Dugesia japonica is a typical planarian species that is widely distributed in the East Asia. Extensive cellular and molecular experimental methods have been developed to identify the functions of thousands of genes in this species, making this planarian a good experimental model for regeneration biology and neurobiology. However, no genome-level information is available for D. japonica, and few gene regulatory networks have been identified thus far.Planarians are non-parasitic Platyhelminthes (flatworms) famous for their regeneration ability and for having a well-organized brain. Xenopus laevis indicated that one of the CNEs in the Djndk gene may be a regulatory element, suggesting that the regulation of the ndk gene and the brain formation mechanism may be conserved between vertebrates and invertebrates.To obtain whole-genome information on this species and to study its gene regulatory networks, we extracted genomic DNA from 200 planarians derived from a laboratory-bred asexual clonal strain, and sequenced 476 Gb of data by second-generation sequencing. Kmer frequency graphing and fosmid sequence analysis indicated a complex genome that would be difficult to assemble using second-generation sequencing short reads. To address this challenge, we developed a new assembly strategy and improved the de novo genome assembly, producing a 1.56 Gb genome sequence  that covers 99.4% of all 19,543 genes in the assembled transcriptome, although the genome is fragmented as 80% of the genome consists of repeated sequences (genomic frequency ≥ 2). By genome comparison between two planarian genera, we identified conserved non-coding elements (CNEs), which are indicative of gene regulatory elements. Transgenic experiments using http://www.planarian.jp.This draft genome and CNE analysis will contribute to resolving gene regulatory networks in planarians. The genome database is available at: The online version of this article (10.1186/s40851-018-0102-2) contains supplementary material, which is available to authorized users. Dugesia japonica (D. japonica) is a typical freshwater planarian species that is widely distributed in East Asia [D. japonica also has a well-organized brain and shows decision-making behavior [Planarian is a common name applied to species of non-parasitic Platyhelminthes (flatworms) of the turbellaria class. Planarians have attracted great interest in the field of regeneration biology for many years . Dugesiaast Asia . This plast Asia . D. japobehavior –6, and ibehavior –10.D. japonica cell-type-specific genes have been isolated [D. japonica’s robust regeneration and brain formation to underlying genes. To achieve a complete understanding of those genes and their regulatory networks, genome-level information on D. japonica is required. However, the unusual number of SNPs that accumulate during asexual reproduction has confounded our efforts to obtain long contigs, although we used the laboratory clonal strain of D. japonica [D. japonica. These results will contribute knowledge essential for further research into gene regulatory networks in planarian.Advances in cellular and molecular biology experimental methods, as well as nucleic acid sequencing technologies, have helped increase our ability to examine the biology of planarians at the molecular level. Using PCR, cDNA libraries, RNA in situ hybridization and immunoscreening, isolated . Highly isolated . Loss-ofisolated . RNA micisolated . Fluoresisolated –17. In aisolated , 19. AllD. japonica strain SSP-9 T-5 was derived from a single individual and maintained in the Agata lab since 2005 [The asexual nce 2005 . The plabase/Cbase = NKmer/CKmer .Kmers were counted in the Illumina Hiseq sequencing data using Jellyfish with the “–C” parameter . Genome de Brujin graph-based method, Allpth-lg [The preliminary genome de novo assembly was performed by llpth-lg , SOAPdenllpth-lg , and Velllpth-lg . To imprllpth-lg . Secondlllpth-lg . Furtherllpth-lg . Thirdlyllpth-lg . Finallyllpth-lg  based onllpth-lg .The independent transcriptome de novo assembly was done by Trinity . The trahttp://www.repeatmasker.org/). One-to-one matched scaffold pairs between the two planarian genomes were identified using NUCmer [Schmidtea mediterranea (S. mediterranea) genome database [Only scaffolds with mRNA evidence were selected by BLAT . Coding g NUCmer . For speg NUCmer  and onlidatabase ) were peβ-actin basal promoter (− 55 to + 53), was previously described as βGFP [D. japonica and S. mediterranea ndk genomic regions were cloned in the actGFP from D. japonica genomic DNA by polymerase chain reaction (PCR) and verified by sequencing. Searches for putative transcription-factor-binding motifs were performed with transcription-factor-binding sites collected from the TRANSFAC and JASPAR databases [Xenopus embryos were generated with a sperm nuclear transplantation method, as described [The reporter plasmid, actGFP, carrying a chicken  as βGFP . Non-codatabases , 39. Traescribed . The fraD. japonica [D. japonica with insert length ~ 35 kb, and 172 fosmids were sequenced by end-sequencing using a first-generation (Sanger) sequencing platform. Totally, 476.48 Gb data were generated . According to these kmer frequency graphs, the estimated D. japonica genome size is ~ 1.52 Gb. The D. japonica genome commonly has 16 chromosomes, twice the number of another planarian species, S. mediterranea . In addition, our previous flow cytometry results also showed that the D. japonica genome size is ~ 1.9 times that of the S. mediterranea genome [D. japonica genome size should be around 1.46 Gb, similar to the genome size estimated from the kmer frequency graph.The a genome . Thus, tD. japonica genomic DNA fosmid library and sequenced them by the Sanger method. We then aligned next-generation sequencing DNA and RNA reads to those fosmid sequences.To display genome features visually, we screened out several individual fosmid clones of certain genes by a colony multiplex qPCR-based 3S3DBC method  from theDjth  insert sequence of the gene jth Fig.  showed tjth Fig. . Alignmejth Fig. . FurtherDe Bruijn graph-based genome methods could be used to assemble the genome. We used Allpath-lg [D. japonica genome would severely interfere with genome assembly. Not surprisingly, the de novo assembled results were fragmented  and its corresponding genome scaffold matched well, although some mismatches existed and large gaps in the scaffold caused by repeated sequences could not align to the fosmid  were generated. All of these sequences were mapped back to the assembled reference genome scaffolds using Bowtie ; 80.6% cmid Fig. .Fig. 4AlD. japonica transcriptome was obtained by assembling Roche 454 RNA sequencing reads derived from our previous results [Moreover, the  results . The ass results  with ideD. japonica genome assembly by comparison with FESs and the transcriptome showed the relative completeness of the draft genome, especially of the coding region. However, due to the heterogenous and repetitive sequences, some gaps and chimeric scaffolds may still exist.Thus, assessment of our http://www.repeatmasker.org/). RepeatModeler was used to build the consensus models of putative interspersed repeats as a new repeated sequence library based on the genome. RepeatMasker was used to search the planarian genome against the combined library Repbase. The two results were integrated to gain a comprehensive analysis of repeats in the D. japonica genome. Approximately 39.7% of the assembled genome sequences were marked as repetitive elements  were aligned against the D. japonica genome with BLAT (identity > 95%) [D. japonica genome and annotated them by Blast2GO annotation [To predict coding genes in the y > 95%) . The besy > 95%)  to help notation . 15,601 The comprehensive gene and protein prediction were performed by MAKER genome annotation program , which eS. mediterranea in Swiss-Prot database, all of which had homologous hits in our D. japonica reference assembly. We separately run Blastx on the unconfirmed 1366 protein sequences of S. mediterranea in Uniprot database and found homologous hits to 1129 (82%) of them. Moreover, from our D. japonica’s genome, we found several conserved critical protein coding genes which were recently reported missing from the genome of S. mediterranea in the report by Grohme et al. [D. japonica a good model animal for further research.So far there are only three experimentally confirmed protein sequences from e et al. , includiD. japonica online genome database (http://www.planarian.jp/). It allows users to download the genome and the transcriptome results, and features a graphical interface for browsing the genome using the JBrowse platform [For easy access to the assembly and annotation data, we prepared a platform , 54. In D. japonica and S. mediterranea exhibit strong morphological, physiological and functional similarity, their evolutionary positions are distant  were found to include transcription factor binding sites , and the chicken ) and the chicken  and Blas chicken  and onlidatabase ), we ideDjndk (a homolog of the vertebrate fgfrl1 gene), was shown to play a crucial role in brain formation during planarian regeneration [Djndk to the brain region are still unknown. To test whether the CNEs we identified by genome comparison are indeed regulatory elements, and with the hope of finding regulators of the Djndk gene, we took the gene Djndk as an example for CNE functional analysis.In 2002, the planarian gene neration , 59. HowD. japonica, which contains all 8 exons of the gene ndk and its flanking sequences on both 5′ upstream and 3′ downstream. Two scaffolds, v31.003636  and v31.014514 , were found from the genome of S. mediterranea  as a representative example for transgenic expression experiments  loci. The 140 bp CNE3 was inserted upstream of the promoter of a β-actin promoter-driven GFP expression vector to form a reporter construct called CNE-actGFP. When this expression vector was injected into Xenopus laevis embryos, the GFP expression pattern was localized in the neural-plate-forming region of the embryo, which was especially evident in the anterior region at the end of gastrulation , confirming the discovery of this reporter-gene-expression regulation by CNE3.There is only one corresponding scaffold 77.80 kbp) in our assembled genome of 7.80 kbp ion Fig. . Such exD. japonica and S. mediterranea, putative transcription factor-binding motifs were identified from perfectly or almost matched sequences . These results suggest that the 140 bp CNE3 might be a regulatory element of the Djndk gene, and that the Jun/Fos-related transcription factor(s) regulates the expression through this region. Accordingly, CNEs are good candidates of regulatory elements in the planarian genome, and further analysis should be done in the future to thoroughly dissect their roles in gene regulatory networks in planarian.By aligning the CNE sequences between ces Fig. . We madeces Fig. . StatistD. japonica draft genome. Our kmer frequency graphs, analysis of fosmid sequences, and our further genome analysis after assembly, taken together with previous heterogenesis observations, indicated that D. japonica possesses a hyper-varying genome structure. Although, the planarian clonal line we used for genomic DNA extraction was derived from a re-cloned planarian individual, we recently found accumulation of large number of mutations during planarian asexual proliferation, suggesting that planarian has a property to avoid complete genome stability to adapt changing environment [D. japonica across Russia, China, Korea, Japan, and other East Asian countries [D. japonica’s genome project and make the genome assembly much more challenging.In this study, we assembled the ironment , which mountries . One posountries . In addide Bruijn graph-based method did not produce good assembly results. Alternative solutions for this problem, such as bacterial artificial chromosome (BAC)-to-BAC sequencing and fosmid-pooling sequencing [D. japonica in the future. Moreover, because genomes extracted from different planarian cells even within one individual are possibly heterogenetic, which increased the complexity of the genome structure of D. japonica analyzed here and hampered the genome assembly, the usage of single-cell or single chromosome sequencing technology will help reduce the complexity and improve the genome assembly in future research.During genome assembly, simply using the SGS short sequencing data and assembling by the quencing  are diffquencing , so we eDjndk gene is a regulatory element that has a binding site for the Jun/Fos-related transcription factors. Previous result showed that Xenopus Djndk homologue (FGFRL-1) was expressed in the anterior part of the neural plate of Xenopus embryos, which will form the brain during development [Djndk gene expression. In addition, CNE3 in planarians has a similar counterpart in the human, mouse, and frog ndk-homologous genes, and thus we expect that the regulation of the ndk gene and even ndk-related aspects of the brain formation mechanism might be conserved between vertebrates and invertebrates.CNEs are well accepted as locations of gene expression regulatory elements. In this research, we also proved that one CNE (CNE3) in the elopment . HoweverD. japonica. Although the draft genome is still fragmented, it covers nearly all gene coding regions and is useful for helping to identify CNEs in the planarians, which will facilitate research on the gene regulatory networks of planarians.We presented the first draft genome of the planarian Additional file 1:Quality control of sequencing reads. (PDF 255 kb)Additional file 2:Repbase annotation of repeat sequences on the fosmid DJF-016O13. (XLSX 15 kb)Additional file 3:De novo assembly by De Bruijn Graph algorithm. (XLSX 10 kb)Additional file 4:Gene ontology annotation. (XLSX 11 kb)


