Toxocara canis, Ancylostoma caninum, Dipylidium caninum, and Echinococcus spp. are well-known zoonotic parasites worldwide, resulting in high public health risks. Poor data about canine helminth species and prevalence are available in Russia, mainly due to the absence of official guidelines for the control of dog parasites. Moreover, the consequent low quality of veterinary monitoring and use of preventive measures, the high rate of environmental contamination by dog feces and the increase of stray dog populations, make the control of the environmental contamination by dog helminths very difficult in this country. This paper reviews the knowledge on canine helminth fauna and prevalence in Russia. Practical aspects related to diagnosis, treatment, and control of parasitic diseases of dogs in Russia are discussed.Dogs are the hosts for a wide helminth spectrum including tapeworms, flatworms, and nematodes. These parasites affect the dog health and cause morbidity and mortality, especially in young and old animals. Some species, as  Dipylidium caninum, Echinococcus granulosus, Ancylostoma spp., and Toxocara canis are common parasites of dogs that can affect humans in different countries around the world. Knowledge about parasite species in domestic dogs, prevalence and intensity of helminth infestations in dog populations, transmission of canine parasites and the seasonal dynamics of parasite infestations are essential for control and prevention of helminthosis in domestic animals and humans.Dogs are the most popular pets worldwide and are infested with many parasites, which may represent a health risk for humans, especially children, the elderly and the immune-compromised -3. For iInvestigation of free-roaming dog populations as part of urban ecology is a major key for the solution of many ecological problems in industrial ecosystems ,5.http://laboratorium.narod.ru/gelm.html) and 10,000 in Omsk [In Russia, 40-50% of household owners keep a dog, and the total dog population amounts to 30 million . Therefo in Omsk . Stray d in Omsk ,10. This2 . Russia consists of seven basic climate zones. The humid continental climate predominates in all parts of the country: European Russia, in the south of West Siberia and in the south of the Russian Far East, including the cities of Moscow and Saint Petersburg, except for the tundra and the extreme southeast.The Russian Federation is the largest country in the world, covering 7,125,200 kmThe Russian Federation is divided into eight large Federal Districts: Northwestern, South, Central, Volga, North Caucasian Federal district, Siberian, Urals, and Far East Federal District. There is lack of data about canine helminth communities and prevalence. The North Caucasian District -17 and CToxascaris leonina, T. canis, D. caninum, E. granulosus and A. caninum, are frequently found in dogs from different Russian regions , Metorchis bilis , Metorchis xanthosomus , Dicrocoelium lanceatum , Diphyllobothrium latum, and Strongyloides stercoralis have only been sporadically reported in Russia. Clonorchis sinensis Looss, 1907 is an endemic species from the Asian Region; it was found in dogs from the Russian Far East [Taenia species found in dogs from Russia, the most common parasites are Taenia hydatigena, followed by Taenia multiceps. Two species, Taenia ovis and Taenia pisiformis, have been found in the North Caucasian District only.In Russia, dogs may be infested with a large number of helminths including cestodes, trematodes, and nematodes. The canine gastrointestinal helminth fauna includes 21 species . There as Tables-. Other hFar East . Among TStrongyloides larva detection techniques and post-mortem examination. Helminth eggs are usually detected in feces by ordinary coprologycal techniques such as Fulleborn’s method and Darling’s method; these methods are present low sensitivity for some helminth species and result in the underestimation of the real prevalence of some parasites [Opisthorchis eggs is also use in some studies  are worldwide parasites of Cyprinidae fishes, and infest fish-eating mammals. In Russia, M. xanthosomus was found in Dagestan and Kabardino-Balkaria Republic, while M. bilis was found in Caucasian Minerals Water , E. canadensis  were found in Russia [However, toxocariasis was recorded in 2.1 per 100,000 people from 2008 to 2012 . InfestaDistrict . The Toxted food  or by dited food . Echinocted food . Higher ted food . The Oreted food . Recentl species . E. grann Russia .D. caninum, which occurs through ingestion feline fleas infected with tapeworms [D. caninum infestation was also registered in Russia. Dipylidiasis was recorded in humans from the Orenburgskii Region [People are infrequently infested with apeworms ,81. D. ci Region , Moscow i Region , and thei Region .Clonorchis Loos, 1907, Metorchis, and Opisthorchis Blanchard 1895, in the family Opisthorchiidae, exploit freshwater snails and fish as the first and second intermediate hosts, respectively. The final hosts, fish-eating birds and mammals, including dogs and humans, are infected by eating fish harboring infective metacercariae [Opisthorchis felineus and Metorchis spp. are major sources of water contamination [O. felineus in people is 70-80% [Liver flukes of the genera ercariae . Feces omination . The lars 70-80% .M. bilis is also found in fish from the Ob-Irtysh basin. The prevalence rate in people from West Siberia is 28.1% [is 28.1% .C. sinensis is endemic to the Far Eastern region and it was also found in dogs and people from China and Korea [C. sinensis is frequently found in people from the Russian Far East [The flatworm nd Korea ,89. C. sFar East .M. lineatus has major veterinary importance, and occasionally, it has been found in people [M. lineatus infestation in humans have not been reported, however dog feces containing proglottids are major sources of environmental contamination.The tapeworm n people . In RussD. latum is a common parasite of fish-eating mammals. Fecal contamination of water is a source of D. latum spread. In the last 10 years, only one report regarding canine infestation from Ivanovo was published. A big center of infestation located in Russia is Baikal Lake. Infestation rates in humans from the Irkutsk Region were 9.6 cases per 100,000 people [0 people .Alaria alata, has specific veterinary importance [A. alata was found in dogs, foxes, wolves and badgers in the Vladimir, Ivanovo and Moscow regions, and in the Volgograd and Astrachan regions, in the North Caucasian District. Prevalence rates were 38.4-48.6% in farm dogs, 46.1-59.3% in stray dogs and 100% in wolves and foxes [Another flatworm, portance , howeverportance ,95. In Rnd foxes .T. canis and E. granulosus, which have a worldwide distribution.Zoonosis is the major veterinary and medical problem. Zoonotic infestations include well-known parasite species such as http://www.capcvet.org/) in the United States and the European Scientific Counsel Companion Animal Parasites (ESCCAP: http://www.esccap.org/) in Europe.However, there are no official guidelines for the control of endoparasite infestations in dogs, such as that provided by the Companion Animal Parasite Council (CAPC: http://docs.cntd.ru/document/1200050554) with State Sanitary and Epidemiological Supervision of the Russian Federation (http://rospotrebnadzor.ru/documents/details.php?ELEMENT_ID=2890). Veterinarians do not have a native source of information for parasite epidemiology, life cycles or control measures [There is scant information about problems of veterinary epidemiology in Russia. Two guidelines for the sanitary and veterinary rules were published by the Veterinary Department of the Ministry of Agriculture of the Russian Federation (measures .C. sinensis and O. felineus infestation in dogs, it is recommend to avoid the feeding of fresh cyprinid fishes [Parasitology monitoring is provided irregularly. Poor living conditions and lack of anti-parasitic medication causes environmental contamination with helminth eggs . Environd fishes .Many dog owners cannot afford preventive measures and will act only when a life-threatening problem is affecting their animals. Furthermore, there are a large number of free-roaming dogs populations in the Russian cities. Government is not able to manage these animals due to the lack of adequate infrastructure and trained personnel to conduct an effective long-term population control program. As a result, pet dogs and cats are usually endangered by a wide range of parasites that may cause disease in them and eventually in their human counterpart.The close contact between pets and humans may involuntarily represent a hazard for humans. Therefore, to avoid the potential risks associated with owning a pet, it is fundamental to maintain pets in good health and protect them from zoonotic pathogens.Therefore, veterinary practitioners and medical physicians should work together toward improving the well-being and general health of both animals and humans.MTV and AVE participated in the draft and revision of the manuscript. Both authors read and approved the final manuscript.


