Most of Multiple Sclerosis (MS) patients undergo disease modifying drug (DMD) therapy at childbearing age. The objective of this prospective, collaborative study, was to assess outcomes of pregnancies fathered by MS patients undergoing DMD.Structured interviews on pregnancies fathered by MS patients gathered in the Italian Pregnancy Dataset were collected; pregnancies were divided according to father exposure or unexposure to DMD at time of procreation. Treatment were compared with multivariable logistic and linear models.Seventy-eight pregnancies fathered by MS patients were tracked. Forty-five patients were taking DMD at time of conception , while 33 pregnancies were unexposed to DMD. Seventy-five pregnancies ended in live-births, 44 in the exposed and 31 in the unexposed group. No significant differences between the two groups were found in the risk of spontaneous abortion or malformations (p > 0.454), mean gestational age (p = 0.513), frequency of cesarean delivery (p = 0.644), birth weight (p = 0.821) and birth length (p = 0.649). In comparison with data of the Italian general population, the proportion of spontaneous abortion and caesarean delivery in exposed pregnancies fell within the estimates, while the proportion of pre-term delivery in the exposed group was higher than expected.Our data indicate no association between paternal DMD exposure at time of conception and risk of spontaneous abortion, adverse fetal outcomes and congenital malformations. Further studies clarifying the role of DMD fathers intake prior and during pregnancy are desirable, to supply guidelines for clinical practice. Multiple Sclerosis (MS) is the most frequent and important inflammatory demyelinating disease of the Central Nervous System (CNS) . Since tOn the other hand, there is a dearth of information on the role of paternal DMD therapy on pregnancy outcome. Potential mechanisms of adverse effects may be due to direct mutagenic actions, impairment of spermatogenesis, or the transfer of chemicals via semen . ExpressThe MS Study Group of the Italian Neurological Society launched a project on pregnancy in MS, establishing a network among the main national MS Centers. The dataset collected included information on pregnancy outcomes mothered and fathered by MS patients.In this prospective, collaborative study, we focused on pregnancy and fetal outcomes related to MS patients who had fathered offspring while being treated with DMD.The Italian Pregnancy dataset has been described in detail elsewhere . In briein utero exposure to toxins or radiations, folic acid supplementation, maternal smoke, alcohol intake, abortion, pregnancy complications, amniocentesis, ecographic alterations), delivery and fetal outcomes . Conception was determined as 14 days after the mother’s last menstrual period. A specific section of the questionnaire investigated major developmental problems and malformations of the babies detected following birth, gathered and up-dated after delivery. In case of any malformation or major developmental problems were identified from the questionnaire, the baby’s clinical charts were reviewed.The information form included a section related to MS patients , a section related to their partners’ pregnancy  and those fathered by MS patients who were not taking any medication, i.e. who had discontinued DMD at least 70 days from conception or who had never been treated (unexposed pregnancies).Data on spontaneous abortion (before 22 completed weeks of gestation), pre-term delivery (before 37 weeks of gestation), cesarean delivery, baby birth weight and length were compared between DMD exposed and unexposed pregnancies; furthermore, the proportions of spontaneous abortion, pre-term and cesarean delivery were compared with data reported in the Italian population , derivedThe study was approved by the Ethic Committee of the University of Florence and a written informed consent was obtained from all patients.Outcomes considered in statistical analysis were spontaneous abortion (yes vs no), pre-term delivery (yes vs no), cesarean delivery (yes vs no), birth weight and length of babies.χ2, Fisher’s exact χ2 and Mann–Whitney U tests when appropriate.Baseline characteristics for treatment groups were reported as frequency (percentage) and mean ± standard deviation (SD), and compared with Pearson’s Multivariable logistic and linear models were used for treatment comparisons, including as confounders factors related to MS fathers , factors related to mothers (previous pregnancies and abortions [yes vs. no]), factors collected for both the father and the mother , and factors related to delivery and fetal outcomes . In particular, a) the multivariable logistic model assessing spontaneous abortion as dependent variable included as confounders: age at conception, education, disease duration, previous pregnancies and abortions (yes vs. no), smoking, alcohol and substance exposure during pregnancy (yes vs. no). b) The multivariable logistic model assessing pre-term delivery as dependent variable included as confounders: age at conception, education, disease duration, previous pregnancies and abortions (yes vs. no), smoking, alcohol and substance exposure during pregnancy (yes vs. no), cesarean delivery, gender of the baby. c) The multivariable logistic model assessing caesarean delivery as dependent variable included as confounders: age at conception, education, disease duration, previous pregnancies and abortions (yes vs. no), smoking, alcohol and substance exposure during pregnancy (yes vs. no), gestational age, cesarean delivery, gender of the baby. d) The multivariable linear model assessing birth-weight as dependent variable included as confounders: age at conception, education, disease duration, previous pregnancies and abortions (yes vs. no), smoking, alcohol and substance exposure during pregnancy (yes vs. no), gestational age, cesarean delivery, gender of the baby. e) The multivariable linear model assessing birth-length as dependent variable included as confounders: age at conception, education, disease duration, previous pregnancies and abortions (yes vs. no), smoking, alcohol and substance exposure during pregnancy (yes vs. no), gestational age, cesarean delivery, gender of the baby.Education missing data were substituted with the mean value of the study sample. Other missing data were deleted from the analysis.Comparisons with Italian population data of proportions of spontaneous abortion, pre-term and cesarean delivery were performed using a one-sample binomial test with a 95% CI.We used the SPSS 20.0 running on Windows .During the study period, a total of 78 pregnancies fathered by MS patients were tracked Figure ; Table 1Forty-five patients (57.7%) were taking DMD around the time of conception (17 subcutaneous interferon Beta-1a (IFNB-1a), 14 intramuscular IFNB-1a, 8 subcutaneous IFNB-1b, 6 glatiramer acetate (GA) (Table The group of 33 unexposed pregnancies included those fathered by MS patients who had never been treated with DMD (27 subjects) or had interrupted DMD intake at least 70 days before conception (within six months in six subjects).Twenty-six patients were classified as smokers, 14 in the exposed and 12 in the unexposed group. Furthermore, 14 patients were customary alcohol consumers, 10 in the exposed and 4 in the unexposed group.In pregnant partners of patients’ group there were five smokers, two in the exposed and three in the unexposed pregnancy group, while no women reported either alcohol intake or drug and toxin exposure during pregnancy. Five women had had previous pregnancies  and two had had previous abortions .A total of 78 pregnancies were analyzed: 45 exposed and 33 unexposed. We observed 75 pregnancies resulting in live-births , 44 in the exposed and 31 in the unexposed pregnancy group. One pregnancy of the exposed group ended in spontaneous abortion, occurring after approximately 12 weeks of gestation  fetal malformations or complications were observed in the exposed group , while four 12.1%) occurred in the unexposed group: Edward Syndrome, Down Syndrome, both leading to intentional therapeutic abortion, one case of ocular malformation (myopia and cataract) and one of renal agenesis (solitary kidney) (Table .1% occurχ2 test p > 0.454).There was no significant association between DMD father exposure and risk of spontaneous abortion or malformations . Pre-term delivery was observed in seven exposed pregnancies (17.1% of live birth deliveries) and three unexposed pregnancies . Pre-term delivery was observed at 32 and at 33 weeks in two cases, at 36 weeks for two babies and at 37 weeks for three babies in exposed pregnancies. The three preterm unexposed pregnancies ended at 35, 36 and 37 weeks respectively.In multivariable analysis, cesarean delivery  was the only predictor of pre-term delivery.Cesarean delivery was performed in 15 exposed (34.1% of live birth deliveries) and 9 unexposed pregnancies  and was associated with lower gestational age  in multivariable analysis.Mean birth-weight of newborns was 3.282 ± 445 g in exposed and 3.328 ± 474 g in unexposed pregnancies (p = 0.821).Mean birth-length was 49.9 ± 2.3 cm in the exposed and 50.2 ± 2.1 cm in the unexposed group (p = 0.649).In multivariable analysis, father’s higher education (school years) and lower age at conception were associated with lower birth-weight Table . Higher Maternal complications occurred in seven out of 44 live birth pregnancies in the exposed group (15.9%) and in one out of 31 live birth pregnancies (3.2%) in the unexposed group , we observed one developmental abnormality (mild speech disorder) in a child of the unexposed group.During the year 2005, in the Italian population, a total of 128.09 spontaneous abortions every 1,000 live-births were observed . HypothePre-term delivery was observed in 6.7% of live births and caesarean delivery in 37.3% . HypotheIncreasing interest has been posed on MS patients parenthood, and in particular on DMD exposure around conception and early pregnancy . PreviouThe information on pregnancy and fetal outcomes of MS-fathers is limited. In a recent study, paternal MS and MS-related clinical factors were not significantly associated with birth outcomes . As for The interest on paternally-mediated adverse pregnancy outcomes comes from the notion that drug intake around conception could cause reproductive toxicity. Three main mechanisms have been described: non-genetic , genetic , and epigenetic  ,17. The in vitro studies [Interferon beta and glatiramer acetate have shown no mutagenic effects on  studies ,20. To dIn the present study we examined 78 pregnancies fathered by MS patients, 45 exposed to DMD therapy at time of conception.We found no significant differences in the risk of spontaneous abortion and malformations, mean gestational age, frequency of cesarean delivery, birth weight and birth length between DMD exposed and unexposed pregnancies. Moreover, comparing data of exposed pregnancies with incidence estimates inferred from the Italian general population , we founAlthough we found no significant difference in preterm delivery rates between exposed and unexposed pregnancies, the proportion of pre-term delivery observed in the exposed group (17.1%) was higher than expected, considering the estimates extrapolated from general population.There were no significant differences in the risk of maternal complications, among which partial placental abruption, preeclampsia, threatened abortion, threatened preterm delivery, and a minor infection. However, the considerable heterogeneity of the observed maternal complications in our small study sample, does not allow to draw definitive conclusions.Eventually, in our study sample, data on possible developmental abnormalities are encouraging, because in a median follow-up period of 1.8 years we only observed the occurrence of mild speech disorders in a child in the unexposed pregnancies group.in utero fetal exposure. Finally, as for the comparison of the Italian population, demographic matching cannot be warranted, since information on age of the mothers was lacking.Some limitations have to be considered in the interpretation of the study’s results. The study sample was relatively small, and some data of patients' partner were lacking . Moreover, since the questionnaire collected very detailed complex medical data directly for the fathers a recall bias cannot be excluded. Furthermore, we had no information on sexual habits, and we cannot infer the influence of paternal exposure during pregnancy related to potential Our data suggest the absence of association between paternal DMD exposure at time of conception and risk of spontaneous abortion, adverse