Streptococcus pyogenes is an uncommon pathogen of purpura fulminans, and the pathogenesis of S. pyogenes-purpura fulminans remains unclear because of paucity of cases. We reported a pediatric case of S. pyogenes-purpura fulminans with literature review of the disease.S. pyogenes-purpura fulminans was made for bacterial isolation from throat and peripheral blood. Intensive therapy led to a survival with amputation of the left distal metatarsal bone, and normal development. The isolated M12 carried no mutation of csrS/R or rgg. Thrombophilia or immunodeficiency was excluded.A 3-year-old boy showed limping, lethargy and acral gangrene within 24 h. A diagnosis of S. pyogenes-purpura fulminans started with shock and coagulopathy. Five patients age < 8 years had no underlying disease and survived. One youngest and two immunocompromised patients died.Twelve-reported cases (9 pediatric and 3 elderly) of Streptococcus pyogenes-acute infectious purpura fulminans is a distinctive rare form of aggressive GAS infections. Streptococcus pyogenes (S. pyogenes) is the causative bacteria of scarlet fever, pharyngitis, impetigo, and cellulitis, as well as rheumatic fever and acute poststreptococcal glomerulonephritis [S. pyogenes is isolated from the throat of 10–30% children with acute pharyngitis, and healthy carriers are the source of outbreak. On the other hand, the most aggressive GAS infections comprise streptococcal toxic shock syndrome (TSS) and necrotizing fasciitis (NF) [ephritis . The groephritis . S. pyogtis (NF) .Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, and rickettsiae [S. pyogenes is an uncommon pathogen of PF in adults, and the pathogenesis of severe streptococcal disease remains unclear.Purpura fulminans (PF) is a life-threatening condition characterized by sudden-onset progressive cutaneous hemorrhage and acral necrosis . It is ckettsiae . S. pyogS. pyogenes infection. The isolated strain of GAS from the previously healthy boy was M12 without high-virulent mutations of csrS/R or rgg [S. pyogenes is a weighty organism of acute infectious PF, occurring as a distinct type invasive GAS infection affecting children and elderly.We herein report a rare case of acute infectious PF caused by R or rgg , 10. TwecsrS, csrR, and rgg genes were determined by automated sequencers, such as an Applied Biosystems 3130xl Genetic Analyzer , as described previously [Total DNA was extracted from bacterial cells and the nucleotide sequences of the eviously . The molPROC exons 1–9) and as described previously [STAT3, PIK3D, and PIK3R by the established method [Genomic DNA was extracted from peripheral blood after obtaining informed consent from the patients. For screening of thrombophilia, after coagulation study, the direct sequencing of polymerase chain reaction (PCR) products was completed for the coding regions of protein C gene , creatinine , total bilirubin , aspartate aminotransferase , alanine aminotransferase , lactate dehydrogenase , creatine kinase , and C-reactive protein . Coagulation studies showed hypofibrinogenemia , prolonged prothrombin time  and activated partial thromboplastin time , and increased levels of fibrinogen degradation products  and D-dimer . Thrombin-antithrombin complex (TAT) was > 120.0 ng/mL (rr: < 4), plasmin α2–antiplasmin complex (PIC) was 25.2 μg/mL (rr: < 0.8) and thrombomodulin 37.7 FU/mL (rr: < 4.5). Plasma activities of protein C  and protein S  were respectively low, and those of antithrombin were normal . The rapid antigen tests were positive for S. pyogenes and negative for Pneumococcus. S. pyogenes, determined later as the serotype M12, was isolated from throat and peripheral blood. Sterile cerebrospinal fluid showed no pleocytosis. These determined the diagnosis of acute infectious PF with S. pyogenes-septic shock. Antibiotic and anticoagulant therapy was started on the intensive care. Next day, leg purpura and acral gangrene extended. Anticoagulation with plasma-derived activated protein C (Anact C®), gabexate mesilate, fresh frozen plasma replacement, and platelet infusion, was intensified by the addition of a direct thrombin inhibitor (argatroban), antithrombin, recombinant thrombomodulin, and low-molecular heparin. The relaxing incisions were then conducted for the compartment syndrome of both legs on the 3rd day of illness. S. pyogenes was also isolated from the debridement tissues, and the histopathology indicated absent necrosis of the fascia and necrotic alteration of the muscle without specific inflammation. The continuous infusion of penicillin G led to the negative blood culture 24 h after the start of antibiotic therapy. Under controlled sepsis and coagulopathy, continuous irrigation and debridement were repeated. On the 13th day, debridement tissues showed necrotic muscle and fascia with chronic inflammatory changes. The histological changes indicated that the necrotic muscle tissues originated from ischemia rather than spreading infection of the fascia. The distal portions of the left metatarsal bone were finally amputated. Repeated skin-autografts resulted in the cosmetic and functional improvement of the extremities. There was no evidence of primary immunodeficiency diseases including asplenia, phagocyte disorders, hypogammaglobulinemia, immunoglobulin G subclass deficiency, complement deficiency or IRAK4/MyD88 deficiency, and absent mutation of STAT3, PI3KD, and PIK3RI genes. The genetic study of protein C or Factor V Leiden excluded heritable thrombophilia. He lives an active life for walking rehabilitation with no delayed development at 6 years of age. The isolated strain possessed T12 and M12 antigens, and emm12.0 gene, but no mutation of csrS/R or rgg.On admission, the comatose patient showed 180/min of tachycardia and unmeasurable blood pressure on the assist ventilation. Light reflex was prompt. The body temperature was 40.1 °C. Capillary refilling time was prolonged over 2 s, while cardiopulmonary sounds were normal. There was no hepatosplenomegaly or lymphadenopathy. Purpura and ecchymoses expanded to the both legs with necrotic toes  PF” and “idiopathic (antibody-mediated) PF”. Neither of TSS or NF typified the present case. M types 1, 3, 12, and 28 are the common isolates associated with shock and multiorgan failure. However, the isolated strain in this case had no high-virulent mutation. The literature review first revealed no patients age 10–50 years, in contrast to the young adult patients with TTS and NF. No previous reports verified the bacteriological property or host predisposition [S. pyogenes-acute infectious PF may exemplify a distinct form of invasive GAS infection heralded by septic shock.position . Shock aS. pyogenes -acute infectious PF remains unclear, because of the paucity of cases. Several toxins released from meningococci or pneumococci trigger the thrombotic action and subsequent hypercoagulability [S. pyogenes-derived superantigens cause TSS in healthy adults, but no thrombogenic toxins of GAS have been identified. S. pyogenes are considered to evolve the aggressive form via the horizontal crosstalk among the bacteria, although the precise mechanism has not been clarified [S. pyogenes that deteriorate local tissue infection. Streptococcal TSS is defined as any streptococcal infection associated with the sudden onset of shock and organ failure. Cytokine storm is crucial for the development of circulatory failure in bloodstream or deep tissue infection. In addition to the excessive T cell activation via superantigens, S. pyogenes exotoxins may induce the production of vasodilator or cardiac depressant leading to shock [S. pyogenes-PF. A higher frequency of TSS has been noticed in S. pyogenes-PF than seen in S. pyogenes-NF [S. pyogenes-PF arises from the vulnerability to impaired circulation and hypercoagulability on toxic or, if any, septic shock (Fig. S. pyogenes-PF may involve the ability to produce circulatory collapse-inducing factors rather than the tissue invasiveness of strain.The pathogenesis of lability . The strlability . S. pyoglarified . The invlarified  suggesteto shock . We demogenes-NF . The phygenes-NF . It is nock Fig. . The GASS. pyogenes-PF. The pathogen was repeatedly isolated from the debridement tissues until the start of continuous PCG infusion with CLDM, that is usually used for the treatment of invasive GAS infection [S. pyogenes-PF.Antibiotic and anticoagulant therapy is the mainstay of acute infectious PF. The treatment goal is the survival free from amputation and neurological deficit. Our patient required repeated debridement of the legs to treat compartment syndrome but not for NF. The histopathologic study showed ischemic necrosis in the subcutaneous tissues and muscles. It may corroborate a pivotal role of the circulatory failure in the pathophysiology of nfection . It accoS. pyogenes-acute infectious PF rarely occurred as the most severe form of S. pyogenes-TSS in healthy young children and the elderly. The first treatment of circulatory failure may be crucial for the outcomes of S. pyogenes-PF.In conclusion,


Burkholderia reimsis BE51, isolated from maize rhizosphere, has a promising biocontrol activity against a set of phytopathogens. Here, we report its draft genome sequence with the aim of providing insight into the potentially produced secondary metabolites and genes related to plant growth-promoting and biocontrol properties. Burkholderia reimsis BE51, isolated from maize rhizosphere, has a promising biocontrol activity against a set of phytopathogens. Here, we report its draft genome sequence with the aim of providing insight into the potentially produced secondary metabolites and genes related to plant growth-promoting and biocontrol properties. Burkholderia contains different members that have been isolated from different ecological niches  using Illumina MiSeq and HiSeq 2500 technology platforms, with 2 to 250-bp paired-end reads, and the mean coverage was 140×. For all software, default settings were used. The nearest reference genome, for Burkholderia lata, was determined using Kraken (http://bio-bwa.sourceforge.net). The reads were assembled by de novo assembly using SPAdes (http://cab.spbu.ru/software/spades/). The draft genome sequences, assembled into 182 contigs with an N50 contig size of 157,936 bp, was estimated at 8,934,495 bp with a G+C content of 66.40%. The gene function prediction was detected using the Rapid Annotations using Subsystems Technology (RAST) server (http://rast.nmpdr.org)  . In siliQMFZ00000000. The version described in this paper is the first version, QMFZ01000000. The SRA accession no. is SRP156928.This whole-genome shotgun project has been deposited at GenBank under the accession no.