Cladolabes schmeltzii, we have found a rather complex organization of connective tissue. Transcripts of genes encoding a wide range of structural proteins of extracellular matrix, as well as various proteases and their inhibitors, have been discovered. All these molecules may constitute a part of the mechanism of connective tissue mutability. According to our data, the extracellular matrix of echinoderms is substantially distinguished from that of vertebrates by the lack of elastin, fibronectins, and tenascins. In case of fission, a large number of genes of transcription factors and components of different signaling pathways are expressed. Products of these genes are probably involved in regulation of asexual reproduction, connective tissue mutability, and preparation of tissues for subsequent regeneration. It has been shown that holothurian tensilins are a special group of tissue inhibitors of metalloproteinases, which has formed within the class Holothuroidea and is absent from other echinoderms. Our data can serve a basis for the further study of the mechanisms of extracellular matrix mutability, as well as the mechanisms responsible for asexual reproduction in echinoderms.Echinoderms are capable of asexual reproduction by fission. An individual divides into parts due to changes in the strength of connective tissue of the body wall. The structure of connective tissue and the mechanisms of variations in its strength in echinoderms remain poorly studied. An analysis of transcriptomes of individuals during the process of fission provides a new opportunity to understand the mechanisms of connective tissue mutability. In the holothurian  Asexual reproduction is the most ancient type of reproduction of organisms that occurs in members of most phyla of modern Metazoa –3. UnlikOne of the animal phyla that are able to reproduce asexually is Echinodermata. These are ancient, exclusively marine animals, which, along with chordates and hemichordates, form the group Deuterostomia. Asexual reproduction is found in some members of Asteroidea, Ophiuroidea, Echinoidea, and Holothuroidea –15. Sea Apostichopus japonicus has been sequenced [Strongylocentrotus purpuratus are described more in detail. This species has a set of genes for ECM components similar to that of other animals: collagens, proteoglycans, laminins, etc. [The body wall in echinoderms consists almost exclusively of connective tissue ,23. Therequenced ,30; howens, etc. ,32. Morens, etc. –37.A noteworthy feature of echinoderm connective tissue is the capability of changing its mechanical properties under the effect of various factors ,39. For Cucumaria frondosa [Paracentrotus lividus [A few hypotheses have been proposed for explaining the mechanisms of MCT changes ,45,48,49frondosa . It was frondosa . Several lividus . Blockin lividus –57. Prot lividus . NeverthCladolabes schmeltzii shows the capability of transverse division [We have recently found that the holothurian division ,59. The division ,21,60. TCladolabes schmeltzii . The animals were collected in Nha Trang Bay, South China Sea near the south part of Hon Tre island . C. schmeltzii are abundant in coastal areas of Vietnam. The species is not endangered or protected. They are invertebrate animals and no specific permissions are required for their collection. In three holothurians that were in the process of division, the area of the body with the constriction formed during fission was taken for the analysis  by the standard method. Treating with DNAase1 (ThermoScientific) was performed with addition of inhibitor of RNAases RiboLock (ThermoScientific). Synthesis and normalization of cDNA, construction of libraries, library quality control and sequencing were carried out by Evrogen JSC. The amplified dsDNA was prepared using the SMART method. To obtain the most complete transcriptome, the was used . SamplesAll reads were filtered and trimmed using the Trimmomatic 0.36 tool with parameters “LEADING:20 TRAILING:20 SLIDINGWINDOW:5:21 AVGQUAL:25 MINLEN:30” . Read paThe assembled sequences have been deposited in the NCBI Transcriptome Shotgun Assembly (TSA) database (GFWR00000000) and NCBI Sequence Read Archive (SRA) . The assembled contigs were used as the input for the BLASTX homology search  against For finding the unique contigs for fission and norm, we used the number of paired-end reads per contig . If thishttp://smart.embl-heidelberg.de/) and NCBI’s conserved domain search tool. The study of potential TIMP sequences was performed with HMMER 3.1 [Crassostrea gigas (outgroup) [S. purpuratus, sea star Patiria miniata, and holothurian Parastichopus parvimensis [C. schmeltzii were used for the analysis. The sequences were filtered by the minimum alignment length of 130 amino acids and verified by the NCBI NR protein database. Alignment was created using COBALT with standard settings [The domain structure of the supposed proteins was determined using the SMART (MMER 3.1  against utgroup) , sea urcvimensis ,75, thresettings . All seqsettings . Then alsettings . Tree cosettings .The results were obtained using the equipment of Shared Resource Center “Far Eastern Computing Resource” of Institute of Automation and Control Processes FEB RAS.A total of 37.6 million Illumina paired-end reads and 237 thousand 454 GS FLX+ single reads were obtained as a result of sequencing of the sample from intact (control) holothurians and 53% raw reads remained after filtration. When sequencing the sample of dividing animals, we obtained a total of 61.8 million Illumina paired-end reads and 32% raw reads remained after filtration. The assembly includes 50959 contigs with a mean length of CDS 516 nt . Of all An analysis of gene ontology showed that the physiological condition of holothurians quite markedly changes in case of fission . Such biA. japonicus. In this species, cathepsin D (aspartyl peptidase) takes an active part in degradation of extracellular matrix of the body wall [Among the molecular functions, a significant increase in peptidase activity during fission should be noted. Genes having a similar function are associated with such GO terms as “aspartic-type peptidase activity”, “zinc ion binding”, and “peptidase activity”. The intensification of peptidase activity during fission and the involvement of aspartyl peptidases in this agree with the previously obtained data on the holothurian ody wall .Among cellular components, we can emphasize the genes grouped under the term “integral component of plasma membrane”, which hints at the variations in receptor complexes of cells at the site of fission. The active transformation of organs and extracellular matrix is indicated by such terms as “basement membrane”, “gap junction”, “laminin-10 complex”, and “extracellular vesicle”. In general, GO annotation shows that many physiological and structural changes occur in the process of asexual reproduction. In holothurians, metabolism, immune and nervous systems are activated.C. schmeltzii. For example, in the Biological process group, they account for about 33%. As is known currently, retrotransposons may participate in regulation of various functions of organism [It is an interesting fact that transcripts of genes, associated with the functioning of viruses and retrotransposons, predominate in tissues of dividing individuals of organism ,82. Theiorganism –85. Our C. schmeltzii, transcripts of the genes of many ECM components such as collagens, proteoglycans, and glycoproteins, which are characteristic of most multicellular animals [tropoelastin in the transcriptome of C. schmeltzii. An analysis of the NCBI databases has shown an absence of sequences close to tropoelastin in echinoderms. An additional search in the genomes of the sea urchin S. purpuratus [A. japonicus [In  animals , have be animals . To our rpuratus  and the aponicus  has alsoaponicus ,87. Thesaponicus ,89. Our C. schmeltzii , network-forming collagens, and multiplexins. Like other echinoderms [C. schmeltzii has genes of fibrillar collagens of the types I/II/III and V/XI. Though the relatively small fragments were found in the transcriptomes of C. schmeltzii, it can be concluded that collagens of this species have a typical structure. Molecules include triple helical domains (Gly-X-Y) and COLFI domain is located at the C-terminus, as in other animals [The total number of types of collagens found in inoderms ,32, C. s animals .C. schmeltzii is represented by collagen IX. In vertebrates, this type of collagen is a component of cartilage, where it is located on the surface of collagen fibrils [The FACIT group in  fibrils . Collage fibrils . In echicollagen XV/XVIII [collagen XV/XVIII were detected in the transcriptome of C. schmeltzii. Collagens XV and XVIII are characterized by the highly interrupted collagenous domain and a large number of sites of binding with chondroitin sulfate and heparin sulfate glycosaminoglycans [The group of network-forming collagens is represented by collagen IV. This type of collagen is a component of basal membranes ,92,94. CXV/XVIII ,32. Fragoglycans . As a reoglycans . In echiC. schmeltzii  and ECM of body wall (collagen XV/XVIII).There are qualitative differences in the expression of collagen genes between intact and dividing hmeltzii . ProductProteoglycans and glycoproteins are multifunctional components of connective tissue, which mediate adhesion, proliferation, differentiation, and migration of various cells –98. The C. schmeltzii, were typical for echinoderms [syndecan, glypican, betaglycan, bamacan , and perlecan (basement membrane-specific heparan sulfate proteoglycan core protein-like). Moreover, in holothurians, we detected products of aggrecan, which had not been found in S. purpuratus [C. schmeltzii lack the products of Secreted modular calcium-binding protein 1, which are present in S. purpuratus. These genes are probably not expressed in tissues of holothurians taken for the analysis.The transcripts of genes of a number of proteoglycans found in inoderms ,32. Thesperlecan is worth special mentioning, as it is expressed only in dividing individuals , indicates a reorganization of epithelia during fission.Of the above-listed proteoglycan genes, ividuals . Its actlaminins, nidogens, fibrillins, fibulins, agrin, dystroglycan, and thrombospondins–were detected in C. schmeltzii. In particular, transcripts of laminins, as well as nidogen/entactin were found   . Thrombohyaluronidase, chondroitin sulfate synthase, chondroitinase , and heparanase genes were found in both dividing and intact individuals of C. schmeltzii  in MCT structures of crinoids, echinoids, and holothuroids are located along collagen fibrils –122. In hmeltzii . In thistransglutaminase and one Lox gene. No products of the transglutaminase were found in C. schmeltzii. Lox transcripts were present in the transcriptome of C. schmeltzii and are found in both dividing and intact individuals  and CUB (C-terminus). Transcripts of four genes are found only in dividing individuals  and 72 kDa type IV collagenase (XM_775263.4). Second, the catalytic domain MT-MMPs contains an insertion of approximately eight amino acids, the so-called MT-loop, which plays an important role in functioning of these proteases [C. schmeltzii, irrespective of presence of TD in them, this insertion is absent.The SMART reveals the transmembrane domain (TD) in four MMPs of hmeltzii . It meanroteases ,139. In C. schmeltzii, four MMPs  and ADAMTSs genes were also detected in C. schmeltzii  site, with which the transcription factors of the Fos and Jun families can interact [MMPs genes and their promoters and judge more objectively about regulation of MMPs expression in these animals. It should be mentioned that transcripts of one of the genes that regulate the expression of MMPs in mammals, transcription factor NF-κB, in C. schmeltzii is found only in dividing individuals (see below).The activity of MMPs can be controlled in