating event, were generated and submitted to genotype and phenotype analyses. The analysis of multiple allelic markers across the genome suggested that each progeny clone inherited at least one full set of chromosomes from each parent, with loss of heterozygosity at some loci, and uniparental retention of maxicircle kinetoplast DNA. Hybrids with DNA contents of approximately 2n, 3n, and 4n were observed. In vivo studies revealed clear differences in the ability of the hybrids to produce pathology in the skin or to disseminate to and grow in the viscera, suggesting polymorphisms and differential inheritance of the gene(s) controlling these traits. The studies, to our knowledge, represent the first experimental confirmation of cross-species mating in Leishmania, opening the way toward genetic linkage analysis of important traits and providing strong evidence that genetic exchange is responsible for the generation of the mixed-species genotypes observed in natural populations. 

Cross-species genetic exchange between visceral and cutaneous strains of Leishmania in the sand fly vector.

In the Indian sub-continent, visceral leishmaniasis (VL), also known as kala azar, is a fatal form of leishmaniasis caused by the kinetoplastid parasite Leishmania donovani and transmitted by the sand fly Phlebotomus argentipes. VL is prevalent in northeast India where it is believed to have an exclusive anthroponotic transmission cycle. There are four distinct cohorts of L. donovani exposed individuals who can potentially serve as infection reservoirs: patients with active disease, cured VL cases, patients with post kala azar dermal leishmaniasis (PKDL), and asymptomatic individuals. The relative contribution of each group to sustaining the transmission cycle of VL is not known.

The Potential Use of Forensic DNA Methods Applied to Sand Fly Blood Meal Analysis to Identify the Infection Reservoirs of Anthroponotic Visceral Leishmaniasis.

Patients with active visceral leishmaniasis are important reservoirs in the anthroponotic transmission cycle of Leishmania donovani. The role of the blood or skin as a source of infection to sand flies remains unclear, and the possible effect of multiple exposures to fly bites on transmissibility has not been addressed.

Increased Transmissibility of Leishmania donovani From the Mammalian Host to Vector Sand Flies After Multiple Exposures to Sand Fly Bites.

The life cycle of the  parasite in the sand fly vector involves differentiation into several distinctive forms, each thought to represent an adaptation to specific microenvironments in the midgut of the fly. Based on transcriptome sequencing (RNA-Seq) results, we describe the first high-resolution analysis of the transcriptome dynamics of four distinct stages of  as they develop in a natural vector,  The early transformation from tissue amastigotes to procyclic promastigotes in the blood-fed midgut was accompanied by the greatest number of differentially expressed genes, including the downregulation of amastins, and upregulation of multiple cell surface proteins, sugar and amino acid transporters, and genes related to glucose metabolism and cell cycle progression. The global changes accompanying post-blood meal differentiation of procyclic promastigotes to the nectomonad and metacyclic stages were less extensive, though each displayed a unique signature. The transcriptome of nectomonads, which has not been studied previously, revealed changes consistent with cell cycle arrest and the upregulation of genes associated with starvation and stress, including autophagic pathways of protein recycling. Maturation to the infective, metacyclic stage was accompanied by changes suggesting preadaptation to the intracellular environment of the mammalian host, demonstrated by the amastigote-like profiles of surface proteins and metabolism-related genes. Finally, a direct comparison between sand fly-derived and culture-derived metacyclics revealed a reassuring similarity between the two forms, with the  forms distinguished mainly by a stronger upregulation of transcripts associated with nutrient stress. The life cycle of  parasites in the sand fly vector includes their growth and development as morphologically distinct forms of extracellular promastigotes found within the different microenvironments of the gut. Based on RNA-Seq, we provide here the first high-resolution, transcriptomic analysis of  insect stages during their cyclical development , from tissue amastigotes ingested with the blood meal to infective, metacyclic promastigotes that initiate infection in the mammalian host. The most extensive genetic reprogramming occurred during the early transformation of amastigotes to rapidly dividing procyclic promastigotes in the blood-fed midgut, with major changes in the abundance of mRNAs for surface proteins and metabolism. The post-blood meal-adapted nectomonad stage was characterized by the downregulation of cell cycle-related genes and the upregulation of stress- and starvation-related genes. Finally, the transcriptome of metacyclic promastigotes shifted to a more amastigote-like profile, suggesting their preadaptation to the intracellular host environment.

The Transcriptome of  Developmental Stages in Their Natural Sand Fly Vector.

For many arthropod vectors, the diverse bacteria and fungi that inhabit the gut can negatively impact pathogen colonization. Our attempts to exploit antibiotic treatment of colonized Phlebotomus duboscqi sand flies in order to improve their vector competency for Leishmania major resulted instead in flies that were refractory to the development of transmissible infections due to the inability of the parasite to survive and to colonize the anterior midgut with infective, metacyclic stage promastigotes. The parasite survival and development defect could be overcome by feeding the flies on different symbiont bacteria but not by feeding them on bacterial supernatants or replete medium. The inhibitory effect of the dysbiosis was moderated by lowering the concentration of sucrose (<30% w/v) used in the sugar feeds to maintain the colony. Exposure of promastigotes to 30% sucrose was lethal to the parasite in vitro. Confocal imaging revealed that the killing in vivo was confined to promastigotes that had migrated to the anterior plug region, corresponding to the highest concentrations of sucrose. The data suggest that sucrose utilization by the microbiota is essential to promote the appropriate osmotic conditions required for the survival of infective stage promastigotes in vivo.

