AbstractPolydora Fenzl and Oocephala H.Rob. Disposition of pores in Polydora seems more asymmetric than in Oocephala. Based on the known relationships within the Vernonieae, some conjectures are made regarding development of the pantoporate condition in the group.Pantoporate pollen, which occurs sporadically in the Monocots and Dicots, has now been found in Asteraceae in two apparently related genera of the tribe Vernonieae,  Vernonieae is notable for remarkable variations in pollen structure, variations in sublophate and lophate forms with varying degrees of reduced perforated tectum in the exine and variations in the attachments of the outer exine to the footlayer. Now a new variation has been encountered during a study of South African Vernonieae that involves the first known examples of pantoporate pollen with non-equatorial pores in the Asteraceae. It is of some interest that one of the genera involved, Polydora  was one in which pollen was first used as a taxonomic character in the Asteraceae by The tribe PageBreakVernonieae are the only examples of pollen in the Asteraceae with perforated tectum partially or completely lacking in non-colpar areas. Also unique to the tribe in the family is the six-porate form of pollen in the southeast Asian genus Camchaya Gagnep., where the pores occur equatorially in three pairs , that is, not all equatorial in position.Already known in the Tribe ee pairs . There ir pollen . Now, fo6) electron sources. To achieve limited increased breakage, material gathered at the base of a centrifuge tube was given a few jabs with a dissecting needle.Specimens examined are from the U.S. National Herbarium in Washington, D.C. Examination of pollen with a light microscope was insufficient to show the position of the pores. Pollen grains in most illustrations were treated with acetolysis (Erdtman 1960), followed by staining with osmium thiocarbohydrazide solutions and sputter coating with gold-palladium . UnacetoOocephala and Polydora, are evidently closely related to each other according to the pollen similarity, and both have pedunculate heads, a characteristic of many genera. Nevertheless, the two genera are easily distinguished from each other. Polydora has an ordinary broadly campanulate capitulum, and an ordinary capillary pappus. Oocephala (egghead) has an egg-shaped capitulum and a plumose pappus.The two genera in which the pantoporate pollen is now being reported are both restricted to sub-Saharan Africa. The two genera, PageBreakOocephala, Oocephala centauroides (Klatt) H.Rob. & Skvarla, comb. nov.   H.Rob. & Skvarla, comb. nov.   H.Rob.  H.Rob. (Polydora bainesii (Oliv. & Hiern) H.Rob. (Polydora chloropappa (Baker) H. Rob; Polydora jelfiae (S. Moore) H.Rob.; Polydora sylvicola (G.V. Pope) H.Rob. (Polydora poskeana (Vatke & Hildebr.) H.Rob. (Polydora steetziana (Oliv. & Hiern) H.Rob. (Polydora serratuloides (DC.) H.Rob.  H.Rob. ; and eig) H.Rob. , 4; Poly) H.Rob. ; Polydor) H.Rob. ; Polydor) H.Rob. ; Polydor) H.Rob. ; and Pol) H.Rob. .Centrapalinae and Erlangeinae that are native to the Paleotropics. A note of interest is the possible difference in the chemical make-up of these muri, since they have been seen to survive the degrading effects of chloral hydrate more completely than the muri and tectum of other Vernonieae when mounted in Hoyer’s solution (The pollen of both genera has a lophate exine pattern that is nearly spherically symmetrical. The pattern consists of a mixture of pentagonal and hexagonal lacunae. Perforated tectum is essentially absent leaving totally exposed muri and their subtending columellae. Both genera also share a mural structure that has been referred to as rhizomate  Figs 1F, 6C, E. solution .Centrapalinae and Erlangeinae. The lacunae in which the pores occur are totally undifferentiated in any other way, being either pentagonal or hexagonal, even in the polar PageBreakPageBreakPageBreakPageBreakPageBreakPageBreakPageBreakregions. The depths of the lacunae usually prevent seeing more than one pore at a time unless the grains are unacetylized and the caps on the pores are preserved (Oocephala and Polydora are particularly helpful. The totally exposed sphere of the pollen footlayer shows the pores very well (Oocephala and Polydora. It was study of the toy ball that led to the conclusion that the pollen of Oocephala could characteristically have seven or eight pores. (Positions of the pores are often difficult to determine in lophate pollen of the reserved  (Robinsoreserved . Thus, tery well , 7C, D. ery well , 7C, 7D.t pores. , 2E.PageBreakOocephala, even in grains with the muri present, pores can be seen in adjacent lacunae (Polydora (Oocephala (Polydora (An exact count of the pores can never be certain, since there is evidence of some uneven distribution of pores in some grains. In a few cases, in  lacunae , 4B, 6D.Polydora –7C, expaPolydora . The totocephala , 2E, andPolydora , but panBromeliaceae, Alismataceae and Araceae. In basal dicots polyporate pollen is mentioned only from the Trimeniaceae (Caryophyllales, Ranunculales, Podostemaceae, Linaceae, Zygophyllaceae and Geraniaceae as achieving the polyporate condition by multiplication of simple furrows that are reduced to pores. In other families such as Malvaceae, Sterculiaceae, Thymelaeaceae, Euphorbiaceae and Malpighiaceae a spiralization of a line joining centers of apertures is suggested. All of these are in derived groups, and polyporate pollen is not basic to any group.Two basic porate conditions of Angiosperm pollen are well known . The foreniaceae . In the Asteraceae, and suggestions here are based on observations of the pollen of related members of the family in the tribe Vernonieae.There have been no previous reports of pantoporate pollen in the Asteraceae has pollen that is basically tricolporate, a condition that seems basic to all the tribes including the Vernonieae (Asteraceae is found in the tribe Vernonieae which has the some of the most destabilized pollen formation of any tribe in the family. The pantoporate pollen has developed within the subgroup of the Vernonieae that already has the most spherically symmetrical forms of pollen in the tribe, the subtribal pair, Erlangeinae and Centrapalinae. It is in these subtribes that the colpi are totally suppressed, and orderly arrangement of the lacunae, even at the poles, is completely lost. These features that have become so spherically symmetrical in these subtribes are among the last layers laid down as the pollen grain matures. The position of the pores is much more basic, being established in some of the innermost PageBreakand earliest laid down layers of the pollen. It is the contention here that the loss of radial symmetry in the Vernonieae pollen started with the outermost and most belatedly deposited layers of the grains, and progressed eventually to the inner and earlier deposited layers.The rnonieae . The mosPolydora and less evident in the more specialized Oocephala. This could trace to the fact that the developing pollen grain has a distal surface that faces outward in the pollen mother cell, and a proximal surface that faces inward toward the center of the tetrad of pollen grains. Thus, the distribution of the pores is influenced by early stages in pollen development, beginning with position in the tetrads or mother cell.There is even a developmental basis for the asymmetry in the distribution of the pores, more obvious in


Taenia solium cysticercosis in pigs from Myanmar are published. The authors found a porcine cysticercosis prevalence of 23.67% in slaughtered pigs, which, as they mention, indicates the presence of human taeniasis and also the risk of acquiring human cysticercosis and, therefore, neurocysticercosis.We have read with interest the paper by Khaing et al. , in whic T. solium taeniasis among the inhabitants of Myanmar leading to a high presence of infective eggs in the environment. However, as far as we know, the presence of T. solium taeniasis, as well as neurocysticercosis, has only been diagnosed in refugees or immigrants from that country but not in people living in Myanmar [The high porcine cysticercosis prevalence detected by the authors means, obviously, that there has to be a high prevalence of human Myanmar –6. T. solium is present in Myanmar; however, our query is whether other human Taenia species might also be present in that country and, in particular, whether T. asiatica could be present in Myanmar as suggested more than 20 years ago by Fan et al. [There is no doubt thatn et al.  and receT. asiatica was described as a new species in 1993 [ Taenia species has practically the same gravid proglottid morphology as Taenia saginata, but its life cycle is just the same as that of T. solium; that is, its intermediate hosts are pigs instead of cattle, the intermediate hosts for T. saginata. The clear liver tropism the cysticerci of T. asiatica presents in pigs is worth mentioning. Regarding its geographical distribution, the species has been found in Taiwan, South Korea, Philippines, Indonesia, Thailand, China, Vietnam, Japan, and Nepal [ T. asiatica was initially considered as an exclusively Southeastern Asian parasite, its finding in Nepal (far from Southeast Asia) and the phenomenon of globalization (it is a parasite with cosmopolitan hosts) have recently led to the notion that the species probably has a wider distribution [ T. asiatica in three more Asian countries, Myanmar, Laos, and Malaysia, is highly suspected [ in 1993 . This thribution , 11. The T. asiatica was detected in a Karen immigrant who moved from Myanmar to Kanchanaburi province, Thailand, in 2005. However, she had already started to expel proglottids in faeces in 1997, when she was still living in Myanmar [ T. asiatica in Myanmar is very likely.Concerning Myanmar, no cases have been confirmed so far, but a case identified asT. asiatica may be found in the definitive (humans) as well as in the intermediatehost (pigs). The detection of T. asiatica taeniasis is by no means an easy task. Taking into account that neither the characteristics of the eggs nor the morphology of the gravid proglottids are specific enough to distinguish T. asiatica from T. saginata, these two species can only be differentiated by means of molecular techniques ) [action)) . Unfortu T. asiatica cysticerci in pigs. Routine meat inspections conducted in pigs in Myanmar include the study of T. solium predilection sites such as the tongue, masseter, brain, shoulder, diaphragm, heart, and skeletal, fore limb as well as hind limb muscles [ T. asiatica are almost exclusively located in the liver, such local inspections would not be sensitive enough to detect infected livers [ T. asiatica cross-reacts even in the most specific immunological method to detect T. solium cysticercosis, the enzyme-linked immunoelectrotransfer blot (EITB) [ T. asiatica cysticercosis.A specific meat inspection should be carried out to detect muscles . Consided livers . In addit (EITB) . Therefo T. asiatica cysticerci can easily be differentiated from those of T. solium due to their smaller size as well as the lack of hooklets or the presence of vestigial ones [ T. asiatica is definitely present in Myanmar or not.For the aforementioned reasons, we urge Khaing and colleagues to take advantage of their research in pigs in Myanmar and carry out a detailed study of the surface as well as the parenchyma of pig livers.ial ones . Molecul


Dr. Sineenart Oota is not included in the author byline. She should be listed as the eighteenth author, and her affiliation is: National Blood Center, Thai Red Cross Society, Bangkok 10330, Thailand. The contributions of this author are as follows: Contributed reagents/materials/analysis tools.