various ways: for example, through regulation of gene transcription, activation of proenzyme by removing the propeptide domain, and interaction with inhibitors . In vertolecules –143. ProC. schmeltzii, there are products of the furin and plasminogen genes , and TIMPs . Producthmeltzii . Transcr fission .TIMPs genes in echinoderms varies within a broad range and may reach 45 in some of species [TIMP-like genes were identified in C. schmeltzii , TIMPs of Synapta maculata (8.m.2049.240066) and Abyssocucumis abissorum (43.aa.8353.697), tensilin of C. frondosa and tensilin2 of C. schmeltzii (see below about tensilins). Thus, out of the 144 analyzed sequences, as many as 41 TIMPs of echinoderms can be considered as closest to TIMPs of vertebrates in their structure. In C. schmeltzii, only three TIMP-like proteins–Cs-TIMP6, Cs-TIMP7, and Cs-TIMP8 –have three features specific to mammalian TIMPs  and the standard C-X-C motif (121 sequences) . Metzincan TIMPs . The resMMPs underwent substantial duplication and divergence after the separation of Ambulacraria and Vertebrata [Thus, most echinoderm TIMPs quite significantly differs in structure from mammal TIMPs. These differences are supposed to be related to “co-evolution” with MMPs. In echinoderms, rtebrata . Accordirtebrata  believe rtebrata ,16. In trtebrata ,146–148,C. schmeltzii. Contigs of Cs-TIMP10 are recorded only from dividing individuals .It is evident that TIMPs play a certain role in asexual reproduction in ividuals . FurtherTIMP-like genes of C. schmeltzii encode proteins, which are close to tensilins of other holothurians. In this connection, they were named Cs-tensilin1 and Cs-tensilin2.One of the key molecules of the mechanism of changing the MCT properties in echinoderms is tensilin. According to Keene, Trotter , this pC. frondosa—two for A. japonicus (tensilin1 and tensilin2) and one for Holothuria forskali—are found in the NCBI databases. Almost all the tensilins, including the tensilins of C. schmeltzii, are combined into a single group on the TIMPs tree  and two proteins of Psolus sp. (11.m.32702.873 and 11.m.26875.385). Hence, they also should be referred to as tensilins.Currently, three proteins close to tensilin of MPs tree . The onlC. frondosa, and one of proteins of A. abissorum (43.aa.8353.697)  , S2 Fig.ividuals . For thiAll the detected tensilins belong to members of relatively young groups of holothurians  and, conTensilins are found only in the species belonging to the order Dendrochirotida and a group of holothurians that were earlier combined into the order Aspidochirotida . Dendrocwnt genes was performed in transcriptomes of C. schmeltzii. We found products of 10 out of the 12 wnts typical of holothurians [wntA, wnt1-7, wnt10, wnt16  chain. This fragment encodes the C-terminus of collagen I/II/III containing the COLFI domain. Closer to the N-terminus of the contig, there is a sequence coding the Gly-X-Y motif, typical of collagens. The sequence encoding the holokinin PLGFLFR is located between it and the COLFI domain. Like that in A. japonicus [C. schmeltzii is distinguished from that of A. japonicus by two amino acids in the middle part. According to Birenheide et al. [collagen I/II/III occur in both intact and dividing individuals of C. schmeltzii. However, the collagen I/II/III itself is required for renewal of connective tissue, both in intact individuals and in asexual reproduction. Also, it is not clear whether the degradation of collagen with the release of holokinin occurs in this case. Nevertheless, this neuropeptide can be considered as a potential participant in the mechanism of changing the properties of connective tissue in holothurians.Holokinin in aponicus , is appaaponicus , it is ne et al. , holokintropoelastin, fibronectins, and tenascins genes were found. In this connection, the structure and the mechanisms of renewal of ECM should have their own unique features that are yet to be clarified. Nevertheless, the main components of connective tissue, characteristic of vertebrates, are present in echinoderms. They have fibrillar collagens, collagens IX and XV/XVIII, fibrillins, fibulins, and thrombospondins. Molecules of these proteins and glycoproteins have a complex tertiary structure and can form both fibers and a three-dimensional network. Moreover, they have sites of binding with polysaccharides and other types of collagens, which allows them to form cross-links between fibrils. Obviously, the complexity of the ECM structure and the variety of intermolecular interactions predetermines also the complexity of the mechanisms of changing the connective tissue properties in echinoderms. Probably, these mechanisms depend not only on the number of cross-links, but also on the composition of ECM and the properties of constituent molecules.An analysis of the available literature and own data has shown that the composition of connective tissue in echinoderms is generally similar to that in vertebrates, but it has a number of significant differences. In echinoderms, no collagen XV/XVIII more as compared to that in intact individuals. This fact shows the importance of this type of collagen in the processes that take place during fission in holothurians, although the specific role of this protein is yet to be established. It is obvious that various proteases are involved in destruction of the body wall. In further studies of MCT transformation mechanisms, attention should be paid to such enzymes as ADAMTS, serine proteases, and cathepsin D. MMPs are apparently also involved in the modification and destruction of connective tissue in echinoderms; however, no qualitative change in their composition at fission was recorded during our study. To identify MMPs involved in ECM transformation, a more detailed study of their functions and dynamics of expression is necessary. The question of the participation of various neuropeptides in the transformation of connective tissue during asexual reproduction in echinoderms remains open. A small number of transcripts of NGIWYamide precursor in dividing individuals can be explained by the fact that this peptide is synthesized and accumulated at an earlier stage, when animal is still preparing for asexual reproduction.Our study has shown that the fission process in holothurians is accompanied by significant physiological changes. The metabolism is increased, the nervous and immune systems are activated, and the structural changes occur in the fission zone. One of the significant indicators of morphological changes is the activation of the genes of laminins and nidogen, which probably an evidence of reorganization of epithelia. In the fission zone, the number of products of C. schmeltzii, of which nine are expressed during fission. In this case, contigs of one of them, Cs-TIMP10, are detected only in dividing individuals. All the above facts confirm the data that TIMPs can participate in the transformation of ECM in echinoderms. Tensilins, being TIMP, apparently represent a separate group of genes, which have formed within the class Holothuroidea and which probably have a specific function, typical only of holothurians. The presence of tensilins can be associated with development of the thick connective-tissue body wall in these animals.It is obvious that various molecules that activate or inhibit proteases are also involved in the transformation of connective tissue. Holothurians have a wide range of these molecules that can regulate the activity of proteases at various levels. One of the well-known inhibitors is TIMPs. A total of 13 TIMP genes are found in One of the distinguishing features of ECM in echinoderms, which is neglected by the existing hypotheses on MCT functions, is the presence of a large number of factors mediating the cell–cell and cell–matrix interactions. In this regard, it would be probably necessary to pay more attention to the structure and functions of fibrillins in echinoderms. It is possible that the fibrillin microfibril scaffold, like that of vertebrates, forms a niche for regulatory factors and mechanosensation. Conducting a signal from the extracellular microenvironment to competent cells can be a part of the mechanisms of MCT mutability.In addition to the genes responsible for transformation of ECM, a large number of factors, which probably regulate the division of body into parts and the preparation of tissues for subsequent regeneration, are expressed in asexual reproduction. We could not identify the genes responsible for determination of the site of division. The most probable candidates for this role are genes of the Wnt familiy, but more studies are needed to confirm this assumption. The difference in the qualitative composition of the expressing transcription factors between intact and dividing holothurians will make it possible in the future to identify factors that regulate asexual reproduction. Moreover, the presence of transcripts of genes involved in regulation of morphogenesis of various tissues and organs may indicate that the preparation of tissues for the subsequent regeneration in holothurian begins immediately during fission.S1 Fig(ZIP)Click here for additional data file.S2 FigTIMP proteins with metzincin-binding interface are denoted by red color, green frame borders group of tensilins.(ZIP)Click here for additional data file.S1 Table(ZIP)Click here for additional data file.S1 File(ZIP)Click here for additional data file.S2 File(ZIP)Click here for additional data file.S3 File(ZIP)Click here for additional data file.


Anthurium is the largest genus of Araceae, with 950 species distributed in the neotropics. Despite the great diversity of the genus, the knowledge of its floral vasculature is based on observations in only two species, viz. A. denudatum and A. lhotzkyanum, with remarkable variation in vascular carpellary supply: carpels are either vascularized by ventral bundles alone or by reduced dorsal bundles in addition to the ventral ones. Our main objective is to test this peculiar variation through a detailed anatomical study of the floral vasculature in taxa belonging to some sections of Anthurium designated as monophyletic groups in recent phylogenies.Anthurium, using both light and confocal laser scanning microscopies.We compare the floral vasculature of 20 neotropical species belonging to distinct sections of A. affine (Anthurium sect. Pachyneurium series Pachyneurium), A. obtusum and A. scandens (Anthurium sect. Tetraspermium), described here for the first time for the genus.The number and position of vascular bundles are constant within the tepals and stamens, regardless of the species and sections studied. However, the gynoecium vasculature exhibits variation between species belonging to the same or different sections. Our results reveal two patterns of vasculature: carpels vascularized by synlateral bundles alone (Pattern A) and carpels vascularized by both dorsal and synlateral bundles (Pattern B). Pattern A is shared by the majority of species studied here and corroborates the previous data in the literature. Pattern B occurs in three species: Anthurium is corroborated here. However, our results in addition to those from the available literature suggest the existence of three patterns  of carpellary vasculature. Based on the recent phylogeny of Anthurium it is possible to notice that the three patterns of carpellary vasculature occur in representatives of Clade B and deserve to be investigated in a larger number of species. Pattern A could be a plesiomorphy for the genus and the occurrence of dorsal bundles could be a derived character. Our data contributes to the taxonomy and to the understanding of the floral evolution of the largest neotropical genus of Araceae.The variation in the supply to the carpels in  Pothos L., Pothoidium Schott, Pedicellarum M. Hotta and Anthurium Schott; the latter is the largest genus of the family, with 950 species  Engler emend. Anthurium