Leishmania development in the sand fly gut leads to highly infective forms called metacyclic promastigotes. This process can be routinely mimicked in culture. Gene expression–profiling studies by transcriptome analysis have been performed with the aim of studying promastigote forms in the sand fly gut, as well as differences between sand fly–and culture-derived promastigotes.HASP/SHERP) cluster, the glycoprotein 63 (gp63), and autophagy genes, which are up-regulated in sand fly–derived promastigotes compared with cultured promastigotes. Because HASP/SHERP genes are up-regulated in nectomonad and metacyclic promastigotes in the sand fly, the encoded proteins are not metacyclic specific. Metacyclic promastigotes are distinguished by morphology and high infectivity. Isolating them from the sand fly gut is not exempt from technical difficulty, because other promastigote forms remain in the gut even 15 days after infection. Leishmania major procyclic promastigotes within the sand fly gut up-regulate genes involved in cell cycle regulation and glucose catabolism, whereas metacyclics increase transcript levels of fatty acid biosynthesis and ATP-coupled proton transport genes. Most parasite's signal transduction pathways remain uncharacterized. Future elucidation may improve understanding of parasite development, particularly signaling molecule-encoding genes in sand fly versus culture and between promastigote forms in the sand fly gut.Transcriptome analysis has revealed the crucial role of the microenvironment in parasite development within the sand fly gut because substantial differences and moderate correlation between the transcriptomes of cultured and sand fly–derived promastigotes have been found. Sand fly–derived metacyclics are more infective than metacyclics in culture. Therefore, some caution should be exercised when using cultured promastigotes, depending on the experimental design. The most remarkable examples are the hydrophilic acidic surface protein/small endoplasmic reticulum protein , especially under stress situations and at differentiation processes. However, transcript and protein levels behave similarly in approximately 60% of cases from a qualitative point of view . Changes in translational efficiency observed in other trypanosomatids strongly suggest that the differences are due to translational regulation and regulation of the steady-state protein levels. The lack of low-input sample strategies does not allow translatome and proteome analysis of sand fly–derived promastigotes so far. Leishmania spp. (Kinetoplastida: Trypanosomatidae) life cycle is digenetic because two hosts are involved: a mammal and a sand fly . The promastigote is the motile stage, which develops within the gut of the invertebrate host and is transmitted to the mammalian host during blood sucking (reviewed in  area)  area) , 7 compaotes/mL) –10; secootes/mL) –14 and iotes/mL) ; and thiotes/mL) , 17, beiotes/mL) , 19, higThe main promastigote forms within the sand fly gut are procyclics, nectomonads, leptomonads, and metacyclics , 20. TheL. infantum promastigotes showed that the peanut lectin–nonagglutinating metacyclic subpopulation (LiPro-PNA−) is more infective than the agglutinating subpopulation (LiPro-PNA+) and the whole population in stationary phase of axenic culture (LiPro-Stat), from which both are isolated [− are less infective  than promastigotes isolated from the sand fly vector Phlebotomus perniciosus (LiPro-Pper) SV [P. perniciosus–L. infantum vector–parasite pair, the metacyclic promastigote proportion in culture [In vitro infection of the human myeloid U937 cell line with isolated . The samPper) SV , 26. San culture , 25 and  culture  is appro culture , 29. Cul culture . TherefoLeishmania spp. life cycle is completed in three dividing phases, which are separated by nondividing transmission stages. One of them is amastigote replication within mammalian phagocyte phagolysosomes. Then, the blood meal phase takes place in the sand fly abdominal midgut. This phase consists of procyclic promastigote replication followed by differentiation to nectomonad promastigotes. This is valid for suprapylarian species, which are grouped within the subgenus Leishmania. Peripylarian species (subgenus Viannia) begin development in the hindgut [Leishmania spp., the term “metacyclic” has been defined as the infective form or the end product of promastigote development within the sand fly vector [According to Gossage and colleagues’ model , based o hindgut . Nectomo hindgut  stress ty vector , a smally vector . Gossagey vector  highlighL. major within P. papatasi is the LPG interaction with gut epithelium galectins. However, the presence of LPG receptors in other sand fly species remains unclear, and LPG-independent development has been reported. In fact, LPG composition is variable across species. The LPG, together with certain proteophosphoglycans (PPGs), may also have a major role in resistance to proteolysis within the gut (reviewed in [Bates  and Dostiewed in ). Once niewed in . Leptomoiewed in , mainly iewed in , which aiewed in , which tiewed in , 41, proiewed in  and/or fiewed in , 43, wheiewed in . This priewed in , 44. In iewed in . CultureAccording to the blocked fly hypothesis, the PSG plug obstructs the SV until it is removed by regurgitation during blood meal intakes . LeptomoL. infantum, whereas no substantial differences have been observed in the case of Leishmania mexicana, and unlike in L. infantum, L. mexicana metacyclics bind slightly tighter than procyclics [The phenotypical features of the different promastigote forms found in the sand fly gut differ between species. Separately studying each form is challenging. For example, the binding ability is strictly stage dependent, as nectomonads and leptomonads are considerably bound to the epithelium according to the different mechanisms mentioned above and further explained in the next section, whereas procyclics and metacyclics are nonbinding forms. Nonetheless, the relative binding ability is variable between different species, and in certain cases, a mild binding tendency has been observed in procyclics and metacyclics. For example, nectomonads bind tighter than leptomonads in ocyclics .Leishmania spp. and the sand fly gut have been revealed [Leishmania spp. [Lutzomyia longipalpis, P. papatasi, and P. perniciosus [Leishmania spp. molecules.Few molecular interactions between revealed . The innrevealed –60. Defenia spp. , 61–65. niciosus –69 reveaLeishmania species. This feature is crucial for vector competence, defining compatible and noncompatible vectors with a given Leishmania species [L. major genome encodes for ISPs that do not have targets in the parasite’s proteome [The blood meal induces digestive enzymes . These are serine proteases –72 like  species –76. At l species . At the  species –80 throuproteome  but haveproteome  and trypproteome . Amastigproteome . Phosphoproteome . For exaLeishmania spp. chitinase. For this reason, the parasite is not able to escape the PM until blood has been digested [Leishmania species are not able to break the PM of all sand fly vector species, and not escaping from the PM leads to parasite elimination through defecation. Therefore, this mechanism contributes to parasite–vector competence [The PM is composed of peritrophins, which contain one or more chitin-binding domains (CBDs), which have been predicted in most cases , 67, 69.digested . Howevermpetence .Leishmania spp. FLAG1/SMP1 flagellar protein [Leishmania species, and permissive, meaning they support development of multiple Leishmania species [Leishmania LPG–sand fly midgut galectin attachment mechanism [L. major Friedlin V1 strain–P. papatasi or P. duboscqi pairs, but other L. major strains are not able to bind. The LPG is composed of a glycosylphosphatidyl inositol (GPI) anchor and a glycan backbone composed of PG units and attached to the anchor through a hexasaccharide core [Leishmania donovani lines optimized for galactosylation pattern [L. major, thus allowing promastigote release from galectins [L. longipalpis and P. perniciosus [L. major and L. mexicana promastigotes in their guts in an LPG-independent manner [Leishmania spp. competence in different vectors [Leishmania spp.–sand fly pairs have been reported, the receptors have not been identified yet (see next section). In summary, it is known that different mechanisms mediate attachment of nectomonad promastigotes to the sand fly gut microvilli, but most remain uncharacterized, and there is controversy about LPG’s roles in different parasite species.Once nectomonads escape the PM, attachment to the gut epithelium is required to avoid clearance and then progressively ascend throughout the gut. It has been shown that nectomonad and leptomonad promastigotes specifically attach to the gut microvilli, and the mechanism depends on the parasite–vector pair , 90. A m protein . Accordi species –94. The echanism , 96. Thiide core . Side-chide core . Monogal pattern . Also, Lalectins . Alternaniciosus , which at manner , 45. How vectors . This st vectors . AlthougL. major proteins of unknown function encoded in the HASP/SHERP gene cluster  [Finally, the sand fly gut conditions may contribute to promastigote differentiation. An acidic environment, nutrient depletion, and probably scarce tetrahydrobiopterin levels induce metacyclogenesis. In this process, endosome sorting and autophagy are essential , as wellroteins) .Leishmania parasites was performed by Nicolle in the Nicolle–Novy–McNeal medium [L. infantum and L. major [+) subpopulation is less infective than the nonagglutinating (PNA−) subpopulation in L. major and L. infantum [L. infantum [L. infantum chagasi [L. donovani strain agglutinates at early differentiation stages when in contact with PNA [L. infantum PNA− promastigotes are more infective than PNA+ promastigotes [L. infantum LPG–L. longipalpis midgut epithelium interaction based on PG receptors. The interaction is based in β1,3-glucosylation, and release is caused by glucose residue removal. The same mechanism was previously described for an Indian L. donovani strain and the vector P. argentipes [Leishmania braziliensis because glucose residue addition leads to ex vivo detachment from L. longipalpis gut explants [L. infantum strains starting at 50 μg/mL [− and PNA+ forms can be isolated in the monogenetic trypanosomatid Crithidia fasciculata [The first axenic culture of l medium . Since tl medium –110. StaL. major , 25, 111L. major ). These L. major . The modL. major , 113. Thinfantum , 24. Howinfantum , includi chagasi , is diff chagasi . The LPGwith PNA , 117, buwith PNA . L. infastigotes  reportedexplants . In summ50 μg/mL . The difciculata , but theL. infantum promastigotes have shown that the LiPro-PNA− metacyclic subpopulation is more infective than the agglutinating LiPro-PNA+ and the whole population in stationary phase of axenic culture (LiPro-Stat), from which both are isolated [− are less infective  than promastigotes isolated from the SV of the sand fly vector P. perniciosus (LiPro-Pper) [P. perniciosus–L. infantum vector–parasite pair, the proportion of metacyclic