Schizaphisgraminum, Rhopalosiphum padi, R.maidis and Sitobion avenae variedsignificantly among the five districts surveyed. The population ofS. graminum was observed more abundant inarid, R. padi in rice, S.avenae in aird and rice, and R.maidis in cotton-I zones. Aphids ended their populationdynamics on 25th March in central mixed-cropped zone and12th April in other three zones. Various species of naturalenemies, mainly Coccinella septumpunctata, C.undecimpunctata, Menochilus sexmaculata,Chrysoperla carnea, Syrphidae and parasitoid mummies wereinconsistently observed in four agro-ecological zones. The population ofC. septumpunctata, was observed moreabundant in rice zone, C. undecimpunctata andC. carnea in cotton-I and arid zones,M. sexmaculata in cotton-I and II zones,Syrphidae in cotton-I zone and parasitoid mummies in rice and arid zones. Therewere no clear relationships between aphid and the natural enemy populations. Thepresent study may serve as a baseline regarding distribution of wheat aphids andtheir natural enemies and the results provided insights for further studies onthe potential top-down  versus bottom-up (fertilization andirrigation regimes) forces in management of wheat aphids in easternPakistan.Aphids are major pests of wheat crop in Pakistan inflicting considerable economiclosses. A better knowledge of landscape scale spatial distribution of aphids andtheir natural enemies could be used to improve integrated pest managementprograms. Therefore, the present study aimed to document spatio-temporalvariations in populations of wheat aphids and their natural enemies in Pakistan.The 2-year survey study was carried out at ten experimental farms located infive districts of four contrasted agro-ecological zones of eastern Pakistan(Punjab area) i.e. District Chakwal in arid zone, Gujranwala in rice-croppedzone, Faisalabad in central mixed-cropped zone, and Khanewal and Multan incotton-cropped zone. The dominant aphid species i.e.  Triticum aestivum L.) is Pakistan’s staple diet and accountsfor 10 percent of value added in agriculture and contributes over two percent to thecountry’s GDP [Wheat  province while, Macrosiphum miscanthi, Siphamydis and Sipha elegans from northern hilly areas(temperate climate) [S. graminum and M.miscanthi, Aphis maidis has been recorded fromthe plains of the Punjab [Several species of aphids have been reported from various agro-ecological zones ofPakistan. climate) –15. In ae Punjab ,17. Pune Punjab . The pope Punjab –21. Multe Punjab , 23.Aphidius ervi, Diaeretiella rapae,A. matricariae, Aphytismelinus, Lysiphlebus fabarum [The infestation of cereal aphids can be reduced by applying various biological,cultural and chemical control strategies . Pestici fabarum –31, besi fabarum ,33. Kno fabarum –36. Temp fabarum –42. HoweTherefore, there is need to conduct a study to investigate tempo-spatial variationsin populations and seasonal dynamics of various aphid species and their naturalenemies across the four agro-ecological zones in eastern Pakistan. These findingscould serve as a baseline for using natural enemies in optimized integrated aphidmanagement strategies in plain areas of the Pakistan.th to 31st in bothgrowing seasons (2010–2011). All agronomic practices were done according tolocal recommendations.The study was conducted at ten experimental farms without use of pesticides infour agro-ecological zones of Punjab, Pakistan during two consecutive wheatgrowing seasons in 2010 and 2011 . The agrth February to 12th April during both the seasons.Three hundred and forty eight sampling points were selected in each of 2-ha plotin zigzag fashion on each observation day and completed within a week from allten locations. On each sampling point, three consecutive tillers of a wheatplant were randomly selected and visually observed for counting species ofaphids  and their natural enemies .Aphid species were morphologically identified using the identification keysdeveloped by the University of Idaho [Weekly visual observations of aphids and their natural enemies were started from8of Idaho . Only thof Idaho .Prior to analysis, data were checked for their conformance to the normaldistribution using Kolmogrov-Simirnov normality test. Per tiller populations ofaphids and natural enemies among the five locations were compared per species(or group of species) using Kruskal-Wallis tests. The means of ranks werecompared using Dunn’s post hoc test. To compare the difference in the means ofpopulation of aphids and natural enemies between the two years, T-test  was applied. The relationship between natural enemy andaphid density was tested using linear regression analysis. The data of allaphids and natural enemies species were pooled to compare the relativeproportion of natural enemies and aphids in four agro-ecological zones during2010 and 2011. XLSTAT computer software was used for all analysis .S.graminum: KW = 3.27, P = 0.51,d.f. = 4; R.padi: KW = 2.97, P = 0.56,d.f. = 4; R.maidis: KW = 3.92, P = 0.42,d.f. = 4; S.avenae: KW = 1.55, P = 0.82,d.f. = 4)  . All theectively.S. avenae, thepopulation of S. graminum, R.padi and R. maidis variedsignificantly  among the five districts . Schizaphis graminum andR. padi were the most abundant at arid andrice zones, respectively. Rhopalosiphum maidis was the mostabundant at cotton zone II whereas it was the least abundant on 22ndMarch during 2011. Sitobion avenae was the most abundant atcotton zone I on the 22nd March  among the five districts of four agro-ecological zones. TheDunn’s post hoc test showed the maximum mean of ranks for Syrphidae in cottonzone I and the minimum for arid zone. In the case of mummies, the maximum meanof ranks was recorded for rice zone and the minimum for cotton zone II , all the other five natural enemies did not