fetal outcomes and congenital malformations. Although we found a higher preterm delivery rate in the exposed group, these data should be interpreted with caution in light of the above considerations. Further studies addressing the issue of MS patients paternity are desirable, to better clarify the role of DMD fathers intake prior and during pregnancy and to supply guidelines for clinical practice purposes.MS: Multiple sclerosis; CNS: Central nervous system; DMD: Disease modifying drug; IFN-beta: Interferon beta; GA: Glatiramer acetate.Dr. Pecori receives research support from Novartis and FISM . Dr. Giannini has nothing to disclose. Dr. Portaccio serves on a scientific advisory board for Biogen-Idec, Merck Serono and Bayer, received honoraria for speaking from Biogen-Idec and Teva, and receives research support from Merck Serono, Biogen-Idec, Bayer Schering Pharma and Sanofi Aventis. Dr. Ghezzi serves on scientific advisory boards for Merck Serono and Teva Pharmaceutical Industries ltd; received honoraria for speaking form Merck Serono, Biogen Idec, Bayer Schering Pharma, and Novartis; serves as a consultant for Novartis, receives research support from Sanofi Aventis, Biogen Idec and Merck Serono. Dr. Hakiki received research support. from Merck Serono, Novartis and Teva. Dr. Pastò has nothing to disclose. Dr. Razzolini has nothing to disclose. Dr. Sturchio has nothing to disclose. Dr. De Giglio has nothing to disclose. Dr. Pozzilli serves on advisory boards for and received speaker honoraria from Sanofi Aventis, Biogen, Bayer Schering Pharma, Novartis, Teva, Merck Serono and research grants from Merck Serono, Sanofi Aventis, Novartis and Biogen. Dr. Paolicelli serves as a consultant for Merk Serono and Bayer Schering Pharma. Dr. Trojano received honoraria for consultancy or speaking from Sanofi-Aventis, Biogen, and Bayer Schering, and research support from Merck Serono and Biogen Idec. Dr. Marrosu serves on a scientific advisory board for Merck Serono, Biogen Idec and Bayer Schering Pharma; has received funding for travel from Biogen Idec, Merk Serono, Bayer Shering Pharma and Sanofi Aventis; serves on the editorial board of Neurological Sciences; has received speakers honoraria from Biogen Idec and Merk Serono; has received research support from Biogen-Idec, Merck Serono, Fondazione Banco di Sardegna. Dr. Patti served on a scientific advisory board for Bayer Shering Pharma, Biogen-Idec, Merck Serono, Novartis, and Sanofi Aventis, received honoraria for speaking from Bayer, Biogen-Idec, Merck Serono, Novartis, and Sanofi Aventis and funding for travel from Merck Serono and TEVA, has received research support from the University of Catania and FISM. Dr. Mancardi has received funding for travel from Biogen Idec, Merk Serono and Bayer Shering Pharma; speaker honoraria from Bayer Schering Pharma, Biogen Idec, Sanofi-Aventis, and Novartis; serves on the editorial board of Neurological Sciences. Dr. Solaro has nothing to disclose. Dr. Totaro has received honoraria for consultancy or speaking from Bayer-Schering Pharma, Biogen Idec, Merck Serono, Novartis Pharma, Sanofi-Aventis, and Teva. DR. Tola has served on scientific advisory boards and received speakers honoraria from Biogen Idec, Sanofi Aventis, Merk Serono and Novartis and has received research support from Sanofi Aventis. Dr. De Luca has nothing to disclose. Dr Lugaresi has served on scientific advisory boards for Bayer Schering, Biogen Idec, Genzyme, and Merck Serono, received travel grants and honoraria from Bayer Schering, Biogen Idec, Merck Serono, Novartis, Sanofi Aventis and Teva Pharmaceutical Industries; has received research support from Bayer Schering Pharma, Biogen Idec, Merck Serono, Novartis, Sanofi Aventis, Teva, Associazione Italiana Sclerosi Multipla (AISM), serves as a Consultant for “Fondazione Cesare Serono”. Dr. Moiola has nothing to disclose. Dr. Martinelli has received speaker honoraria and funding for travel from Biogen-Idec, Merck Serono, Bayer Schering Pharma, Novartis and Sanofi Aventis and has served as a consultant to Bayer Schering Pharma, Sanofi Aventis and Teva Pharmaceutical Industries. Dr. Comi serves on scientific advisory boards for Bayer Schering Pharma, Merck Serono, Teva Pharmaceutical Industries Ltd, Sanofi Aventis, Novartis and Biogen-Idec; has received speaker honoraria from Teva Pharmaceutical Industries Ltd, Sanofi Aventis, Serono Sumposia International Foundation, Biogen-Idec, Merck Serono, Novartis, Bayer Schering and Sanofi Aventis. Dr. Amato serves on scientific advisory boards for, has received speakers honoraria and research support from Biogen-Idec, Merck Serono, Bayer Schering Pharma and Sanofi Aventis and serves on the editorial board of BMC Neurology.CP: Drafting/revising the manuscript. Study concept or design. Analysis or interpretation of data. Acquisition of data. MG: Drafting/revising the manuscript. Study concept or design. Acquisition of data. EP: Drafting/revising the manuscript. Study concept or design. Analysis or interpretation of data. Acquisition of data. Statistical analysis. Study supervision. AG: Study concept or design. Acquisition of data. Study supervision. BH: Drafting/revising the manuscript. Study concept or design. Acquisition of data. LP: Drafting/revising the manuscript. Study concept or design. Acquisition of data. LR: Study concept or design. Acquisition of data. AS: Study concept or design. Acquisition of data. LDG: Study concept or design. Acquisition of data. CP: Study concept or design. Acquisition of data. Study supervision. DP: Study concept or design. Acquisition of data. MT: Study concept or design. Acquisition of data. Study supervision. MGM: Study concept or design. Acquisition of data. Study supervision. FP: Study concept or design. Acquisition of data. Study supervision. GLM: Study concept or design. Acquisition of data. Study supervision. CS: Study concept or design. Acquisition of data. Study supervision. RT: Study concept or design. Acquisition of data. Study supervision. MRT: Study concept or design. Acquisition of data. Study supervision. GDL: Study concept or design. Acquisition of data. AL: Study concept or design. Acquisition of data. LM: Study concept or design. Acquisition of data. VM: Study concept or design. Acquisition of data. GC: Study concept or design. Acquisition of data. Study supervision. MPA: Drafting/revising the manuscript. Study concept or design. Analysis or interpretation of data. Study supervision. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2377/14/114/prepub


Animal chlamydiosis, caused by different chlamydial species, is characterized by clinical or subclinical disease manifestations in cattle, buffalo, ovine, caprine and wild animal species. Animal chlamydiosis often remains underdiagnosed or undiagnosed, and its status in many parts of India is still unknown. Hence, the present study was conducted to determine the seroprevalence of animal chlamydiosis amongst ruminant livestock species of five states of India.viz. cattle (n=430), buffaloes (n=429), sheep (906) and goats (n=362), were tested by agar gel precipitation test for chlamydiosis between 2002 and 2011. Precipitating antigen was prepared from locally isolated strain of Chlamydia psittaci after treatment with sodium deoxycholate.Totally, 2127 randomly selected serum samples collected from ruminant livestock species The chlamydial seroprevalence detected amongst ruminants in five states of India was: Himachal Pradesh: Cattle-10.90%, sheep-10.60% and goats- 22.46%; Punjab: Cattle-1.45%; Andhra Pradesh: Cattle-2.80%, buffaloes-0.93%, sheep-8.90% and goats-9.46%; Maharashtra: goats-8.33%; Jammu and Kashmir: sheep-12.50%. The mean seroprevalence values of each animal species are: Cattle-4.65%, buffaloes-0.93%, sheep-9.82% and goats-19.33%.The results indicate the endemic nature of animal chlamydiosis across five states in India. Hence, it requires further extensive studies in other parts of India also using chlamydial species-specific diagnostics to ascertain overall countrywide prevalence of the disease. The zoonotic nature of the chlamydiae of ruminant origin further adds significance to such prevalence studies. Chlamydiales, family Chlamydiaceae were grouped into two genera, Chlamydia and Chlamydophila, containing nine species [Chlamydia) with 11 species has been adopted by International Committee on Systematics of Prokaryotes [Chlamydia. Amongst ruminants, Chlamydia abortus, Chlamydia pecorum and Chlamydia psittaci cause clinical infections [C. abortus causes abortion, infertility and weak offspring in sheep, goats, cows and buffaloes, whereas, C. pecorum is associated with chronic conditions such as pneumonia, polyarthritis, conjunctivitis, enteritis, encephalomyelitis, metritis and mastitis amongst ruminants. C. psittaci mainly causes “avian chlamydiosis” amongst 471 reported avian species [C. psittaci, C. abortus, Chlamydia caviae and Chlamydia felis are confirmed zoonotic pathogens [Chlamydiae are obligate intracellular bacterial pathogens, responsible for diverse disease conditions in animals, birds and humans -3. Chlam species . But reckaryotes  and is ckaryotes -6. Animafections -9. C. ab species  but can athogens .Chlamydial diseases amongst ruminants are known in India since long time ,14 but aHence, the present study was undertaken to investigate the seroprevalence of animal chlamydiosis in five states of India.Prior consent of the owners of the animals was taken before collection of blood samples for serum extraction. Proper ethical considerations related to animal handling were observed and ensuring not to cause any injury during sampling. The other institutes that submitted the serum samples for diagnosis of the chlamydiosis addressed the animal ethics issues according the local regulations.The study was conducted during the period of 2002-2011 using 2127 serum samples collected randomly or submitted to the Department of Veterinary Microbiology for chlamydial diagnosis from populations of ruminant livestock species from five different states such as Himachal Pradesh, Punjab, Jammu and Kashmir, Andhra Pradesh and Maharashtra. The detail of sample collection is shown in C. psittaci strain from the placenta of an aborted sheep that was multiplied in McCoy cell line. The cells were maintained in Eagle’s minimum essential medium (Gibco) containing 5% fetal bovine serum and 10 mg/ml of gentamicin and 1 mg/ml of cycloheximide and incubated at 37°C in 5% CO2. The EBs were harvested 3-4 days after inoculation and were partially purified. AGPT antigen was prepared by treating these EBs with sodium deoxycholate as reported earlier [Chlamydial group specific antigen was prepared from locally isolated  earlier .C. psittaci as described earlier [Positive serum used in test proper was raised in rabbit using above-mentioned strain of  earlier . BrieflyThe AGPT test was performed as described earlier  with minThe hypothesis that the seroprevalence of chlamydiosis in animal populations of different states is same was tested using cChlamydia spp. Though, AGPT is less sensitive but does not require any specialized infrastructure and can easily be performed under in house conditions. AGPT is reported to detect chlamydial complement fixation antibody titer of more than 8, which is considered advantageous in ruling out weak reactors in case of field studies [Chlamydial infections have been reported from different livestock species globally ,23,24. I studies .et al. [et al. [Analysis of disease-related data from five states revealed that seroprevalence was highest, i.e. 19.33% amongst goats. However, seroprevalence of chlamydiosis amongst goats of different states of India showed variations from 8.33% in Maharashtra to 22.46% in Himachal Pradesh, and the detail is shown in et al.  using AG [et al.  also rep [et al. . In this [et al. ,18,27-29The results of the present investigation indicated that chlamydial infections are endemic amongst ruminants in studied areas of five states of India, which may represents one of the causes of abortion, pneumonia, enteritis, arthritis and other clinical manifestations. Therefore, large-scale screening of native livestock species along with characterization of involved indigenous chlamydial strains is urgently required. Thus, integrated strategies are required to be adopted for control and prevention of sporadic chlamydial infection cases or disease outbreaks keeping in view the zoonotic importance also.RC was associated with overall planning and execution of the research work. SG, BB and PM carried out laboratory work. SV and MS analyzed the data and drafted and revised the manuscript. All authors read and approved the final manuscript.