promastigotes in culture [In vitro infection experiments of the human myeloid U937 cell line with isolated . The samro-Pper) , 26. San culture , 25 and  culture  is appro culture , 29. Cul culture . TherefoConsidering how challenging working with promastigotes from the gut is, the cost–benefit balance presumably tilts to axenic culture in principle, but this is not as clear when considering results obtained by means of transcriptome analysis. Alternative methods for isolation of metacyclic promastigotes, like centrifugation in Percoll gradient, have been described, which are out of the scope of this review.Leishmania research is also available [Microarrays are dense molecular probe matrixes on a solid surface. DNA microarrays contain thousands of genes, gene fragments, and/or noncoding sequences that are hybridized with one or more labeled nucleic acid sample  for different purposes, such as gene expression profiling. In this case, total RNA or mRNA samples are directly labeled, amplified and labeled, or reversely transcribed in order to obtain directly or indirectly labeled cDNA. The fluorescent labels enable measuring the relative levels of each target sequence once emission signals have been acquired with a specialized scanner . Bioinfovailable .RNA sequencing (RNA-seq) is a high-throughput approach based in Next Generation Sequencing (NGS) that consists of genome-scale amplification and NGS of short cDNA fragments generated from RNA samples. For this purpose, double-stranded cDNA is generated and PCR amplified, incorporating appropriate linkers for NGS. The primers used in all steps and the PCR conditions are designed according to the desired fragment size range, which is typically between 0.1 and 1 Kbp. The products are fragmented and subjected to NGS in any of the platforms commercially available  . AlternaNowadays, transcriptome analysis is a routine technical approach thanks to the development of the DNA microarray technology during the mid-1990s, which has been extensively used during the last 2 decades and is being replaced by RNA-seq. At this point, it is important to remark that the condition for a technical approach to be valid is reliability rather than novelty. Both DNA microarray hybridization analysis and RNA-seq are reliable for gene expression profiling or DGE analysis, although RNA-seq is a more powerful and robust approach , 124. MiLeishmania spp. is adapted to respond very quickly to different environments [− promastigotes using microarray analysis [L. major RNA input was 5–20 ng [Before execution of a DGE analysis, biological samples must be examined to determine whether they are appropriate to address the proposed hypothesis. For example, the main features of metacyclic promastigotes are high infectivity and morphology . Therefore, metacyclic promastigotes can be identified for downstream DGE by infection experiments  7, 25, , 25, 26 ronments . Consideanalysis . In factanalysis –129. The 5–20 ng . The bas 5–20 ng .L. donovani RNA (1 ng) samples embedded in a human RNA amount 1,000 times larger, although these samples should be sequenced more deeply to reach the same coverage as pure Leishmania spp. RNA [The presence of tissue from the sand fly host should be minimized when isolating the biological sample. Microarray cross-hybridization controls were performed to select specific hybridization conditions and remove the few cross-hybridizing spots found from analysis , 25, 26.spp. RNA . Once anspp. RNA . Unfortuspp. RNA  datasetsspp. RNA ). mRNA cspp. RNA ). Modulaspp. RNA .L. infantum promastigotes obtained from experimentally infected P. perniciosus within the vector [L. major from P. duboscqi [L. infantum is responsible for zoonotic visceral leishmaniasis in the Mediterranean Basin and South America, whereas L. major is responsible for cutaneous leishmaniasis in the Old World. Their different affinity for sand fly vector species and in key developmental processes  is a probable cause of obtaining mismatched DGE. Second, most samples and comparisons are not equivalent. For example, intracellular L. infantum amastigotes obtained in vitro from the myeloid human U937 cell line [L. major amastigotes obtained from mice footpad lesions (LmAM) [L. infantum (LiPro-Pper) and L. major sand fly metacyclic promastigotes (LmSFMP). In the first case [L. infantum metacyclics in the P. perniciosus gut even 8–15 days after blood feeding from infected dogs. As mentioned above, this is dependent on the parasite–vector pair. In summary, all populations analyzed in the studies listed in L. major [− with LiPro-Pper [L. major–P. papatasi and L. major–P. duboscqi pairs .Four DGE analyses of e vector , 26, 69 duboscqi  have bees (LmAM) . As it crst case . Howeverrst case  did not L. major . For thePro-Pper  are not Pro-Pper  or cultuPro-Pper . For exaPro-Pper , but it Pro-Pper . ConsistPro-Pper . Not onliewed in ). Anothegipalpis  but may L. infantum, the number of DEGs found in the direct comparison of LiPro-Pper with LiPro-PNA− was 285 at the cutoff expression values mentioned above [L. infantum datasets, which reflects the above-mentioned differences found in infectivity between these promastigote populations (LiPro-Pper > LiPro-PNA− > LiPro-Stat). All L. major and L. infantum datasets are different because different stages have been compared in each case. For example, the LmSFMP/Lm-SFPP DGE analysis is not comparable to the LiPro-Pper/LiPro-Stat study because cultures in stationary phase mostly contain nectomonads and metacyclics [L. infantum comparing heterogeneous populations of sand fly promastigotes (LisfPro) [P. perniciosus, with the heterogeneous promastigote populations in axenic culture (LiacPro), we observed approximately 950 genes up-regulated ≥2-fold, which is 2.0 to 3.6 times higher than expected compared with the previous DGE datasets about more homogeneous promastigote populations showing approximately 300 DEGs each [Leishmania spp., including homogeneous and heterogeneous populations . In summary, global concordances and differences between studies on sand fly–derived promastigotes have been found, but comparative interpretation of studies should be cautious, considering different biological comparisons, sample source origin and preparation, and technical approaches.The across-experiment comparison of LmSFMP/LmSFPP and LmCMP/LmPro-Log  is presued above , comparaed above . Most DEacyclics  and probLisfPro)  taken frEGs each , 25, 125L. infantum [L. major [The microenvironment influences the parasite’s differentiation processes , 125. StL. major , 139–142L. major , 125 andL. major .Cell cycle–related genes are generally down-regulated in LmSFMP and LmSFNP compared with LmSFPP and LmAM, which is in agreement with the replicative or nonreplicative status of these stages . Steady-− versus LiPro-Pper, and LmSFMP/LmSFPP versus LmCMP/LmPro-Log) [L. infantum heterogeneous populations composed of all promastigote development forms from the sand fly (whole-gut preparations) and culture (growth curve mixtures), we also observed that gp63 and autophagy genes were up-regulated [HASP/SHERP cluster. As mentioned above, these genes are essential for metacyclogenesis at least in L. major. In fact, Inbar and colleagues’ [LPG3, a gene essential for biosynthesis and assembly of GPI-anchored glycoconjugates, reaches its expression peak in LmSFPP. Sand fly–derived populations enriched in metacyclics (LiPro-Pper) are more infective than stationary-phase cultures (LiPro-Stat) and metacyclics obtained from those populations (LiPro-PNA−) [HASP/SHERP gene cluster up-regulation in sand fly–derived promastigotes compared with cultured promastigotes supports that metacyclogenesis is more successful in the sand fly gut than in culture. Therefore, the microenvironment exerts an important influence in differentiation [Many genes involved in metacyclogenesis (see below) are highly up-regulated in heterogeneous populations of sand fly-derived promastigotes (LisfPro) compared with cultured promastigotes (LiacPro)  but not Pro-Log) , 25, 125egulated , as wellro-PNA−) , 25. Autntiation .L. major [SHERP up-regulation in LmSFNP and LmSFMP, reaching maximum levels in LmSFMP. L. infantum transcriptome analysis is also in agreement with the role in metacyclogenesis, but SHERP transcripts are less abundant in LiPro-Pper than in LiPro-Stat [SHERP is not differentially expressed between LiPro-Pper and LiPro-PNA−, indicating that different microenvironments do no influence SHERP expression in L. infantum [HASP-A1 is also down-regulated in LiPro-Pper versus LiPro-Stat [L. infantum sfPro versus acPro (heterogeneous populations) transcriptome analysis is also consistent with the previous studies because SHERP is up-regulated in sfPro . Interspecies comparison should be cautious, as previously mentioned. SHERP data are concordant between L. major and L. infantum with the previously established idea about essentiality for metacyclogenesis, but simultaneously, transcriptome analysis has revealed specific differences.SHERP is essential for metacyclogenesis in L. major . Inbar aL. major  revealedPro-Stat , indicatinfantum . Stationinfantum , whereasPro-Stat , leadingL. infantum and L. major [−). According to relative infectivity (LiPro-Pper > LiPro-PNA− > LiPro-Stat), sand fly metacyclics are “more metacyclic” than culture metacyclics. These findings are consistent with the considerable energy requirements for high motility ascribed to metacyclic promastigotes [Genes involved in fatty acid biosynthetic processes are up-regulated in sand fly–derived metacyclics in both L. major , 125, buL. major . CertainL. major . Genes istigotes .− promastigotes because this is a direct comparison [Confrontation of the transcriptomes and infectivity of sand fly–derived promastigotes with cultured promastigotes  is in agmparison  and thesTrypanosoma cruzi epimastigotes [A considerable number of the DEGs are involved in signal transduction and gene expression regulation at the posttranscriptional, translational, and posttranslational levels between cultured and sand fly–derived promastigotes , 25. Howstigotes , 144 shoL. major promastigotes with sand fly gut–derived L. major exosomes leads to greater footpad lesions in mice [Promastigotes constitutively secrete exosomes to the sand fly gut lumen. Coinoculation of cultured  in mice . These e in mice –150. The in mice : gp63, w in mice ; HSP70 [ in mice  and HSP8 in mice ; calpain in mice ; trypare in mice ; and sur in mice . Transcr in mice .