differ significantly among thefive districts . The Dunn’s post hoc test showedthe maximum mean of ranks of C. septumpunctatain rice zone and the minimum in the mixed zone . There was no significant relationship between populations of theaphids and their natural enemies in both seasons (linear regressions: bothP>0.05). The relative proportion between cumulativeaphids and natural enemies population in five zones showed that the highestrelative proportion of natural enemies to aphids was observed in rice and aridzones during 2010, and rice and mix zones during 2011. The natural enemies wererelatively more abundant during 2010 than 2011  [In present study, the peak activity period of all the aphid species lasted from11n (2005)  reportedn (2005)  reportedthFebruary, peaked during last fortnight of March followed by a sharp or gradualdecline until the 12th April. Seasonal dynamic pattern of aphids variedacross latitude as several previous studies have documented slightly differenttrends in Multan and Peshawar districts. Aslam et al. (2004) [rd week of January and increased exponentially and peaked on the3rd week of March followed by a sudden decline after the4th week of March until the 1stweek of April. Similarly,Khattak et al (2007) [nd week of January and attained its peak on the first two weeks ofMarch. Another study from Peshawar suggested that aphid population started toincrease in the last week of December, remained low populations in January andpeaked in the 1st week of March [Seasonal dynamics of aphids followed almost similar pattern in both growing seasons(2010 and 2011), i.e. the population started to grow from the 15. (2004)  reportedl (2007) reportedthMarch to 1st April in 2010 while 29th March to 5thApril in 2011. Similar findings have been reported by Saleem et al. (2009) [th week of March,syrphids in the 3rd week of March, chrysopids in the 2nd weekof April and parasitoids in the 3rd week of March. The fluctuation ofnatural enemies was more abrupt than aphids in 2010 season compared to 2011 season.The populations of Syrphidae and mummies varied significantly among the fivedistricts in season 2010 while only C.septumpunctata varies significantly in season 2011. Syrphidflies are efficient predators of aphids and exponentially decrease their population[Population dynamics of natural enemies attained their peak from the 18pulation, 62. Thepulation, 64. In pulation, 65–69. pulation–73. Howepulation, behaviopulation, 37, 75,pulation. Thesefpulation.The present study for the first time established a baseline data on distribution andseasonal population dynamics of wheat aphids and their natural enemies in fouragro-ecological zones in eastern Pakistan. Further research is warranted to examine(i) the top-down vs. bottom-up forces in wheat agro-ecosystems in order to developsustainable IPM, and (ii) side effects of commonly used insecticides on the behaviorand physiology of those potential natural enemies.


Soil transmitted helminths (STH) infect >1.5 billion people. Mass drug administration (MDA) effectively reduces infection; however, there is evidence for rapid reinfection and risk of potential drug resistance. We conducted a randomized controlled trial in Bangladesh  to assess whether water, sanitation, hygiene and nutrition interventions, alone and combined, reduce STH in a setting with ongoing MDA.In 2012–2013, we randomized 720 clusters of 5551 pregnant women into water treatment, sanitation, handwashing, combined water+sanitation+handwashing (WSH), nutrition, nutrition+WSH (N+WSH) or control arms. In 2015–2016, we enrolled 7795 children, aged 2–12 years, of 4102 available women for STH follow-up and collected stool from 7187. We enumerated STH infections with Kato-Katz. We estimated intention-to-treat intervention effects on infection prevalence and intensity. Participants and field staff were not blinded; laboratory technicians and data analysts were blinded.A. lumbricoides, 9.2% for hookworm and 7.5% for T. trichiura. Most infections were low-intensity. Compared to controls, the water intervention reduced hookworm by 31% , prevalence difference [PD] = -2.83 ) but did not affect other STH. Sanitation improvements reduced T. trichiura by 29% , PD = -2.17 ), had a similar borderline effect on hookworm and no effect on A. lumbricoides. Handwashing and nutrition interventions did not reduce any STH. WSH and N+WSH reduced hookworm prevalence by 29–33% , PD = -2.63 ; N+WSH: PR = 0.67 , PD = -3.00 ) and marginally reduced A. lumbricoides. Effects on infection intensity were similar.Prevalence among controls was 36.8% for In a low-intensity infection setting with MDA, we found modest but sustained hookworm reduction from water treatment and combined WSH interventions. Impacts were more pronounced on STH species with short vs. long-term environmental survival. Our findings suggest possible waterborne transmission for hookworm. Water treatment and sanitation improvements can augment MDA to interrupt STH transmission.NCT01590095. Soil-transmitted helminths (STH) are associated with a large disease burden worldwide. Mass administration of deworming drugs for preventive chemotherapy is the cornerstone of global strategy for STH control, but treated individuals often rapidly become reinfected, and there is also concern about emerging drug resistance. Interventions to treat drinking water, wash hands at critical times and isolate human feces from the environment through improved sanitation could reduce STH transmission by reducing the spread of ova from the feces of infected individuals into the environment and subsequently to new hosts. Nutrition improvements could reduce host susceptibility to infection. Existing evidence on the effect of these interventions on STH is scarce. In a setting with ongoing