sect. Pachyneurium Schott encompasses approximately 120 species (A. series Pachyneurium (Schott) Croat and A. series Multinervia Croat, which correspond to Clades 9 and 12 and to Clade 11, respectively, in the phylogeny of Anthurium sect. Tetraspermium Schott is a reduced group, formed by only four species  (A. lhotzkyanum Schott (A. sect. Urospadix)   and A. lospadix) . In A. d& Lauche , Barabé,& Lauche . These cAnthurium, specifically in traditionally recognized groups such as the four previously described, is expected to point out new anatomical data that may be used in their delimitation. Thus, the present study proposes a comparative analysis of floral vasculature of Anthurium species, with emphasis on gynoecial aspects, seeking to answer the following questions: (1) Are the patterns of carpellary vasculature previously reported for Anthurium found in the taxa analyzed here; (2) Do the floral vascular characteristics aid in the delimitation of Anthurium sect. Dactylophyllium, A. sect. Pachyneurium series Pachyneurium, A. sect. Tetraspermium and A. sect. Urospadix?The study of floral vasculature in more representatives of Anthurium, mainly distributed in Brazil, were selected for the present study , samples were dehydrated through a n-butyl alcohol series , embeddeFor study using confocal laser scanning microscopy (CLSM), samples were embedded in polyethylene glycol (PEG 1500) , and secDiagrams were produced using CorelDRAW X7 (Corel Corporation) software.Anthurium and is divided into two subtopics: “Pattern A: Carpels vascularized by only synlateral bundles,” and “Pattern B: Carpels vascularized by synlateral and dorsal bundles.”The results are presented in two main topics: “Floral organography” and “Floral vasculature.” In the first topic a single floral description is presented for all species herein studied because they generally share many anatomical aspects; differences, when present, are emphasized throughout the text. The second topic includes the description of the floral vascular supply in Anthurium are sessile ) ) , parenchA. obtusum and A. scandens (A. sect. Tetraspermium) does the formation of an apical septum occur, situated between the opening of the stylar canal and the ovarian locules. The locules are filled with mucilage (A. obtusum and A. scandens (A. sect. Tetraspermium), in which two ovules form per locule.The ovary is superior and bicarpellary, with a septum separating the locules  and 2C. scandens    divergesNext, each of the two internal complexes (c1’)  and 4B dEach of the two carpellary complexes (cc)  and 6D dThe placental complex (pl) extends through the ovarian septum  and 8B, Dorsal bundles were not observed in the carpels.A. affine (A. sect. Pachyneurium series Pachyneurium), A. obtusum and A. scandens (A. sect. Tetraspermium). As in pattern A, the vasculature of the axis of the spadix diverges to the flower, branching into two   and 9B dA. obtusum and A. scandens (A. affine (Each complex (c2’) diverges into two carpellary traces (ct)  and 10A.scandens , the dor. affine , it is l. affine .At the base of the flower, each of the other two vascular complexes (c1’)  and 5B oEach of the two carpellary complexes (cc)  and 9D dA. obtusum and A. scandens  extends through the ovarian septum  and 10E scandens  and 5J, . affine  and 11A.Anthurium sect. Dactylophyllium, A. sect. Pachyneurium series Pachyneurium, A. sect. Tetraspermium and A. sect. Urospadix, broaden the knowledge on floral vasculature of this neotropical genus of Araceae and reveal the homogeneity of this characteristic in the stamens and tepals, irrespective of the species and sections studied.Our results, based on the analysis of 20 species belonging to Anthurium, as reported by previous studies . In this species, two ventral bundles and two dorsal bundles were observed at the base of the ovary. In the transverse section of the median plane of the ovary, only placental bundles were observed, though without knowledge of their origin, whether they were formed from ventral bundles or from dorsal bundles of the carpels. This pattern was not observed in any of the species studied in the present work.The first description of floral vasculature in Anthurium lhotzkyanum (=A. augustinum) (A. sect. Urospadix). In the gynoecium of this species, two ventral complexes and two placental bundles were observed both at the base and in the median plane of the ovary. In tranverse sections at the height of the style, the ventral bundles were observed surrounding the stylar canal. There is no report of dorsal bundles of the carpels. This pattern of gynoecium vasculature was corroborated by the majority of the species we analyzed that belong to A. sect. Dactylophyllium, and in some species of A. sect. Pachyneurium series Pachyneurium and A. sect. Urospadix, here called as Pattern A. However, in the present study another terminology is proposed to refer to this ventral vascular supply of the carpels which will be further discussed.The second description of the floral vasculature of the genus was presented by A. sect. Pachyneurium series Pachyneurium (A. affine) and to all of the studied species of A. sect. Tetraspermium (A. obtusum and A. scandens). These three species all have carpels vascularized by both synlateral and dorsal bundles.For the first time for this genus, our data show the existence of a third pattern of carpellary vasculature, called as Pattern B. In our sample, the occurrence of this pattern is restricted to only one of the studied species of In angiosperms, each carpel is generally vascularized by three bundles: one follows along its median plane, called dorsal or median bundle; and the other two continue along its margins, called lateral or ventral bundles e.g., . This paPothos (Anthurium denudatum (Lysichiton camtschatcensis (L.) Schott  Engl.  Schott , Spathipii Regel , and Zam.) Engl. , in whic.) Engl. . Althoug.) Engl. , ventral.) Engl. , synlate.) Engl. , and sep.) Engl. . This va.) Engl. .Anthurium, the characteristics of the bundles to vascularize the margins of the carpels, either branching (Pattern A) or not (Pattern B) in the style, suggest that they resemble (in position and function) those bundles located opposite the ovarian septum described in previous studies. Anthurium also presents a set of vascular bundles located in the ovarian septum that derive the traces that supply the ovules. In previous studies, this vascular supply is referred to as ventral bundle , a genus previously included as early-divergent in Araceae, carpels are supplied by vascular bundles of a central column , A. obtusum and A. scandens (A. sect. Tetraspermium), also studied in the present work.On the other hand, in species of A. obtusum and A. scandens (A. sect. Tetraspermium) are the synlateral bundles located in the ovarian wall. This variation in the location of the synlateral bundles in A. sect. Tetraspermium may be related to the supply to the apical and ovarian septa found in this group, in addition to the existence of two ovules per locule. The apical septum was reported for A. scandens by Also in relation to the floral structure, another highlight regarding the heterogeneity of the carpellary vasculature is the variation in the location of the synlateral bundles. In the majority of the species studied here, the synlateral bundles are located between the margin of the carpel and the ovarian wall. However, only in Anthurium is to emphasize the variation in terms of the origin of the dorsal and synlateral bundles. Our results provide the first evidence that the carpellary bundles in Anthurium possess a mixed nature; they originate from two distinct vascular complexes and present a relationship intrinsic to the branching, or not, of the external complex (referred to here as c1) of the floral base into an additional complex, referred to here as c2.More important than highlighting the existence of these three patterns of carpellary vasculature in Anthurium follow three main patterns: Pattern A, in which the gynoecium is vascularized only by synlateral bundles, as verified in the majority of the species of A. sect. Dactylophyllium, A. sect. Pachyneurium series Pachyneurium and A.sect. Urospadix —observed in all of the studied species of A. sect. Tetraspermium —might represent synapomorphies of this section.While analyzing the patterns of vasculature described here, and the possible use of the vascular characteristics for better delimitation of the sections of Anthurium is divided into Clades A and B that contain the large majority of the species. The three patterns of carpel vasculature occur in Clade B and deserve to be investigated in a larger number of species, particularly from A. sect. Pachyneurium, since this section presents this carpel variation and was already designated as being more than one group of species, both by works of classical taxonomy (Anthurium species, given that it is also found in other species of A. series Pachyneurium, and that the appearance of dorsal bundles is derived within the genus. However, the lack of samples from other sections precludes a clear distinction between these two patterns and reinforces the importance of vasculature data in the genus.In the evolutionary hypothesis presented in the Bayesian analysis by taxonomy  as well


Leishmania and is transmitted by sand flies. The state of Espírito Santo (ES), an endemic area in southeast Brazil, has shown a considerably high prevalence in recent decades. Environmental niche modelling (ENM) is a useful tool for predicting potential disease risk. In this study, ENM was applied to sand fly species and CL cases in ES to identify the principal vector and risk areas of the disease. Sand flies were collected in 466 rural localities between 1997 and 2013 using active and passive capture. Insects were identified to the species level, and the localities were georeferenced. Twenty-one bioclimatic variables were selected from WorldClim. Maxent was used to construct models projecting the potential distribution for five Lutzomyia species and CL cases. ENMTools was used to overlap the species and the CL case models. The Kruskal–Wallis test was performed, adopting a 5% significance level. Approximately 250,000 specimens were captured, belonging to 43 species. The area under the curve (AUC) was considered acceptable for all models. The slope was considered relevant to the construction of the models for all the species identified. The overlay test identified Lutzomyia intermedia as the main vector of CL in southeast Brazil. ENM tools enable an analysis of the association among environmental variables, vector distributions and CL cases, which can be used to support epidemiologic and entomological vigilance actions to control the expansion of CL in vulnerable areas.Cutaneous leishmaniasis (CL) is caused by a protozoan of the genus  Leishmania Ross, 1903 and is transmitted by insects from the subfamily Phlebotominae ..23].L. intermedia, L. migonei, L. choti, L. lenti, and L. whitmani, excluding L. longipalpis, which is a vector of visceral leishmaniasis .The AUCs were greater than 0.80, indicating excellent discrimination for whitmani . The modL. intermedia, L. whitmani, L. migonei, L. choti, and L. lenti, respectively  presented cutaneous lesions, 49 (3.3%) presented mucocutaneous lesions, and 159 (10.8%) presented mucosal lesions. The model constructed in the current study shows that this disease occurs in the south-central region of the state of ES and is limited to areas below 1,119 metres; however, only 17 (4.76%) of 357 occurrence localities were related in elevation above 850 metres .The model was considered excellent, with an AUC of 0.817 ± 0.020. The variables BIO4, BIO12, slope, BIO14, and BIO5 contributed 70.31% of the model construction .L. intermedia model showed significantly higher overlap, indicating that it is more associated with the disease transmission than secondary vectors