− promastigotes are more infective than PNA+ in both L. major [L. infantum [L. major, whereas the parasite-interaction mechanisms remain unknown in all other species. LPG-independent promastigote development has been demonstrated in permissive vector species (see "Sand fly–Leishmania interactions" section). However, highly infective (therefore metacyclic) promastigotes isolated using the PNA-negative selection procedure is possible in L. infantum [P. perniciosus. Alternative unknown mechanisms participate in recognition because LPG is not strictly required for development, and the importance of this molecule is relegated to L. major only [Leishmania species. Unknown PG receptors recognize the LPG in the sand fly gut [L. major development stages (see "Sand fly–Leishmania interactions" section), and presumably in L. infantum because both contain the key repeated  motif in the LPG structure [− and PNA+ subpopulations could be isolated in the monoxenous parasite C. fasciculata [C. fasciculata may lead to raising other approaches for searching LPG interactions and alternative functions in different Leishmania species. High-throughput comparative metabolomics approaches may be useful to answer these questions, but not transcriptomics approaches. Bearing these considerations in mind, we suggest that the role of the modified LPG at this stage may not be necessarily the same between species, as already shown for the unmodified LPG at earlier stages. Consequently, we postulate that the “metacyclic status” of PNA− from L. infantum may not be necessarily the same as for PNA− from L. major, as the molecular markers and infection mechanisms may be different depending on the species. This is not surprising, because each species complex causes different pathology, and accurate measurements comparing metacyclic promastigote infectivity of each species are not possible. The peanut lectin has different affinity for LPG from a distinct origin, as different substitutions of the molecule disaccharide backbone are found depending on the species (see "The axenic culture model: Strengths and limitations" section). In any case, L. major [L. infantum PNA− promastigotes [+ promastigotes.Metacyclic promastigotes are defined by morphology, but their molecular features are not entirely known. PNA separation is effective to obtain highly infective promastigotes because PNAL. major  and L. iinfantum , but theinfantum , 156, whjor only . However fly gut , 117, whtructure . Variaticiculata , a fact L. major  and L. istigotes  have beeHASPA1, HASPA2, HASPB, respectively, LinJ.23.1200, LinJ.23.1220, and LinJ.23.1240), one out of three membrane-bound acid phosphatases (LinJ.28.2850), all three apical membrane antigen 1  proteins, and the META domain–containing protein 2  gene. Both small hydrophilic surface protein–encoding gene copies  are not included in the LisfPro versus LiacPro DEG set according to the 2-fold threshold value imposed, but they still show statistically significant approximately 1.5-fold higher levels in sfPro versus acPro [SHERP is clearly up-regulated in L. major metacyclics (LmSFMP versus LmSFPP and LmCMP versus LmPro-Log) and, to a lower extent, in nectomonads (LmSFNP versus LmSFPP) [L. infantum. Although the specific SHERP expression profiles are different, both are concordant with SHERP essentiality in metacyclogenesis [L. infantum and L. major metacyclics differentially regulate SHERP expression . Interestingly, both SHERP genes are up-regulated in LiPro-Stat versus LiPro-Log of this species according to microarray analysis [SHERP compared with procyclics. SHERP is a good metacyclogenesis marker but not a metacyclics marker because it is overexpressed in more than one promastigote form (nectomonads and metacyclics). The data suggest that the SHERP gene expression patterns are similar between L. major and L. infantum, except for the promastigote form reaching the maximum expression levels, which peak earlier in L. infantum than in L. major. This would not be surprising whenever confirmed in the future given the different biological affinity for vectors and different developmental processes of both species, resulting in different disease progression in mammalian hosts. These observations are in agreement with the fact that metacyclic promastigote features and behavior may vary between species and are not entirely known. For example, they are highly infective, or more infective than other promastigote forms, but by how much? Which molecules are true markers of metacyclics in each species?When comparing the heterogeneous populations LisfPro and LiacPro by slRNA-seq, a group of genes directly involved in metacyclogenesis was found to be highly up-regulated (≥4-fold) , which sus acPro . Whereas LmSFPP) , 136, diogenesis . Culturees LinJ.2.2850, alanalysis . This isMETA1 gene was described to be expressed specifically at the metacyclic stage in culture, but the high-throughput DGE studies of L. infantum and L. major have not confirmed this result at the transcript level in sand fly–derived promastigotes [Leishmania spp. genomes encode for hypothetical proteins, most of unknown biological role in the parasite. These observations provide an idea of how little is known about development within the sand fly vector.The stigotes , 25, 125Elucidation of processes involving the unknown relationship between external stimuli from the microenvironment, the parasite’s uncharacterized sensing and intracellular signaling mechanisms, and the unusual gene expression regulation mechanisms found in these organisms  maL. donovani, Lahav and colleagues [8 cells  in the case of Saccharomyces cerevisiae [3 and the average volume of a Leishmania spp. cell is approximately 65–75 μm3, about 10 times more promastigotes or amastigotes would be required in principle.In an experiment combining DGE analysis by means of DNA microarrays and quantitative proteomics with polysome profiling in lleagues  observedrevisiae . As the T. brucei are more extensive than indicated by transcriptome profiling [9 parasites per sample were used to generate the ribosome-footprinting and the fragmented-mRNA library. Jensen and colleagues [T. cruzi [Leishmania spp. promastigotes obtained from the sand fly so far. In fact, as many as approximately 104 infected sand flies would be required to obtain enough promastigotes for a replicate of a ribosome-profiling experiment, and many more sand flies would be required for ribosome profiling of more homogeneous populations—for example, approximately 106 for metacyclics.Ribosome profiling is a more specific high-throughput approach for measurement of translational efficiency. Protection of mRNA sequences by ribosomes is quantified by means of NGS from a ribosome-footprinting library combined with a fragmented-mRNA library . The firrofiling , 161. Inlleagues  also mapT. cruzi , in whic8Leishmania spp. cells for both two-dimensional electrophoresis-based strategies [Typical samples for proteome analysis require approximately 1–2 × 10rategies  and quanrategies . AlthougHASP/SHERP cluster, the gp63 metalloprotease family, and autophagy genes are overexpressed in sand fly metacyclic promastigotes compared with cultured promastigotes. Differential expression of several genes involved in gene expression regulation, signaling, and metabolic processes between sand fly–derived and cultured promastigotes supports an important microenvironmental influence differentiation. Elucidating signal transduction pathways in these parasites may substantially improve understanding of the relationships between promastigotes and the different microenvironments in the sand fly gut (Metacyclic promastigotes are distinguished by morphology (rapid-swimming forms with an elongated flagellum) and high infectivity. No molecular markers are available. Metacyclics can be isolated by negative selection with PNA, as confirmed by infection experiments. Caution should be exercised when using cultured promastigotes depending on the experimental design, and when comparing studies. Transcriptome analysis has revealed the crucial microenvironmental role in parasite development in the sand fly gut because substantial differences and moderate correlation between cultured and sand fly–derived promastigotes have been found. In fact, sand fly–derived metacyclics are more infective than metacyclics in culture, and genes involved in metacyclogenesis such as the  fly gut . UnfortuLeishmania spp. promastigote forms? (2) Are the multiple roles of the LPG different between species, causing different types of leishmaniasis? (3) Are there truly stage-specific markers? (4) Are they different between species? (5) How different are canonical signal transduction cascades and those of Leishmania spp.? (6) Are there developmentally regulated changes in Trans-splicing? If so, what implications would they have? (7) How can relative protein levels be analyzed in sand fly–derived promastigotes?The main outstanding questions are: (1) What are the molecular features of the different Metacyclic promastigotes are highly infective forms, but no markers are available.Accurate description of samples compared by means of high-throughput strategies and caution when comparing different studies are essential and are particularly important for samples obtained from the sand fly because different vector and parasite pairs are considered.Transcriptome data and infection experiments support that sand fly–derived promastigotes are more infective than cultured ones.Sand fly–derived promastigotes are more infective than cultured promastigotes to in vitro–cultured human phagocytes, which combined with transcriptome profiles, supports that metacyclogenesis is more successfully completed in the sand fly gut.Leishmania infantum and L. major promastigotes derived from the sand fly gut confirm that the hydrophilic acidic surface protein (HASP), the small hydrophilic endoplasmic reticulum protein SHERP, and the glycoprotein 63 (gp63) genes are involved in metacyclogenesis and already increased in nectomonad promastigotes and thus are not metacyclic promastigote markers.Transcriptome analysis in Differential expression of several genes involved in gene expression regulation, signaling, and metabolic processes between sand fly–derived and cultured promastigotes supports an important influence of the microenvironment in differentiation.Studying the translatome and the proteome is not feasible in sand fly–derived promastigotes so far. Transcriptomics is the only alternative, and interpretation of the results should be cautiously discussed because transcript levels do not always reflect protein levels.Alcolea PJ, Alonso A, Gomez MJ, Postigo M, Molina R, et al. Stage-specific differential gene expression in Leishmania infantum: from the foregut of Phlebotomus perniciosus to the human phagocyte. BMC Genomics. 2014;15:849.Alcolea PJ, Alonso A, Dominguez M, Parro V, Jimenez M, et al. Influence of the microenvironment in the transcriptome of Leishmania infantum promastigotes: Sand fly versus culture. PLoS Negl Trop Dis. 2016;10(5):e0004693.Alcolea PJ, Alonso A, Degayon MA, Moreno-Paz M, Jimenez M, et al. In vitro infectivity and differential gene expression of Leishmania infantum metacyclic promastigotes: negative selection with peanut agglutinin in culture versus isolation from the stomodeal valve of Phlebotomus perniciosus. BMC Genomics. 2016;17:375.Alcolea PJ, Alonso A, Baugh L, Paisie C, Ramasamy G, Sekar A, et al. RNA-seq analysis reveals differences in transcript abundance between cultured and sand fly-derived Leishmania infantum promastigotes. Parasitol Int. 2018;67(4):476–80.Inbar E, Hughitt VK, Dillon LA, Ghosh K, El-Sayed NM, et al. The transcriptome of Leishmania major developmental stages in their natural sand fly vector. MBio 2017;8(2):e00029-17.