mass drug administration, we assessed the effect of individual and combined water, sanitation, handwashing and nutrition interventions on STH infection in children. Approximately 2.5 years after the initiation of interventions, we found reductions in STH infection from water treatment and sanitation interventions; there was no reduction from the handwashing and nutrition interventions. While the reductions were modest in magnitude compared to cure rates achieved by deworming drugs, they indicated sustained reduction in environmental transmission. The reductions were more pronounced for STH species that do not have long-term environmental reservoirs. These findings suggest that water treatment and sanitation interventions can augment mass drug administration programs in striving toward elimination of STH. Ascaris lumbricoides (roundworm), 465 million with Trichuris trichiura (whipworm), and 439 million with Necator americanus and Ancylostoma duodenale (hookworms) [A. lumbricoides, 82% for T. trichiura and 57% for hookworm within 12 months post-treatment [Over 1.5 billion people globally are infected with soil transmitted helminths (STH), specifically 819 million with okworms) . DewormiA. lumbricoides but not T. trichiura and hookworm [A. lumbricoides, in soil [Water, sanitation and hygiene improvements could potentially complement MDA programs in reducing STH transmission . Two syshookworm . Handwashookworm . Two trihookworm ,13. It i in soil . While s in soil .A. lumbricoides but not other STH in a Kenyan trial [Combined water, sanitation and hygiene improvements targeting multiple transmission routes might achieve a larger impact by complementing the primary barrier of sanitation with the secondary barriers of water treatment and handwashing ,17. Schoan trial . A recenan trial .The effect of nutrition on STH infections is also poorly understood. Impaired immune function from nutritional deficiencies could increase host susceptibility to STH infection or exacerbate infection severity while nutritional supplements could also increase infection severity as excess nutrients are available for pathogens ,21. A syWe conducted a cluster-randomized trial  in Bangladesh to assess the impact of individual and combined water, sanitation, handwashing (WSH) and nutrition interventions on child diarrhea and growth (primary and secondary outcomes) . The triThe trial was conducted in the Gazipur, Mymensingh, Tangail and Kishoreganj districts of central rural Bangladesh, which were selected because they had low groundwater arsenic and iron  and no other water, sanitation, hygiene or nutrition programs. The majority of households in the study area relied on untreated tubewell water for drinking, and while most households had access to on-site sanitation, it was common for latrines to drain into the environment, and animal feces were also commonly observed in the compounds ,24.A. lumbricoides but have lower cure rates for T. trichiura; for hookworm, albendazole has a high cure rate while mebendazole has a modest cure rate [Since 2008, the Bangladesh Ministry of Health has implemented a school-based MDA program that provides deworming to school-aged children while pre-school-aged children receive deworming through the Expanded Program on Immunization (EPI). The school-based MDA offers a single dose of mebendazole biannually while the EPI uses albendazole . In a siure rate ,26. A 20ure rate . WASH BeThe WASH Benefits trial enrolled pregnant women in their first or second trimester intending to stay in their village for 24 months post-enrollment, with the objective of following the birth cohort (“index children”) born to them. Field staff screened the study area for pregnant women and collected the global positioning system (GPS) coordinates of the compounds they lived in. Eight neighboring eligible women were grouped into clusters using their compounds’ GPS coordinates. Cluster dimensions were chosen such that one field worker could visit all participants in a cluster in one day. A minimum 1-km buffer was enforced between clusters to minimize spillovers of infections and/or intervention behaviors between study arms. Every eight adjacent clusters enrolled formed a geographic block. An off-site investigator (BFA) used a random number generator to block-randomize clusters into study arms, providing geographically pair-matched randomization. Participants and field staff were not blinded as interventions entailed distinct hardware; blinded technicians enumerated STH outcomes and blinded analysts  independently replicated data management and analysis. Details of the study design have been previously described . The stuStudy arms included (1) water treatment: chlorination with sodium dichloroisocyanurate (NaDCC) tablets coupled with safe storage in a narrow-mouth lidded vessel with spigot, (2) sanitation improvements: upgrades to concrete-lined double-pit latrines and provision of child potties and sani-scoops for feces disposal, (3) handwashing promotion: handwashing stations with a water reservoir and a bottle of soapy water mixture at the food preparation and latrine areas, (4) combined water treatment, sanitation and handwashing (WSH), (5) nutrition improvements including exclusive breastfeeding promotion (birth to 6 months), lipid-based nutrient supplements (6–24 months), and age-appropriate maternal, infant, and young child nutrition recommendations (pregnancy to 24 months), (6) nutrition plus combined WSH (N+WSH), and (7) a double-sized control arm with no intervention . FurtherThe WSH interventions aimed to reduce children’s early-life exposure to fecal pathogens. Bangladeshi households are clustered in compounds shared by extended families; in our study, the compound containing the household where the index child