Mycobacterium cosmeticum; however, growth and biochemical features indicate that it is distinctly different. Here, we highlight for the first time an unusual NTM that is a probable cause of ear infection.We describe the identification and characterization of a novel nontuberculous mycobacterium (NTM), isolated from an ear swab of an adult male patient with chronic otitis externa. Genetically, the bacterium is most closely related to  Mycobacterium abscessus complex are typical causative agents [ M. avium [ M. fortuitum [ Pseudomonas aeruginosa and Staphylococcus aureus being most commonly implicated [ Mycobacterium spp.Nontuberculous mycobacteria (NTM) are known albeit uncommon causes of otic infections (otitis media and otomastoiditis). Members of thee agents , 2, althM. avium  and M. fortuitum  have alsplicated , 6; howe M. cosmeticum-like bacterium (NTM165). The isolate was obtained from an ear swab of a 39-year-old male patient with chronic otitis externa. No other microorganism grew from the specimen and NTM165 was diagnosed to be clinically significant. As this was a referral sample from a local polyclinic, we were unable to obtain further clinical details with regard to history and treatment. Ziehl-Neelsen staining indicated that the bacilli were acid-fast. After 3 days of growth at 37°C on Middlebrook 7H10 agar plates, NTM165 colonies were smooth and off-white color. No growth was observed at 42°C. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-ToF MS) identification of NTM165, with full protein extraction procedures, was performed as described by the manufacturer . A result score of 1.727 matching Mycobacterium cosmeticum was generated; however, this was an inadequate score to confidently define the species. The isolate was sent to a local reference tuberculosis laboratory for further identification by high-performance liquid chromatography (HPLC) analysis. Evaluation of the mycolic acid pattern by HPLC indicated that NTM165 had a profile similar to the M. fortuitum complex but provided no further identification. Mycobacterial commercial DNA probe assays, GenoType Mycobacterium CM/AS  and INNO-LiPA MYCOBACTERIA v2 , yielded no identification for the NTM.In this report, we describe the isolation of a novel rpoB gene and hsp65 genes [ M. cosmeticum type strain LTA-388 (NCBI Reference Sequence NR_025787). It has been proposed that a genotypic difference of >1% is sufficient to be named as a new species [ hsp65 had a 99% (411/414 nt) DNA identity to M. cosmeticum (GenBank accession number AY449731.1) whilst the closest match to the rpoB sequence was M. canariasense (GenBank accession number KJ720535.1) . The ITS sequence was also searched against the GenBank database and the closest result was a 94% identity to M. neoaurum (GenBank accession number CP006936.2). The absence of M. cosmeticum and M. canariasense ITS sequences in the GenBank database likely accounted for M. neoaurum as the best match.For molecular identification, the mycobacterial DNA was extracted according to the DNeasy Blood and Tissue Kit  protocol for Gram-positive bacteria. The full length 16S rRNA, 16S-23S internal transcribed spacer (ITS), and partial65 genes , 8 were  species  and wher M. cosmeticum and M. canariasense, which were also highly susceptible to antimycobacterial drugs [Biochemical characterization of NTM165 was performed as described by Metchock et al. . The isoal drugs , 13. M. cosmeticum. However, subsequent molecular analysis showed that NTM165 was genetically dissimilar to the closest matches of M. cosmeticum and M. canariasense, having <100% identity for the 16S rRNA, hsp65, and rpoB gene sequences. Additionally, comparative biochemical assays and growth features showed NTM165 was uniquely different to another related Mycobacterium. For the identification of M. canariasense, there were reported difficulties with its accurate identification using HPLC, matching it to M. fortuitum [MALDI-ToF provided the first clues that this organism was highly similar toortuitum . Our exportuitum –14. M. cosmeticum were obtained from cultures of a sink drain in a nail salon and from a granulomatous lesion of a female mesotherapy patient in Venezuela [ M. cosmeticum was further demonstrated in additional cases whereby the bacterium was responsible for pulmonary disease, catheter-associated bacteremia and most recently as a colitogenic agent [ M. canariasense, akin to M. cosmeticum, is an opportunistic pathogen linked to bacteremia in patients with underlying malignant diseases [The first reported strains ofenezuela . The patic agent , 17. M. diseases , 13, 18. M. cosmeticum-like NTM, which is believed to be the etiologic agent of the infection. In this study, we highlight the importance of the use of a multiple-pronged approach for the identification and characterization of the microorganism, in particular, the importance of sequencing housekeeping genes to further differentiate closely related mycobacterial species.In this report, we describe for the first time the isolation from an ear swab sample of a patient with chronic otitis externa, a novel hsp65, rpoB, and ITS sequences of NTM165 have been deposited in GenBank under the respective accession numbers of KP012254, KP012255, KP012256, and KP012257.The 16S rRNA,


Coastal cliff-tops are specific saline environments, where only highly specialized halophytes can thrive. Limonium spp. are commonly found in these ecological conditions, many of them being considered as threatened or with an unknown conservation status. The habitat requirements of Limonium multiflorum, an apomictic halophyte endemic to western Portugal, were investigated. Results showed the species narrow habitat specificity as well as its intolerance to competition with invasive alien plants. We conclude that in situ conservation of this rare and vulnerable species emerges as a priority in order to ensure that its biodiversity is not lost. Limonium are commonly found. Among them, the aneuploid tetraploid  Limonium multiflorum, endemic to the west coast of Portugal, is an interesting case study for investigating the ecology and conservation of a halophyte agamospermic species. Although it is listed in the IUCN red list of threatened species, information on its population size or rarity, as well as its ecology, in some respects is still unknown. Field surveys in the largest known population were performed  in order to determine habitat requirements and conservation status. A total of 88 quadrats were monitored, 43 of which contained at least one L. multiflorum individual. For each sampled quadrat, four abiotic and four biotic variables as well as two spatially derived variables were recorded. Principal component analysis and cluster analysis showed narrow habitat specificity for this species which appeared to be intolerant to competition with invasive alien plants. We conclude that in situ conservation in a local ‘hotspot’ of this rare and vulnerable species emerges as a priority in order to ensure that biodiversity is not lost.Coastal areas and other saline environments are major contributors to regional and global biodiversity patterns. In these environments, rapidly changing gradients require highly specialized plants like halophytes. In European coastal cliff-tops, rocky and sandy seashores, and saltmarshes, typical halophytes from the genus  Along tL. multiflorum  is found at this site, within an area of ∼0.6 km2, inside a Site of Community Importance (SCI Sintra/Cascais PTCON0008) for the Mediterranean biogeographical region. This cape consists of gently folded limestone, forming a rocky shoreline with low cliffs and numerous deep incisions along fault lines or abraded mylonitic rocks   and the remaining 45 were randomly selected quadrats containing no individuals from this species (absence quadrat) , cobble (Cobble), coarse sand (CoarseSd) and fine sand (FineSand) , were recorded. Four biotic variables related to vegetation cover (Coverage) as well as invasive non-native species (INNS) cover, dead organic matter (lignified or not) (DOM) cover and litter (Residues) cover were also estimated (expressed in %) in each quadrat. To facilitate data collection, each quadrat was then sub-divided into sub-quadrats of 1 m2, and the percent coverage value obtained for the sub-quadrats of the same 100 m2 quadrat were averaged. Two spatial variables, distance from coastline (Dist_coa) and mean quadrat slope (Mean_slo), were also derived using the ArcGIS® 10.0 software, by ESRI.Field surveys used a grid of 10 × 10 m quadrats which was overlaid over the aerial photographs of the site and later transferred to the site using global positioning system (GPS) receivers. A total of 88 quadrats were surveyed, 43 of which contained at least one at) Fig. . The ranat) Fig. . In quadL. multiflorum habitat requirements we performed a principal component analysis (PCA) on the resulting data matrix using abiotic, biotic and spatially derived variables. In order to discriminate the vegetation structure and composition in the monitored quadrats, a cluster analysis using Euclidean distance and Ward clustering method (L. multiflorum. To ensure that the group of quadrats (clusters) had the same influence from the environmental variables, Kruskal–Wallis tests of variance were performed  while control quadrats did not show any individual from this species (absence). Considering the presence quadrats, L. multiflorum mean coverage was very low (about 0.67 %). Only one of the variables measured in each quadrat fitted a normal distribution (variable ‘Mean_slo’), and the other nine failed to do so, even after a logarithmic transformation. Therefore, the remaining analyses were performed using the original (untransformed) values. Remarkably, visual inspection of PCA revealed that in the first two represented ordinations, quadrats showing the presence of L. multiflorum individuals were highly correlated with rock formation (variable ‘RockForm’) and non-correlated with high coverage (variable ‘vegetation cover’)  Fig. . Consider’) Fig. . The firL. multiflorum presence. The data obtained revealed the existence of four floristic groups among the sampled quadrats  were found. Although in both Groups 1 and 2, ‘INNS’ percentages were greater than those in the other groups, Group 2 differed in terms of fine sand percentages (variable ‘FineSand’).A cluster analysis was performed