Propionibacterium acnes, recently renamedCutibacterium acnes, along with the other major pathophysiological factors of increased seborrhea, hyperkeratinization of the pilosebaceous unit, and inflammation, has long been implicated in the pathogenesis of acne. Recent advances have contributed to our understanding of the role ofP. acnes in acne. Although there are no quantitative differences inP. acnes of the skin of patients with acne compared with controls, theP. acnes phylogenic groups display distinct genetic and phenotypic characteristics,P. acnes biofilms are more frequent in acne, and different phylotypes may induce distinct immune responses in acne.P. acnes plays a further important role in the homeostasis of the skin’s microbiome, interacting with other cutaneous commensal or pathogenic microorganisms such asStaphylococcus epidermidis,Streptococcus pyogenes, andPseudomonas species. In the era of increasing antimicrobial resistance, the selection of acne treatment targetingP. acnes and the prevention of antibiotic resistance play a key role in improving outcomes in acne patients and public health.The skin commensal Corynebacteria,Propionibacteria, andStaphylococci1.The cutaneous microbiome exists in a finely tuned equilibrium in healthy skin that when perturbed may lead to various inflammatory skin diseases. The three most commonly observed cutaneous genera arePropionibacterium acnes has been implicated in the pathophysiology of prostate cancer2, sarcoidosis3, infective endocarditis4, infections involving prosthetic devices 5, and acne, the last of which is the focus of this review.P. acnes is a Gram-positive, non-spore-forming human skin commensal that prefers anaerobic growth conditions7. It is a member of the normal skin microbiota along withP. avidum,P. granulosum, andP. humerusii8. TheP. acnes genome is 2.5 Mb in size and has been completely sequenced. It has genes encoding metabolic enzymes, enabling it to survive in microaerophilic conditions, but also lipases that degrade the lipids of the pilosebaceous follicle, providing the bacterium with the energy it needs9. Recently, a taxonomic reclassification was proposed in whichP. acnes was renamedCutibacterium acnes to account for genomic adaptive changes and to differentiate it from otherPropionibacteria species. In particular, specific lipase genes were identified encoding for triacylglycerol lipase and lysophospholipase able to degrade sebum lipids10. However, it has been proposed that it is taxonomically valid to continue to use the genus namePropionibacterium for the cutaneous group within dermatology specialties for a range of different reasons, including to avoid confusion with the previous name,Corynebacterium acnes11.P. acnes concerning taxonomy, the role of different phylotypes andP. acnes biofilm in acne pathophysiology, and the targeting ofP. acnes with appropriate acne treatments and the respective implications in the homeostasis of the skin’s microbiome and the emergence of antimicrobial resistance.In this review, we describe the characteristics ofP. acnes has been classified into three phylotypes (phylogroups) based on gene sequences or biological characteristics (lipase activity): I, II, and III12. These phylogroups in turn have been split into distinct subspecies known asP. acnes subsp.acnes,P. acnes subsp.defendens, andP. acnes subsp.elongatum, respectively, to denote distinct phylogenetic, genomic, and phenotypic characteristics as well as their association with different clinical diseases, including acne and progressive macular hypomelanosis14.With regard to taxonomy,P. acnes subsp.acnes has been described. Extracellular enzymes include RNase, neuraminidase, hyaluronidase, acid phosphatase, lecithinase, and lipase. The bacterial cells ferment glucose, and lactic acid is produced from fermentable carbohydrates in variable quantities. The major long-chain fatty acid produced is 13-methyltetradecanoic acid14. Gene sequence analysis ofP. acnes on the basis of the genesrecA andtly revealed further phylogenetic subdivisions within the type I clade: the types IA, IB, and IC. Higher-resolution methods provided additional differentiation of IA strains into types IA1 and IA213. SoP. acnes is subdivided into six phylotypes: IA1, IA2, IB, IC, II, and III15. Multi-locus sequence typing and single-locus sequence typing (SLST) identified further subgroups among phylotypes, called clonal complexes.The subspeciesP. acnes phylogroups have been associated with specific diseases and distinct virulence, biochemical, and immunological characteristics that will be discussed in the following section.TheP. acnes has been regarded as an important member of the cutaneous microbiota. It has been linked to the inflammatory skin condition acne vulgaris for more than 100 years. The four major pathophysiological factors implicated in the pathogenesis of acne include the role ofP. acnes, increased seborrhea, hyperkeratinization of the pilosebaceous unit, and inflammation16.P. acnes colonization of the skin is necessary but not sufficient for the establishment of acne pathology.P. acnes dominates the microbiota of pilosebaceous units and accounts for 87% of clones in patients with acne and in individuals without acne17.P. acnes has been reported to represent more than 30% of the facial microbiota in patients with acne1, but another study of 55 patients with facial acne reported lower rates (less than 2%) of sampled bacteria18. These results should be interpreted with caution given the role of the sampling methodologies used. Different sampling methods, such as swab, scrape, cyanoacrylate gel biopsy, and needle biopsy, are used to collect skin bacteria for testing. Each technique targets different skin structures and anatomical sites. The sampling of superficial and intra-stratum corneum bacterial populations is considered quite straightforward. However, the sampling of hair follicle populations has proven more difficult and a skin biopsy may be required. The use of tape-stripping for hair follicle sampling in acne can be misleading, as multiple superficial and intra-stratum corneum microbial populations are sampled but bacteria may reside in a deeper part of the hair follicle19. This area is inaccessible with the above-mentioned sampling methodologies, providing very little material from inside the hair follicle and making it difficult to standardize8.P. acnes sampling with bacterial culture may not reliably distinguish betweenP. acnes populations with possibly variable pathogenic potential20.P. acnes in the skin of patients with acne compared with controls17, its phylogenic groups display distinct genetic and phenotypic characteristics in acne10 and different phylotypes are known to induce distinct immune responses in acne12. DifferentP. acnes types have been isolated from acne vulgaris, and the type III strains have been associated with progressive macular hypomelanosis, underscoring the importance of genetic division ofP. acnes and suggesting the involvement of specificP. acnes phylotypes in the pathophysiology of acne21.Although there is no quantitative difference ofP. acnes isolates has revealed distinct profiles in patients with acne  suggested thatIn vitro growth ofP. acnes biofilms demonstrated the composition of the extracellular polymeric substance (EPS) matrix ofP. acnes biofilm with extracellular DNA, proteins, and glycosyl residues as well as upregulated mRNA expression of Christie–Atkins–Munch-Peterson (CAMP) factor 126.P. acnes biofilm in acne patients compared with controls. A case-control study in facial biopsies showed that follicularP. acnes was more frequently demonstrated in samples from acne patients compared with matching controls. Furthermore,P. acnes from acne samples more frequently formed biofilms in the sebaceous follicles compared with control samples20. Although similar biofilms have also been observed in skin diseases other than acne, such as folliculitis, folliculitis decalvans, and hidradenitis suppurativa, these were seen in terminal hair follicles29.Only one study has investigatedP. acnes modulates the differentiation of keratinocytes and increases local inflammation, it is regarded as an etiological agent of both the microcomedone (a structure invisible to the naked eye) in the early stages of acne and of the inflammatory acne lesions30. The different target activities ofP. acnes in acne are summarized inAsP. acnes shows complex interactions with key events implicated in the pathogenesis of acne. It interacts with the innate immunity, including Toll-like receptors (TLRs), antimicrobial peptides (AMPs), protease-activated receptors (PARs), and matrix metalloproteinase (MMP), and upregulates the secretion of pro-inflammatory cytokines, including interleukin-1a (IL-1a), IL-1β, IL-6, IL-8, IL-12, tumor necrosis factor-alpha (TNF-α), and granulocyte-macrophage colony-stimulating factor (GM-CSF), by human keratinocytes, sebocytes, and macrophages31. Moreover, the production of AMP , cytokines (IL-1α), and MMP was associated with the increased expression of the G-protein-coupled receptor PAR-2 in keratinocytes from acne-affected skin32.P. acnes extracts are directly able to modulate the differentiation of keratinocytes by inducing b1, a3, a6s, aVb6 integrin expression, and filaggrin expression on keratinocytes, changes seen in the development of acne lesions33. Interplay betweenP. acnes and macrophages in the perifollicular dermis can induce IL-1β32, which in turn may further activate the NLRP3-inflammasome pathway in antigen-presenting cells and myeloid cells19. Recentin vitro studies have revealed thatP. acnes can induce IL-17 production by T cells (Th1/Th17)31. Clusters of CD3+ cells have been demonstrated in the vicinity of theP. acnes-positive comedones, cells that were absent from the surrounding inflamed lesions. These findings in early acne stage further support the role ofP. acnes in the initiation of inflammation34.P. acnes releases extracellular vesicles (EVs) which also induce cellular responses via TLR2 signal cascades. TheseP. acnes-derived EVs induce IL-8 and GM-CSF and decrease epidermal keratin-10 and desmocollin, contributing to the development of acne lesions35.et al. showed that acne-associatedP. acnes phylotypes induced distinct cytokine patternsin vitro in peripheral blood mononuclear cells from healthy individuals, including higher levels of inflammatory interferon-gamma (IFN-γ) and IL-17, suggesting a mechanism of inducing acne via both Th1 and Th17 pathways12. On the other hand,P. acnes phylotypes associated with healthy skin induced higher levels of IL-10. Moreover, there were different expression patterns between phylotypes; acne-associated phylotypes showed higher expression of an adhesion protein, whereas phylotypes associated with healthy skin showed higher expression of a cell surface hydrolase. These identified immune responses and proteomes of differentP. acnes strains provided deeper