lived (“index household”) had an average of 2.5 households (range: 1–11). The interventions targeted the compound environment as we expected this to be the primary exposure domain for young children . InterveThe delivery of interventions was initiated around the time of index children’s birth. Local women hired and trained as community health promoters visited intervention arm participants on average six times per month to deliver intervention products for free, replenish the supply of consumables , resolve hardware problems and encourage adherence to the targeted WSH and nutrition behaviors; health promoters did not visit control arm participants . The heaWe assessed STH infections in children living in WASH Benefits compounds approximately 2.5 years after intervention initiation. Households with no live birth or index child death were excluded from intervention promotion and subsequently follow-up. The following children were eligible to enroll in the STH assessment: (1) all index children (aged 30 months on average at follow-up), (2) up to two other children per enrolled compound aged 3–12 years, living either in the index household or another household in the compound, enrolled in the preferential order of sibling of index child, child living in index household, or child living in another household in the compound, depending on availability.Giardia duodenalis, Cryptosporidium spp., and Entamoeba histolytica were measured at baseline among children aged 18–27 months (the anticipated age range for index children at the time of the STH assessment) to assess baseline balance on parasite infections between arms [We did not measure pre-intervention STH prevalence as index children were not born at the time and also because detection of any infections would ethically have required treatment and thus prevented us from testing our hypotheses under the typical conditions of our study population. Randomization was expected to ensure baseline balance in STH outcomes across study arms. Additionally, protozoan infections with een arms .To measure STH outcomes, field staff distributed sterile containers to primary caregivers of enrolled children, instructed them to collect stool from the following morning’s defecation event, and retrieved the containers on the morning of defecation. If any enrolled child was absent or failed to provide a specimen, field staff returned to the household twice before classifying them as lost to follow-up. After the completion of stool collection in a given compound, all compound members were offered a single dose of albendazole.A. lumbricoides, hookworm and T. trichiura. Two slides were prepared from each stool sample and enumerated within 30 minutes of slide preparation [Specimens without preservatives were transported on ice to the field laboratory of the International Centre for Diarrhoeal Disease Research, Bangladesh  and analyzed on the same day. Laboratory staff were trained at the icddr,b parasitology laboratory using the Vestergaard Frandsen protocol to perform double-slide Kato-Katz and enumerate ova of paration . 10% of Primary caregivers of children provided written informed consent. Children aged 7–12 years provided written assent. The protocol was approved by human subjects committees at University of California, Berkeley (2011-09-3652), Stanford University (25863), and the icddr,b (PR-11063). A data safety monitoring committee at icddr,b oversaw procedures.https://osf.io/v2c8p/) in August 2016 before data analysis began.WASH Benefits was registered at ClinicalTrials.gov (NCT01590095) in April 2012 before trial enrolment began in May 2012; this registration lists the trial’s primary and secondary outcomes . The trial design was published in June 2013 before the STH follow-up began in May 2015 and lists STH under tertiary outcomes in Appendix 3 . The preA. lumbricoides, ≥2,000 epg for hookworm, and ≥1,000 epg for T. trichiura) [Our pre-specified outcome measures were the infection prevalence, infection intensity and moderate/heavy infection prevalence for each STH species and for any of the three target species (“any STH”). For each species, we classified stool samples with any ova as positive. We quantified infection intensity in eggs per gram (epg) by multiplying the sum of egg counts from the two duplicated slides by 12 . We defined moderate/heavy intensity infections based on WHO categories  . We asseichiura) .A. lumbricoides, 50% for hookworm, 39% for T. trichiura, and 18% for any STH.WASH Benefits was designed to detect effects on child length and diarrhea with a planned sample size of 5040 pregnant women . We assuWe compared STH outcomes in 1) individual and combined water, sanitation, handwashing and nutrition arms vs. controls (primary hypothesis), (2) combined vs. single WSH intervention arms, and (3) N+WSH vs. WSH and nutrition arms. We estimated prevalence ratios (PR), prevalence differences (PD) and fecal egg count reductions  between arms. We estimated FECRs using geometric and arithmetic means; while geometric means prevent extreme data points from skewing means, arithmetic means are more sensitive to high infection intensities thought to correlate with higher morbidity burden and transmission [ individuRandomization led to extremely good covariate balance , and ourOur primary analysis included all enrolled children of all ages. As different interventions were implemented at index child, index household and compound levels, we also conducted subgroup analyses for the following three categories of children: (1) index children, (2) other children living in index household, and (3) children living in other households in compound. The subgroup analysis for index children was pre-specified and the analysis for other children living in the index household