After publication of this work , we noteThe authors declare that they have no competing interests.YC and MN conceived the study. YC and MN participated in the design of the study. YC, MI, HI, NO, and MN participated in data interpretation, and drafted the manuscript. All authors read and approved the final manuscript.


Anisakis simplex sensu stricto and Pseudoterranova decipiens. Recently, however, DNA sequencing of larvae from 15 of 16 anisakiasis patients confirmed the cause to be Anisakis pegreffii infection. Molecular analysis should be performed for all extracted larvae.Anisakiasis in humans in South Korea has been considered to be caused exclusively by the larvae of  Anisakis and Pseudoterranova, and the source of infection is marine fish or squids harboring these larvae , originally recovered from a Mediterranean monk seal ; however, molecular techniques can easily distinguish the 2 types of larvae , South Korea , and some European countries  where people eat raw or undercooked fish  genes.In South Korea, Anisakis type I larvae were removed from the stomach of 16 patients referred to the Department of Parasitology and Tropical Medicine, Seoul National University College of Medicine, Seoul, South Korea, from 2000 through 2013 ; nested PCR and nucleotide sequencing were performed on the ITS region  according to procedures reported previously  with those of the A. pegreffii sequence available in GenBank (accession no. AB277823), whereas their identity with A. simplex s.s. (accession no. AB277822) was 99.2% (A. simplex s.s. (accession no. AB277822) and 99.2% identity with those of A. pegreffii (accession no. AB277823). On the basis of these results, A. pegreffii nematode infection was diagnosed for 15 of the 16 patients, and A. simplex s.s. infection was diagnosed for only 1 patient.Of the 26 as 99.2% . The remA. pegreffii nematode infection in humans in South Korea, making this the third country  in which this infection in humans has been identified. This high proportion of A. pegreffii nematode infections in humans is surprising and suggests that most cases of anisakiasis in humans in South Korea may be caused by A. pegreffii rather than A. simplex s.s. larvae. To confirm the source of infection, molecular analyses of Anisakis larvae extracted from human patients are required in South Korea.Our results confirm the presence of A. pegreffii nematodes was first documented in Italy by use of PCR-based restriction fragment length polymorphism (PCR-RFLP) analysis  was not expected because in Japan (Kyushu and Hokkaido), which are geographically close to South Korea, Anisakis larvae from humans are mostly A. simplex s.s. (99/100 larvae from 84/85 patients); only 1 larva was identified as A. pegreffii , including Kyushu and Fukuoka (close to South Korea), showed more A. pegreffii than A. simplex s.s larvae (Anisakis larvae species that infect humans in South Korea and Japan.The markedly high proportion of A. simplex s.s. larvae for human patients was compared with that of A. pegreffii (A. simplex s.s. larvae had greater potential than A. pegreffii larvae to survive acidic gastric juice and to penetrate the human stomach, small intestine, and colon. However, further studies are needed to elucidate this finding.When the pathogenic potential of A. simplex s.s. and A. pegreffii larvae to elicit gastroallergic reactions. These reactions are characterized by urticaria on the arms and abdomen and by angioedema or anaphylaxis when the live parasite attempts to penetrate the gastric mucosa (Anisakis nematode allergy among patients in South Korea (Another clinicopathologic concern associated with anisakiasis in humans is the potential for A. pegreffii over A. simplex s.s. nematode infection among humans with anisakiasis in South Korea. The study highlights the need to perform molecular analysis for each larva extracted from human patients in this country.Our study demonstrates the predominance of


Bactrocera dorsalis species complex currently harbors approximately 90 different members. The species complex has undergone many revisions in the past decades, and there is still an ongoing debate about the species limits. The availability of a variety of tools and approaches, such as molecular-genomic and cytogenetic analyses, are expected to shed light on the rather complicated issues of species complexes and incipient speciation. The clarification of genetic relationships among the different members of this complex is a prerequisite for the rational application of sterile insect technique (SIT) approaches for population control.The Bactrocera dorsalis complex were cytologically characterized. The taxa under study were B. dorsalis s.s., B. philippinensis, B. papayae, B. invadens and B. carambolae. Mitotic and polytene chromosome analyses did not reveal any chromosomal characteristics that could be used to distinguish between the investigated members of the B. dorsalis complex. Therefore, their polytene chromosomes can be regarded as homosequential with the reference maps of B. dorsalis s.s.. In situ hybridization of six genes further supported the proposed homosequentiallity of the chromosomes of these specific members of the complex.Colonies established in the Insect Pest Control Laboratory (IPCL) , representing five of the main economic important members of the B. dorsalis s.s. as reference maps for all these five biological entities is proposed. Present data provide important insight in the genetic relationships among the different members of the B. dorsalis complex, and, along with other studies in the field, can facilitate SIT applications targeting this complex. Moreover, the availability of 'universal' reference polytene chromosome maps for members of the complex, along with the documented application of in situ hybridization, can facilitate ongoing and future genome projects in this complex.The present analysis supports that the polytene chromosomes of the five taxa under study are homosequential. Therefore, the use of the available polytene chromosome maps for  Bactrocera dorsalis complex species is a group of true fruit flies belonging to Tephritidae, with great economic importance. Following the most recent taxonomic revisions, this complex is currently harboring approximately 90 morphological similar taxa [B. dorsalis s.s., B. philippinensis, B. papayae and B. carambolae. In 2003, an addition to the complex was made: B. invadens was detected in Kenya, and initially was considered a morphological variant of B. dorsalis s.s. [B. dorsalis complex [The lar taxa ,2. Among complex . Ever si complex .B. papayae with B. philippinensis; however there is also an ongoing debate about the species status of other important pests of the complex.In recent years, accumulating data cast doubt on the 'actual' number of different species in the complex. Research performed by different laboratories points to a possible overestimation in the number of discrete taxa in the complex and the need of another taxonomic revision to incorporate the synonymic status of different species. This research includes morphological/morphometric studies -10, behaB. dorsalis complex is not just a scientific question regarding evolution and speciation. It is also important for the agricultural economies of countries that heavily rely on fruit exports. The first aspect refers to quarantine measures. The current taxonomy leads to the implementation of certain quarantine policies; therefore it is critical to be as accurate as possible, when assessing species limits of economic important pest populations. As a characteristic example, the description of B. invadens as a separate species within the B. dorsalis complex prompted additional fruit export restrictions in many African countries, leading to increased economic losses [Ceratitis capitata [B. dorsalis complex, initially developed for the control of B. dorsalis s.s. [B. carambolae with B. dorsalis s.s. sterile flies point in such a direction.The delimitation of species within the c losses ,12. The capitata -23. In plis s.s. -26. The lis s.s. ,28, showDrosophila, based on mitotic and polytene chromosomes, were the first to detect interspecific inversions' differences [Drosophila species [Species limits can sometimes be obscure, and speciation can be driven by a variety of forces. Among them, chromosomal rearrangements (mainly inversions), are considered as key factors in Diptera speciation, especially in sympatric populations . Early cferences ,31. Sturferences  and Dobzferences  first sh species -38 and m species -41 suppo species ). More r species ,43. A po species , that caDrosophila species [repleta species group [Ceratitis [Bactrocera [Dacus [Rhagoletis [Anastrepha [Taking into account the above, it is evident that cytogenetic analyses can help in resolving species boundaries within species complexes. This has been well documented in different  species , such as species  and the es group . In resperatitis , Bactrocctrocera -53, Dacua [Dacus , Rhagoleagoletis -57,57 anastrepha  is valuaB. dorsalis complex have also demonstrated the importance of adequate and well characterized samples: when exploring species limits and characters that may overlap, it is important to develop well organized and comprehensive sampling schemes [Studies in  schemes ,16. SincB. dorsalis s.s., B. philippinensis, B. papayae, B. invadens and B. carambolae, through the analysis of their mitotic complements and the comparison of their polytene chromosomes with the published reference maps for B. dorsalis s.s. [1 bidirectional hybrids  were also analyzed, aiming at the detection of fixed chromosomal rearrangements among the parental colonies. We focused on these hybrids since: a) B. invadens is the only member of the complex originating from Africa, and its current recognition as a distinct species within the complex has severe quarantine consequences and b) B. carambolae is considered to be more clearly differentiated from the other four members of the complex [in situ hybridization was performed using unique genes, attempting to: a) provide diagnostic landmarks for the polytene chromosome arms, b) reveal small chromosome rearrangements undetectable by microscopic observation and c) test the utility of B. dorsalis complex polytene chromosomes and polytene maps for future mapping experiments.In the present study, we tried to identify chromosomal differences between five of the main agricultural pests of the complex, namely lis s.s. . As worklis s.s. ,11-13,16 complex ,11,16. FB. dorsalis s.s. (Saraburi - Thailand and Nakhon Si Thammarat - Thailand), one of B. philippinensis (Philippines), one of B. papayae , one of B. invadens (Kenya) and one of B. carambolae  were analyzed. In addition, the two following F1 bidirectional hybrids were analyzed: a) B. dorsalis s.s. (Saraburi strain) × B. carambolae and b) B. dorsalis s.s. (Saraburi strain) × B. invadens.Colonies representing the five economic important members of the complex currently established at the IPCL were used. Specifically, two colonies of oC). Chromosomes were stained with Giemsa . Chromosome slides were analyzed at 100 × magnification, using a phase contrast microscope (Leica DMR), and photographs were taken using a CCD camera . At least 15 good quality preparations (each one representing one larva) per sample and at least 10 well spread nuclei per preparation were analysed.Chromosome preparations were made, as described in . Brain trd instar larvae, as described in [Polytene chromosome preparations were made from 3ribed in . Larvae in situ hybridization were made from salivary glands of 1-4 day-old pupae, as described in [Polytene chromosome preparations for ribed in . Six hetribed in . HybridiB. dorsalis s.s., B. philippinensis, B. papayae, B. invadens and B. carambolae) show five pairs of autosomes and one pair of heteromorphic sex chromosomes (XX/XY). The autosomes have been numbered II to VI according to descending size order [All the members of the complex analyzed here , previously found in B. dorsalis s.s. [A comparison of the polytene elements of all analyzed strains with the reference map of lis s.s.  revealedlis s.s. , was alsB. dorsalis s.s. samples  and F1 B. dorsalis s.s. × B. carambolae hybrids  was performed. The analysis of chromosome preparations of the hybrids did not reveal signs of fixed chromosome differences between the parental strains, evident from the perfect synapses among the parental homologous chromosomes  in situ hybridization of selected genes confirmed that there are no differences among the five members of the complex based at least on the limited number of probes tested. The in situ results also provided characteristic landmarks for the recognition of the polytene arms and demonstrated the utility of polytene chromosomes and reference maps of the complex for in situ mapping projects.The main findings of the present study can be summarized as follows: a) mitotic karyotypes of the five members of the complex presented form A, the typical one for rding to  represenB. dorsalis species complex includes at least eight economic important pests [B. dorsalis s.s. [a priori suitable for mass rearing and release purposes. These strains have to exhibit a number of traits, such as genetic stability and good productivity in the laboratory or in mass rearing conditions over a number of generations as well as male mating competitiveness in the field. In respect to this, cytogenetic knowledge of the chromosomal events generating GSSs, along with standard Quality Control (QC) measures are of major importance for the assessment of the abovementioned traits [The nt pests  that inflis s.s. -26. Howed traits .B. dorsalis s.s., B. papayae, B. philippinensis and B. invadens), favour the 'universal' application of the B. dorsalis s.s. GSSs against all the above members of the complex. This is very important, considering the effort required in generating GSSs through classical genetic methods. In respect to this, the recent study of [B. dorsalis s.s. GSS against B. carambolae, after several generations of crosses aiming to integrate this strain to B. carambolae genomic background.The resolution of biological relationships among the different entities of species complexes is of high importance, since SIT application without this knowledge could jeopardize the effectiveness of such programs, especially in areas where different members of the complex overlap. This information is very useful in respect to the selection of appropriate laboratory strains for release purposes. The findings of the present study along with other studies that support a single species scenario ,13,16,17study of  presenteB. dorsalis s.s.