insight into how specificP. acnes phylotypes influence the pathogenesis of acne12. In a follow-up study, Agaket al. reported differential effects of acne-affected skin- and healthy skin-associated lineages ofP. acnes on CD4+ T-cell and Th17 cell responses and suggested thatP. acnes strains express different antigenic components on their surface structure, possibly explaining the higher IL-17 levels induced in acne-affected skin-associatedP. acnes strains36.YuP. acnes has been implicated in lipogenesis and sebum production, as it stimulates the sebaceous glands and sebum synthesis via the corticotropin-releasing hormone (CRH)/CRH receptor pathway37. Expression of the complete CRH system has been described in acne; a study in biopsies from the facial skin of patients with acne reported a stronger expression of CRH in sebocytes of acne-involved skin compared with non-involved and normal skin38. In particular, CRH augments the synthesis of sebaceous lipids and induces IL-6 and IL-8 release by sebocytes, mediated by the CRH receptor39.Furthermore,P. acnes has a role in its cytotoxicity, as mutations of CAMP diminishedP. acnes colonization and inflammation in mice40.P. acnes CAMP factor can induce cell death of sebocytes in sebaceous glands, resulting in amplification of the inflammation response41. In addition, a study reported that theP. acnes surface protein CAMP factor 1 stimulated keratinocytesin vitro by interacting directly with TLR242.A recent study reported that a secretory CAMP factor ofP. acnes and can generate reactive oxygen species that induce inflammation in keratinocytes and result in acne lesions. Johnsonet al. showed that acne-associatedP. acnes strains produced more porphyrins than health-associated strains isolated from individuals and that vitamin B12 supplementation significantly increased porphyrin production in the acne-associated strains only43. Another study showed that theP. acnes vitamin B12 biosynthesis pathway was downregulated in acne patients compared with healthy individuals. Furthermore, intramuscular vitamin B12 supplementation repressed its own biosynthesis inP. acnes and promoted increased porphyrin production in healthy subjects44.Porphyrins are secreted byP. acnes population that differ in their ability to degrade HA and could be involved in the pro-inflammatory responses seen in acne. One variant is present inP. acnes type IA strains and is associated with acne, and the other one is in type IB and II strains and is associated mainly with soft and deep tissue infections. HA fragments interact with cell surface receptors such as CD44 and TLR2 and induce the inflammatory response45.Hyaluronic acid (HA) lyase is a ubiquitous enzyme with two distinct variants in theP. acnes has an intriguing role in the homeostasis of the skin’s microbiome, interacting with other cutaneous microorganisms such asStaphylococcus epidermidis,Streptococcus pyogenes, andPseudomonas species. In the microbiome of healthy skin,S. epidermidis may limit the overcolonization withP. acnes strains and reduceP. acnes-induced IL-6 and TNF-α production by keratinocytes. On the other hand,P. acnes may limit the proliferation ofS. aureus andS. pyogenes by promoting triglyceride hydrolysis and propionic acid secretion. As a result, an acidic pH is maintained in the pilosebaceous follicle. A change of the microbiome composition may lead to a disturbed skin barrier and inflammation. In acne, a modified profile ofP. acnes is noticed; different phylotypes differ between patients with and without acne46. Hallet al. showed in cutaneous samples that whenP. acnes was present,Pseudomonas species typically were not, and vice versa47. Interestingly, antibiotic treatment for acne that decreasesP. acnes colonization on the skin may also result in Gram-negative folliculitis caused byPseudomonas48. Megyeriet al. recently proposed thatP. acnes strains may be implicated in antimicrobial defense pathways by triggering a local increase in the autophagic activity of keratinocytes49.Apart from its target activities in acne,P. acnes is a worldwide problem, and rates of resistance increased from 20% in 1979 to 64% in 2000; rates for tetracyclines were lower compared with rates for clindamycin and erythromycin50. A study of 664 patients in the UK, Spain, Italy, Greece, Sweden, and Hungary reported that the prevalence ofP. acnes resistance rates ranged from 50.8% to 93.6% to any antibiotic  and that all included dermatologists who specialized in treating acne were colonized with resistantPropionibacteria51.The antibiotic resistance ofin vitro antibiotic susceptibility patterns ofP. acnes among different countries is recognized56. A possible explanation is the fact that there are different antibiotic-prescribing habits among the countries and even different concomitant topical agents used. In studies from Korea, the UK, Colombia, Mexico, Hong Kong, Hungary, and Spain,P. acnes antibiotic resistance was noted in 36.7%, 55.5%, 40%, 75.5%, 54.7%, 51%, and 94% of patients with acne, respectively , are indicated for moderate to severe inflammatory papulopustular acne and acne affecting the trunk . The durP. acnes was modified; oral isotretinoin, though not an antibiotic, correlated with a reduction in the numbers ofP. acnes, including isolates resistant to antibiotics, that were cultured from the cheeks, but there was no effect inP. acnes sampled from other anatomic sites67.A study of 56 patients with cystic or severe acne vulgaris treated with oral isotretinoin (1 mg/kg per day) reported that the colonization of the skin with. acnes, underlying its role in the pathogenesis of acne68. The mode of action of PDT includes not only photodynamic damage of the sebaceous gland but also the photodestruction ofP. acnes70.Emerging off-label therapeutic modalities for acne, such as topical photodynamic therapy (PDT) with photoactivation of aminolaevulinic acid (ALA) or methyl aminolaevulinic acid (MAL), target PP. acnes was investigated, and relevant studies initially stopped in 2011, as effectiveness in humans with acne was not shown71. Interestingly, a recent study reported the efficacy of CAMP factor antibodies in the neutralization of the acne inflammatory response inex vivo acne models; the incubation ofex vivo acne skin explants from acne patients with monoclonal antibodies (mAbs) to theP. acnes-secreted CAMP factor diminished the amounts of pro-inflammatory cytokines IL-8 and IL-1β40. The authors also proposed that the injection of the mAb to CAMP factor directly into acne lesions may prove to be beneficial40.The potential for vaccination againstP. acnes in the pathogenesis of acne. Although there is no quantitative difference ofP. acnes among patients with acne and healthy individuals,P. acnes phylogenic groups may display distinct genetic and phenotypic characteristics. Different phylotypes may induce distinct immune responses, and theP. acnes biofilm has been reported more frequently in patients with acne. Furthermore,P. acnes plays important roles in the homeostasis of the skin’s microbiome, interacting with other cutaneous microorganisms such asS. epidermidis,S. pyogenes, andPseudomonas species. Non-antibiotic approaches targetingP. acnes without inducing antibiotic resistance may improve patient outcomes in acne while avoiding public health issues.Significant progress has been made in understanding the role of


Rhipicephalus sanguineus (sensu stricto) is reputed to be the most widespread tick of domestic dogs worldwide and has also been implicated in the transmission of many pathogens to dogs and humans. For more than two centuries, Rh. sanguineus (s.s.) was regarded as a single taxon, even considering its poor original description and the inexistence of a type specimen. However, genetic and crossbreeding experiments have indicated the existence of at least two distinct taxa within this name: the so-called “temperate” and “tropical” lineages of Rh. sanguineus (sensu lato). Recent genetic studies have also demonstrated the existence of additional lineages of Rh. sanguineus (s.l.) in Europe and Asia. Herein, we assessed the biological compatibility between two lineages of Rh. sanguineus (s.l.) found in southern Europe, namely Rhipicephalus sp. I  and Rhipicephalus sp. II .The brown dog tick Rh. sanguineus (s.l.) were collected in southern Portugal and southern Italy. Tick colonies were established and crossbreeding experiments conducted. Morphological, biological and genetic analyses were conducted.Ticks morphologically identified as Rhipicephalus sp. I females presented mtDNA genotype of either Rhipicephalus sp. I or Rhipicephalus sp. II, suggesting the occurrence of paternal inheritance or mitochondrial heteroplasmy. While biologically compatible, these lineages are distinct genetically and phenotypically.Crossbreeding experiments confirmed that ticks from the two studied lineages were able to mate and generate fertile hybrids. Hybrid adult ticks always presented the same genotype of the mother, confirming maternal inheritance of mtDNA. However, larvae and nymphs originated from Rh. sanguineus (s.l.) studied herein are biologically compatible and genetic data obtained from both pure and hybrid lines indicate the occurrence of paternal inheritance or mitochondrial heteroplasmy. This study opens new research avenues and raises question regarding the usefulness of genetic data and crossbreeding experiments as criteria for the definition of cryptic species in ticks.The temperate lineages of The online version of this article (10.1186/s13071-018-2941-2) contains supplementary material, which is available to authorized users. Ticks are external parasites of great medical and veterinary significance, causing incalculable losses to the livestock industry and a great burden on companion animals and human populations around the world , 2. ClimRhipicephalus sanguineus group, which is an assembly of 17 morphologically similar tick species, including Rh. sanguineus (sensu stricto) [Rh. sanguineus (s.s.) was believed to be a single taxon, even considering its poor original description and the inexistence of a type-specimen [Rh. sanguineus (s.s.) is designated, ticks assigned to this taxon should be referred to as Rh. sanguineus (sensu lato) [Rh. sanguineus (s.l.) [Rhipicephalus sp. I”, which is present in some temperate countries, such as Italy and Greece [Rh. sanguineus (s.l.) has implications, not only from a taxonomic perspective but also from a medico-veterinary standpoint. Indeed, ticks currently identified as Rh. sanguineus (s.l.) are vectors of various bacteria , protozoans (e.g. Babesia vogeli and Hepatozoon canis) causing diseases in dogs and/or humans [Rh. sanguineus (s.l.) for E. canis may vary [Taxonomy and systematics of ticks have traditionally been based on morphological features. In the last three decades, the widespread use of genetic data and phylogenetic analysis has revolutionized both taxonomy and systematics of the Ixodida , but genstricto) , 6. For specimen , 6. Howesu lato) , 6. Indes (s.l.) . Additiod Greece . The pred Greece . The exir humans . For insmay vary .Rh. sanguineus (s.l.) are known to occur: the so-called temperate lineage  and Rhipicephalus sp. I [crt gene) [Rhipicephalus sp. I and Rhipicephalus sp. II shared crt intron-present and intron-absent alleles and one Rhipicephalus sp. I individual from Putignano  showed both alleles, which could support the occurrence of a heterozygous genotype and ongoing gene flow. Alternatively, incomplete lineage sorting or past gene flow could explain the observed pattern at the crt gene locus. Within this context, the main objectives of this study were: (i) to characterize morphologically and molecularly the pure Rhipicephalus sp. I and Rhipicephalus sp. II tick lines; (ii) to verify the biological compatibility between ticks from these two lineages by performing crossbreeding experiments; and (iii) to assess the fertility of pure and hybrid tick lines.In Europe, at least two genetic lineages of us sp. I , 19. Howus sp. I . The posrt gene) . In factRhipicephalus sp. I and Rhipicephalus sp. II were originally collected from sheltered dogs in Putignano  and privately-owned dogs living in Faro , respectively. In the above-mentioned collection sites, only these genotypes have been found in previous studies [Ticks used in this study originated from Portugal and Italy. In particular, engorged females genetically identified (see section “Genetic study”) as  studies , 19, 23.1 and F2, respectively.Larvae  originated from wild-caught, engorged females were defined as “wild type”. Ticks generated from males and females belonging to the same lineage were defined as “pure tick lines”, whereas ticks obtained by crossing different lineages were defined as “hybrid tick lines”. The first and second laboratory generations of crossed tick lines were designated as FThroughout the study, all ticks were maintained in a laboratory incubator under controlled conditions of temperature, relative humidity and light, and fed on naïve rabbits, as described elsewhere .Unfed larvae and nymphs (10–20 days of age) from pure progenies were killed with warm water (50 °C) and placed in vials containing 70% ethanol. Then, they were mounted on glass slides using Hoyer’s solution  and exam2) (Table Crossbreeding experiments were carried out and the fertility of hybrid tick lines was assessed until the second generation (F2) Table . The folRhipicephalus sp. I and Rhipicephalus sp. II, as well as larvae, nymphs, males and females from laboratory pure and hybrid tick lines , were used for genetic analysis. Genomic DNA was extracted from individual specimens using a commercial kit , following the manufacturer’s instructions. Partial cytochrome c oxidase subunit 1 (cox1) gene sequences (472 bp) were amplified using primers and PCR conditions described elsewhere [2, 10 mM Tris-HCl (pH 8.3), and 50 mM KCl, 250 μM of each dNTP, 50 pmol of each primer and 1.25 U of AmpliTaq Gold . Approximately 100 ng of genomic DNA (with the exception of the no-template control) were added to each PCR. Amplified products were examined on 2% agarose gels stained with GelRed  and visualized on a GelLogic 100 gel documentation system . Amplicons were purified and sequenced, in both directions using the same primers as for PCR, employing the Big Dye Terminator v.3.1 chemistry in an automated sequencer . The cox1 gene sequences were aligned using the ClustalW program [http://blast.ncbi.nlm.nih.gov/Blast.cgi).Wild type ticks belonging to the lineages lsewhere . Each re program  and comp1 ticks  of Rhipicephalus sp. I and Rhipicephalus sp. II, by analysis of variance (ANOVA). Morphometric data generated was also analysed through discriminant analysis to classify F1 ticks into different groups, based on a series of correlated variables (measurements). A structure matrix was generated for F1 larvae, nymphs, and adults  to highlight those variables that have the strongest correlations with the canonical function and that could help to discriminate between group 1 (G1) and group 2 (G2) (pure tick lines). The canonical function was then used to predict group membership and the success of assignment into the right group was expressed in percentage of correct classification. Statistical analysis was performed using SPSS for Windows, version 13.0.The mean differences of measurements were compared between F1 ticks belonging to G1 and G2 are provided in Tables Morphometric data obtained from FRhipicephalus sp. I males were able to mate with Rhipicephalus sp. II females, and vice versa, generating fertile hybrids. Detailed data from biological parameters recorded for pure and hybrid tick lines (G3 and G4) are provided in Table 1 and F2 females from all groups showed similar patterns in terms of feeding success, engorgement weight, pre-oviposition period, oviposition period, egg-mass weight produced and blood meal conversion index. However, with regard to hybrids, engorged F2 females were heavier than those of F1, although they did not produce greater egg masses. Indeed, they presented lower blood meal conversion index as compared with F1 females. The minimum egg incubation period and egg hatchability were also similar across generations (Table 2 larvae (80%) and nymphs (95.3%) from the hybrid line with females of Rhipicephalus sp. I  close to unity in all groups .Crossbreeding experiments showed that ns Table . No noti2 larvae 0% and nycox1 sequences were generated and analysed [Additional files Rhipicephalus sp. I  or Rhipicephalus sp. II (GenBank: KC243891) retrieved from GenBank, confirming the genetic identity of the ticks used in this study. No ambiguous single nucleotide polymorphisms were detected for the sequence obtained from G1 and G2 offspring specimens.In total, 122 partial 1 ticks from pure and hybrid lines showed the maternal mtDNA as expected, with the exception of larvae and nymphs originating from Rhipicephalus sp. I females, which showed either the Rhipicephalus sp. I or Rhipicephalus sp. II genotype. A high percentage of nucleotide identity (99–100%) was recorded by comparing all F1 tick sequences with the reference strains, for each group and developmental stage examined.All immature and adult FRh. sanguineus (s.l.), namely Rhipicephalus sp. I and Rhipicephalus sp. II. Phenotypically, these lineages are very similar, but morphometric analysis revealed differences for some measurements, especially for larvae and nymphs  had already been suggested as reliable morphological characters for separating Rh. sanguineus (s.l.) and R. turanicus [In the present study, we conducted morphometric, biological and genetic comparisons between two temperate lineages of hs Table . In facths Table . Scutal uranicus . AltogetRh. sanguineus (s.l.) revealed that these ticks could mate and generate viable hybrids [Rh. sanguineus (s.l.) have been separated for quite some time; this hypothesis is also supported by the differences found in their mitochondrial genomes [Previous studies using ticks belonging to the tropical and temperate lineages of  hybrids , 29. Mos genomes . A recen genomes .Rhipicephalus sp. I males were able to mate with Rhipicephalus sp. II females, and vice versa, generating fertile hybrids. While this may suggest that these lineages are conspecific, previous studies have shown hybridization to be possible in some tick species, under both laboratory [Our experiments confirmed that boratory , 32 and boratory . TherefoRhipicephalus sp. I and Rhipicephalus sp. II are genetically quite divergent, i.e. up to 7, 10.4 and 12.5% for 16S rRNA, 12S rRNA and cox1 genes, respectively [cox1 sequences) between Rhipicephalus sp. I and Rh. guilhoni, Rh. pusillus, Rh. turanicus, and tropical lineage of Rh. sanguineus (s.l.) were 10%, 11.1%, 11.7% and 12.3%, respectively [Rhipicephalus. The ability of ticks from different species to mate and generate fertile hybrids has been previously demonstrated in the laboratory, for instance, with Rh. appendiculatus and Rh. zambeziensis [It is worth nothing that, while morphologically similar and biologically compatible, ectively . To put ectively . These feziensis . AltogetRhipicephalus sp. I and Rhipicephalus sp. II are different evolutionary entities [Rhipicephalus sp. I being found in Africa (north of the Sahara) and south-eastern Europe, and Rhipicephalus sp. II being predominantly found from the middle to the western part of Europe [Rhipicephalus sp. I reported in the south (Puglia and Sicily) and Rhipicephalus sp. II in both the south (Sicily) and the north (Verona) [Other researchers have recognized that entities , 35. Indf Europe , 23, 35.(Verona) , 21. Thi(Verona) , 37.Rh. sanguineus (s.l.) for transmitting E. canis [Rhipicephalus sp. I and Rhipicephalus sp. II for human pathogens, including the bacterium R. conorii, the main causative agent of Mediterranean spotted fever.The occurrence of hybrids in sympatric zones as well as their impact (if any) in the occurrence of certain pathogens should be investigated. For instance, a study evaluated the vector capacity of ticks from four populations  of E. canis . The stuRhipicephalus sp. II and the cross between Rhipicephalus sp. II females and Rhipicephalus sp. I males generated larvae, nymphs and adults presenting the same mtDNA genotype of their female progenitor. On the other hand, the cross between Rhipicephalus sp. I females and Rhipicephalus sp. II males generated larvae and nymphs presenting either Rhipicephalus sp. I or Rhipicephalus sp. II mtDNA genotypes. This suggests the occurrence of paternal leakage  or mitochondrial heteroplasmy of parental females . This hypothesis opens up new research avenues concerning mitochondrial inheritance and heteroplasmy in ticks and should be investigated in future studies.Both the pure line of Rhipicephalus sp. I females may suggest that the persistence of paternal mtDNA or heteroplasmy may vary across tick developmental stages. For instance, it has been shown that heteroplasmy frequency changes between tissues of the same individual and between generations in humans [Interestingly, adult ticks from all groups presented mtDNA of their mothers. The finding of paternal mtDNA in larvae and nymphs and the absence in adults descending from n humans . It is an humans . These hRh. sanguineus (s.l.) studied herein are biologically compatible and genetic data obtained from both pure and hybrid lines suggest the occurrence of paternal inheritance or mitochondrial heteroplasmy. This study opens new research avenues and raises question regarding the usefulness of genetic data and crossbreeding experiments as criteria for the definition of cryptic species in ticks.The temperate lineages of Additional file 1:Table S1. Group, stage, generation and genotype of ticks genetically identified in this study. (DOCX 18 kb)Additional file 2:c oxidase subunit 1 (cox1) gene sequences generated in this study. (FAS 60 kb)Partial cytochrome