vs. the rest of the compound was added post-hoc. We also conducted a pre-specified subgroup analysis by child age (pre-school-age vs. school-age) as well as additional pre-specified subgroup analyses by deworming status, household size, wealth, housing materials, and baseline sanitation conditions .Individuals that were lost at follow-up or failed to submit a specimen were classified as missing. To assess if the likelihood of missing data was differential by study arm and/or covariates, we compared the percentage of missing observations between arms and the enrollment characteristics of those with available vs. missing specimens. We also assessed the balance of baseline covariates between arms for households captured at follow-up. We conducted a complete-case analysis and an inverse probability of censoring-weighted (IPCW) analysis re-weighting the measured population to reflect the original enrolled population  .Fieldworkers identified 13279 pregnant women in the study area . BetweenAverage age at follow-up was 30 months (range: 22–38) for index children and 7 years (range: 3–12) for non-index children. Caregivers reported that 60% of index children and 67% of non-index children had been dewormed in the six months prior to our data collection. For index children, the predominant source (86%) of deworming drugs was individual purchase at a pharmacy. For non-index children, the predominant sources of deworming drugs were schools (48%) and pharmacies (44%), and approximately half of children were reported to be dewormed through the MDA program. The average time since deworming was 9 weeks (range: 0–24) for both index and non-index children. The percentage of dewormed children and the average time since deworming was balanced across trial arms . CaregivA. lumbricoides, 9.2% (n = 142) for hookworm, 7.5% (n = 115) for T. trichiura, and 43.4% (n = 664) for any STH. The geometric mean egg count among controls was 5.2 epg (143.4 epg among positive samples) for A. lumbricoides, 0.6 epg (119.8 epg among positive samples) for hookworm and 0.4 epg (115.4 epg among positive samples) for T. trichiura. Most infections were low-intensity. Moderate/heavy infection prevalence among controls was 4.2% (n = 65) for A. lumbricoides, 0.1% (n = 2) for hookworm and 0.4% (n = 6) for T. trichiura. The ICC for any STH infection was 0.18 for children within the same compound  and 0.08 for children within the same cluster of compounds . The quality control measures indicated high interrater reliability  for iability .T. trichiura prevalence by 29% , PD = -2.17 ) from a control prevalence of 7.5% and achieved a similar borderline reduction on hookworm but had no effect on A. lumbricoides. Single handwashing or nutrition interventions did not reduce the prevalence of any STH compared to controls; there was a borderline increase in A. lumbricoides prevalence in these arms , PD = -2.83 ) from a control prevalence of 9.2% but had no effect on other STH . The sanese arms .A. lumbricoides by 7–10% compared to controls , PD = -2.47 ; N+WSH: PR = 0.90 , PD = -3.79 ) but had no effect on T. trichiura , PD = -2.63 ) and N+WSH by 33% , PD = -3.00 ) compared to controls. WSH and N+WSH also marginally reduced richiura .A. lumbricoides more than handwashing alone , PD = -6.21 ; this reflects the increased A. lumbricoides prevalence in the handwashing arm. We found no other benefit from combined WSH vs. its individual components , PD = -7.31 ); hookworm: PR = 0.65 , PD = -3.29 ) but did not achieve any reduction compared to WSH ; we note that these analyses should be considered exploratory as they had limited statistical power.Subgroup analyses on index children, other children in the index household and children in other households in the compound yielded findings consistent with those using pooled data from all children. Non-index children had higher infection prevalence , consistcompound ; howevercompound . Our fulA. lumbricoides or T. trichiura. These findings suggest possible waterborne hookworm transmission, and chlorine water treatment with safe storage is effective in reducing this transmission. While infections of A. lumbricoides or T. trichiura are transmitted by ingesting embryonated ova, hookworm larvae infect hosts by penetrating skin; however A. duodenale can also be transmitted by ingesting larvae [A. duodenale several times more frequently than N. americanus, suggesting that A. duodenale could be the dominant hookworm species in the region [A. lumbricoides or T. trichiura. The safe storage container with a narrow mouth and lid would also reduce STH contamination of stored drinking water by eliminating contact with hands, which are known reservoirs of ova and larvae [A. lumbricoides or T. trichiura were not reduced by our water intervention, potentially suggesting that the hookworm reduction is due to chlorine rather than safe storage; there are scarce data on the effectiveness of chlorine on hookworm. Alternatively, waterborne transmission could be more important for hookworm in this setting than for A. lumbricoides or T. trichiura.In all intervention arms that included water treatment , we found a significant reduction in hookworm but not in g larvae . A studye region . STH ovae region , suggeste region . While ce region , fragiled larvae . An obsed larvae . Storaged larvae . HoweverT. trichiura and achieved a borderline reduction in hookworm but had no effect on A. lumbricoides. While the other two arms with a sanitation component  reduced hookworm to a similar degree as the single sanitation intervention, T. trichiura was not affected in these arms; the reduction in the sanitation arm for this species could thus be a chance finding and should be interpreted cautiously. Two previous sanitation trials in India found no impact