such as L. migonei and L. whitmani (p < 0.05), with no difference between the latter two species (p > 0.05), followed by L. lenti and L. choti, most likely without epidemiological importance in the transmission.Values of Schoener's D range from 0 (no overlap) to 1 (perfect overlap). This indicator was compared between species to evaluate which one is most involved in disease transmission . The KruThis is the first study to build and associate environmental niche models using algorithms between vector species and autochthonous CL cases in endemic areas of south-eastern Brazil.Leishmania infantum  spatial distribution have been studied [Lutzomyia longipalpis and cases of visceral leishmaniasis in Mato Grosso do Sul [Environmental modelling research focused on public health is under considerable development in Brazil and worldwide. In Portugal, Spain, France, and Italy, studies have mapped the environmental risk of visceral leishmaniasis, using the potential distribution of canine leishmaniasis and their vectors ,29,30. I studied . Aparici studied . Almeidao do Sul . Nieto ao do Sul . Petersoo do Sul . Almeidao do Sul .L. intermedia was the species most associated with the occurrence of CL in the state of ES, followed by the secondary vectors L. migonei and L. whitmani. However, the species L. lenti and L. choti were not considered important vectors of the disease in that region.The current study revealed that Lutzomyia intermedia has been indicated as the main vector of CL in the state of ES [Leishmania (Viannia) braziliensis Vianna, 1911 [te of ES ,10. Thisna, 1911 ,11,44–46na, 1911 –9,10,47.na, 1911 ,47.Lutzomyia migonei has been considered a secondary vector of CL in anthropic environments, with occurrence at altitudes above 750 m in ES [L. migonei is the most common species found inside the domicile [ m in ES . This spdomicile ,47–48.Lutzomyia whitmani has also been considered a secondary vector of CL, with high density in the endemic areas of the states of São Paulo, Minas Gerais, and ES [, and ES ,49–50. T, and ES ,45,47. TLeishmania braziliensis is observed in areas of remaining forests due to the larger number of reservoirs and vector species, mainly L. intermedia and L. whitmani. However, in peri-urban areas, L. intermedia is the only vector, and domestic dogs are the reservoirs, a situation that reduces the diversity of the protozoa in this area. This reinforces the idea that L. whitmani is a connecting vector between the preserved and anthropic environments.Rocha et al.  suggesteLutzomyia lenti and L. choti have no association with the occurrence of CL. Despite its occurrence in all regions of Brazil, with a wide distribution and abundance, L. lenti has not been reported as an important vector in endemic areas of the state of ES. This species has been found in the mid-western region of the country in areas surrounding houses in the state of Mato Grosso do Sul and Goiás, mainly in domestic animal shelters [Lutzomyia choti is a species with heterogeneous habitats and is present in both wild environments and areas surrounding houses. However, no records exist of natural infection in this species by L. (V.) braziliensis. In addition, this species has not been observed in the main endemic areas of the state of ES. Brandão-Filho et al. [shelters –55. A poshelters . Lutzomyo et al.  suggesteRegarding the ecological niches constructed in this study, slope was a relevant variable in the occurrence of all species of phlebotomine sand flies. Areas with steep slopes have dimples, which allow a greater variability of water and organic matter accumulation, as well as a reduction in sunlight and wind exposure, thereby creating a greater diversity of habitats; this diversity in habitats contributes to the maintenance of food and shelter conditions for these insects .Lutzomyia whitmani is limited to the western region of the state, which has lower precipitation rates, higher elevations, steep slopes, and low temperatures. Lutzomyia intermedia is found in warmer areas with lower slope gradients and lower elevations. In addition, this species is less susceptible to temperature and rainfall variations. Lutzomyia migonei presents a distribution between L. intermedia and L. whitmani and is concentrated in the mid-western region of the state of ES.L. intermedia was collected at 1,123 m above sea level but at low density, suggesting the importance of vector density in the occurrence of disease cases in a particular region.Elevation appears to be inversely proportional to the abundance of phlebotomine sand flies ,11,51,57L. whitmani was more abundant in colder and drier months , whereas L. intermedia was more abundant in the warmer months of the year . These data are in agreement with previous studies performed by Souza et al. [The precipitation and maximum temperature of the warmest month were also relevant to the construction of the models. Temperature and humidity have positive effects on the activity and abundance of phlebotomine sand flies, which depends on the species ,58. In ta et al.  and Costa et al. .L. whitmani, with the latter being more tolerant to variations in temperature and precipitation [The variables of precipitation seasonality and temperature seasonality were considered important in the occurrence of pitation ,49.The temporal mismatch between the species distribution data (1997–2013), the cases data (1978–2013), and climate data (1950–2000) did not significantly affect the modelling results because during the last 50 years, there have been no important environmental changes in peridomiciles in endemic areas of CL in ES. Furthermore, the geographic expansion of the disease occurred from the migration of rural populations to the outskirts of the cities in the 1980s, carrying infected dogs from the western area to east of ES, where vector species were already adapted to the peridomicile ,14.A low sandfly frequency in some areas of the state did not influence the species environmental modelling because this method can provide a measure of potential species occurrence in areas not covered by biological surveys and consequently, has become an interesting tool for healthy planning .Maxent presented a strong performance compared with different methods because it performed well and remained stable with respect to the prediction accuracy and the total area predicted, indicating that Maxent can compensate for small species occurrence data sets .The results of the current study indicate that modelling and geoprocessing tools enable a reliable analysis of the association between geo-climatic variables, geographic distribution of vectors, and CL cases in the state of ES. The definition of areas at potential risk for CL transmission allows us to make available relatively accurate information at the regional level that can guide entomological and epidemiological surveillance activities to control the geographic expansion of this endemic disease in vulnerable locations.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Plasmodium infections in assumed “minor”, non-vector, and cryptic and novel species is reviewed. Their importance in terms of evading current control and elimination strategies and therefore maintaining malaria transmission is emphasized.Entomological indices and bionomic descriptions of malaria vectors are essential to accurately describe and understand malaria transmission and for the design and evaluation of appropriate control interventions. In order to correctly assign spatio-temporal distributions, behaviors and responses to interventions to particular anopheline species, identification of mosquitoes must be accurately made. This paper reviews the current methods and their limitations in correctly identifying anopheline mosquitoes in sub-Saharan Africa, and highlights the importance of molecular methods to discriminate cryptic species and identify lesser known anophelines. The increasing number of reports of  Anopheles, remain the most dangerous animals on the continent.Over the past fifteen years concerted efforts to control malaria have led to reduction of incident cases globally by more than a third, with deaths due to malaria dropping by 60% . Even grAnopheles genus of mosquitoes occurs globally and the majority of the 460 or so species are not vectors of malaria; only an estimated 30 to 40 species worldwide are regularly associated with Plasmodium transmission to humans. This mosquito genus, which falls under the subfamily Anophelinae was first classified by Theobald in 1940, but to date much of the taxonomy is not completely resolved [Anopheles or Cellia subgenera, and it is in these two that the Old World vectors of malaria have been grouped.The resolved . Of the An. gambiae complex [An. gambiae sensu stricto widely spread across Africa and Madagascar [An. coluzzi common in west Africa with a distribution extending into central Africa and Angola [An. arabiensis broadly distributed across much of the continent [An. melas and An. merus, found in the coastal regions of west and east Africa, respectively [An. funestus s.s., a member of the An. funestus species complex, is likely the main species driving transmission in southern and some parts of east Africa, but occurs across much of the continent and in some areas be a much more dominant vector than An. gambiae [An. nili s.s., a sibling species in the An. nili group, and An. moucheti are also considered important vectors in some areas of west and central Africa, particularly along rivers and in humid, densely forested areas [Reviews of literature from a wide range of studies conducted on anopheline species composition led to the publication of predictive distribution maps of the dominant vector species of malaria in Africa, with only eight assumed to play a major or important role in transmission, including five species within the  complex . The fivdagascar , An. cold Angola , An. araontinent , and theectively . An. fun gambiae . An. niled areas . These eThe implication of particular species of mosquitoes in malaria transmission requires demonstration that there is spatial and temporal overlap of that anopheline species with human malaria cases, that contact between the mosquito and people and ultimately human blood feeding takes place, and that the mosquito is found to harbor malaria parasites . To demoIn establishing each of the associations for vector incrimination, it is essential to accurately assign the species to anopheline specimens. In the middle of the 20th century, extensive studies across much of Africa were undertaken to describe the morphological features unique to particular anopheline species. Field entomologists such as Gillies, Coetzee, Leeson, Coluzzi, Ramsdale, De Meillon and Giles created morphological keys for the vast majority of anophelines species found on the continent ,19. ThesAnopheles gambiae species complex which falls in the Pyretophorus Series, consists of eight reproductively isolated yet morphologically similar species; An. gambiae Giles 1902, An. coluzzii Coetzee and Wikerson 2013, An. arabiensis Patton 1905, An. amharicus Hunt, Coetzee and Fettene, An. quadriannulatus Theobald, An. bwambae White 1985, An. melas Theobald 1903, and An. merus Dönitz 1902, and An. comorensis Brunhes, le Goff and Geoffroy [An. gambiae sensu lato is often referred to as the most efficient vector of malaria globally, in fact only five of these sibling species are considered major vectors. An. bwambae, for example, has such a limited geographical distribution, being found only in the geothermal springs of western Uganda [An. quadriannulatus and An. amharicus is assumed to preclude them from transmission. Furthermore, the five species that do transmit malaria demonstrate different behaviors and may occupy different ecological/transmission niches necessitating specific tailoring of interventions. An. arabiensis, for example, demonstrates more exophagic and zoophagic