[B. carambolae. In that study, samples derived directly from infested fruits and characterized as B. carambolae based on morphological, geographic and host criteria, were reported to possess × chromosomes larger than the autosomes (form E karyotype). Our analysis does not confirm this report. A recent cytogenetic study on a B. carambolae colony derived from Malaysia also presented a typical form A karyotype for this species [Our analysis of mitotic chromosomes shows that all members of the complex studied here exhibit the same karyotype, described previously as form A . This foalis s.s.,61,63-65 species .Given that a) geographic origin and plant host alone cannot be regarded as absolute taxonomic criteria ,10,19 anB. dorsalis complex have shown that there is considerable variability in size and ratio of the X chromosome arms [A. fraterculus complex [Previous studies on mitotic karyotypes of the ome arms ,63-65. X complex . Τhe hig complex -58,68,69B. dorsalis species complex is based on the following observations: i) absence of fixed chromosomal rearrangements in comparison to the reference map of B. dorsalis s.s.; ii) absence of differences among the parental homologous chromosomes in the two hybrids studied; iii) identical heterochromatic mass of the centromeric regions of each chromosome element in all taxa; iv) common characteristic asynapsis of the chromosomal region 73-74 and v) in situ localization of each of six genes on the same chromosomal region in all taxa analyzed.The proposed chromosomal homosequentiality of the five members of the in situ mapping of unique genes show that chromosomal rearrangements characterize different species [B. dorsalis s.s. and B. tryoni, a species outside, but closely related to the B. dorsalis complex, clearly shows at least one fixed inversion in polytene arm 2R that differentiates the two species . A similar observation has been reported for another Thailand B. dorsalis s.s. population [B. dorsalis complex could provide important insight in the involvement of chromosomal rearrangements in speciation within this species group.Even though fixed rearrangements were not found in the polytene chromosome of the species studied, polymorphic inversions were observed in the two pulation . Althougpulation . The cytB. dorsalis s.s. × B. carambolae hybrid, in respect to the B. dorsalis s.s. × B. invadens hybrid, may indicate that the B. carambolae genome has small differences compared to the other members of the complex. Current literature tends to support B. carambolae as a discrete entity within the complex, but closely related to the others [B. carambolae to a) mate, although with reduced compatibility, b) produce viable and fertile progeny in the lab and c) produce hybrids carrying intermediate characteristics with other members of the complex [B. carambolae and the other members of the complex. Therefore, this is a case most likely representing incipient rather than complete speciation, a phenomenon also observed in the A. fraterculus complex [The minor polymorphic asynapses observed in all samples most probably represent differential gene expression of the two parental homologous chromosomes. However, the presence of small, undetectable (with microscopic observation) rearrangements, such as inversions, deletions or insertions of repetitive or heterochromatic material, cannot be excluded. Indeed, even small inversions can alter the control of regulatory elements and lead to differential gene expression (puffing activity) . Thus, te others ,13,16,19 complex ,28,70,71 complex ,69.B. dorsalis species complex. Our data are in accordance with other recent studies questioning the currently accepted number of discrete species within this complex, since no fixed chromosomal differences were found. This outcome is of major importance for SIT applications targeting the different members of the complex. Currently, there are efforts towards genome/transcriptome sequencing of the B. dorsalis complex [in situ hybridization of genes on the polytene chromosomes, highlight the importance of cytogenetics in gaining more insight regarding organization of newly generated contig sequences and chromosomal localization of genes of specific interest.The present study sheds important light in the delimitation of species boundaries within the  complex ,73 that No competing interests exist.AAA, AGP, AZ conceived the experiments. AAA, ED, AGP, PMT, AZ performed the experiments. AAA, ED, AGP, KB, PMT, AZ performed the analysis. AAA, ED, AGP, KB, PMT, AZ wrote the manuscript. All authors read and approved the manuscript.


The following information is missing from the Funding section: Funding was also provided by the National Science Centre, decision no. DEC-2013/09/B/ST6/02317.Additionally, one of the affiliations for the third author is not indicated. Boleslaw K. Szymanski is also affiliated with the Wroclaw University of Technology, Poland.


The hormonal family of vasoinhibins, which derive from the anterior pituitary hormone prolactin, are known for their inhibiting effects on blood vessel growth, vasopermeability, and vasodilation. As pleiotropic hormones, vasoinhibins act in multiple target organs and tissues. The generation, secretion, and regulation of vasoinhibins are embedded into the organizational principle of an axis, which integrates the hypothalamus, the pituitary, and the target tissue microenvironment. This axis is designated as the prolactin/vasoinhibin axis. Disturbances of the prolactin/vasoinhibin axis are associated with the pathogenesis of retinal and cardiac diseases and with diseases occurring during pregnancy. New phylogenetical, physiological, and clinical implications are discussed. CleavagVasoinhibin species are detected in the rat , 73, mouThe presence of vasoinhibins in the pituitary gland and plasma reflect their hormonal nature, that is, their generation and secretion by an endocrine gland into the circulation. However, vasoinhibins can also act locally at their production site, as demonstrated in rodent studies in which antiproliferative and proapoptotic actions of vasoinhibins were observed in the anterior pituitary gland . In caseWhether the effects of vasoinhibins in the above-mentioned target tissues are due to their action on the vessel system in these tissues or on a tissue-specific cell-type is not always clear. The vasoinhibin effect in the heart, however, in which vasoinhibins utilize the cardiomyocyte to exert their effect on the heart's vasculature, points to the possibility of a complex interplay between both, vascular and nonvascular cells. Furthermore, the proportion as to which the total amount of vasoinhibins present in a target tissue is of systemic or local origin is unknown.2+/calmodulin-activation of endothelial nitric oxide synthase (eNOS) -signal transducer and activator of transcription (STAT) pathway, the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, and the mitogen-activated protein kinase (MAPK) pathway . Of notee (eNOS) . They ale (eNOS) , the acte (eNOS) , 23. Thee (eNOS) . These vKd = 10 nM) than to PAI-1 (1 μM) or uPA-PAI-1 (0.5 μM). While the difference in affinity may suggest a higher Kd for the natural multicomponent PAI-1-uPA-uPA receptor complex, the contribution of other vasoinhibin binding sites, for example a new cell surface receptor, cannot be disregarded and continues to be an unresolved question.A target  and freqA role further downstream in the vasoinhibin-signaling cascade, downstream from a potential cell surface receptor or signaling via PAI-1-uPA-uPAR, is described for the vasoinhibin-miR146a (microRNA-146a) circuit. It appears from the studies in PPCM models, that vasoinhibins mediate part of their antiangiogenic effects via induction of miR146a in endothelial cells. If exposed to vasoinhibins, HUVEC proliferation is reduced via upregulation of miR146a and HUVEC release miR146a-loaded exosomes that can be absorbed by neonatal rat cardiomyocytes . In thesNo data exist about the half-life of vasoinhibins in the circulation. The extent as to which the liver and the kidney contribute to the clearance of vasoinhibins is also unknown. However, vasoinhibins were detected in the urine of women with preeclampsia , 65, poiNew physiological and clinical implications of the prolactin/vasoinhibin axis arise from the governing principle of the growth-restricting effects of vasoinhibins. Vasoinhibins mediate their growth-restricting effects via the inhibition of angiogenesis, which reduces tissue growth due to the lowering of oxygen and nutrient availability. Physiological tissue growth and angiogenesis is fundamental in pregnancy, where angiogenesis is a prerequisite for growth of the placenta and therefore fetal growth.During pregnancy, PRL level progressively rise to ∼20–40 ng/ml at the end of the first trimester, 50–150 ng/ml by the end of the second trimester, and 100–400 ng/ml at term , perhapsVasoinhibins are generated in the human placenta with higher rates observed in placentas from women with preeclampsia  and TypeProlactin and growth hormone (GH) are phylogenetically related, they evolved from a common ancestral gene , and shaIt is unclear if the differences in molecular mass impact the structure and function of vasoinhibins. Also, it remains to be shown whether and to what extent each of the various proteases contributes to the physiological, endogenous level of vasoinhibins, and how the synthesis of different vasoinhibins is modified in disease conditions. Accordingly, the total composition and full identity of endogenous vasoinhibins in the circulation and at the target-tissue levels, including their interplay, has yet to be determined. The impact of glycosylation of PRL on vasoinhibin physiology is unknown. It is possible, however, that glycosylation alters the proteolytic cleavage of PRL and may thus impact vasoinhibin generation, as well as the action and clearance of these peptides . These uAnother major unresolved question is the normal concentration of endogenous vasoinhibins in the human circulation. This is owing to the lack of a quantitative assay, with which circulating vasoinhibin levels and reference ranges could swiftly be established. The only method for the evaluation of endogenous vasoinhibins, which is considered relatively reliable is immunoprecipitation and Western blotting. However, this method is only semiquantitative and subject to significant interassay variability. Mass spectrometric techniques were successfully adapted to determine vasoinhibins in sera ; howeverProlactin is a pleiotropic hormone whose functions are classified into seven categories: water and electrolyte balance, growth and development, endocrinology and metabolism, brain and behavior, reproduction, immunoregulation and protection, and actions associated with pathological disease states . As vasoProlactin, GH, and PL have evolved from a common ancestral gene. On the basis of sequence comparisons of tetrapod hormones, the divergence is located approximately 392 million years ago . Two modXenopus tropicalis, and none in the zebrafish and the spotted gar  in body fluids. The latter is a major demand for studying the involvement of vasoinhibins in pregnancy-associated diseases, diabetic complications, and cancer and for their implications in risk stratification, diagnosis, and treatment. Naturally, resolving the solution structure of vasoinhibins by NMR spectroscopy, possibly by analyzing recombinant human vasoinhibins, would enhance our understanding of the structure-function relationships of the vasoinhibin family and produce valuable insights into their mechanism of action. Upon successful resolution of these issues, the knowledge of the role of vasoinhibins in health and disease will be greatly enhanced and the development of a sustainable classification of vasoinhibins that provides orientation in future biomedical research, will commence.National Council of Science and Technology of Mexico .This study was supported by the No conflicts of interest, financial or otherwise, are declared by the author(s).Author contributions: J.T. and C.C. conception and design of research; J.T., D.R.-B., C.F.P., and C.C. analyzed data; J.T. and C.C. interpreted results of experiments; J.T., T.B., D.R.-B., G.M.d.l.E., and C.C. prepared figures; J.T. drafted manuscript; J.T., T.B., D.R.-B., C.F.P., M.H., G.M.d.l.E., and C.C. edited and revised manuscript; J.T., T.B., C.B., D.R.-B., C.F.P., M.H., G.M.d.l.E., and C.C. approved final version of manuscript.