using the dataset of quadrats showing ats Fig.  and 2. ECarpobrotus edulis with a coverage percentage >65 % whereas Cluster 2 presented a moderate coverage of C. edulis (<35 %). In this cluster significant mean coverage was observed for psammophilous species like the perennials Armeria welwitschii and Elymus farctus subsp. boreo-atlanticus. As for Cluster 3, significant frequencies of the perennial Limonium ovalifolium and the therophyte Parapholis incurva were revealed. Finally, Cluster 4 which had the greatest coverage percentage of L. multiflorum (0.835 %) of all clusters appeared to be associated with chasmophytic species such as C. maritimum, and other perennials like Frankenia laevis and Inula crithmoides.Vegetation composition also varied between clusters. Cluster 1 was dominated by L. multiflorum grows on sea-cliffs preferentially up to 50 m asl in karstic crevices within exposed rocks more or less filled with substrate, or on shallow soil above the rock strata and on scree slopes. The presence of L. multiflorum was favoured by the presence of rock formation, cobble, low percentage of vegetation coverage, low frequency of invasive non-native species and closest distance to coast. In fact, as pointed out by L. multiflorum's preference for salt-rich rock crevices seems to be a strategy to use such locations as refuges, thus avoiding competition with many other coastal plants that are unable to colonize such habitats. Furthermore, as it happens in endemic Limonium spp., populations tend to be fragmented and usually show low population sizes as a consequence of many social and economic activities like urbanization, tourism and traffic . Cluster 3 is typically formed by species of rocky substrate, with a high slope and direct influence of wind loaded with salt. Here L. ovalifolium, C. maritimum, A. welwitschii, F. laevis, L. multiflorum, P. coronopus form the perennial community Limonietum multifloro-virgati  nutrient-rich droppings are frequent , in our study it seems to compete mainly with species showing preference for sand substrates. Remarkably, some littoral species show strong resilience to C. edulis invasion, like the narrow endemic cliff-species Armeria pseudoarmeria   and diverse karyological polymorphisms were found within and among populations, with greater variability in the Raso cape population  , both unaxa e.g. , and eveLimonium spp.  and other areas under legal protection , in the past decades no information on population size along its distribution range has been available. However, we hypothesized that population decline might have occurred due to increasing urban development in the coastal areas and the impact of tourism. For instance, in Raso cape, sightseeing tourists, sports fishing and other socio-economic activities, together with competition associated with invasion by non-native species are cumulatively leading to population decline . Notably, methylation-sensitive amplified polymorphism (MSAP) data from L. multiflorum natural populations reveal low levels of genetic/epigenetic diversity  and to perform selective removal and control of invasive species  but population reinforcements by planting newly produced specimens using seed stock that originated from the same natural populations (e.g. Raso cape) appears to give better results  have also been conducted   (PEST-OE/AGR/UI0240/2011). A.D.C.  and A.S.R. (SFRH/BD/62542/2009 grant) were supported by FCT.Our work was funded by A.D.C. and P.A. designed and coordinated the study. M.D.E.-S. coordinated the fieldwork and A.D.C., M.D.E.-S., A.P.P. and A.S.R. performed the fieldwork. A.F. and V.S. processed the raw data and carried out statistical analysis. J.C.C. and M.D.E.-S. performed the phytosociological analysis. A.D.C. and P.A. drafted the manuscript. All authors read and approved the manuscript.None declared.


Coccinella septempunctata L. and Hippodamia variegata Goeze are the most effective predators upon Aphis gossypii Glov., which is an economically important pest of melon. Understanding their likelihood to engage in reciprocal predation is a key point for conservation of biological control. Here, we aim to investigate, under laboratory conditions, the level of IGP between the two above mentioned aphidophagous species. (2) Fourth-instars of the two species were isolated in petri dishes with combinations of different stages of the heterospecific ladybird and different densities of A. gossypii. The occurrence of IGP events was recorded after six hours. (3) C. septempunctata predated H. variegata at a higher rate than vice versa . Higher density of the aphid or older juvenile stage of the IG-prey (22% of fourth instars vs. 74% of eggs and second instars) reduces the likelihood of predation. (4) To our knowledge, IGP between C. septempunctata and H. variegata was investigated for the first time. Results represent a baseline, necessary to predict the likelihood of IGP occurrence in the field.(1) Intraguild predation (IGP) can occur among aphidophagous predators thus reducing their effectiveness in controlling crop pests. Among ladybirds,  Generalist predators, including coccinellids, are very effective in pest suppression . In factAphis gossypii Glov. has a worldwide distribution and is a major economically important pest of Cucurbitaceae, including melon [A. gossypii to develop resistance to insecticides [A. gossypii infestations [Coccinella septempunctata L. and Hippodamia variegata (Goeze), may provide an efficient control over the population of this pest. However, before considering these natural enemies for conservation biological control purposes, it is necessary to determine their likelihood to establish intraguild interactions, with unpredictable consequences for disruption of aphid biological control . Theiewed by ). In parC. septempunctata and H. variegata adults collected from melon crop field in Central Italy and reared on an ad libitum diet of Aphis fabae Scopoli. Aphis gossypii winged females were collected from the field and populations were established in the laboratory on Hibiscus syriacus L. plants.Coccinellid cultures were established from C. septempunctata or H. variegata (within 8 h after ecdysis) were isolated in 15 mL glass test tubes (1.5 cm diameter), containing a strip of filter paper, closed with cotton wool and arranged horizontally. Larvae to be screened as IG-predator were starved for 12 h (water provided on cotton wool) to induce a constant level of hunger [i.e., H. variegata if the starved larva was C. septempunctata and vice versa) with, respectively, 0, 5, 10, 25, 50, 100 or 200 A. gossypii individuals. Filter papers were moistened in order to provide a good environment for larvae and to avoid insects from hiding below the paper. Whether or not each starved larva completely ate the IG-prey item was recorded after six hours. The larvae were further allowed to feed for additional 18 h to ensure they were able to entirely consume during this interval the amount of aphids provided. The experiment was replicated seven times for each combination of IG-predator, IG-prey and aphid density, with a total of 294 observations. Insect rearing and experiments were performed in a controlled environmental chamber at 25 ± 1 °C, 70% ± 5% RH, and 14 h L:10 h D photoperiod.Fourth-instars of either f hunger . SubsequC. septempunctata to eggs at different aphid densities. To identify the minimum adequate model, we used backward elimination, starting with a model that included the IG-predator species, initial aphid density and IG-prey stages and all the interactions between variables. Model selection was performed based on penalized likelihood ratio test [Logistic regression was performed to analyze the relationship between IGP frequency and the variables: IG-predator species, IG-prey stage, initial aphid density, and the interactions between them. To account for data separation, logistic regression models were fitted by applying Firth’s correction ,30,31, wtio test , with patio test .The best-fitted model retained the principal effect of the IG-predator species, the IG-prey juvenile stage and the aphid density, without any interactions between them .C. septempunctata (H. variegata (vs. 41% of larvae), with second-instars , both in petri arena [et al. [Coccinella transversoguttata Brown and Hippodamia convergens Guérin-Méneville, dominant native species in agricultural fields in Eastern Washington and Northern Idaho, were often the prey of C. septempunctata.Our results revealed that iewed by ). Coccinri arena  and in sri arena . Snyder, [et al. , under lC. septempunctata upon H. variegata larvae could be explained by the higher dimension of larval instars of the former species, although other behavioral aspects should be studied , while H. variegata was predominant on cotton (in June–July). In addition to voltinism, the abundance of the two species could be explained for example by their fecundity . Asybehavior  or by thbehavior ,39. In tbehavior  and furtvent IGP . Moreovee season ,43 by lae season ,45. For , et al.  revealedpunctata ), and bypunctata . When colay eggs . Moreovee leaves . Finallye leaves .Harmonia axyridis . This ladybird acted as asymmetric IG-predator under laboratory conditions [C. septempunctata over H. variegata in small arenas might be predictive of field situations remains to be elucidated. In addition, our results suggest that IGP could be relevant also at higher aphid density. Recently it has been demonstrated that IGP among coccinellids could be common in nature even at high aphid densities [H. variegata has been proposed as a candidate for augmentative programs against A. gossypii [C. septempunctata populations wherever the two ladybirds co-occur. Conservation approaches might consider the seasonal movement of C. septempunctata from arable to vegetable crops [C. septempunctata adults in the vegetable early after the maturation of the cereal in June [The useful and predictive potential of two-dimensional arena experiments has been demonstrated recently for another species, nditions ,55,56,57nditions ,61,62,63gossypii ,65, our le crops . In this in June  and thisC. septempunctata and H. variegata when they share a common prey, the aphid A. gossypii. We are presently conducting field investigations on the predator guild that regulates A. gossypii populations in melon crop and we are developing molecular gut content analyses on field-collected individuals. In this respect, our results would represent a baseline to predict the likelihood of predation in the field and investigate trophic relationships in coccinellid assemblages and their role in pest suppression.In conclusion, our study has value in evaluating the potential of IGP between