behaviors and is more desiccation tolerant than its sibling An. gambiae s.s.These dichotomous keys exist for adults, larvae and eggs. However, some species of mosquitoes can only be distinguished from one another at certain life stages , thus neGeoffroy ,20,21,22n Uganda , that itThe use of morphological keys requires reasonably well preserved, intact specimens. Lack of key features such as legs, wings, setae or scales may mean that the key cannot be followed to a definitive identification. Collections from field traps are often damaged and missing critical morphological features. There have been efforts to create computerized visual guides that allow for a combination of physical features of a specimen to be entered, with software that will then generate likely candidate identities . UnfortuAn. gambiae, An. arabiensis and An. melas were found to show different constituents of cuticular and internal hydrocarbons by gas chromatography. Such assays require only a single specimen for discrimination of species [An. gambiae and An. funestus species complexes, have demonstrated differences in chromosome inversion frequency and associations of particular karyotypes with environmental conditions and ecological zones for the different sibling species [An. gambiae s.s. in west Africa; the Bamako, Mopti, Savanna, Forest and Bissau forms, with the first three often reported in sympatry [An. funestus have indicated as many as 17 different chromosomal forms [An. funestus group [An. gambiae sibling species has shown differing frequencies of gene expression for esterases and can be performed on males and females of any stage [With the recognition of species that cannot be easily identified by morphological methods, and the disadvantages of relying on accurate identification by microscopy, focus turned to biochemical and molecular methods that do not require fully intact or complete specimens. Sympatric samples of  species ,31,32. T species ,35,36,37sympatry  and simial forms ,39. Howeus group ,40. An iny stage . Again, An. gambiae complex [An. quadriannulatus A and B specimens, which are found in the southern and eastern Africa region and Ethiopia, respectively, and now considered to be reproductively isolated [An. gambiae s.s. from west Africa [An. gambiae is now considered a reproductively isolated species and has been renamed An. coluzzi Coetzee and Wilkerson [An. quadriannulatus forms, A and B. An. quadriannulatus is now assigned to specimens found in southern and eastern Africa formerly species A, and An. amharicus Hunt, Coetzee and Fettene is given to those species found in Ethiopia, previously named An. quadriannulatus B. Further studies from West Africa have also revealed what may be cryptic subpopulations of An. coluzzi with divergent behaviors and vectorial capacity for malaria [These early cytogenetic and allozyme studies set the stage for what is now the basis of the most common methods for species discrimination, polymerase chain reaction (PCR) amplification of DNA. These techniques have the advantage of being able to use intact or fragments of mosquito specimens of any life stage or sex. Specimens may be collected and stored under a much wider variety of conditions , as long as the DNA does not become too degraded. DNA is extracted from the mosquito and primers are used to bind to species-specific regions of the target DNA. The resulting DNA region is amplified using PCR and the resulting amplicons visualized by electrophoresis on agarose gels. PCR-based assays for the two dominant African vector species complexes have been developed. The Scott et al. PCR method is the most popular and used by laboratories worldwide to discriminate the most important sibling species of the  complex . It usesisolated . A PCR-Rt Africa ,44. Thist Africa ,46. Othet Africa . Based oilkerson . These s malaria .An. gambiae complex, members of the An. funestus group are morphologically difficult to distinguish at the adult stage. This group falls in the Myzomyia series, and has several subgroups, namely the Funestus Subgroup of Afrotropical species An. aruni, An. funestus s.s., An. parensis and An. vaneedeni, the Rivulorum Subgroup which includes the Afrotropical species An. brucei, An. fuscivenosus, and An. rivulorum, and the Minimus Subgroup of the Asian mosquitoes An. fluviatilis, An. flavirostris, An. minimus A, C, and E, as well as the Afrotropical An. leesoni [An. funestus s.s. and An. rivulorum [An. funestus s.l. and is capable of discriminating between five species of the group; An. funestus s.s., An. rivulorum, An. leesoni, An. parensis, and An. vaneedeni. Since its publication, specific primers have been designed and added to this PCR to identify other cryptic species within this group that appear to be divergent from the previously identified members, such as An. rivulorum-like [An. funestus-like from Malawi [An. funestus s.s. makes it a dominant vector across much of Africa, however despite being endophilic, An. funestus-like is considered to be a non-vector as it has not been found with human blood or malaria parasites [An. rivulorum, An. rivulorum-like [An. vaneedeni [An. leesoni, are largely zoophilic and although they have been shown to carry sporozoites [An. longipalpis, also in the Myzomyia Series but not in the Funestus group, resembles An. funestus in the adult stage and due to its similar endophilic nature is commonly mistakenly identified as the vector in collections [An. longipalpis is a complex with at least two cryptic species, Type A and Type C, and these PCR-based assays are able to distinguish these types as well.As with the  leesoni ,49. Variivulorum  promptedivulorum . This PCrum-like  found inrum-like  and An. m Malawi  and possm Malawi . Discrimarasites . Real-tiarasites ,53. An. rum-like , An. vananeedeni  and An. rozoites ,57, theylections . This prlections  and a PClections . FurtherPrimers targeting the internal transcribed spacer 2 (ITS2) region of ribosomal DNA and the cytochrome oxidase 1 (COI) region of mitochondrial DNA are popular targets for additional PCR-based assays developed to discriminate other anopheline species that do not fall in the Gambiae or Funestus complexes. ITS2 is a non-coding nuclear gene with conserved primer binding sites and is more variable than coding genes so can be used for fine resolution phylogenetic analyses and well as construction of diagnostic tools . COI is All PCR-based assays utilize primers that bind to specific known sequences of DNA, thus creating diagnostic assays that positively and accurately identify mosquito sample to species. Unfortunately, this approach is limited to species and complexes for which sequence data is available from specimens that have been reliably identified using morphology and phenotypes first. Therefore, although extensive amounts of sequence data exist for targeted regions of DNA, such as the ITS2 and COI, and even for a few anopheline genomes ,65, the The value of combining both morphological and molecular tools to mosquito studies has long been recognized . Using bAn. gambiae and An. funestus, as these are considered the dominant vectors of malaria, thought to account for 95% of malaria transmission and have generally comprised the bulk of routinely conducted indoor collections across the African continent [An. gambiae s.l. and An. funestus s.l. across Africa [The majority of entomological studies for malaria in Africa focus on identifying the members of two major species complexes, ontinent ,69. Howeontinent . Insectis Africa ,72 and ts Africa ,73,74,75s Africa ,77 to avAn. gambiae s.s. mosquitoes were reported to be caught in much higher proportions outdoors, and that greater exposure occurred both indoors and outdoors prior to midnight than observed a decade earlier [An. funestus demonstrated a shift in peak biting times from their typical midnight/early morning foraging times to just before sunrise, as well as demonstrating an increase in exophagic behavior following scale-up to universal coverage of LLINs [An. funestus s.s. were caught by human landing catch indoors and outdoors after sunrise to 11:00 in the morning as vector control coverage increased, although the small sample size limits how generalizable these results may be [An. gambiae s.s. to An. arabiensis and in some areas increasing dominance of An. funestus in others [On Bioko Island, Equatorial Guinea,  earlier . This fo earlier . The vec earlier . In Beniof LLINs . In Senes may be . Shifts n others ,83,84,85n others .An. gambiae s.s. was reported to demonstrate more exophagic behaviors on Bioko Island, a recent study set out to compare infection rates in individuals who spent more time outdoors than others [In studying malaria mosquito species composition and determining their role in transmission it is important to sample mosquitoes where and when human-vector contact occurs; simply showing mosquitoes rest and forage either indoors or outdoors does not demonstrate exposure unless the human activity overlaps with that of the mosquito ,88,89,90n others . InfectiThe more recently documented changes in current vector populations are not novel. Following the large scale house-spraying exercises of the Global Malaria Eradication Program (GMEP) between 1955 and 1969, the emergence of insecticide-resistance and persistence of some exophilic vectors were some of the reasons given for the failure of the program to achieve global eradication ,93. It iThe question of whether malaria can be maintained by the presence of these lesser documented anophelines was raised following the GMEP and at the time, in the pre-molecular era, extensive studies were conducted dissecting numerous specimens from twelve or so different species to examine their salivary glands for sporozoites. This laborious process demonstrated a number of species harboring sporozoites but at low frequencies. It was assumed at the time that the role of these species in malaria transmission was “negligible” . HoweverAn. gambiae, An. arabiensis, An. funestus, An. nili, and An. moucheti represented almost 90% of the collection, malaria parasites were found in nine secondary malaria vectors: An. ovengensis Awono-Ambene et al., An. carnevalei Brunhes et al., An. coustani Laveran, An. hancocki Edwards, An. marshallii Theobald, An. paludis Theobald, An. pharoensis Theobald, An. wellcomei Theobald, and An. ziemanni Grünberg. Infection rates in these secondary vectors were overall lower than the presumed major vectors, but parasites or their proteins were found repeatedly over time and across sites in An. pharoensis and An. ovengensis. In some areas where biting rates were high, EIRs were estimated as high as 70 infected bites per person per year from An. ovengensis making this species the likely major vector locally. An. ovengensis was first established as a new member of the An. nili group in 2004 and has been found in forested areas, often sympatric with An. moucheti and An. gambiae in Cameroon, rarely resting indoors and demonstrating both exo- and endophagic behaviors [An. pharoensis is commonly found in Sudanese and Sahelian regions, but has been implicated in malaria transmission in many parts of Africa such as Nigeria, Guinea-Bissau, Mauritania, Senegal, Egypt, Ethiopia, Chad, Kenya, Tanzania, and possibly Zambia [A review of archived anophelines collected over a 5 year period in Cameroon showed the presence of 21 different species, and while the known major vectors; ehaviors . It has y Zambia ,111,112.An. coustani Laveran, An. symesi Edwards, An. paludis Theobald, An. tenebrosus Dönitz, An. caliginosis De Meillon, and An. ziemanni Grünberg. Low parity rates and a long gonotrophic cycles of An. tenebrosus were assumed to prevent this species from being a competent vector and infections have not been detected, however infections were found in other species of the group albeit at low levels. One exception is the Congolese member