Bactrocera fruit fly species are economically important throughout the Pacific. The USDA, ARS U.S. Pacific Basin Agricultural Research Center has been a world leader in promoting biological control of Bactrocera spp. that includes classical, augmentative, conservation and IPM approaches. In Hawaii, establishment of Bactrocera cucurbitae (Coquillett) in 1895 resulted in the introduction of the most successful parasitoid, Psyttalia fletcheri (Silvestri); similarly, establishment of Bactrocera dorsalis (Hendel) in 1945 resulted in the introduction of 32 natural enemies of which Fopius arisanus (Sonan), Diachasmimorpha longicaudata (Ashmead) and Fopius vandenboschi (Fullaway) were most successful. Hawaii has also been a source of parasitoids for fruit fly control throughout the Pacific region including Australia, Pacific Island Nations, Central and South America, not only for Bactrocera spp. but also for Ceratitis and Anastrepha spp. Most recently, in 2002, F. arisanus was introduced into French Polynesia where B. dorsalis had invaded in 1996. Establishment of D. longicaudata into the new world has been important to augmentative biological control releases against Anastrepha spp. With the rapid expansion of airline travel and global trade there has been an alarming spread of Bactrocera spp. into new areas of the world . Results of studies in Hawaii and French Polynesia, support parasitoid introductions into South America and Africa, where B. carambolae and B. invadens, respectively, have become established. In addition, P. fletcheri is a candidate for biological control of B. cucurbitae in Africa. We review past and more recent successes against Bactrocera spp. and related tephritids, and outline simple rearing and release methods to facilitate this goal. Bactrocera genus is particularly important throughout the Pacific. It consists of at least 440 species distributed primarily in tropical Asia, Australia, and the South Pacific . A. AF. ariFopiusarisanus occurs from south India to Taiwan, and has been introduced and established in Australia, Cook Islands, Costa Rica, Fiji, Hawaii, Mauritius, Samoa, Tonga, Reunion, and Israel . In augmentative releases of P. fletcheri against melon fly, numbers of B. cucurbitae emerging from fruits placed inside field treatment cages were reduced by up to 21 fold and numbers of parasitoids were increased by 11 fold [P. fletcheri into ivy gourd patches throughout the Kailua-Kona area, parasitism rates were 4.7 times higher in release plots compared to those in control plots. However there was no significant reduction in emergence of flies from fruits. Similarly, in releases of P. fletcheri in zucchini plots in Waimea, there was an increase in parasitoid recovery rates, but no reduction in melon fly damage. Fopius arisanus was also tested as an augmentative tool in small plots of guava in Waimea where the existing population of F. arisanus was low. Levels of parasitism were increased, but infestation was not reduced [The role of parasitoids were tested in the Hawaii AWPM fruit fly program at three levels of application: (1) conservation; (2) augmentative releases; and 3) classical releases [ classica 11 fold . In openF. arisanus and D. longicaudata into South America and Africa, where B. carambolae and B. invadens have become established, respectively [F. arisanus and D. longicaudata evolved in areas where the B. dorsalis complex species are indigenous and their success is likely to be higher on species of that complex than other fruit flies. Given F. arisanus attacks the egg stage and forages on ripening fruits, while D. longicaudata attacks third instar larvae and tends to forage among fallen fruits [F. arisanus and D. longicaudata could also be released against B. latifrons, which has recently invaded the African continent, and the peach fruit fly, B. zonata (Saunders), in Africa and the Indian Ocean region . Pystta


Troglostrongylus has grown due to the increased number of unexpected cases of infections with Troglostrongylus brevior in domestic cats from Mediterranean Europe, likely due to a spill-over from wild reservoirs. Thus, there is a merit to increase our knowledge on the occurrence of this parasite in felids from European regions. The present paper describes lung lesions associated with T. brevior infection in the endangered Eurasian lynx (Lynx lynx) from Bosnia and Herzegovina.In the past few years the interest of the scientific community on lungworms of the genus T. brevior were found in tracheal scraping. The histopathological examination revealed multifocal to coalescing areas, centered on bronchi and bronchioles, and expanded alveoli filled with necrotic debris, degenerated inflammatory cells, mostly neutrophils and macrophages, and multiple cross sections of parasite larvae and thin-walled morulated eggs of lungworms. The paraffin-embedded lung samples were molecularly positive for T. brevior.The carcass of an illegally killed 3-year-old male Eurasian lynx was presented for necropsy at the Faculty of Veterinary Medicine of Sarajevo (Bosnia and Herzegovina). Grossly, multiple, multinodular, consolidated and firm, tan to grey areas, occupying the caudal third of caudal lung lobes, were observed. At cut section, the catarrhal fluid was draining from the airways. Larvae of T. brevior in the Eurasian lynx and the associated host lung pathology. Given its pathogenic potential and the lack of data on troglostrongylosis in lynx populations, the occurrence and impact of Troglostrongylus spp. on wildlife health as well as the role of L. lynx as reservoir of infection for other felids, should be further investigated.This paper describes the first record of  Troglostrongylus Vevers, 1923 (Metastrongyloidea: Crenosomatidae) encompasses four species of parasitic nematodes that have been described for the first time from the respiratory system of wild felids, i.e. Troglostrongylus troglostrongylus Vevers, 1923, T. brevior Gerichter, 1949, T. subcrenatus Railliet and Henry, 1913 and T. wilsoni Stough, 1953 [T. brevior from the queen to the kittens has recently been hypothesised [The genus T. brevior has been described in domestic cats from islands of Spain [T. brevior may contribute to more severe lung pathology when co-infections with A. abstrusus occur [Felis silvestris silvestris) is the natural reservoir of T. brevior with prevalences of infection of up to 71.4 % [T. brevior burden and body condition index [T. brevior may actually cause moderate to severe lung lesions in infected F. s. silvestris [Although regarded as a parasite of wild felids , 2, 4, iof Spain , Italy [of Spain , 7 and Gof Spain  and fromof Spain –12. In tof Spain , 8–12. Fus occur . In Italo 71.4 % –15. A ston index . Nonethelvestris .T. brevior in both domestic and wild cats [Troglostrongylus spp. in another endangered European wild felid, i.e. the Eurasian lynx (Lynx lynx), are available. Indeed, wild Lynx spp. are frequently infected with T. wilsoni, i.e. the bobcat (Lynx rufus) in several regions of the United States [Felis canadensis) in northern Ontario [T. brevior in an Eurasian lynx illegally killed in Bosnia and Herzegovina.Despite an apparent spreading of ild cats , 13–15, d States , 17 and  Ontario . Given tc. 3-year-old male Eurasian lynx was presented for necropsy at the Department of Pathology, Faculty of Veterinary Medicine in Sarajevo. The lynx was shot by poachers in Central Bosnia, in the Fojnica municipality  on the eastern slopes of the Vranica Mountain. The animal was in a good body condition. Tissue samples from lungs, kidneys, intestine, stomach, liver and spleen, were fixed in 10 % neutral buffered formalin overnight, embedded in paraffin, and cut at 3 to 6 μm sections. Deparaffinised sections were stained with hematoxilin and eosin and examined under a light microscope. Tracheal mucosa was scraped and examined under the light microscope. Genomic DNA was extracted from three paraffin-embedded lung tissue samples where morulated eggs and embedded nematode first stage larvae were analysed as previously described [In February 2014, the carcass of a escribed , moleculT. brevior were detected  and the negative result for A. abstrusus in a very sensitive DNA-based assay [T. brevior. In any case, the presence of A. abstrusus in the animal examined here cannot be ultimately ruled out because the genomic DNA of A. abstrusus obtained from parrafin-embedded tissue could have been too fragmented to provide binding sites from specific primers or because A. abstrusus was not present in the three examined samples.The lesions observed in the lung of the present case are almost identical to those described in a kitten co-infected with  brevior . No adulrenchyma . In caserenchyma . A mixeded assay . Additioed assay , 10, 11 ed assay , and lesed assay . In the T. wilsoni have been described in Lynx spp., apart from the proteinaceous fluid noted in the lung of one bobcat in Alabama, United States [T. brevior to cause lesions and to hamper the respiratory system of this wild felid. Hence, further studies are warranted to investigate the occurrence of lungworms in this endangered felid species and if and how they may represent a threat for host's health and welfare. In fact, as the domestic cat, the Eurasian lynx could be affected by an overflow of pathogenic lungworms that are usually harboured by wildcats [No lesions caused by d States . Althougwildcats .T. brevior in an European lynx and the associated lung pathology. Given its pathogenic potential and the lack of data on Troglostrongylus spp. in lynx populations, the occurrence and impact of lungworms on Eurasian lynx health and welfare should be further investigated. Moreover, further studies are necessary to better elucidate the epizootiology of T. brevior in felids other than Felis spp., in order to understand whether the infection in the European lynx is due to bridging infections [This paper describes for the first time infection with fections , 4 or, afections , 4, 13,


Mycoplasma bovis is not clear and attempts to prevent M. bovis-induced disease by vaccines have been largely unsuccessful. In this investigation, joint samples from 4 calves, i.e. 2 vaccinated and 2 non-vaccinated, of a vaccination experiment with intraarticular challenge were examined. The aim was to characterize the histopathological findings, the phenotypes of inflammatory cells, the expression of class II major histocompatibility complex (MHC class II) molecules, and the expression of markers for nitritative stress, i.e. inducible nitric oxide synthase (iNOS) and nitrotyrosine (NT), in synovial membrane samples from these calves. Furthermore, the samples were examined for M. bovis antigens including variable surface protein (Vsp) antigens and M. bovis organisms by cultivation techniques.The pathogenesis of caseonecrotic lesions developing in lungs and joints of calves infected with M. bovis antigens in necrotic tissue lesions was associated with expression of iNOS and NT by macrophages. Only single macrophages demarcating the necrotic foci were positive for MHC class II. Microbiological results revealed that M. bovis had spread to approximately 27% of the non-inoculated joints. Differences in extent or severity between the lesions in samples from vaccinated and non-vaccinated animals were not seen.The inoculated joints of all 4 calves had caseonecrotic and inflammatory lesions. Necrotic foci were demarcated by phagocytic cells, i.e. macrophages and neutrophilic granulocytes, and by T and B lymphocytes. The presence of M. bovis-infected calves, is involved in the development of caseonecrotic joint lesions. Only single macrophages were positive for MHC class II indicating down-regulation of antigen-presenting mechanisms possibly caused by local production of iNOS and NO by infiltrating macrophages.The results suggest that nitritative injury, as in pneumonic lung tissue of  Mycoplasma bovis. The. TheMycons occur –8]. Fur. FurMycoS and NO ,7],9].,[9].Myco.,[9].MycM. bovis induced disease. Certain vaccines give partial protection from respiratory disease and reduce the spread of M. bovis to internal organs including