Its molecular function has been hypothesized to be related to anti-apoptosis and the proliferation of immune cells. The Trojan gene has been localized onto the Z sex chromosome. The adjacent two genes also show significant homology to Trojan, suggesting the existence of a novel gene/protein family. Here, we characterize this Trojan family, identify homologues in other species and predict evolutionary constraints on these genes. The two Trojan-related proteins in chicken were predicted as a receptor-type tyrosine phosphatase and a transmembrane protein, bearing a cytoplasmic immuno-receptor tyrosine-based activation motif. We identified the Trojan gene family in ten other bird species and found related genes in three reptiles and a fish species. The phylogenetic analysis of the homologues revealed a gradual diversification among the family members. Evolutionary analyzes of the avian genes predicted that the extracellular regions of the proteins have been subjected to positive selection. Such selection was possibly a response to evolving interacting partners or to pathogen challenges. We also observed an almost complete lack of intracellular positively selected sites, suggesting a conserved signaling mechanism of the molecules. Therefore, the contrasting patterns of selection likely correlate with the interaction and signaling potential of the molecules. The immune system protects an individual from pathogens in the surrounding environment. Driven by a constant need to adapt to novel pathogen challenges, genes of the immune system are often forced to evolve faster compared to other genes . As a reIn jawed vertebrates, the immune system can be divided into innate, which presents the first line of host defense and adaptive, which provides a more sophisticated means of fighting pathogens ,4. PositGallus gallus) protein from an embryonic day 13 (E13) thymus cDNA library. The molecule is a leukocyte-specific, cell surface protein that we named "Trojan" and characterized previously [The molecular tools of host defense involve a variety of proteins many of which are already known, while others are yet to be discovered. Aiming to identify novel proteins related to the immune system, we cloned a previously unknown chicken  of about 1.5 Kb. It translates to a 494 amino acids long, type I transmembrane protein that is likely to be glycosylated. The extracellular part of Trojan is predicted to have a signal peptide, followed by a complement control protein (CCP) domain and a pair of fibronectin type III (FN3) domains. The cytoplasmic tail of Trojan is short and has a region of four positively charged amino acids and two putative serine phosphorylation sites.As described previously, Trojan nucleotide sequence was mapped to the Z sex chromosome of the chicken genome . HoweverIn this paper, we present a detailed analysis of the Trojan gene family in chicken and other avian and non-avian species. The recent advances in the chicken genome assembly and gene annotations , allowed us to identify the proteins coded by the two Trojan-related genes. With the rapid accumulation of genomic sequence data from numerous species, we also found homologous sequences in ten other avian species, as well as three reptiles and West Indian ocean coalecanth fish. Since the majority of these genome assemblies were in the form of separate scaffolds, we performed manual scaffold assembly and gene modeling. Obtaining the predicted Trojan-like genes allowed us to perform phylogenetic analyzes of the family members and determine the pattern of evolutionary selection they have been subjected to. We found strong evidence of positive Darwinian selection mostly in the extracellular domain, while other parts of the proteins appeared to be under purifying negative selection. These contrasting evolutionary patterns likely correlate with the role of the protein domains and cytoplasmic tails.Mystran gene is annotated to have 26 exons, stretching over 26,5 Kb of the genomic positive strand sequence. It is followed by the 5,4 Kb gene of Trojan, which has 10 exons and resides on the opposite strand. Thracian is also found on the negative strand upstream from Trojan, has 10 exons and spans a ∼6,7 Kb genomic region. The total genomic range of the family covers about 36 Kb and is bordered by genes RUSC2 (RUN and SH3 domain containing 2) and TESK1 (testis-specific kinase 1). Detailed coordinates of the genes and their accession numbers can be found in the Materials and Methods section.The Trojan gene sequence is found on chicken chromosome Z in the NCBI  genome database. The neighboring two genes code for putative proteins bearing significant homology to Trojan and to each other . In the in silico analyzes using the RNAseq data present in the Ensembl database  . One SFMTrojan is 494 amino acids long, with a glycosylated extracellular region that bears a CCP domain followed by two FN3 domains . Its shoThracian is predicted to be a 493 amino acid long protein, that has pairs of CCP and FN3 domains within its glycosylated extracellular part. The cytoplasmic tail has three SFM : an immuWe also predicted intrinsically disordered (ID) region binding sites in all three family members. They were found almost exclusively between, or at the border of the identified domains . For MysMystran, Trojan or Thracian from ten other avian species , Corvus brachyrhynchos (american crow), Cuculus canorus (common cuckoo), Falco peregrinus , Ficedula albicollis (collared flycatcher), Geospiza fortis (medium ground finch), Meleagris gallopavo (wild turkey), Melopsittacus undulatus , Opisthocomus hoazin  and Taeniopygia guttata (zebra finch). For many species, we found the genes covering more than one scaffold, leaving the sequence split and sometimes incomplete. When required, we manually joined scaffolds using the orientation of the genes in chicken as a reference to direct the assembly. We then used chicken Mystran, Trojan and Thracian to model the gene homologues from these species. Overall, the homologous genes appeared to have the same positions and orientations as in the chicken: Mystran followed by Trojan and Thracian on the opposite strand  that code for a putative protein phosphatase and a transmembrane protein. Using their deduced protein sequences, we modeled homologous genes in two reptilian species—Chelonia mydas (green sea turtle), Chrysemys picta (painted turtle) and one fish species—Latimeria chalumnae   or Danio rerio (zebra fish).We performed further sequence similarity searches and identified gene regions bearing homology to the Trojan family in non-avian species . First, lecanth) . Our seaRUSC2 and TESK1. Among them were Mus musculus (mouse) and Homo sapiens (human), but no trace of Mystran, Trojan or Thracian genes was found.Searches against mammalian sequences databases did not return hits with a significant identity score. We also searched the genomes of mammalian species, for any evidence of the Trojan family between genes To characterize the proteins encoded in the modeled genes, we predicted their topology organization. Avian Mystran, Trojan and Thracian homologues showed strong resemblance to their chicken counterparts . In someAll proteins with modeled cytoplasmic tails had a variety of intracellular SFM, but covering them all is beyond the scope of this paper. However, it is worth noting that the SFM found in chicken were also predicted in other species .Mystrans showed the highest overall identity, due to their conserved cytoplasmic tails but had a high diversity within their extracellular regions  Figs. ThA. carolinensis were predicted to code for proteins with the same domain types as in chicken  gene tree. The family members identified from avian species were analyzed along with the related members from reptiles and fish. After testing several substitution models in Phylogenetic estimation using ML (PhyML), we selected DCMut to construct the tree  from galloanseres species. Their atypical position in the tree is likely an effect of gene conversions between family members (see below). Mystran from A. plathyrhynchos, however, was clustered with the rest of the phosphatases. The rest of the Trojans formed another major group (63%) with Thracian from M. undulatus intertwined. It probably clustered there due to its incomplete genomic sequence, resulting in an incorrectly predicted peptide. The third major group was formed by the Thracian orthologues (98%).Mystrans, Trojans and Thracians from avian species formed three major and one minor cluster . Almost all Mystrans were clustered together (87%), with the exception of those from C. canorus is likely a result of gene duplication. Using the CODEML program from the Phylogenetic Analysis using Maximum Likelihood (PAML) suite, we estimated the duplication event to have occurred around 44.3 to 46.2 MYa. The estimates were calculated under local or global clock models of nucleotide substitution, calibrated with dates from the fossil record. The two Trojan genes formed their own minor subgroup (100%), within the clustered Trojans.The identification of a second Trojan gene in Mystran and Trojan of G. gallus, which covers almost 350 extracellular amino acids. The second largest fragment was between the two Trojan genes from C. canorus, and accounted for about 300 amino acids. Gene conversions detected using default program settings are listed in We then investigated all avian species for gene conversion events between their Trojan-like genes, using the GENECONV program. Focusing on silent sites only, we detected gene conversions in 6 species, listed in Mystran, Trojan and Thracian for evidence of positive evolutionary selection, often observed for immune-related genes. Each set of genes was analyzed by the CODEML program from the PAML suite. We compared model M8A (ω ratio varies between sites according to a beta distribution and ωs = 1 is added to the beta distribution) versus M8 (adds a discrete class to the beta distribution ωs > 1) to test the hypothesis of positive selection , Trojan  and Thracian . The bordering