species, An. paludis, where infection rates by dissections where documented as high as 10% [Plasmodium spp. However, recently in the central highlands of Madagascar where malaria epidemics had been observed, Plasmodium infection rates determined by circumsporozoite protein (CSP) ELISA and confirmed by PCR as high as 9.5% were recorded in An. coustani collected in animals stables attached to houses. A small number of infected specimens of this species were also found indoors [An. coustani, with some degree of anthropophily, were predominant in the early evening at rates 20-fold that of the assumed primary vector. The authors suggested the resurgence associated with the malaria epidemics could be attributed to the lack of efficacy of the IRS program against this outdoor foraging species that rested in non-human shelters, which were not targeted for spraying. Plasmodium infections, albeit in a low number of samples, have been recorded in An. coustani in south-western Ethiopia [An. coustani were found to be harboring parasites.Another species complex common across most parts of Sub-Saharan Africa is the Coustani group. Members of this group are largely exophagic and are caught in large numbers next to animals although some degree of anthropophagy has been documented, likely due to close interaction of communities with animals they tend . This grh as 10% ,114. At  indoors . Biting Ethiopia , althougEthiopia ,116. In An. squamosus has recently been recognized as a vector of P. falciparum [Plasmodium infection rates for this species are very low, suggesting they might have a “negligible” role, infection levels at the household or collection level can be quite high. In the absence of well-recognized vector species, all data suggest that this under-valued species is critically important in sustaining transmission and perhaps in preventing total elimination. This pattern is becoming more frequently reported across the African continent. Studies published in the last two years have documented low levels of Plasmodium infections in other assumed ‘non-vector’ species such as An. quadriannulatus, An. theileri, and An. rufipes [An. funestus s.s. a further three “unknown” species of anophelines may contribute to malaria transmission in the highlands of western Kenya [Emerging or newly recognized malaria vectors are appearing in regions where malaria has been dramatically reduced but not eliminated. In an area of southern Zambia targeted as an elimination zone, lciparum . Althoug rufipes ,56, manyrn Kenya ,96. GeneAn. funestus and An. gambiae species complexes. Increasing surveillance to include other anopheline species may identify a larger problem at hand. In any case, whether this proportion be 10% or 50%, without also tackling this ‘residual’ transmission and the vector species involved, malaria elimination goals will likely not be achieved. In the western lowland of Kenya it was recently determined that despite a long history of deployment of LLINs and IRS and evidence of insecticide resistance in the vectors, the majority of the exposure to malaria vectors still occurs indoors [An. funestus, An. gambiae and An. arabiensis foraging behaviors indicated that only An. arabiensis had demonstrated a shift towards increased exophagy since expansion of vector control, although indoor biting earlier in the evening was documented in both An. funestus and An. arabiensis. The authors argue that indoor control needs to be prioritized as outdoor transmission accounts for less than 10% of exposure. Whilst malaria has reduced substantially following mass distribution of nets, prevalence and incidence rates have plateaued since 2009. Outdoor exposure, albeit of a low level, and early indoor exposure may account for the lack of continuing impact since 2009 in this part of Kenya. It is important to acknowledge that interventions must tackle exposure both indoors and outdoors, and programs need to closely monitor changes in mosquito behaviors to adequately design and re-deploy appropriate interventions [A review of exposure to malaria vectors by Huho et al. (2013) from six sites across west, east and southern Africa reported that the vast majority of human exposure to anopheline mosquitoes still occurs indoors even after indoor directed interventions have been rolled out . The aut indoors . Studiesventions ,120,121.A number of vector control methods exist that can target both indoor and outdoor resting and foraging populations depending where and when they are deployed . Some meSuccessful malaria control is dependent on rapid and accurate identification of the vectors involved. In many regions of malarious Africa, successful control and decline of well-recognized and long-studied malaria vectors has resulted in reduced malaria transmission, but not zero transmission. In some of these regions, residual malaria transmission appears to be maintained by what were secondary, minor, or novel cryptic vectors. Unfortunately, tools for surveillance and positive identification of many of these anophelines is severely lacking. There is a similar absence of knowledge about the basic biology, bionomics and behavior of these vectors which is critical to implementation of appropriate control measures. Although some specific tools are lacking, the programmatic needs are clear. Better surveillance tools targeting anopheline vectors that forage outdoors are needed and attention should be paid to all specimens collected, rather than the “usual suspects”. Improved sampling targeted to epidemiologically relevant sites will provide insight into the behavior and bionomics of these vectors and more accurately determine human-vector contact. Additionally, new molecular tools are necessary for rapid identification of these understudied secondary and cryptic anopheline species. These molecular tools, however, must build on and complement morphological and biological studies that are absolutely necessary to define these species and species complexes. As the cost of sequencing continues to fall, whole genomic sequencing approaches aimed at molecular documentation of these understudied and novel taxa would seem an ideal approach. Although molecular costs are becoming less of a limitation, sustained funding and support is required to establish and maintain personnel for such complementary biological and molecular archiving efforts, both for surveillance and research endeavors. This capacity both in terms of the technology and workforce to employ these tools on the African continent where the need is most dire, needs to be a prime focus.


Naganishia albida  is a non-neoformans cryptococcal species rarely isolated as a human pathogen.N.albida as the causative agent of infection. The minimum inhibitory concentration (MIC) analysis was carried out for the isolated agent, and the patient was treated using 100 mg daily of oral itraconazole.Herein, we present the case of a 26-year-old Iranian man with a superficial cutaneous lesion in the axilla. The initial treatment for pityriasis versicolor by clotrimazole was unsuccessful. We performed skin sampling based on the standard protocol and conducted further investigations by the conventional laboratory tests and molecular analysis of the skin samples. All the mentioned analyses revealed N. albida can be the causative agent of some superficial infections. This is the first report on the successful detection and treatment of a superficial skin infection due to N. albida by oral itraconazole. Cryptococcus spp. are basidiomycetous yeasts considered as the responsible agents for a wide range of diseases, among which C.neoformans and C. gattii are mentioned as the most common pathogenic species . In addition, N. albida is an opportunistic and encapsulated yeast found on human skin . Although N. albida rarely causes any diseases, there have been some case reports, including cases of meningitis, peritonitis, fungaemia, pulmonary and cutaneous infections, and keratitis (eratitis .N. albida has never been reported as the causative agent of a superficial skin infection (Moreover, cases of encephalitis, disseminated cryptococcosis, and pneumonia have been reported , 20-29. nfection . As can N. albida. In addition, treatment choices are limited for cryptococcal infections. The first choice of treatment for these infections is the combination of amphotericin B and flucytosine [N. albida is not well-defined, and amphotericin B has been mentioned to have limited efficacy in the treatment of N. albida [N. albida shows various responses to different antifungal treatments. Therefore, there is no common treatment protocol for infections caused by this fungus.To the best of our knowledge, the case described here is the first report of a pityriasis versicolor-like superficial infection due to cytosine , 12. Non. albida . N. albiN. albida is only sensitive to amphotericin B and itraconazole [N. albida, which led the therapy to change to itraconazole [N. albida using a mean treatment period of 56 days [Recently, it has been indicated that conazole , 30, 31.conazole . Besides 56 days . In the present case, after MIC approval, we chose 100 mg daily of itraconazole in a 10-day treatment period, which resulted in successful treatment. The three-month follow up of the patient revealed no relapse of the infection and no evidence of any clinical manifestations at the involved site. Furthermore, several factors such as the anatomical location of involvement, the power of immune system in host, and tissue damages can determine the proper medication and the required treatment duration for cryptococcal infections due to non-neoformans species , 17, 33.N. albida was found to have the ability to cause superficial infections. The case presented here is the first report of successful detection and treatment of infection by this yeast. N. albida was detected by sequencing of ITS1-4 rDNA in this report and treated using oral itraconazole.In conclusion,


The authors of the original article  would liThe revised author list includes authors Masayoshi Itoh, Hideya Kawaji, Timo Lassmann, Carsten O. Daub, Erik Arner, Piero Carninci, Yoshihide Hayashizaki and Alistair R. R. Forrest, as well as their affiliations.The following updates to Author contributions’, Acknowledgements and Funding have also been included below:PZ, TH, DS and DG generated samples for the time series. MI and PC generated the CAGE libraries. YH, COD, ARRF and PC managed and organized the FANTOM5 project. ED, TL, HK, EA and PZ performed data analysis. PZ performed biological validation experiments. PZ, DG, ED and WH wrote the manuscript. The authors read and approved the final manuscript.We thank J. Yeung, J. Cairns, S. Tremblay, A. Poon, J. Wilking for support and suggestions on experimental design and manuscript preparation. We thank F. Lucero Villegas for animal management. We thank M. Larouche, D. Rains and J. Boyle for technical support. We thank Dora Pak and Anita Sham for management support and Miroslav Hatas for systems support. We would like to thank all members of the FANTOM5 consortium for contributing to generation of samples and analysis of the data-set and thank GeNAS for data production. We thank GenomeBC, National Institutes of Health, Natural Sciences and Engineering Research Council of Canada, NeuroDevNet, MEXT Japan and University of British Columbia for funding support.The efforts of PZ, TH, DS and DG were supported by GenomeBC and National Institutes of Health, Natural Sciences and Engineering Research Council of Canada. FANTOM5 was made possible by a Research Grant for RIKEN Omics Science Center from MEXT to YH and a grant of the Innovative Cell Biology by Innovative Technology (Cell Innovation Program) from the MEXT, Japan to YH. It was also supported by Research Grants for RIKEN Preventive Medicine and Diagnosis Innovation Program to YH and RIKEN Centre for Life Science Technologies, Division of Genomic Technologies .