the joints but other attempts have largely been unsuccessful  only onlyM. bM. bovis-infected calves [M. bovis-infected calves.The antibodies used in this study for immunolabelling of macrophages detect calprotectin, which is a heterodimer of two calcium-binding proteins. Calprotectin is present in the cytoplasm of neutrophilic granulocytes and reactive tissue macrophages and is secreted extracellularly from stimulated cells or is released as a result of cell disruption or death . Calprotd calves . Recent d calves . FurtherM. bovis, following infection of a joint spreads to other joints causing necrotizing and inflammatory changes. The findings that the presence of M. bovis antigens in necrotic foci is associated with marked local production of iNOS and NT strongly suggest that in the development of these joint lesions nitritative injury is involved as in necrotizing lung lesions of M. bovis- infected calves. Also, the results of this study revealed that, as in pneumonic animals, M. bovis is evading the immune response and that the responses both in vaccinated and non-vaccinated calves do not protect them from the development and spread of arthritic lesions. Further investigations are needed to study the interference of M. bovis with the specific and innate immune responses of the host.The results of this study demonstrate that The authors declare that they have no competing interests.M. bovis antigens, T and B lymphocytes, and MHC class II. KH conducted the immunohistochemistry of iNOS, NT, S100A8, and S100A9. VRD, KH and MH-T performed the analysis and interpretation of the histological and immunohistochemical data. VRD and MH-T drafted the manuscript. All authors revised and finally approved the manuscript.FP designed and carried out the experimental infection, performed the necropsies, and carried out the bacteriological examinations. DLG selected and collected the tissues samples. VRD performed the histopathological and immunohistochemical examination for


The protein-protein interaction network (PIN) is a useful tool for systematic investigation of the complex biological activities in the cell. With the increasing interests on the proteome-wide interaction networks, PINs have been reconstructed for many species, including virus, bacteria, plants, animals, and humans. With the development of biological techniques, the reconstruction methods of PIN are further improved. PIN has gradually penetrated many fields in biological research. In this work we systematically reviewed the development of PIN in the past fifteen years, with respect to its reconstruction and application of function annotation, subsystem investigation, evolution analysis, hub protein analysis, and regulation mechanism analysis. Due to the significant role of PIN in the in-depth exploration of biological process mechanisms, PIN will be preferred by more and more researchers for the systematic study of the protein systems in various kinds of organisms. Protein–protein interaction (PPI) refers to the physical binding of two or more proteins as responses to different disturbances and circumstances, which provide considerable adaptability for biological cells to adapt flexibly to the changing environmental conditions . Based oIn this review, the development of PIN in the last fifteen years is discussed, including the reconstructed PINs for different organisms, techniques for rebuilding a PIN, and applications of PINs on function annotation, subsystem investigation, evolution analysis, hub protein analysis, and regulation mechanism analysis.The study of PINs has covered a wide range of lifeforms, from viruses to humans, with tens of PINs reconstructed . SystemaEscherichia coli, one of the best studied model organisms, were explored with two different networks [et al. [E. coli proteins and finally obtained 716 stringent interactions involving 83 essential proteins and 152 non-essential proteins. A core sub-network was found broadly conserved across prokaryotes in this PIN, which is composed of 154 interactions and 71 proteins conserved in more than 125 genomes obtained by BLAST homologous searching in different organism genomes. This network provides an available access to the study of protein interaction in prokaryotes. Arifuzzaman et al. reconstructed the second PIN of E. coli with an analogous method as Butland et al. The PIN is composed of 16,050 interactions among 2667 proteins, including 798 uncharacterized proteins [et al. The common proteins were connected by 3088 and 5030 interactions in the two networks, respectively. However, only 218 common interactions were found in those interactions. This indicates that large differences may exist between PINs of the same organism reconstructed from different data sources and experimental strategies. However, both networks show an obvious scale-free property. Additionally, the connectivity of essential genes in Arifuzzaman’s network was consistent with the conclusion that proteins with high essentiality intend to have many interactions [PINs of prokaryote mostly focus on bacterium, including several model organisms and the pathogens for plants, animals, and humans. For several more concerned organisms, multiple PINs were reconstructed by different research groups with the various approaches for the further investigation of latent information in the cell. For example, the protein–protein interactions of networks ,11. Butl [et al.  detectedBacillus subtilis, an important industrial bacterium, started from a small network consisting of 91 interactions among 69 proteins [B. subtilis prove that the extension of current PIN is an efficient way for network improvement and a larger scale of information mining.The PIN of proteins  and was proteins . The finet al. [Helicobacter pylori, a pathogen that causes various gastroduodenal diseases in animals and humans. With querying to the Uniport database [H. pylori infection response was reconstructed, which was composed of 808 interactions and 604 proteins.For the pathogens, the detection of differentially-expressed proteins after infection is a specific approach for the reconstruction of a disease- or immune-related PIN. Kim et al.  filtereddatabase , the difdatabase  and the database . With thStaphylococcus aureus [Treponema pallidum [Many studies of PINs for prokaryotes focus on the pathogens, such as s aureus  and Treppallidum , which iSaccharomyces cerevisiae is the best-characterized organism, with over 90% of its proteins having been screened and the related interactions identified [S. cerevisiae were reconstructed based on a large number of datasets accumulated in the large-scale identification of protein interactions. Schwikowski et al. analyzed 2709 interactions encompassing 2039 proteins in S. cerevisiae and diagramed the set of links within a large protein network [et al. [S. cerevisiae. The largest sub-network in the PIN includes the vast majority of the proteins (87%) and interactions (94%). Ho et al. [PINs for eukaryotes are much more numerous than those for prokaryotes. There has been a lot of effort to explore the eukaryotic protein-protein interaction maps through high-throughput methods. However, no global maps have been fully characterized. entified ,21,22. T network . They de [et al.  built a o et al.  detectedPlasmodium falciparum were reconstructed based on in vivo [in silico methods [et al. [P. Falciparum. The reconstructed computational models have a larger scale but might contain a lot of false positive items [PINs for pathogens also exist in the eukaryote. Several PINs of  in vivo  or in si methods ,27,28,29 [et al. , includive items .et al. [Arabidopsis thaliana by aligning with S. cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and Homo sapiens, including 1159 high-confidence, 5913 medium-confidence, and 12,907 low-confidence interactions. The confidence levels were identified based on three factors: (1) the number of datasets from which the interaction was predicted; (2) the kinds of experiments supporting the interaction; and (3) the number of species where the interaction was found. They found that the interacted proteins tend to locate in the same subcellular location based on the distribution analysis of interaction pairs. This hypothesis supplied a valuable reference for the prediction of protein functions, novel complexes and pathways, as well as providing more information on the known protein complexes and pathways. Cui et al. created an interactome of A. thaliana and collected the interactions into a database named Arabidopsis thaliana Protein Interactome Database (AtPID) [Arabidopsis. Moreover, the computational prediction is shown to be a significant complement to in vivo experiments in the discovery of novel proteins. Furthermore, De Bodt et al. [A. thaliana by identifying the orthologous groups from S. cerevisiae, C. elegans, D. melanogaster, and H. sapiens. With the filter of GO biological process similarity, GO cellular component similarity, and the Pearson correlation coefficient, the confidence of interactions was improved. Ultimately, they obtained a filtered interactome of 18,674 interactions among 2233 proteins. However, less than 15% of the filtered interactions were covered by the PIN reconstructed by Geisler-Lee et al. and, in general, a quite small overlap of the filtered interactions with AtPID can be observed, which indicates the quite different results from the use of different interaction databases and different reconstruction techniques. Lin et al. [Arabidopsis PIN inferred from multiple pieces of evidence, such as homologous interactions, annotation, co-expression, co-localization, and co-evolution, presenting a predicted dataset, namely PAIR (Predicted Arabidopsis Interactome Resource). The dataset holds 149,900 potential molecular interactions, which are expected to cover about 24% of the entire interactome with about 44% precision compared with reported experimental interactions [PINs for plants were mostly reconstructed in the past eight years. As the data obtained from high-throughput technologies in plants is far from depicting the global PPI maps in a plant cell, most PINs for plants are reconstructed with the computational methods, which predict PPIs by sequence alignment ,31 or inet al.  identifi (AtPID) . The dat (AtPID) . AtPID pt et al.  found 51n et al.  providedractions .Populus trichocarpa was built with a genetic algorithm  which included 10,969 high-confidence co-complex membership interactions involving 2297 Drosophila proteins.The PINs for animals mostly focus on the model organism, such as nogaster ,41, C. e elegans , and H.  sapiens ,43,44. G [et al.  reconstret al. [H. sapiens containing about 8100 available Gateway-cloned open reading frames and 2800 protein interactions, 78% of which were verified through an independent co-affinity purification assay. The CCSB-HI1 dataset revealed more than 300 novel protein interactions, including 100 disease-related proteins. This research plays a critical role in the human interactome project. Rob et al. [H. sapiens protein interaction network containing 6643 interactions and 2235 proteins. With in-depth mining of this dataset, they uncovered some novel, previously-unknown, protein interactions and associations between pathways. It is a major step toward studying human disease in the future. The third proteome-scale human interaction network is HI-II-14, which was reconstructed based on literature and validated with different experiments [Due to the increased attention to the health problems, human PINs are usually correlated to diseases. Rual et al.  reconstrb et al.  reconstreriments . HI-II-1et al. [Eriocheir sinensis eyestalk, Y-organ, and hepatopancreas, based on the transcriptome sequencing and proteomes of six model organisms including D. melanogaster, C. elegans, H. sapiens, Rattus norvegicus, Mus musculus, and S. cerevisiae. It is the first large-scale PIN for an aquatic crustacean. This map was used as an effective tool for the function annotation of proteins, extraction of signal sub-network, and evolutionary analyses.In addition to the model organisms, certain