genes are RUSC2 (GeneID: 431657) and TESK1 (GeneID: 429878). The CDS and amino acid sequences of Mystran (RefSeq: XM_003642970.2) and Thracian (RefSeq: XM_003642971.2) were downloaded from the database for the further analyzes. For Trojan, the database annotated two transcriptional variants: Trojan-X1 (RefSeq: XM_003642914.2) and Trojan-X2 (RefSeq: XM_004937133.1). Using the online bl2seq tool http://blast.ncbi.nlm.nih.gov/) to compare the two sequences, Trojan-X1 was found to be 99% identical to the Trojan clone reported previously [The Trojan family was identified from the chicken genome database annotations (v4.0) at the NCBI on chromosome Z: http://www.ensembl.org/) genome browser (release 75 – February 2014) was searched with the NCBI gene coordinates of Mystran (chicken Z: 8862119..8883723), Trojan (chicken Z: 8883937..8889333) and Thracian (chicken Z: 8891672..8898353). The following RNASeq alignments were selected: brain, breast, cerebellum, fibroblasts, embryo, heart, kidney, liver, macrophages, testes, somites. Their read values were plotted using Gnumeric spreadsheet (https://projects.gnome.org/gnumeric/).The Ensembl (http://genome.ucsc.edu/) and EBI (European Bioinformatics Institute: http://www.ebi.ac.uk/). The genomic sequences to which we found similarity hits were collected as chromosomal regions or whole scaffolds, depending on the level of database completion.The Mystran, Trojan and Thracian amino acid sequences from chicken were used in BLASTP and BLAT searches against the genomic/translated databases at NCBI, UCSC , driven by UniPro Ugene [http://www.ebi.ac.uk/Tools/psa/genewise/), with the following settings: global mode, modeled splice sites, synchronous model, algorithm 623.The CDS of chicken Mystran, Trojan and Thracian were aligned to the collected avian genomic sequences using Spidey (ro Ugene . This prro Ugene  (http://A. carolinensis (http://genes.mit.edu/GENSCAN.html). This predicted two lizard genes homologous to the Trojan family from the regions. Their deduced amino acid sequences were run in a second similarity search that gave homology hits to genomic sequences of other non-avian species. We then modeled these other non-avian genes after the proteins from A. carolinensis, using the same strategy as for the avian species. GeneWise settings were the same as above, except for C. mydas where a flat model was used.Scaffold GL343585.1 from linensis  that shoThe exon/intron organization of the modeled genes was determined by aligning their CDS to the genomic sequences. Graphical representation of the genes and scaffold assemblies was rendered by the GenomeTools software package .RUSC2 and TESK1. Among the species searched were Homo sapiens (RefSeq: NC_000009.11) and Mus musculus (RefSeq: NC_000070.6).We created HMM3 (hidden Markov models) profiles from the sequences of Mystrans, Trojans and Thracians by HMMER3, included in UniPro Ugene. We performed searches in several mammalian genomes between the genes MSA of the family members CDS were generated for each species by Clustal Omega , driven http://www.bork.embl.de/pal2nal/). The alignments were then analyzed for recombination by GENECONV, considering only silent-site polymorphisms (option: /r).For a pair of CDS, amino-acid polymorphisms may have arisen as a result of strong evolutionary selection, becoming clustered in the alignment. This would artificially elevate the significance of fragments detected elsewhere by GENECONV. For this, the program provides the option to focus on silent polymorphic sites only and examine the synonymous changes within codons. Given the positive evolutionary selection identified by our other experiments we decided to use this approach, too. The avian family members CDS were codon aligned following their translated MSA, by PAL2NAL (http://whttp://www.timetree.org/). The duplication time of the two Trojan genes from C. canorus was then estimated by CODEML, part of the PAML suite [A tree based on the most recent avian phylogeny  was consML suite . Global http://smart.embl-heidelberg.de/) database in normal mode with detection for outlier homologues, PFAM domains and signal peptides. Putative serine, tyrosine and threonine phosphorylation sites were predicted with NetPhos (http://www.cbs.dtu.dk/services/NetPhos/). N-glycosylation sites were predicted by NetNGlyc (http://www.cbs.dtu.dk/services/NetNGlyc/). Searches for short functional motifs were performed at ELM (Eucaryotin Linear Motif: http://elm.eu.org/) [http://pfam.sanger.ac.uk/generate_graphic/), while cytoplasmic tails were drawn by PROTTER (http://wlab.ethz.ch/protter/).Domain organization of the proteins was predicted using the SMART (simple modular architecture research tool: eu.org/) . SchematPairwise alignments between the chicken family members were performed by Jalview  with defhttp://www-nmr.cabm.rutgers.edu/bioinformatics/disorder/), which utilizes a vast variety of disorder prediction tools. Potential protein binding regions were identified by ANCHOR [http://anchor.enzim.hu/).Disordered regions in chicken Mystran, Thracian and Trojan were predicted at the Dismeta server  versus M8 . The tests were compared (2ΔL) in Gnumeric and the chidist formula  was used to calculate their likelihood estimates . The postmean ω values estimated by Bayes Empirical Bayes (BEB) of the selected model were plotted in Gnumreic using the amino acid numbering of chicken Mystran, Thracian and Trojan.We created separate codon alignments of avian Mystran, Thracian and Trojan CDS by PAL2NAL. Alignments were based on the protein MSA generated earlier and were independent for each gene set. We then analyzed the codon alignments in conjunction with trees based on the latest avian phylogeny  by the CMYS_ANAPL, TRO_FALPE and THR_FICAL from the Mystran, Trojan and Thracian gene sets, respectively. We also performed two extra analyzes of Trojan, excluding TRO2_CUCCA—a sequence bearing a gene conversion fragment with TRO1_CUCCA. Analysis was done with or without TRO_FALPE present in the MSA.Since we observed a certain level of heterogeneity of the MSA used above, we did alternative runs of CODEML, with the most divergent sequences omitted. These were http://caps.tcd.ie/) [We used CAPS2   with deftcd.ie/) , while iS1 FigA. platyrhynchos (ANAPL), C. brachyrhynchos (CORBR), C. canorus (CUCCA), F. peregrinus (FALPE), F. albicollis (FICAL), G. fortis (GEOFO), M. gallopavo (MELGA), M. undulatus (MELUN), O. hoazin (OPPHO), T. guttata (TAEGU). B) Non avian species: A. carolinensis (ANOCA), C. mydas (CHEMY), C. picta (CHRPI), L. chalumnae (LATCH).Regions used for gene prediction are indicated as blank boxes showing the gene direction. Exon organization of the predicted genes is presented as filled fragments. Scaffolds are shown as light-gray pointed boxes, indicating their assembly and orientation. Gray triangles on scaffolds’ sides indicate preceding, successive and connecting sequence segments. Gene names combine the respective homologue: Mystran (MYS), Trojan (TRO), Thracian (THR), Protein phosphatase (PP) or Transmembrane protein (TP) and the corresponding species abbreviation. A) Avian species: (PDF)Click here for additional data file.S2 FigA. platyrhynchos (ANAPL), C. brachyrhynchos (CORBR), C. canorus (CUCCA), F. peregrinus (FALPE), F. albicollis (FICAL), G. fortis (GEOFO), M. gallopavo (MELGA), M. undulatus (MELUN), O. hoazin (OPPHO), T. guttata (TAEGU). A) Mystrans; B) Mystrans, excluding cytoplasmic tails; C) Trojans; D) Thracians;Amino acid sequence identity is shown as a percentage. Names combine Mystran (MYS), Trojan (TRO) or Thracian (THR) and the corresponding species abbreviation. Species: (PDF)Click here for additional data file.S3 FigA. platyrhynchos (ANAPL), C. brachyrhynchos (CORBR), C. canorus (CUCCA), F. peregrinus (FALPE), F. albicollis (FICAL), G. fortis (GEOFO), M. gallopavo (MELGA), M. undulatus (MELUN), O. hoazin (OPPHO), T. guttata (TAEGU). Non-avian species: A. carolinensis (ANOCA), C. mydas (CHEMY), C. picta (CHRPI), L. chalumnae (LATCH). A) MSA of avian Mystrans; B) MSA of avian Trojans; C) MSA of avian Thracians; D) MSA of non-avian Protein phosphatases; E) MSA of non-avian Transmembrane proteins.Amino acid similarity is indicated by a color saturation scale. The domain organization of the first sequence is shown on top as a reference; SP (gray): signal peptide, CCP (orange): complement control protein domain, FN3 (green): Fibronectin type III domain, PTP (light blue): protein tyrosine phosphatase domain, TM (blue): transmembrane region. Names combine Mystran (MYS), Trojan (TRO), Thracian (THR), Protein phosphatase (PP) or Transmembrane protein (TP) and the corresponding species abbreviation. Avian species: (PDF)Click here for additional data file.S4 FigMystran, Trojan and Thracian analyzed without the sequences that appeared too divergent . B) Trojan analyzed with TRO2_CUCCA omitted. C) Trojan analyzed with TRO2_CUCCA and TRO_FALPE omitted.Amino acid postmean ω values are mapped onto the protein topologies. Non selected sites are shown in blue, selected sites with probability below 90% are shown in light blue and selected sites with probability greater than 90% are shown in orange. Sites with probability greater than 95% and 99% are indicated by one or two red dots, respectively. Domain types and other topology properties are shown in the legend. The Mystran CCP domain is shown in gray scale, as it was predicted slightly below threshold, but had the expected position. A) (PDF)Click here for additional data file.S1 TableA. platyrhynchos (ANAPL), C. brachyrhynchos (CORBR), C. canorus (CUCCA), F. peregrinus (FALPE), F. albicollis (FICAL), G. fortis (GEOFO), M. gallopavo (MELGA), M. undulatus (MELUN), O. hoazin (OPPHO), T. guttata (TAEGU).The gene converted fragments between sequence pairs (Sequence I and Sequence II) are given with respect to their unaligned offsets and lengths within each sequence. “BC KA P-values”: Bonferroni-corrected KA . Names combine Mystran (MYS), Trojan (TRO) or Thracian (THR) and the corresponding species abbreviation. Species: (PDF)Click here for additional data file.S2 TableAmino acids from chicken Mystran, Trojan and Thracian with Bayesian posterior probabilities to belong to site-class under positive selection are listed. Probability: >90%, >95% (*) or >99%(**), as inferred by Bayes-Empirical-Bayes (BEB).(PDF)Click here for additional data file.