Leishmania (L.) infantum is the causative agent in an endemic focus of canine leishmaniasis in the Mont-Rolland district . In this area, the transmission cycle is well established and more than 30% of dogs and 20% of humans are seropositive for L. infantum. However, the sand fly species involved in L. infantum transmission cycle are still unknown. Between 2007 and 2010, 3654 sand flies were collected from different environments  to identify the main L. infantum vector(s). Nine sand fly species were identified. The Phlebotomus genus  duboscqi and Phlebotomus (Ph). rodhaini) was markedly under-represented in comparison to the Sergentomyia genus  adleri, Se. clydei, Se. antennata, Se. buxtoni, Se. dubia, Se. schwetzi and Se. magna). Se. dubia and Se. schwetzi were the dominant species indoor and in peridomestic environments, near humans and dogs. Blood-meal analysis indicated their anthropophilic behavior. Some Se. schwetzi specimens fed also on dogs. The dissection of females in the field allowed isolating L. infantum from sand flies of the Sergentomyia genus . It is worth noting that one Se. dubia female not engorged and not gravid revealed highly motile metacyclic of L. infantum in the anterior part of the midgut. PCR-based diagnosis and sequencing targeting Leishmania kinetoplast DNA (kDNA) highlighted a high rate of L. infantum-positive females . More than 2% of these positive females were unfed, suggesting the parasite survival after blood-meal digestion or egg laying. L. infantum prevalence in Se. schwetzi was associated with its seroprevalence in dogs and humans and L. infantum prevalence in Se. dubia was associated with its seroprevalence in humans. These evidences altogether strongly suggest that species of the Sergentomyia genus are probably the vectors of canine leishmaniasis in the Mont-Rolland area and challenge one more time the dogma that in the Old World, leishmaniasis is exclusively transmitted by species of the Phlebotomus genus. Leishmania transmission belong either to the Phlebotomus genus (Old World) or to the Lutzomyia genus (New World). In the rural community of Mont-Rolland , Leishmania infantum is the causative agent in an endemic focus of canine leishmaniasis. Recent surveys revealed more than 30% of dogs and 20% of humans with a positive serological test for Leishmania in this community. However, the sand fly species involved in L. infantum transmission were still unknown. Between 2007 and 2010, we carried out a study in this community to identify the sand fly species responsible for L. infantum transmission. We collected nine species belonging mainly to Sergentomyia genus and in low proportion to Phlebotomus genus. The abundance around dogs and humans, the detection of live and mature parasites in anterior midgut, the high rate of L. infantum-positive females using molecular analyses and the identification of dog and human blood in the fed females incriminates Se. schwetzi and Se. dubia as possible vectors of L. infantum. This hypothesis is strongly supported by statistical analyses performed to compare the prevalence of infected sand flies with the seroprevalence data in humans and dogs.Leishmaniases, neglected tropical vector-borne diseases, remain today a problem of public health. Classically, the sand flies involved in  Leishmania parasites that live in a wide range of ecosystems and may have different clinical manifestations   , and Ph. (Anaphlebotomus) rodhaini]  .Leishmania in sand flies and in humans and/or dogs. The supporting observations are: iii) demonstration that the incriminated sand fly commonly feeds on the reservoir host(s); iv) presence of the sand fly species in places where the Leishmania and the caused disease are found; v) demonstration that the sand fly species can support the flourishing development of the parasite; and vi) demonstration that the sand fly species can transmit the parasite by bite [Leishmania should be seen in the anterior midgut and on the stomodeal valve of naturally infected female flies. Concerning Criterion 3, the fly species under study must be attracted to and readily bite humans and any reservoir host. Criterion 4 deals with the evidence of a strong ecological association between flies, humans and any reservoir hosts and Criterion 5 concerns the demonstration of the experimental transmission. The two last criteria 6 and 7 are based on mathematical modeling of transmission and of links between disease prevalence and biting.According to Killick-Kendrick , the ess by bite . These c by bite  deals wiL. infantum vectors in the Mont-Rolland community.In the present work, we based our approach on several of these criteria to identify the Sergentomyia genus. Indeed, the two Phlebotomus species were poorly represented . Phebotomus duboscqi sand flies were caugh mainly in farming areas in the sandy ecosystem, where canine leishmaniasis is less frequent [Ph. rhodlaini, a previous study showed that this species is probably underestimated because of unsuitable traps and could play a role in L. donovani transmission between animal reservoir hosts [Ph. rhodaini collected reflect the population of this species in the environments where L. infantum is transmitted.In this study, almost all the sand fly specimens captured around the habitats of dogs and humans belonged to the frequent , Fig 1).ir hosts . Rodent Sergentomyia dubia, Se. schwetzi, and to a lesser extent Se. magna, were the most abundant species captured indoors and in peridomestic environments, around infected dogs and serologically positive humans. As previously reported [Se. dubia, the vector of the gecko leishmaniasis in Senegal, showed the most endophilic behavior. This species was significantly associated with the indoor environment where the majority of blood-fed and gravid females were also caught. Blood-meal analysis confirmed that Se. dubia feeds frequently on humans , although it was also well represented indoors. Blood-fed and gravid females were more numerous in peridomestic and farming areas, suggesting a more exophilic behavior. In accordance with previous studies [Sergentomyia magna was present in the different sites of capture. Although slightly more abundant in farming areas, its presence in peridomestic areas and indoors was not negligible, suggesting a regular contact with dogs and humans. One of the Se. magna females had fed on humans.reported , Se. dubn humans . These r studies , the res studies , 25. SerSergentomyia species were very rare or almost absent indoors and in peridomestic areas, although the blood-meals of one Se. clydei and two Se. buxtoni, caught indoor, were of human origin. Sergentomyia clydei, Se. antennata and Se. adleri were most abundant in farming areas. In agreement with previous studies [Se. buxtoni was mainly collected in the sylvatic area, suggesting a very exophilic behavior and feeding preferences focused on wild animals.The other  studies , 12, 25,Se. dubia and 0.79% of Se. schwetzi were infected with living L. infantum promastigotes of which two nongravid and unfed individuals (one S. schwetzi and one S. dubia) had mature forms in the anterior midgut. Natural infections in unfed specimens strongly suggest that the parasites have overcome the main barriers to metacyclogenesis  in the sand fly midgut [Sergentomyia species by L. infantum. It is consistent with the prevalence of L. infantum infection in many sand fly vectors [L. infantum by dissection in Se. dubia and Se. schwetzi individuals is an important finding.The finding of naturally infected sand flies is essential to incriminate a vector and also to study the infection rates in endemic areas , 19, 21. vectors –29. As t vectors . TherefoL. infantum kDNA in Se. dubia , in Se. schwetzi (4.19%) and also in Se. magna (3.64%). These percentages are in agreement with the infection rates reported in proven vectors by Aransay et al. [et al. [Leishmania DNA in a sand fly does not prove the vector competence [Leishmania kDNA was due to Leismania ingested while feeding, or to the presence of well-established or developing parasites [Se schwetzi, 4.44% of Se. dubia and 2.76% of Se. magna) may reflect the survival of parasites (or persistence of DNA) in these sand fly species. Indeed, the presence of Leishmania kDNA in these females strongly suggests that the parasites were ingested several days before the capture and that they had started their developmental cycle. However, we cannot exclude the persistence of DNA without any infective role, especially because the sand flies were caught in the vicinity of infected dogs. Nevertheless, the microscopic observation of mature L. infantum promastigotes in unfed females strongly suggests that they are not only carrying parasite DNA, but that they could be competent vectors. None of the other species was found to be infected with L. infantum both by dissection and PCR testing.The results of the nested PCR diagnostic assay confirmed the dissection data. They revealed the presence of y et al.  and Kish [et al. . Neverthmpetence , 32. Indarasites , 30, 32.Sergentomyia genus is still not considered to be involved in leishmaniasis transmission [Leishmania DNA and in one of them live parasites of L. major in various Sergentomyia and Spelaeomyia species, suggesting that this genus could play a role in Leishmania transmission [Currently, phlebotomine sand flies remain the exclusive proven vectors of leishmaniasis, although other insects, such as midges, are suspected to act as leishmaniasis vectors  and few smission ; howeversmission , 36–40Phlebotomus genus, which contains the classical vectors of L. infantum in the Old World. However, in the Mont-Rolland area, the two species belonging to the Phlebotomus genera (Ph. duboscqi and Ph. rodhaini) were not abundant and not infected by L. infantum parasites. These results are in agreement with previous works showing that Ph. duboscqi, the main L. major vector, is refractory to infection by L. infantum [Our working hypothesis was that the vector species should belong to the infantum .Sergentomyia species satisfied several criteria for vector incrimination. First, Se. dubia, Se. schwetzi and, to a lesser extent, Se. magna were the most abundant species captured indoors and in peridomestic environments surrounding infected dogs and serologically positive humans. Sergentomyia dubia showed an endophilic and anthropophilic behavior, feeding frequently on humans and Se. schwetzi a more exophilic and opportunistic behavior. Sergentomyia magna was moderately collected in the different sites, but displayed also a regular contact with dogs and humans. Sergentomyia dubia and Se. schwetzi females were found to be infected with Leishmania parasites both by dissection and by PCR-based diagnostic assay. Se. magna was found to be infected with Leishmania only by PCR. The isolates obtained from the dissections were successfully cultured and characterized as L. infantum, similarl to the parasites isolated from the dogs in this region [L. infantum survived after digestion of the blood-meal or egg laying. These data suggest that L. infantum can develop in Se. dubia and in Se. schwetzi. Nevertheless, a recent experimental study on the susceptibility of Se. schwetzi to L. donovani, L. infantum and L. major reported that this sand fly species is refractory to infection by L. infantum because the parasites were defecated with the blood remnants [Sergentomyia capacity to transmit Leishmania, it is important to work with parasites and sand flies collected from the area in which the transmission is suspected. Indeed, the sand fly-Leishmania interactions could be the results of a specific and recent co-evolution in that area and the ability of Sergentomyia species to transmit L. infantum could be region-specific. On the other hand, in the study by Sadlova et al. [Se. schwetzi colony was established from specimens collected in north-western Ethiopia and the L. infantum strain was isolated in Turkey from a sand fly specimen belonging to the Ph. tobbi species [Se. dubia or Se. magna.Conversely, some s region . Furtherremnants . It is ea et al. , the Se. species . No expeSe. dubia and Se. schwetzi were strongly associated with humans and dogs (indoor and peridomestic environments); the probability of infection was higher indoors and in peridomestic environments for Se. dubia sand flies and in peridomestic areas for Se. schwetzi; the PCR-positive Se. schwetzi and Se. dubia specimens were significantly correlated with the seroprevalence data in dogs and/or humans.Our hypothesis is also supported by the results of the statistical analyses that demonstrate a strong ecological association between flies, humans and hosts , 3 = L. major (5ASKH), 4 = L. tropica (K27), 5 = L. tarentolae. B) 1 = L. infantum (ITMAP 263), 2 = Sergentomyia dubia female infected by L. tarentolae, 3 = Se. schwetzi female infected by L. infantum, 4 = Se. dubia female infected by L. infantum, 5 = negative control (no template).A) Reference strains: 1 = negative control, 2 = (TIF)Click here for additional data file.S1 TableSergentomyia species were studied, the Phlebotomus captures being too rare. The “Isolated” environment was taken as the baseline. The effect of the environment is measured in terms of odds-ratio (OR) for the proportion of captures of the studied species. The P-values correspond to the test of the null hypothesis OR = 1. Post-hoc tests were conducted, where the hypotheses tested are equality of two odds-ratios.Only the (DOCX)Click here for additional data file.