important economical species have also attracted the interests of some researchers. Hao et al.  proposedE. coli), as well as ϕ29 (from B. subtilis), are among the best-studied bacterial viruses. An abundance of work has been done on learning the function of protein-protein interactions in the life cycles of phages [E. coli, have been investigated [et al. [E. coli [E. coli transcriptional network and λ proteins, which opened a new door for the thorough understanding of biological regulation mechanisms. Recently, the research on pathogen-host interactions attracts many interests with several virus-human PINs reconstructed [Bacteriophages T7, λ, P22, and P2/P4  and DNA-binding domain (BD). AD and BD are separated, firstly, with BD fused to the interest protein as bait and AD fused to another protein as prey. The interaction between bait and prey can elicit the reconstitution of AD and BD to function together as a transcription factor, which can direct the expression of the reporter gene downstream [in vivo, it shows the natural environment, to some extent, in the cell. Researchers can detect weak and transient interaction due to the accumulative effect of gene product [Y2H was first proposed in 1989 . It is awnstream . In addiwnstream , the stuwnstream , and thewnstream . The sys product .S. cerevisiae, D. melanogaster, E. coli, P. Falciparum, C. elegans, T. pallidum, C. jejuni, and H. sapiens, especially for the PINs reconstructed before 2006. Interestingly, the different PINs generated based on Y2H for the same organism usually have quite a low overlap. For example, the PIN of S. cerevisiae constructed by Ito et al. [et al. [Many PINs were reconstructed based on Y2H, including PINs of o et al.  has only [et al. , which rin vitro. The captured proteins are then digested into peptides and detected with MS. The bait protein can be endogenous or a protein fused to an “epitope tag”. The limitation of this method is that the result is usually influenced by the proteins co-purified from affinity purification, improper folding and mislocalization [in vivo interactions. TAP/MS is also based on the use of tags attached to the terminus of the target proteins. The genes encoding tags and target proteins are carried by a retrovirus to transfer and express in a host cell. Then, the target protein complexes are isolated by two steps of affinity purification. Coupled with mass spectrometry, the target proteins, and their interactions are identified [etc., which have been described in a previous review [Mass spectrometry (MS) is usually used coupled with an affinity purification method for the detection of protein interactions, such as affinity purification coupled with mass spectrometry (AP/MS), tandem affinity purification coupled with MS (TAP/MS), and co-affinity purification coupled to mass spectrometry (coAP/MS). Different from the pull-down method, which fixes the “bait” protein indirectly through tags, AP/MS directly immobilizes the “bait” protein on a solid support to capture target proteins from a soluble phase lization . TAP/MS entified . coAP/MSentified . Howevers review , can alsDPiM [S. cerevisiae [E. coli [Mycoplasma pneumonia [E. coli PINs were reconstructed based on the TAP/MS method by Butland et al. [et al. [E. coli ORF rather than the larger SPA tags at the C-terminal of ORFs used in the former work. Furthermore, an overproduction system with multi-copy plasmid clones was used in the work of Arifuzzaman et al. to avoid sensitivity problems but, in the mean time, it lost stoichiometry between bait and prey proteins. The scales of the two PINs are quite different, and the overlap is rather low. As the two PINs were reconstructed with different tags and overproduction system, it may indicate that these two factors have a significant influence on the final result, with the different tags may affect the conformation of proteins [The DPiM  was recorevisiae , E. coli[E. coli , and Mycneumonia . Two of d et al.  and Arif [et al. , respectproteins  and the Although the experimental method has detected thousands of PPIs in various model organisms, the current size of the interactome detected experimentally usually constitutes quite a small part of the whole genome size . MeanwhiArabidopsis PIN combined with four reference model organisms, S. cerevisiae, C. elegans, D. melanogaster, and H. sapiens [P. Falciparum also includes the dataset obtained by comparing proteins of P. Falciparum with previously-known sequences in other organisms [Arabidopsis and E. sinensis. However, the interolog-based method is incapable of detecting the PPIs with non-conserved proteins. Interestingly, the reconstruction of the H. pylori PIN is based on the distant homology search of interologs between bacteria and human [Interologs are defined as the protein-protein interactions that are conserved in two species . The int sapiens . The homrganisms ,27,28. Tnd human , which mA. thaliana, P. trichocarpa, M. musculus, H. sapiens, and S. cerevisiae. The PIN of A. thaliana reconstructed by ENTS includes more than twice as many predicted interactions as the PIN reconstructed by Geisler-Lee et al. [et al. [Genetic algorithms are a search heuristic that mimics the process of natural evolution to find an optimum solution to a problem. In recent years, the genetic algorithm has usually been used in the prediction of PPIs based on the features of interacted proteins, such as the residue profiles  or domaie et al.  and De B [et al. . By cont [et al.  and less [et al. . HoweverInteractions predicted by computational methods compensate for the lack of PPIs obtained from experiments to some extent . HoweverWith the accumulation of PPI information obtained from different methods, a series of databases have been established to collect and manage the PPIs or PINs from various organisms. Moreover, the International Molecular Exchange (IMEx) consortium was founded with 16 major public interaction data providers for the purpose of establishing a non-redundant set of protein-protein interactions available in a common website . Proteinin vivo experiment. However, the interaction between an unknown protein with a well-characterized protein can supply a major clue to the function of the former. From this point of view, PINs supply a much more convenient, and relatively reliable way, for the detection of protein functions. Although further verification is still needed for the predicted protein functions, the PINs are of considerable value to predict functions for novel proteins and a step forward to complete our understanding of mechanistic information in proteomes. Here, we elaborated the main methods and cases of protein annotation with PINs in the following sections.Although the number of fully-sequenced organisms is increasing and the technique in analyzing the structure of proteins is developing, many proteins remain functionally uncharacterized. Since proteins usually function as complexes, it is hard to find the function of a given protein without knowing what kind of other proteins it interacts with. Function annotation of proteins is a complex and onerous mission by et al. demonstrated that the function and cell localization of interacting proteins have clustering features [S. cerevisiae by analyzing the function of adjacent proteins and retaining the top three highest frequency function annotations. Alexei et al. [S. cerevisiae reconstructed by Schwikowski et al. and the accuracy of the prediction was 60%–70% for the proteins with two neighbors. Hishigaki et al. [S. cerevisiae proteins with the accuracies of 72.7%, 63.6%, and 52.7%, respectively, based on the ontology annotation from Yeast Proteome Database [et al. [E. sinensis, which made up 76% of all the unknown proteins in the global PIN.As classified and unclassified proteins interact in a vast complex network, PINs naturally serve as a platform to place functionally-unclassified proteins in a biological context, which makes it possible to predict the function of unclassified proteins according to their neighborhood relationship . Schwikofeatures . Based oi et al.  further i et al.  extendedDatabase . Hao et  [et al.  took theet al. [Modularity is a common property of most networks. The nodes with similar features in a network intend to be classified into the same module/cluster based on different algorithm. For PINs, the proteins located in the same cluster usually reflect their similar function or membership in a complex. Therefore, the modularity feature of PINs has been used in the identification of protein complexes and the function of proteins. Guruharsha et al.  used DPiet al. . This meet al. [S. cerevisiae PIN. Additionally, three sub-networks consisting of proteins involved in autophagy, spindle pole body function, and vesicular transport were also extracted from the global PIN of S. cerevisiae [et al. created several sub-networks for different purposes with the PIN of maize [S-adenosyl methionine synthase sub-network, were also investigated, which can be applied in the analysis of specific responses to pathogens. The signaling transduction sub-network composed of 2039 interactions was extracted from E. sinensis and seven classical signaling pathways were found in it [Investigation of the subsystems by extracting and analyzing the sub-networks is one of the main applications of PINs. Subsystems are easily identified with PIN according to the function or pathway annotation of proteins in the network. A sub-network concerning a specific function is a good platform for deep investigation of the correlated subsystem. Ho et al.  gained arevisiae . Bryan eof maize . Two hignd in it . The appnd in it . The difnd in it . With thnd in it . The cloPINs are composed of a vast number of interactions which may have interologs in many other organisms, in particular for the PINs reconstructed based on the interologs. Therefore, PINs can serve as an evolutionary analytical tool for further understanding of the evolution routes of some specific sub-networks .C. elegans has been used for the evolutionary analysis and shed light on the assumption that new cellular functions rely on the interactions between evolutionarily new and ancient proteins, which is in line with the classical evolutionary theory [E. sinensis shows that various pathways have different evolution origins with the speculation that Hippo, Jak-STAT, mTOR, and Wnt pathways may grow and mature in the primitive and bilateria stages, respectively [The PIN of y theory . Additioectively . Furtherectively .S. cerevisiae can lead to the apoptosis with three-fold more possibility compared to non-hub genes [et al. revealed two types of hubs, party hubs and date hubs. The former bind most of their neighbors simultaneously, and the latter interact with their different neighbors at different times or locations. In a model with organized modularity, date hubs organize the proteins to connect biological processes or modules to each other, whereas party hubs function inside modules, which has been proved by both in silico studies of network connectivity and in vivo genetic interactions [et al. [Hub proteins refer to a small, but significant, proportion of proteins which interact with many partners. Biological PINs are particularly robust to the random removal of proteins, but are significantly influenced by the removal of hub proteins. It is demonstrated that the knockouts of hub-related genes in ub genes ,104,105.ub genes ,106,107 ub genes . Han et ractions . Howeverractions . In some [et al.  also dem [et al. . Further [et al. , which iH. pylori PIN [B. subtilis [In the lori PIN , with ansubtilis . The GoHsubtilis . The grosubtilis ,115. As E. coli was analyzed with a phage-host interaction network [As interactions in a PIN are not limited in a single organism, the PIN can be a useful analytical tool for the mutual influence between two or more organisms, in particular for the relationship between pathogen/bacteriophage and their hosts. The regulation between phage λ and  network  and 78 s network ) and tarin vivo experiment to some extent, the quality of the reconstructed PINs is still far from satisfaction. The generation of interactions from protein structures might be a trend to invalidate the existing PPIs or predict new PPIs. Second, the PINs show the static situation of the interactions, whereas PPIs in a cell are time-variant. Therefore, the reconstruction of dynamic PINs is another challenge to further study. With these limitations, much more effort is still needed to make PIN a more efficient tool for the research on interactomes and proteomes in the cell.With the development of high-throughput techniques, PIN attracts more and more interest for the study of interactomes in the model or non-model organisms. The systematic property makes PIN a valuable tool for the large-scale study of interactomes, and the network nature makes it a significant platform for the detailed analysis of certain functions and processes based on the specific sub-networks. In this review, we detailed the published large-scale PINs in the past fifteen years and summarized the related reconstruction methods and applications. The urgent problem in the further development of PINs is to present a cell model which can reflect the true biological process dynamically and precisely. There exist two essential problems in the way to achieve this goal. First, the data from a high-throughput experiment, such as Y2H and TAP/MS, is not quite stable against the disturbances. Although the computational predictions for PPIs are complementary to the