Grallator type (sensus lato), thus representing relatively small trackmakers. Such ichnofaunas supplement the skeletal record from this unit that lacks theropods thus far, proving a larger diversity of dinosaur faunas in that region. Sauropods are represented by medium to large sized and narrow and wide-gauge groups, respectively. The latter correspond with earlier discoveries of titanosauriform skeletons in the same unit. Previous records of ornithischian tracks cannot be positively confirmed. Purported occurrences are re-evaluated here, the trackways and imprints, except of a single possible specimen, re-assigned to theropods. Palecologically the Tuchengzi ichnofauna is characteristic of semi-arid fluvio-lacustrine inland basins with Upper Jurassic-Lower Cretaceous deposits in northern China that all show assemblages with abundant theropod and sauropod tracks and minor components of ornithopod, pterosaur and bird tracks.New reports of dinosaur tracksites in the Tuchengzi Formation in the newly established Yanqing Global Geopark, Beijing, China, support previous inferences that the track assemblages from this formation are saurischian-dominated. More specifically, the assemblages appear theropod-dominated, with the majority of well-preserved tracks conforming to the  The Middle-Upper Jurassic Yanliao Biota and the Lower Cretaceous Jehol Biota in northeastern China have been the subject of intense palaeontology research for over a decade due to exceptionally well-preserved fossils such as feathered dinosaurs, angiosperms, primitive mammals, etc. , 2.There is a large temporal gap between the record of the Yanliao Biota and the Jehol Biota . This trChaoyangsaurus youngi and Xuanhuaceratops niei ); The sedimentary environment of the Tuchengzi Formation is fluvial-lacustrine . CharactMonocraterion isp. and Scoyenia isp. , . T. T38]. Tv = 0.25g0.5. SL1.67. h-1.17, where g = gravitational acceleration in m/sec, SL = stride length, and h = hip height—estimated as 4.5 and 4.9 times foot length, using the ratio for small and large theropods proposed by Thulborn ).).v = 0.2At the QJDILL site , a trackAccording to Olsen , Weems , and LocCarrano and Wilson  provide The theropod tracks at Yanqing tracksite can be divided into two morphologies: Morphotype A and B; the former is represented by three different track sizes, the latter by two different track sizes.Morphotype A Figs , Table 1These tracks are divided into small (length 5.5 cm), medium-sized (length ~11–20 cm) and large (length 26–30 cm) tridactyl tracks, with an average TL/TW ratio of 1.5. Morphotype A is characterized by weak to moderate mesaxony , which is typical for footprints of the ichno- or morphofamily Eubrontidae Lull 1904 .Eubrontes, Anchisauripus and Grallator from the Late Triassic-Early Jurassic, but it commonly has a wider divarication of digits II–IV (average 58°) compared with Eubrontes (10°–40°) . Y. Y12]. Yr ssatoi . MatsukaAnchisauripus, however, it is insufficient for a confident identification. The AT is 1.00 (IVPP VC 15815 G) and 0.63 (IVPP VC 15815 B and F). Presumably it represents two different morphologies of theropod tracks, as inferred for the Qianjiadian tracksite, and possibly two different ichnospecies . M. M17]. Mang site : see Locang site  for furtTherangospodus isp., and one large-sized track to Megalosauripus isp. The Therangospodus assignment was based on the morphology: medium-sized (averaging 28 cm in length), elongate, asymmetric theropod tracks with coalesced, elongate, oval digital pads that are not subdivided into discrete phalangeal pads, and with an AT of 0.55 . H. HTheranTherangospodus isp., and fifteen tracks with a low TL/TW ratio (1.2–1.3) to cf. Ornithopoda. For the Shangyi theropod tracks, the mean TL/TW ratio is 1.7 (N = 38), and the AT is 0.69 (N = 36), which is close to Qianjiadian Morphotype B. The Shangyi theropod tracks also lack a developed metatarsophalangeal region. This is similar to the condition seen in the Qianjiadian Morphotype B. However, most Shangyi theropod tracks are stouter than Qianjiadian Morphotype B, which probably suggests the morphological difference in the pes of Grallator type trackmakers.Shangyi tracksite, Zhangjiakou City, Hebei Province . Xing etGrallator isp. were discovered in the Yixian Formation of the Sihetun tracksite  is  is Brontntopodus  or to nantopodus . GloballBrontopodus , , w, wBrontoauropods . It suggauropods .Deltapodus isp. When Xing et al. , . Bo. Bo6]. BGrallator–Eubrontes plexus and well known from Lower Jurassic strata. The presence of ornithopods is possibly indicated by isolated occurrences but remains a rather scarce element. Theropod trackmakers were small to medium-sized individuals that probably belonged to different taxonomic groups of coelurosaurs including functionally didactyl dromaeosaurs. The sauropod trackways belong to medium and large-sized animals that represent both narrow-gauge and wide forms, the latter being attributed to titanosauriforms whose skeletons are known from the same unit. The track-record from the Tuchengzi Formation is important because it supplements the scarce bone record that thus far lacked theropods. Stratigraphically it fills a gap in the dinosaur record representing strata that are slightly older than the famous Jehol Biota from Liaoning Province.Theropod and sauropod trackways from the Jurassic-Cretaceous boundary Tuchengzi Formation support the former view of saurischian dominated assemblages in East Asia during this time interval . This mi


Nilaparvata lugens) is one of the most destructive rice plant pests in Asia. N. lugens causes extensive damage to rice by sucking rice phloem sap, which results in hopper burn (complete death of the rice plants). Despite its importance, little is known about the digestion, development and defense mechanisms of this hemimetabolous insect pest. In this study, we aim to identify the serine protease (SP) and serine protease homolog (SPH) genes, which form a large family in eukaryotes, due to the potential for multiple physiological roles. Having a fully sequenced genome for N. lugens allows us to perform in-depth analysis of the gene structures, reveal the evolutionary relationships and predict the physiological functions of SP genes.The brown planthopper  and SPH (25) genes in N. lugens SP and SPH genes, which give insights into clarifying the potentially functional roles of these genes in the biological processes including development, digestion, reproduction and immunity.Our findings provide comprehensive gene sequence resources and expression profiles of the The online version of this article (doi:10.1186/1471-2164-15-507) contains supplementary material, which is available to authorized users. Nilaparvata lugens Stål (Hemiptera: Delphacidae) is a typical phloem sap feeder and is considered to be one of the most devastating pests for rice throughout Asia. It sucks sap from rice phloem and transmits plant viruses, which can lead to a dramatic reduction in yield and cause significant economic loss[N. lugens management is the use of chemical insecticides; however, the overuse of insecticides has caused planthopper resurgence and environmental risks[Bacillus thuringiensis) toxins exist.The brown planthopper mic loss. The firal risks. In receal risks, 4. RNAial risks. The taral risks. CurrentSerine proteases (SPs) in the chymotrypsin (S1) family constitute one of the largest gene families of multifunctional enzymes that play important roles in various physiological processes, including digestion, development and the immune response. They arDrosophila melanogaster[Apis mellifera[Bombyx mori[N. lugens genome and obtained gene annotation information (Zhejiang University N. lugens genome project team). N. lugens genome is the first characterized genome of a monophagous sap-sucking arthropod herbivore. The N. lugens genomic information allows the global analysis of SP and SPH genes in this insect species. In our previous study, we performed transcriptome sequencing and gene expression analysis using the next-generation high-throughput Illumina technology, which provided the detailed gene expression information regarding the developmental stages, wing dimorphism, sex differences, immune responses and tissue specificity in N. lugens[N. lugens genome sequence coupled with the available transcriptome datasets generated the comprehensive information of SP and SPH genes, which presents an overview of the gene structures, evolutionary relationships and the expression specificity of these genes. These data could be useful in identifying the potential target genes for insect pest management.Genome-wide analyses of SP and SPH genes have been performed in Diptera nogaster, Hymenopellifera and Lepibyx mori. However. lugens, 12, 13.N. lugens strain was originally obtained from local rice fields in the Huajiachi Campus of Zhejiang University, Hangzhou, China. The insects employed in this study were the offspring of a single female and were reared at 26 ± 0.5°C with 50 ± 5% humidity on fresh rice seedlings under a 16:8 hour light:dark photoperiod as previously described[The escribed, 13.N. lugens 5th instar nymphs were immunized with a microinjection of heat-killed Escherichia coli K12 or Bacillus subtilis  using the FemtoJet Microinjection System . Nymphs were collected at different time points  after infection for the bacteria-induced expression experiment as previously described[escribed.N. lugens were dissected under a Leica S8AP0 stereomicroscope . The fat body, midgut, salivary gland, male reproductive system, ovary and carcass (the remaining body) were isolated and quickly washed in cold diethylpyrocarbonate (DEPC)-treated NaCl/Pi solution  and immediately frozen at -80°C as previously described[N. lugens individuals were dissected and used for RNA extractions.For studies of tissue-specific expression, the N. lugens nymphs or adults using RNAiso plus based on the manufacturer’s protocol . As previously described[N. lugens transcriptomic sequences (accession number: SRX023419) that have been submitted to the Sequence Read Archive (SRA) database (http://www.ncbi.nlm.nih.gov/sra), as shown in an Additional fileN. lugens 18 s rRNA gene as an internal control (GenBank accession no. JN662398) was analyzed using the following primers: 5′-CGCTACTACCGATTGAA-3′ (sense) and 5′-GGAAACCTTGTTACGACTT -3′ (antisense). The use of reference genes as internal controls is the most appropriate normalization strategy for achieving the reliable qRT-PCR assay[N. lugens 18 s rRNA gene has been validated for their stably expressions in N. lugens tissues, developmental stage and immune-induced individuals[N. lugens 18 s rRNA. An NTC sample was run to detect any contamination and to determine the degree of dimer formation. The relative quantitative method  was used to evaluate the relative differences in the transcript levels[t = the Ct of SP gene - the Ct of the 18 s rRNA gene.Total RNA was extracted from escribed, 13, theCR assay. In our ividuals, 13. In t levels. Namely,N. lugens genome sequence based on the KEGG , Swissprot  and Trembl  annotations. Predicted coding sequences of SP and SPH genes were used as reference sequences to match the N. lugens transcriptomic sequences using the tBLASTX algorithm with a cut-off E-value of 10-10. The deduced protein domains and signal peptides were determined