on STH; however, both studies entailed community-level programs with relatively low adherence [The WASH Benefits sanitation intervention with concrete-lined double-pit latrines, potties and scoops for feces management reduced dherence ,13. WASHdherence . In strudherence . Low adhdherence . Also, cdherence . Finallydherence ,45; it iA. lumbricoides could also be due to its prolonged survival in soil, providing a persistent reservoir to sustain infection [T. trichiura ova for months [A. lumbricoides ova can survive in soil for several years in warm and saturated conditions [A. lumbricoides in 67% and T. trichiura in 36% of courtyard soil samples; hookworm ova were not detected in soil (the protocol was not designed to detect hookworm larvae). Among positive samples, 70% of A. lumbricoides and 88% of T. trichiura ova developed larvae when incubated  [T. trichiura whose larvae/ova are shorter-lived in the environment than those of A. lumbricoides, which is consistent with our findings. The effect of sanitation on A. lumbricoides infections may not be apparent until existing ova in the environment from pre-intervention contamination are naturally inactivated.The lack of sanitation impact on nfection . Hookworr months . In contnditions . A pilot viable) . Soil fr viable) ,48. Any T. trichiura intensity. Previous hygiene programs have reduced STH infections [A. lumbricoides ova in 51%, T. trichiura ova in 23% and hookworm larvae in 26% of fingernails [T. trichiura intensity but not prevalence was reduced in the handwashing arm; if the intervention reduced but did not eliminate T. trichiura ova on hands, this could lead to a reduced worm burden without affecting prevalence.Handwashing did not reduce STH infection except for a modest reduction in fections –11. Two fections ,10, whicgernails , and naigernails . This coA. lumbricoides compared to controls.We found no added benefit from combining WSH interventions compared to individual interventions. While we did not power the study to statistically detect differences between combined vs. individual intervention arms, the effect estimates suggest that combined WSH and N+WSH achieved a similar magnitude of reduction in hookworm prevalence as the individual water and sanitation interventions. One possible explanation is that combined interventions might have lower user adherence as they require more complex behavior change . HoweverA. lumbricoides prevalence in the nutrition arm. A recent study found increased hookworm in school children receiving micronutrient-fortified rice [Lipid-based nutrient supplements, breastfeeding and complementary feeding promotion did not reduce STH prevalence/intensity alone or in combination with WSH, even when the analysis was restricted to index children directly receiving this intervention. There was a borderline increase in ied rice . Other sied rice . WASH Beied rice ,52. One ied rice ,21. Chroied rice . HookworA. lumbricoides), demonstrating ongoing transmission and suggesting that MDA alone is unlikely to break STH transmission in this setting.STH control policies, which currently emphasize MDA programs, could be strengthened by complementing these with water, sanitation and hygiene interventions . ApproxiAgainst this backdrop, we found a 30% relative reduction in hookworm prevalence from water treatment and combined WSH interventions, as well as a borderline reduction of similar size from sanitation improvements. While the reductions were comparable in magnitude to the reductions in child diarrhea and protozoan infections achieved by WASH Benefits ,31 and oA. lumbricoides, which was more common . This is consistent with a school-based trial in Kenya that found reduction in A. lumbricoides  but not the more prevalent hookworm  from a combined water, sanitation and hygiene intervention [It is also possible that the effect of water, sanitation and hygiene on STH depends on background transmission intensity. In our study, WSH interventions had more pronounced effect on hookworm, which was relatively rare , than on rvention . These fA. lumbricoides, 53% for hookworm, 80% for T. trichiura for double-slide Kato-Katz for low-intensity infections [A. lumbricoides and high specificity for T. trichiura and hookworm . Assuming non-differential misclassification by arm, imperfect sensitivity and specificity would bias our estimated intervention effects toward the null  so there was only sufficient statistical power to detect relatively large effects on hookworm and T. trichiura reduction in the sanitation but not WSH and N+WSH arms could indicate random error. However, most observed reductions followed consistent patterns that are unlikely to be explained by chance. Hookworm prevalence and intensity showed internally consistent reductions of similar size in all arms with a water or sanitation component, while the only intervention that achieved a borderline reduction in A. lumbricoides was combined WSH—the most biologically plausible intervention to reduce environmental transmission.We conducted multiple comparisons, increasing the risk of chance findings; the A. lumbricoides given its long survival in soil [A. lumbricoides elimination.Another limitation is that we assessed STH outcomes 2.5 years after intervention initiation, which is a relatively short period of time to assess impact on  in soil . This tiEnvironmental conditions such as temperature, humidity and soil type affect the fate and transport of STH ova ,46 and iA. lumbricoides and T. trichiura; this could be because of the short survival of hookworm in soil, precluding persistent environmental reservoirs of ova from pre-intervention contamination. Our findings suggest that drinking water can potentially be an overlooked transmission route for hookworm and that water treatment