Babesia and Theileria species in both domestic and wild animals in Xinyang city, Henan province, where tick-borne diseases have recently been reported. This study aims to analyze the distribution patterns of piroplasms infections in animals, and assess their potential threat to humans in Central China.Piroplasms are kinds of tick-borne parasitic apicomplexan protozoa, which are detrimental to humans and animals in tropical and subtropical areas around the world. Up until now, there has been a limited amount of reliable information available about the prevalence of piroplasms infections in wild animals in China. Therefore, we have investigated the infections of Babesia spp. and Theileria spp. were detected by polymerase chain reaction (PCR) and identified by sequencing and phylogenetic analysis. Moreover, the characteristics of detected piroplasms in different animal hosts were compared between the two study regions.Blood samples were collected from sheep, dogs and hedgehogs in two regions, including Shihe District and Luoshan County, of Xinyang city, Henan province from August to December 2012. Babesia spp. was only detected in the two dogs. Theileria spp. was detected both in the sheep and the hedgehogs, and the total positive rate of Theileria spp. in the sheep and the hedgehogs was 57.53% and 13.82%, respectively. Sequencing and phylogenetic analysis revealed that the Theileria spp. detected in the sheep and the hedgehogs were very close to T. lunwenshuni cloned from a small ruminant and Theileria spp. isolated from a febrile hospitalized patient in China.A total of 227 blood samples were collected from 73 sheep, two dogs and 152 hedgehogs. Babesia and Theileria infections were detected in both domestic and wild animals in Xinyang city, Henan province in Central China, thus warranting further studies in these regions. Please see Additional file Babesia or the Theileria species. These protozoan parasites are transmitted by vector ticks and can infect many different species of domestic and wild animals. Some species of the protozoan parasites are also pathogenic to humans [Piroplasmosis is a tick-borne disease which is caused by infection with apicomplexan protozoa, such as the o humans -3.B. caballi and T. equi were detected in horses mainly in the northeastern and northwestern parts of China [T. buffeli, T. sergenti, B. bigemina, B. bovis, B. ovata and B. orientalis were detected in cattle and buffalo in Central and southern China [T. ovis, T. uilenbergi, T. luwenshuni and B. motasi were detected in sheep and goats mainly in Gansu, Ningxia, Qinghai, Henan, Inner Mongolia and Jilin provinces [B. gibsoni and B. canis vogeli were detected in dogs in Jiangsu, Chongqing, Guangdong, Guangxi, Hainan and Zhejiang provinces [B. microti infections were reported in animals and humans in the northeastern and southern parts of China [B. microti in Taiwan, Beijing, Zhejiang and Heilongjiang provinces [Theileria spp. in Hubei province [T. annulata was highly pathogenic to cattle [Theileriosis and babesiosis are distributed widely in China. For instance, of China -6. T. burn China -9. T. ovrovinces -13. B. grovinces ,15. B. mof China ,17. Howerovinces -21 and Sprovince . Some spo cattle . Some oto cattle .Babesia spp., such as B. microti and B. divergens, are the major pathogenetic species of piroplasms causing human infections. However, some Theileria species can also cause severe acute diseases in humans as human theileriosis [Theileria equi antigens by indirect fluorescent antibody test (IFAT) [Theileria species (18S rRNA GenBank accession number HQ844673) by Liu et al. [T. lunwenshuni.leriosis ,26. Prevu et al.  which waAlthough piroplasmosis is one of the most prevalent diseases of domestic animals, the epidemiology and transmission characteristics among vector ticks and animal hosts are still unclear, and the situation would become even more complex if wild animals were acting as the natural reservoirs.Babesia spp. and Theileria spp. in both domestic and wild animals in Xinyang city where tick borne diseases were recently reported, in order to analyze the distribution patterns of piroplasms infections in animals and assess their potential threat to humans in Central China.Thus, we investigated the infections of The National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention issued the ethical and institutional approval documents for this study.This study was carried out in Luoshan County and Shihe District of Xinyang city in Henan province from August to December 2012  according to the manufacturer’s instructions. Molecular detection of A et al. . AliquotCryptosporidium fragile (EU1627541) was included in the tree as an outgroup.The obtained 18S rRNA gene sequences were edited using the BioEdit 7.0.9 software. The pairwise nucleotide percent identity of the new sequences was calculated using MegAlign software . The neighbor-joining tree was constructed using the MEGA 5.05 package . DistancTheileria spp. in domestic and wild animals were tested by χ2-test, which was performed in SPSS 18.0.Differences in the positive rates of Theileria spp. in the two regions. Babesia spp. was only detected in the two dogs.Out of 227 samples, 63 (21 hedgehogs and 42 sheep) were detected positive for Theileria spp. in the hedgehogs and sheep in Shihe District were 37.25% and 50.94%, respectively (see Table Theileria spp. in the hedgehogs and sheep in Shihe District (P>0.05). However, there was statistically significant difference in the positive rate of Theileria spp. in the hedgehogs and sheep in Luoshan County. The positive rate of Theileria spp. in the hedgehogs and sheep in Luoshan County was 1.98% and 75.00%, respectively. The positive rate of Theileria spp. in the sheep was 37.88 times  higher than that in the hedgehogs. The total positive rate of Theileria spp. in the hedgehogs and sheep in these two regions were 13.82% and 57.53%, respectively. The total positive rate of Theileria spp. in the sheep in these two regions was 4.16 times  higher than that in the hedgehogs  higher than that in Luoshan County  cloned from a small ruminant in China and Theileria sp. (HQ844673) isolated from a febrile hospitalized patient in China  cloned from a dog in Taiwan.The obtained sequences were edited and assembled to a final length of 350 base pairs (bp). All the T. luwenshuni. This result suggests that T. luwenshuni was the predominant Theileria species in both hedgehogs and sheep in the two studied regions. This study is the first to report on the identification of T. luwenshuni in small wild mammals in Central China. Other Theileria species, e.g. T. uilenbergi and T. ovis, have been reported in the southeastern parts of China [In this study, we found that 27.11% (61/225) samples from sheep and hedgehogs were positive for of China , but werT. lunwenshuni was transmitted by Haemaphysalis qinghaiensis[T. lunwenshuni did not show any clinical symptoms or signs of the disease. However, if vector ticks feed on these sub-clinical carriers, they can be infected and transmit the pathogen to exotic animals [It was reported that ghaiensis, and smaghaiensis, with highaiensis. In the  animals , and eveTheileria spp. detected in this study was very close to the Theileria sp. isolated from a febrile hospitalized patient in Suizhou city of Hubei province. In addition, Suizhou city is adjacent to Shihe District geographically. Thus, whether the pathogens were different strains of the same species, whether they would be pathogenic to different species of host animals, or whether they could be a potential threat to human beings is still unclear, and this finding warrants further studies.One exciting finding of this study was, as revealed by the phylogenetic analysis, that the Theileria spp. in the sheep in the two study regions was much higher than that in the hedgehogs, indicating that domestic animals might play a more essential role in the transmission of piroplasmosis. This result was supported by the following facts: First, the population density of domestic animals is much higher than that of wild animals. In fact, sheep husbandry is very common in the rural areas of the study regions. A total of 818.4 thousands of sheep were raised in Xinyang city, based on the data from the Henan Statistical Yearbook 2012. These huge numbers of sheep posed a high risk of exposure and infection to livestock, and increase the economic burden on animal husbandry and farmers in these regions. Second, the habits of animals are indicative of their exposures to infections. Although the population size of hedgehogs in these regions is unavailable, the living habits of hedgehogs are definitely different from domestic animals as hedgehogs are not gregarious animals [Theileria spp. in the hedgehogs in Shihe District was much higher than that in Luoshan County, it also indicated that the population density of sheep in Shihe District might be much higher than that in Luoshan County. Sheep might live in closer contact with hedgehogs in Shihe District due to the increased environmental and social economic activities.In this epidemiological study on piroplasms infection, it is important to understand the role of the different host animals in the transmission cycle -36. We f animals -40. ThisBabesia spp. was only detected in dogs. This could be attributed to only one transmission season, as well as to the limited number of host species that was taken into account. However, babesiosis had been reported in livestock in Henan province [B. microti, which could cause babesiosis in animals and humans. Therefore, it is necessary to carry out more studies on the epidemiology of Babesia spp. and Theileria spp. infections in more species of domestic and wild animals, as well as on the infection status of vector ticks in these regions.In this study, province . Previouprovince  and rodeprovince  collecteBabesia spp. and Theileria spp. infections were detected in both domestic and wild animals in the study regions of Central China. Further studies are needed to estimate the impacts to local animal husbandry by piroplasms infection and to establish biological measures to control the vector ticks.The authors declare that they have no competing interests.ZC conducted the field sampling, performed the laboratory work, generated experimental data, and wrote the manuscript. QL, X-NZ and B-LX had a substantial role in the conception of the study, guidance of the practical work and writing of the manuscript. F-CJ helped with the sample collection. All authors read and approved the final manuscript.Multilingual abstracts in the six official working languages of the United Nations.Click here for file