by using Pfam (http://pfam.xfam.org/), SMART (http://smart.embl.de/) and InterProScan (http://www.ebi.ac.uk/Tools/pfa/iprscan/) as previously described[The SP and SPH genes were searched against the escribed.N. lugens development and sex genes in our previous study[Owing to the accomplishment of the gene expression profiles of the differences between the us study, we are Apis mellifera ; Aedes aegypti (http://www.vectorbase.org/GetData/Downloads/); Acyrthosiphon pisum (http://www.inra.fr/aphidbase/); Drosophila melanogaster (ftp.flybase.org/genomes/Drosophila_melanogaster/dmel_r5.27_FB2010_04/);Trypsin-like genes were identified in the available genomes of the following insect species: Anopheles gambiae (ftp.vectorbase.org/public_data/organism_data/aaegypti/Geneset/); Tribolium castaneum (ftp://ftp.ncbi.nih.gov/genomes/Tribolium_castaneum); Culex quinquefasciatus (http://www.vectorbase.org/GetData/Downloads/); and Bombyx mori (ftp://silkdb.org/pub/release_2.0/), based on the KEGG (ftp://ftp.uniprot.org/pub/databases/uniprot/) and Swissprot (ftp://ftp.uniprot.org/pub/databases/uniprot/) annotations as previously described[escribed.N. lugens SPs and SPHs were aligned with the best-matched homologs of other insect species using the ClustalX program[http://www.megasoftware.net/)[The functional serine protease domains of the  program. The phyre.net/). HomologN. lugens genome sequence based on the KEGG, Swissprot and Trembl annotations, which were validated using the tBLASTX algorithm with a cut-off E-value of 10-10 , respectively , Coleoptera  and Lepidoptera , but not in Hymenoptera or Hemiptera. Apis mellifera has few trypsin-like genes (40), which could reflect its colonial feeding strategy that alleviates the pressure on the digestion system for individual insects. Among all genome-sequenced insects, two plant phloem sap-feeding Hemiptera insects, N. lugens (31) and Acyrthosiphon pisum (34), possess the least number of trypsin-like genes, accounting for only about one-fifth of that in Diptera insects. N. lugens and A. pisum have evolved to survive on a nutritionally imbalanced diet of phloem sap, i.e., simple sugars and amino acids. This imbalanced diet is compensated by the intracellular symbionts, which provide essential nutritional components that are absent in phloem. It is likely that the abundant digestion enzymes are not necessary when these insects utilize the phloem sap as their nutrition source. Therefore, it might be a reasonable strategy to reduce the number of digestive proteases in these insect species when compared to the necessity of abundant trypsin-like serine proteases in leaf-feeding silkworm, the grain-feeding red flour beetle and the polyphagous dipteran insects.A comparison of the genome-available insect species revealed that the trypsin-like genes could have undergone a major expansion in Diptera . Bacteria-triggered hemolymph clotting was mediated via the coagulation cascade, which consists of three principle serine protease zymogens, namely clotting factor C, clotting factor B and a proclotting enzyme. Clotting factor C acts as a biosensor to respond to lipopolysaccharide (LPS), a major cell wall component of gram-negative bacteria, and to activate clotting factor B, which in turn converts the proclotting enzyme to a clotting enzyme, which leads to hemolymph clotting. In contrast to non-insect arthropods, little is known about the molecular basis of hemolymph coagulation in insects.Serine proteases play very important roles in the innate immune responses of invertebrate animals. These proteases are mainly involved in the processes of melanin formation and hemolymph coagulation against the infections of foreign pathogens. Melanin formation is mediated by the prophenoloxidase (proPO) activating cascade, which has been extensively studied in many insect species–21. Seve cascade, 22. TheN. lugens genome. This gene features two complement control protein (CCP) domains. The CCP module, also known as the SUSHI domain, contains approximately 60 amino acid residues and has been identified in complement factors in the mammalian blood coagulation system. The N. lugens clotting factor C like gene consists of a signal peptide, three consecutive low-density lipoprotein receptor class A domains (LDLa), two CCP domains and a serine protease domain with the catalytic triad . These results indicate that proclotting enzyme 1 quickly responded to the invasion of foreign bacteria and may have a role in the host defense response. Despite the functions of the putative N. lugens clotting factors are not understood, the identification of these candidate genes makes it worthwhile to carry out further functional analyses because they provide us with a more comprehensive grasp and a better understanding of insect immune mechanisms.In this study, we identified a limulus clotting factor C like gene in the  Figure A. We fou Figure A. Phylog Figure B. The N.ent work. In this Figure . In contInsects depend on extracellular serine protease cascades to achieve their various physiological processes.N. lugens genome revealed a series of serine protease genes that include one nudel like, one gastrulation defective (Gd), 12 snakes and 15 easters , snake and easter constitute the major components of the extracellular signal cascade in the Toll-Dorsal pathway. In insects, the best-characterized function of the Toll-Dorsal pathway is the establishment of the dorsal-ventral axis in early embryonic patterning. This pathway also contributes to other processes at later developmental stages, such as immune response, morphogenetic movements and muscle development. A searcN. lugens nudel like gene, which has an identical domain structure and significant sequence similarity to the A. pisum nudel like gene, consists of a signal peptide, four consecutive LDLa repeats at its N-terminus and a serine protease domain at the C-terminus  only contain a signal peptide and a serine protease domain but lack the clip domain. The predicted protein products of the N. lugens easter gene have approximately 300–400 amino acids and show significant sequence identities with the Harpegnathos saltator Easter protease  was exclusively expressed in 5th instar nymphs while the other (GenBank accession no. KJ512091) was observed to be specific to male adults. A transmembrane serine protease gene (GenBank accession no. KJ512109), a homolog of the A. pisum ovochymase 1 gene, showed much higher expression levels in 2nd instar nymphs than 5th instar nymphs and eggs. The other two transmembrane serine protease genes, (GenBank accession no. KJ512111 and KJ512110), which showed high sequence similarities to the Tribolium castaneum ovarian serine protease and ovochymase 2, respectively, were highly expressed in female adults or eggs, implying their potential reproduction-associated functions. The widely different expression patterns suggest that N. lugens serine proteases may have multiple functions during the developmental process. To confirm the gene expression data from high-throughput Illumina sequencing, we selected sixteen genes to analyze their development- and sex-specific expressions using qRT-PCR. As a result, the expression patterns of these genes are coincident with the expression profile  database (http://www.ncbi.nlm.nih.gov/sra). The accession number of the N. lugens transcriptomic dataset is SRX023419. The accession numbers of the N. lugens gene expression profile datasets are as follows: eggs (SRX023493), 2nd instar nymphs (SRX023492), 5th instar nymphs (SRX023494), macropterous female adults (MFA) (SRX023495), macropterous male adults (MMA) (SRX023496) and brachypterous female adults (BFA) (SRX023497). The transcript sequences of N. lugens SP and SPH genes were submitted to National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). The accession numbers of these sequences are as follows: KJ512060-KJ512142, KC355213-KC355215 and KC355219-KC355225. We also provide the nucleotide and protein sequences of SPs/SPHs as an Additional filehttp://treebase.org/treebase-web/user/submissionList.html).The supporting data in this study have been submitted to the open access repositories. The Additional file 1: Table S1: Gene-specific primers used in qRT-PCR analysis. (DOCX 25 KB)List S1. The nucleotide and deduced protein sequences of N. lugens SP and SPHs. (TXT 146 KB)Additional file 2:


To the Editor: Cysticercosis-like human infections with the tapewormTaenia crassiceps, which infects foxes as terminal hosts, have beenreported (T. martis (marten tapeworm) in awoman.The patient was a 43-year-old German woman who sought care during July 2010, after 4 daysof perceiving flashing lights in her visual field and a paracentral scotoma in her lefteye. Visual acuity in both eyes was 20/20. Examination of the left fundus revealed amobile subretinal tumor at the temporal upper retinal branch vessel with adjacentintraretinal and subhyaloid bleeding . The subTaenia solium,Echinococcus multilocularis,E. granulosus,Dirofilaria immitis, Strongyloides spp., and Toxocaracanis. Fecal testing results were negative for worm eggs. Images fromultrasonography of the liver and magnetic resonance imaging of the head wereunremarkable. The patient’s travel history included—in addition tosouthern European countries—trips to Nepal and Thailand 15 years previously. The patient reported no other symptoms at that time. Laboratory evaluation found noeosinophilia or elevation of total IgE. Serologic testing results were negative forantibodies against the following parasites: At the time of examination, the patient lived in a small village near Freiburg (imBreisgau) in southwestern Germany. She grew vegetables in the family garden, whichwas next to a forest. Her 3 children and husband did not report any health problems.For the past 6 years, the family had owned a dog, which received antiparasiticmedications on a regular basis; recent checks for intestinal parasitic infectionfound no ova.T. solium; systemic antiparasitic therapy was started. The size of the larvadiminished ; NADH dehydrogenase subunit 1; and the cytochrome-c oxidase subunit 1 but substantially lower identity with T. crassiceps(91%–97%) and T. solium (87%–89%) tapeworms.The removed cyst showed the characteristic macroscopic and histologic features of acysticercus bladder wall (T.martis tapeworm. T. martis tapeworms (cestodes) liveand produce eggs in the intestines of definitive hosts, weasels (familyMustelidae), which also includes pine martens, stone martens,polecats, badgers, wolverines, and stoats (T. martis tapeworms probably occur worldwide whereversuitable definitive and intermediate hosts are present (T. martis tapeworms (Thus, molecular methods unequivocally identified the larva as that of a T. crassiceps tapeworms were immunosuppressed (Although nearly all patients who had cysticercosis-like infections caused byT. solium tapeworm. However, the specificdiagnosis of T. martis tapeworm infection was possible only by useof molecular methods. Thus, human infections with T. martis andother animal tapeworms might occur at times but might be misdiagnosed as T.solium cysticercosis. For therapy, the rules and considerations areprobably the same as those for T. solium cysticercosis, asdescribed (The clinical and histologic appearance of the organism in this patient suggestedcysticercosis caused by a