and sanitation interventions can augment MDA programs in striving towards breaking STH transmission.In a setting with ongoing MDA and low-intensity infections, we found modest but sustained reductions in hookworm prevalence and intensity from water treatment and combined WSH interventions. There was no STH reduction from handwashing and nutrition improvements. Intervention effects were more pronounced on hookworm than on S1 Text(PDF)Click here for additional data file.S2 Text(PDF)Click here for additional data file.S3 Text(PDF)Click here for additional data file.S4 Text(PDF)Click here for additional data file.S5 Text(PDF)Click here for additional data file.S6 Text(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.S7 Table(PDF)Click here for additional data file.S8 Table(PDF)Click here for additional data file.S9 Table(PDF)Click here for additional data file.S10 Table(PDF)Click here for additional data file.


Leishmania DNA in the phlebotomine population of Oiapoque . Phlebotomines were captured in anthropized and wild environments in the outskirts of Oiapoque municipality, using CDC light traps installed in vertical (ground/canopy level) and horizontal (peridomicile/extradomicile/forest-edge/forest) strata. Captured specimens were identified according to their morphology. Females were processed for Leishmania DNA detection and characterization using a multiplex polymerase chain reaction targeting kinetoplast DNA (kDNA) and the phlebotomine cacophony gene. The kDNA positive samples were characterized by cloning and sequencing the Leishmania 234 bp-hsp70 gene. Among the 3957 phlebotomine specimens captured, 26 pooled female samples were positive for Leishmania (Viannia) spp. DNA. Sequencing analysis allowed species-specific identification of L. (V.) braziliensis DNA in Trichophoromyia ininii, Bichromomyia flaviscutellata, Nyssomyia umbratilis, and Evandromyia infraspinosa, and L. (V.) guyanensis DNA in Ny. umbratilis. A pooled sample of Ny. umbratilis was positive for both L. (V.) braziliensis and L. (V.) guyanensis DNA. The present study provided additional information regarding ACL ecology in Oiapoque, highlighting the presence of L. (V.) braziliensis DNA in different phlebotomine species. The epidemiological implications of these findings and the determinant incrimination of L. (V.) braziliensis as proven vectors in that region must be clarified. In this regard, studies on Leishmania spp. infection and suggestive anthropophilic behavior of associated phlebotomines need to be prioritized in entomological surveillance.In the border region between Brazil and French Guiana, American cutaneous leishmaniasis is a worrisome public health issue, and entomological studies are required there to better identify classical and putative emerging transmission patterns. The present study aimed to detect and characterize  Leishmania (Kinetoplastida: Trypanosomatidae) parasites, the causative agents of American cutaneous leishmaniasis (ACL) [5'-GTG GCC GAA CAT AAT GTT AG-3') and 3Llcac (5'-CCA CGA ACA AGT TCA ACA TC-3')] . The secLeishmania (Viannia) (5’-TAA TTG TGC ACG GGG AGG CCA-3') [Lutzomyia longipalpis females, after 72h of feeding on rabbit blood containing 2 × 105L. braziliensis parasites/mL.To optimize detection sensitivity, the amplification products were additionally subjected to dot-blot hybridization using a biotinylated probe specific for  CCA-3') . The hybLeishmania-positive sample was subjected to a second, semi-nested-PCR assay targeting the hsp70 gene. This gene region validated for distinguishing different species of Leishmania present in Brazil, with broad coverage of the L. (Viannia) subgenus. In the first PCR step, a 234 bp fragment of hsp70 was amplified using the oligonucleotides 5'-GGA CGA GAT CGA GCG CAT GGT-3' and 5'-TCC TTC GAC GCC TCC TGG TTG-3' [5'-GGA GAA CTA CGC GTA CTC GAT GAA G-3' [Escherichia coli DH5α cells using the pGEM T-Easy Vector kit vector (Promega), according to the manufacturer's recommendations.The DNA recovered from each G TTG-3' . In the http://www.ncbi.nlm.nih.gov/VecScreen/VecScreen.html). Sequences were compared to those available in the BLASTnucleotide database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) using the BLASTN algorithm.Sanger sequencing was performed with the RPT01A-PDTIS, Fiocruz-RJ sequencing platform (ABI 3730XL Applied Biosystem) , using tt-test to compare diversities between the different sites sampled with equal effort , using the Past software version 3.22 ,[The Shannon-Wiener (H) diversity index was estimated and evaluated with a  Norway),. SignifiCapturing and processing invertebrate fauna (phlebotomines) were authorized by the Sistema de Autorização e Informação em Biodiversidade—SISBIO (Biodiversity Authorization and Information System), under protocol No. 44524.Evandromyia infraspinosa, Nyssomyia umbratilis, and Trichophoromyia ininii which were the most frequent, accounting for 2837 (71.7%) of the total number captured. Greatest diversity was observed, for the wild environment, in the ground level (H = 1.907) and, for the anthropized environment, in the peridomicile (H = 1.809)  . When co= 1.809) .Leishmania spp. DNA detection. Engorged phlebotomines were excluded from the infection assays due to the risk of detecting a non-established infection in flies that had recently consumed blood from an infected source.Phlebotomine DNA extraction was considered successful in 551 samples that amplified the cacophony gene; and thus appropriate for Leishmania (Viannia) spp. DNA. In the anthropized environment, Leishmania (Viannia) spp. DNA was detected in forest-edge samples of Psychodopygus squamiventris maripaensis (n = 1) and Bichromomyia flaviscutellata (n = 3), and in forest samples of N. umbratilis (n = 1) and Th. ininii (n = 