Ixodes ariadnae has been discovered, adding to the two known ixodid tick species (I. vespertilionis and I. simplex) of bats in Europe.Recently a new hard tick species, I. ariadnae clustered closest to I. simplex, whereas according to their 16S rDNA gene they were closest to I. vespertilionis. The subolesin gene of I. ariadnae had only 91% sequence homology with that of I. ricinus, and is the longest known among hard tick species.Scanning electron microscopic comparison of adult females of these species shows morphological differences concerning the palps, the scutum, the Haller’s organ, the coxae, as well as the arrangement and fine structure of setae. Molecular analysis of 10 geographically different isolates revealed 90-95% sequence homology in the 12S and 16S rDNA genes of bat tick species. Based on 12S rDNA sequences, genotypes of I. ariadnae may have a long evolutionary history.The present study illustrates the morphology and clarifies the phylogenetic relationships of the three known bat tick species that occur in Europe. According to its subolesin gene  Ixodes simplex) and the long-legged bat tick (Ixodes vespertilionis) [I. vespertilionis may feed on humans [In Europe and in the majority of the Old World, for more than a century two ixodid ticks were known to be highly specialized to bats, i.e. the bat tick (ilionis) . Recentlilionis) . The sign humans  and has n humans .I. ariadnae [I. vespertilionis (e.g. its relatively large size and long legs) and I. simplex . However, it was beyond the scope of the latter study to illustrate these similarities and differences with appropriate scanning micrographs of all three bat tick species. In addition, although the most important gene that is used for barcoding (species identification) among ticks [I. ariadnae was shown to differ considerably from those of I. vespertilionis and I. simplex [In the description of ariadnae  it was png ticks , i.e. th simplex , examinaIxodes vespertilionis and I. ariadnae were collected in Hungary; I. simplex was collected in Serbia. For scanning electron microscopy (performed as in [I. vespertilionis) or are unknown (I. ariadnae) [The morphology of two adult females of each of the three bat tick species was compared: ed as in ) femalesriadnae) .I. vespertilionis, four of I. ariadnae, and two of I. simplex [Total nucleic acid (TNA) was extracted as described . Ten TNA simplex . From th simplex -9. PCR p simplex  and Maxi simplex .Authorization for bat capture was provided by the National Inspectorate for Environment .I. vespertilionis are the longest and narrowest  in I. ariadnae , and for I. simplex only other genes have been published [Recently it has been suggested that for molecular identification of tick species sequencing of the COI gene should be the first method of choice, and analysis of 12S and 16S rDNA genes can be performed as complementary tests ,7,8. Priublished . TherefoBecause it is more likely that DNA samples of different geographical origin will show sequence divergence, as demonstrated for both the COI  and 12S/I. ariadnae and I. vespertilionis is of similar magnitude to that between I. ariadnae and I. simplex . With bootstrap analyses, isolates/genotypes of the same bat tick species always clustered together, but separately from isolates of other bat tick species. Based on its 12S rDNA sequences I. ariadnae clustered closest to I. simplex , as contrasted to the other two bat tick species (with up to five nucleotide differences)  Figure . In lineI. ariadnae (KM455971). The subolesin gene of I. ariadnae clustered the closest to  that of I. hexagonus  in comparison with that of I. hexagonus and I. ricinus, respectively.The third target of molecular analyses in this study was the subolesin gene. It has an evolutionarily conserved function/sequence and plays a role (among the others) in immunity, development and reproduction of ixodid ticks . Sequencnae KM45571. The sI. ariadnae has the longest known subolesin gene/protein (with 191 amino acids). Taking into account, that according to phylogenetical analyses subolesin sequences may have evolved from longer sequences in Ixodes spp. to shorter ones in other tick genera [I. ariadnae is an ancient tick species. The importance of the latter finding, i.e. a new type of subolesin (protective) gene is further increased by the fact, that this gene is regarded as one of the most likely candidates among the targets of vaccines to control tick-infestations [These results also imply, that among all the hard tick species for which the subolesin gene and translated protein was reported so far (in : 10 speck genera , it is pstations .I. ariadnae as a separate species, and illustrate the morphological and phylogenetic differences between the three known European bat tick species. According to its subolesin gene I. ariadnae may have a long evolutionary history.These results support the taxonomic status of


Karel's innovations and patents span more than four decades.In the 1970s, the development of his extensive model of the human circulation among other things led to the first method and algorithm for the beat-to-beat computation of cardiac output from the pressure waveform, the so-called Wesseling cZ method , which wIn the 1980s, research on continuous, noninvasive finger blood pressure, building on the original volume clamp principle patent of Jan Peňáz , led to The major limitation of Finapres, measuring blood pressure at the finger, was solved by R&D in the 1990s, and the development of brachial blood pressure waveform reconstruction , 11, licThe R&D efforts in this period also resulted in a battery-operated, ambulatory, 24-h blood pressure device, called Portapres, which was provided to many research groups around the world but also was the basis for a series of contracts with space agencies  for the development of several generations of space-qualified “specials” which were used in space shuttle missions, aboard the Russian space station Mir, and now, with Cardiopres, in routine use on the International Space Station.Further R&D in this period led to a new concept of modeling the real-time flow waveform from pressure, resulting in the second generation method and algorithm for the beat-to-beat computation of cardiac output from the pressure waveform, the so-called Modelflow method , which wA series of innovations and patents have led to widely available devices and methods that have changed the world of noninvasive hemodynamic monitoring for good. When we continue the R&D and product development in this field, we stand on the shoulders of a giant. (J.J.S.)The shape of the arterial pressure wave changes on its way to the periphery due to pulse wave reflections, pulse wave distortions, and resistance to pressure. During the development of Finapres, Karel realized at an early stage that peripheral and, especially, finger pressure measurements are sensitive to such effects , 23, 38.Karel was a physicist with a keen eye for the needs of physicians and physiologists. Across institutional borders, he collected a group of professionals consisting of physicists interested in physiology and medicine, and physicians and physiologists with a sense for physics. It has been a delight to participate under the guidance of Karel Wesseling. (W.J.W.B.)When we learned to know each other in 1969, Karel and I were both interested in the physical basis of arterial function. We discussed about the arterial windkessel model , where KBlood pressure has long been known to rise because of emotions or physical exercise and to drop during rest. With the advent of a method to measure blood pressure noninvasively on a beat-to-beat basis during daily life, this variability itself has become the subject of much research.Earlier analysis of cardiovascular variability was restricted to that of heart rate (HRV) . It was How can blood pressure be regulated and still exhibit the extent of variability that is observed during a normal day? Wesseling proposed the “baromodulation” hypothesis , 37 to eIt was a pleasure and a privilege to have known Karel for some 35 years, in particular sharing his years at the Academic Medical Center. When he showed his experimental finger-pressure set-up with loudspeaker magnet, I never dreamed it would ever fly as high as it did, eventually. (J.M.K.)Knowledge of the physiological mechanisms underlying disturbances in cardiovascular control mechanisms is of direct relevance for clinicians: it is the key to taking an intelligent history and understanding the appropriate emphasis for the physical examination. Only then is the doctor able to diagnose the condition and reassure a worried patient by explaining it in lay terminology. In addition, mechanistic insight is a crucial factor for the design and the evaluation of therapeutic measures.In this context, the Finapres or volume clamp method developed by Peňáz and Wesseling , 35 withI had the good fortune to cooperate with Karel Wesseling in the Finapres evaluation studies since the early 1980s , the cloKarel Wesseling's contributions to cardiovascular physiology are immensely important and wide ranging. He will be remembered by innumerable physiologists, anesthesiologists, autonomic nervous function specialists, and most recently, a growing number of cardiovascular epidemiologists. Karel's development of the volume clamp method for noninvasive finger arterial blood pressure measurement into a true cardiovascular “leatherman” tool has opened the field of population studies for serial hemodynamic observations. Given the gap in cardiovascular health between the wealthy and the poor, and more intriguingly between various ethnicities in the world, there is a genuine need to study the origins, for example, of the impressive differences in stroke prevalence between white and black people, or the devastating coronary atherosclerosis in people of Asian descent. Understandably, conventional risk factor analysis has been monopolizing field studies for decades. Karel has now enriched our tool kit with reproducible arterial blood pressure curves that closely follow changes in blood pressure during simple circulatory challenges, and pulse contour-derived stroke volume and vascular resistance. Systemic hemodynamics can now easily be estimated outside the laboratory and related to rates of conventional risk factors to better detect and follow these inequalities from their early start. For instance, preliminary findings in our ongoing population survey in Dutch Suriname comparing healthy Creole and Hindustani subjects show interesting differences in blood pressure regulation during changing posture: in the face of similar rises in blood pressure, in Creole subjects, resistance increases more than in Hindustani subjects, while left ventricular contractility does not change, whereas contractility drops in Hindustani subjects .More accurate cardiovascular risk profiling, made possible by Karel's gentle, probing, independent mind, should ultimately broaden the evidence we need to obtain better-focused guidelines for those who suffer disproportionally. (G.A.v.M.)“die meisten Organe gar nicht Druck, sondern Stromvolumen brauchen [most organs don't need pressure but flow].” By modeling aortic flow from pressure, Wesseling and coworkers Professor Wesseling passed away on September 4, 2014, in The Hague, The Netherlands. He leaves behind his wife Hanny, their three children, and a grandson whom he unfortunately did not live to see. While we are thankful to have had the opportunity to work with Karel for so many years, we are saddened by the loss.We will miss his original way of thinking, his wit, and his friendship. His intellectual heritage in the field of physiology and noninvasive hemodynamic monitoring will live on. (B.E.W.)B. E. Westerhof and J. J. Settels are employed by Edwards Lifesciences BMEYE.B.E.W. conception and design of research; B.E.W. prepared figures; B.E.W., J.J.S., W.J.W.B., N.W., J.M.K., W.W., G.A.v.M., and J.J.V.L. drafted manuscript; B.E.W. edited and revised manuscript; B.E.W., J.J.S., W.J.W.B., N.W., J.M.K., W.W., G.A.v.M., and J.J.V.L. approved final version of manuscript.