The midgut microbiota plays an essential role in sand fly vector competence for Leishmania major.

Inflammatory monocytes can be manipulated by environmental cues to perform multiple functions. To define the role of monocytes during primary or secondary infection with an intra-phagosomal pathogen we employed Leishmania major-red fluorescent protein (RFP) parasites and multi-color flow cytometry to define and enumerate infected and uninfected inflammatory cells in the skin. During primary infection, infected monocytes had altered maturation and were the initial mononuclear host cell for parasite replication. In contrast, at a distal site of secondary infection in mice with a healed but persistent primary infection, this same population rapidly produced inducible nitric oxide synthase (iNOS) in an IFN-γ dependent manner and was critical for parasite killing. Maturation to a dendritic cell-like phenotype was not required for monocyte iNOS-production, and enhanced monocyte recruitment correlated with IFN-γ dependent cxcl10 expression. In contrast, neutrophils appeared to be a safe haven for parasites in both primary and secondary sites. Thus, inflammatory monocytes play divergent roles during primary versus secondary infection with an intra-phagosomal pathogen.

Divergent roles for Ly6C+CCR2+CX3CR1+ inflammatory monocytes during primary or secondary infection of the skin with the intra-phagosomal pathogen Leishmania major.

Hybrid genotypes have been repeatedly described among natural isolates of Leishmania, and the recovery of experimental hybrids from sand flies co-infected with different strains or species of Leishmania has formally demonstrated that members of the genus possess the machinery for genetic exchange. As neither gamete stages nor cell fusion events have been directly observed during parasite development in the vector, we have relied on a classical genetic analysis to determine if Leishmania has a true sexual cycle. Here, we used whole genome sequencing to follow the chromosomal inheritance patterns of experimental hybrids generated within and between different strains of L. major and L. infantum. We also generated and sequenced the first experimental hybrids in L. tropica. We found that in each case the parental somy and allele contributions matched the inheritance patterns expected under meiosis 97-99% of the time. The hybrids were equivalent to F1 progeny, heterozygous throughout most of the genome for the markers that were homozygous and different between the parents. Rare, non-Mendelian patterns of chromosomal inheritance were observed, including a gain or loss of somy, and loss of heterozygosity, that likely arose during meiosis or during mitotic divisions of the progeny clones in the fly or culture. While the interspecies hybrids appeared to be sterile, the intraspecies hybrids were able to produce backcross and outcross progeny. Analysis of 5 backcross and outcross progeny clones generated from an L. major F1 hybrid, as well as 17 progeny clones generated from backcrosses involving a natural hybrid of L. tropica, revealed genome wide patterns of recombination, demonstrating that classical crossing over occurs at meiosis, and allowed us to construct the first physical and genetic maps in Leishmania. Altogether, the findings provide strong evidence for meiosis-like sexual recombination in Leishmania, presenting clear opportunities for forward genetic analysis and positional cloning of important genes.

Whole genome sequencing of experimental hybrids supports meiosis-like sexual recombination in Leishmania.

Sand flies are the natural vectors for the  species that produce a spectrum of diseases in their mammalian hosts, including humans. Studies of sand fly/ interactions have been limited by the absence of genome editing techniques applicable to these insects. In this report, we adapted CRISPR (clustered regularly interspaced palindromic repeat)/Cas9 (CRISPR-associated protein 9) technology to the  sand fly, a natural vector for , targeting the sand fly immune deficiency (IMD) pathway in order to decipher its contribution to vector competence. We established a protocol for transformation in  and were able to generate transmissible  mutant alleles for Relish (Rel), the only transcription factor of the IMD pathway. While the maintenance of a homozygous mutant stock was severely compromised, we were able to establish in an early generation their greater susceptibility to infection with  Flies carrying different heterozygous mutant alleles variably displayed a more permissive phenotype, presenting higher loads of parasites or greater numbers of infective-stage promastigotes. Together, our data show (i) the successful adaptation of the CRISPR/Cas9 technology to sand flies and (ii) the impact of the sand fly immune response on vector competence for  parasites. Sand flies are the natural vectors of  parasites. Studies of sand fly/ interactions have been limited by the lack of successful genomic manipulation of these insects. This paper shows the first example of successful targeted mutagenesis in sand flies via adaptation of the CRISPR/Cas9 editing technique. We generated transmissible  mutant alleles of , a gene known to be essential for the control of immune response in other insects. In addition to the expected higher level of susceptibility to bacteria, the mutant flies presented higher loads of parasites when infected with , showing that the sand fly immune response impacts its vector competence for this pathogen.

Ehud Inbar

Laboratory of Parasitic Diseases,
National Institute of Allergy and Infectious Diseases,
Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases

National Institutes of Health

Sand Fly (Phlebotomus papatasi) Embryo Microinjection for CRISPR/Cas9 Mutagenesis

Ehud Inbar

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Laboratory of Parasitic Diseases,National Institute of Allergy and Infectious Diseases,Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases

National Institutes of Health

Sand Fly (Phlebotomus papatasi) Embryo Microinjection for CRISPR/Cas9 Mutagenesis

Laboratory of Parasitic Diseases,
National Institute of Allergy and Infectious Diseases,
Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases