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Genomic analysis of two phlebotomine sand fly vectors of Leishmania from the New and Old World [1]
['Frédéric Labbé', 'Eck Institute For Global Health', 'Department Of Biological Sciences', 'University Of Notre Dame', 'Notre Dame', 'Indiana', 'United States Of America', 'Maha Abdeladhim', 'Vector Molecular Biology Section', 'Laboratory Of Malaria']
Date: 2023-05
Phlebotomine sand flies are of global significance as important vectors of human disease, transmitting bacterial, viral, and protozoan pathogens, including the kinetoplastid parasites of the genus Leishmania, the causative agents of devastating diseases collectively termed leishmaniasis. More than 40 pathogenic Leishmania species are transmitted to humans by approximately 35 sand fly species in 98 countries with hundreds of millions of people at risk around the world. No approved efficacious vaccine exists for leishmaniasis and available therapeutic drugs are either toxic and/or expensive, or the parasites are becoming resistant to the more recently developed drugs. Therefore, sand fly and/or reservoir control are currently the most effective strategies to break transmission. To better understand the biology of sand flies, including the mechanisms involved in their vectorial capacity, insecticide resistance, and population structures we sequenced the genomes of two geographically widespread and important sand fly vector species: Phlebotomus papatasi, a vector of Leishmania parasites that cause cutaneous leishmaniasis, (distributed in Europe, the Middle East and North Africa) and Lutzomyia longipalpis, a vector of Leishmania parasites that cause visceral leishmaniasis (distributed across Central and South America). We categorized and curated genes involved in processes important to their roles as disease vectors, including chemosensation, blood feeding, circadian rhythm, immunity, and detoxification, as well as mobile genetic elements. We also defined gene orthology and observed micro-synteny among the genomes. Finally, we present the genetic diversity and population structure of these species in their respective geographical areas. These genomes will be a foundation on which to base future efforts to prevent vector-borne transmission of Leishmania parasites.
The leishmaniases are a group of neglected tropical diseases caused by protist parasites from the Genus Leishmania. Different Leishmania species present a wide clinical profile, ranging from mild, often self-resolving cutaneous lesions that can lead to protective immunity, to severe metastatic mucosal disease, to visceral disease that is ultimately fatal. Leishmania parasites are transmitted by the bites of sand flies, and as no approved human vaccine exists, available drugs are toxic and/or expensive and parasite resistance to them is emerging, new dual control strategies to combat these diseases must be developed, combining interventions on human infections and integrated sand fly population management. Effective vector control requires a comprehensive understanding of the biology of sand flies. To this end, we sequenced and annotated the genomes of two sand fly species that are important leishmaniasis vectors from the Old and New Worlds. These genomes allow us to better understand, at the genetic level, processes important in the vector biology of these species, such as finding hosts, blood-feeding, immunity, and detoxification. These genomic resources highlight the driving forces of evolution of two major Leishmania vectors and provide foundations for future research on how to better prevent leishmaniasis by control of the sand fly vectors.
Funding: The work was supported by two grants from the National Human Genome Research Institutes U54-HG003079 to WCW and U54-HG003273 to SR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Data Availability: Sequenced reads sequence reads were deposited in the NCBI SRA under Bioproject accession number PRJNA20279 for Lutzomyia longipalpis and PRJNA20293 for Phlebotomus papatasi. The GeneBank Ph. papatasi Ppap_1.0 assembly accession number is GCA_000262795.1 and the Lu. longipalpis Llon_1.0 assembly accession number is GCA_000265325.1. Both genome assemblies and associated annotations are hosted on VectorBase.
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Introduction
Phlebotomine sand flies are a group of blood-feeding Diptera that vary widely in their geographic distribution, ecology, and the pathogens they transmit. They serve as vectors for several established, emerging, and re-emerging infectious diseases, transmitting protist, bacterial and viral pathogens. The most important of the sand fly transmitted pathogens belong to the genus Leishmania which cause a spectrum of disease in humans known as leishmaniasis, that account for an estimated 2.4 million disability-adjusted life-years (DALYs) [1] and 40,000 deaths annually [2]. These statistics are likely to be underestimated due to misdiagnosis, underreporting, and lack of surveillance systems in many of the affected countries. Political instability, urbanization, and climate change are expanding Leishmania-endemic regions and increasing the risk of epidemics world-wide [3]. These factors coupled with the increase of visceral disease and HIV co-infection, have led the World Health Organization to classify leishmaniasis as one of the world’s epidemic-prone diseases [4].
Leishmaniasis occurs worldwide, in 98 countries over five continents, with 310 million people at risk of contracting the infection [2]. Leishmaniasis is a collective term for a group of distinct clinical manifestations ranging from mild, often self-resolving cutaneous lesions that can lead to protective immunity, to disseminated lesions that do not heal spontaneously, to destruction of the mucous membranes of the nose, mouth, and pharynx, to life-threatening visceral disease. The clinical profile depends on a variety of factors, including vector biology, host immunity, and parasite characteristics; with the Leishmania species that causes the infection being the primary determinant. The two primary clinical forms are cutaneous leishmanisis (CL) and visceral leishmaniasis (VL). The primary Leishmania species that cause CL are Leishmania major, Leishmania infantum, Leishmania tropica, and Leishmania aethipica in the Old World and Leishmania amazonensis, Leishmania braziliensis, Leishmania guyanensis, Le. infantum, Leishmania mexicana, and Leishmania panamensis in the New World. VL is primarily caused by Leishmania donovani in Asia and Africa and Le. infantum in the Middle East, central Asia, South and Central America, and the Mediterranean Basin.
There are approximately 35 proven, and an additional 63 suspected, vectors of at least 40 different Leishmania species to humans [5,6]. Phlebotomus species are the primary Leishmania vectors in the Old World and Lutzomyia species are responsible for transmitting leishmaniasis throughout the Americas [7]. There is a close ecological association, if not co-evolutionary relationship [8,9], between Leishmania species and their specific vectors such that generally a single sand fly species transmits a single Leishmania species under natural conditions. Some sand flies, however, can transmit a range of Leishmania species under experimental conditions [10]. This difference has given rise to the concept of “restricted” and “permissive” vectors [11]. For example, Phlebotomus papatasi is a restrictive vector, transmitting only Le. major parasites [12]. Lutzomyia longipalpis (s.l.) is considered a permissive vector in laboratory conditions, but only transmits Le. infantum naturally [12].
These vectors are part of the Diptera which is an extremely species-rich and ecologically diverse order of insects and contains the vectors of many of the most important pathogens of man and his domesticated animals. Both phlebotomine sand flies (family Psychodidae) and mosquitoes (Culicidae) are specified as members of distinct infra-orders within the suborder Nematocera. While the Nematocera grouping is paraphyletic, the relationships between infra-orders remains to be elucidated [13]. Some studies generated topologies with Psychodomorpha (sand flies) and Culicomorpha (mosquitoes and black flies) as sister groups [14], whereas, others place sand flies nearer to the muscoid flies (Ephydroidea) [15]. The internal relationships within the assemblage that includes Psychodidae also remains a matter of debate [16].
It is postulated that the close evolutionary relationship between sand fly species and the Leishmania species that they transmit may have epidemiological implications for leishmaniasis [17]. For example, there are three primary zymodemes of Le. major that have limited geographical distributions such that the prevalent zymodeme in a particular area overlaps with the distribution of one primary population of Ph. papatasi [18]. Ph. papatasi has a wide geographical distribution, ranging from Morocco to the Indian subcontinent and from southern Europe to central and eastern Africa. Given the wide ecological and geographic distribution of Ph. papatasi populations [19], coupled with the low dispersal capacity of these sand flies [12], it is likely that there is limited gene flow between populations and significant genetic structuring between populations. While previous studies demonstrated relatively low genetic differentiation between Ph. papatasi populations separated by large geographical distances [9,20], more recent studies have identified genetic differentiation between geographically separated populations [18,21–24] and local differentiation [25]. Microsatellite analysis, in particular, revealed two distinct genetic clusters of Ph. papatasi (A & B) with further substructure within each population that correlated with geographical origin (A1-5 and B1 &2) [18,23].
While elucidating the drivers leading to reproductive isolation and speciation remains a challenge, there is strong evidence that Lu. longipalpis is undergoing incipient speciation in Brazil with various levels of differentiation between siblings of the complex [26]. The Brazilian populations of Lu. longipalpis can be divided into three groups based on analysis of their primary copulatory songs which start during mating immediately after the male clasps the female. The males of one group produce Burst-type mating songs the second, more heterogeneous group, has populations which produce different subtypes of Pulse-type songs. The third group, “mix-type” has characteristics from the other Burst and Pulse types but has sufficient significant differences in all measured characteristics to enable them to be differentiated from the other types [27–29]. Acoustic communication in insects is mostly associated with attraction and/or recognition during courtship, prior to copulation. In Lu. longipalpis (s.l.), sound production starts when copulation has commenced and contributes to insemination success indicating that it is directly linked to reproductive success [30].
Male Lu. longipalpis produce sex-aggregation pheromones, volatile chemicals that attract females to male selected mating sites over long distances [31]. Analysis of structure and quantity of these chemicals indicates that there are at least 5 different pheromone types possibly representing cryptic species of Lu. longipalpis in South and Central American countries [32–34] and analysis of molecular correlates [single nucleotide polymorphisms (SNPs) and copy number variation (CNVs)] in the chemosensory genome confirms that these populations have significant genetic differences [35]. The structures of the sex-aggregation pheromones of members of the complex that have been elucidated fall into 2 classes; diterpenes, which have the molecular formula C 20 H 32 and molecular weight (mw) 272 gmol-1 and methylsesquiterpenes with the molecular formula C 16 H 32 and mw 218 gmol-1 [32]. One of the diterpenes, has been characterized as sobralene (SOB) [36] and two of the methylsesquiterpenes as 3-methyl-α-himachalene (3MαH) and (S)-9-methylgermacrene-B (9MGB). These compounds are found only in populations of Lu. longipalpis.
Although the sex-aggregation pheromones of Lu. longipalpis (s.l.) share a biosynthetic origin the methylsesquiterpenes are derived from a 15-carbon precursor, farnesyl diphosphate and six of the seven enzymes of the mevalonate-pathway, plus enzymes involved in sesquiterpenoid biosynthesis, have been found in 9MGB-producing Lu. longipalpis [37] whereas the diterpenes are derived via a 20-carbon precursor, geranylgeranyl diphosphate [38].
Crossing experiments between sympatric and allopatric populations of different members of the Lu. longipalpis species complex revealed reproductive isolation due to both pre-mating and copulatory mechanisms [39,40]. Hickner et al. 2020 provided genomic insights into the chemoreceptor genome repertoire underlying behavioral evolution of sexual communication in the Lu. longipalpis populations, but whole-genome analyses could improve the identification of loci related to critical traits such as vectorial capacity, host preference, and insecticide resistance [35].
Despite the potential importance for influencing Leishmania development and survival in the gut, the sand fly immune response is poorly studied. To date, work has been largely restricted to the study of defensins [41–44]. However, gene depletion via RNAi of the negative regulator of the Immune Deficiency (IMD) pathway caspar [45] led to a reduction in Leishmania population in the gut of Lu. longipalpis. While the knockout of relish, the transcription factor of the IMD pathway, resulted in the increase of Leishmania and bacteria in Ph. papatasi [46].
Adaptation to hematophagy presents many challenges to insects, including avoiding the physiological responses of the host that interfere with obtaining a blood meal, digestion of the blood, and excretion of the excess water contained in the blood meal. Sand flies have evolved a complex cocktail of pharmacologically active salivary molecules to facilitate blood feeding that have been extensively characterized [47].
Many important aspects in sand fly biology such as hematophagy and host seeking are controlled by the biological clock [48]. In Lu. longipalpis, the main clock genes and their expression pattern throughout the day have been previously characterized [49,50]. However, the molecular regulation of circadian rhythms is poorly understood in sand flies. Yuan et al. 2007 proposed three clock models based on the presence of the cryptochrome (CRY) proteins, CRY1 and CRY2 [51]. In the Drosophila clock model, only CRY1, which acts as a blue-light photoreceptor [52], is present. In the butterfly model, CRY1 also acts as a photoreceptor and CRY2, which is a mammalian–like transcriptional repression, dimers with PER to repress CLK/CYC activity. In the bee model, there is only CRY2, which seems to act as a repressor together with PER and some other molecule that is not CRY1 that acts as photoreceptor.
A central inquiry of evolutionary biology is elucidating drivers of speciation, however, defining species boundaries and identifying the genetic architecture that leads to reproductive isolation has been a challenge. Understanding of the mechanisms of vectorial capacity, adaptation to changing ecological environments, and insecticide resistance has epidemiological consequences for the integrated management of sand fly populations that is the cornerstone of leishmaniasis control [53]. To begin to explore the driving forces of evolution of two important phlebotomine sand fly vectors from the Psychodidae family (Phlebotominae subfamily), Ph. papatasi and Lu. longipalpis (s.l.), that exhibit distinct distributions, behavior, and pathogen specificity, we sequenced and analyzed their whole-genomes using comparative genomics approaches. We manually curated a number of gene families with key roles in processes such as immunity, blood-feeding, chemosensation, detoxification, and circadian biology to provide a basis for studying and understanding sand flies as Leishmania vectors. Moreover, as a better understanding of the population structure of geographically separated vector populations is necessary, we also assessed the population structure of Ph. papatasi and Lu. Longipalpis by collecting and sequencing individual field-collected specimens sampled over a large geographical range in the Middle East and North Africa, and Brazil, respectively. Our results provide significant advances in our understanding of the genetics underlying the population structure and provide a foundation for future molecular comparative studies of these two medically important vectors.
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