2). In the wild environment, Leishmania (Viannia) spp. DNA was detected in B. flaviscutellata (n = 1) and E. infraspinosa (n = 2) caught at ground level, in N. umbratilis from ground (n = 4) and canopy (n = 9) levels, and in P. s. maripaensis from ground (n = 1) and canopy (n = 2) levels.Twenty-six samples were positive for Leishmania spp. DNA: P. s. maripaensis (18.1%), N. umbratilis (5.8%), Bi. flaviscutellata (7%), E. infraspinosa (2.8%) and T. ininii (1.2%). Leishmania species identification was successful in 8 of the 26 positive samples, with coverage varying from 80–97% and identity from 88–100%. It was not possible to characterize the others due to cloning failures resulting from low DNA levels or incompatibility with the available sequences in the database.The minimum rate of DNA detection  was calculated for each the five species captured and positive for L. (V.) braziliensis DNA in one T. ininii (10 specimens) sample from the forest-edge, one of B. flaviscutellata (6 specimens) from the forest, two of N. umbratilis  from canopy level, and two of E. infraspinosa  from ground level. Two samples of N. umbratilis (10 specimens each) from the canopy level were positive for Leishmania DNA, one was identified as L. (V.) guyanensis and the other contained DNA from both L. (V.) braziliensis and L. (V.) guyanensis.Sequencing analyses are summarized in In Brazil, ACL presents three characteristic epidemiological patterns: 1) Sylvatic, where transmission occurs mainly in primary forest environments; considered a zoonosis of wild animals with occasional human cases in areas of recent colonization due to the penetration of the man into the wild. 2) Occupational/recreational, associated with disordered forest exploration and with the clearing of forests for the construction of roads, hydroelectric power plants, village settlements, timber extraction, agricultural and military training activities, and ecotourism. 3) Rural and peri-urban in colonization areas, related to the migratory process, occupation of slopes and clusters in urban centers associated with secondary or residual forests . Of thesLeishmania DNA detection within the phlebotomine population.As hypothesized, the phlebotomine species composition observed in this study were highly similar to that recently presented in our previous study . TherefoH = 1.907; p < 0.05), where CDCs are usually set spatially congruent with the flight level of majority of phlebotomine species. On the other hand, canopy stratum comprise a ecological subsystem with fewer number of species with close relationship with arboreal vertebrates, such as occur with N. umbratilis and sloths and birds [H = 1.809; p < 0.05) can be attributed to the search for shelter and offer of blood of domestic animals [In the wild environment, diversity was significantly higher in the ground . Among these, it is important to highlight  forests , and may forests ,30. Seve forests –34; howe studies –37. Therironment .Leishmania spp. be combined with dissection and attempted parasite isolation. Present results support our previous “dissection-based” information about Leishmania parasites in the Oiapoque environment [Molecular tools have been gradually replacing the traditional technique of phlebotomine dissection and provide several advantages, particularly with respect to sensitivity and specificity  allowingironment .The minimum detection rate observed by the molecular method (4.7%) was higher than that demonstrated in our early experience in Oiapoque using the dissection method (0.78%) , which iIt is important to note that these values do not necessarily reflect the risk of human infection; other factors should be considered in assessing the risk of exposure to ACL agents, such as the degree of anthropophilia of their potential vectors .Viannia. Unfortunately, the best targets for genotyping, such as Hsp70, are not good detection targets due to low copy number. Therefore, effective detection and identification of Leishmania spp. from phlebotomines requires a primary detection step targeting kDNA, followed by a subsequent genotyping step using another molecular target, such as hsp70. Even so, the efficiency is reduced, due to the low amount of target DNA, and then we consider some identity values lower than 90%. Although we were unable to characterize 18 of the 26 positive samples, the sequence of eight pools showed compatible coverage and identity to contemplate characterization at species-specific level; providing important information in the context of Amazonian/Guianan ACL.The cloning and sequencing stages are important because phlebotomines present a complex biological material and although kDNA detection provides high sensitivity, it does not allow identification to species level. Using this method, identification is possible at the level of genus and subgenus and this is a particular problem with the subgenus L. (V.) guyanensis DNA was detected in several samples of N. umbratilis, supporting the whole context that this natural vector x parasite relationship that has been discussed in the course of our past experience in Oiapoque [Oiapoque  and baseOiapoque ,10,41–45L. (V.) braziliensis DNA was detected in four different phlebotomine species: T. ininii, B. flaviscutellata, E. infraspinosa, and N. umbratilis. In Brazil, L. (V.) braziliensis transmission is associated with the highest number of known vectors, where there are 17 phlebotomine species with proven or potential links to its transmission; seven of which are only associated by molecular findings [L. (V.) braziliensis strains [findings . The wid strains  may cont strains .L. (V.) braziliensis DNA has not been found previously in any of these four phlebotomine species. Conversely, B. flaviscutellata and N. umbratilis play well-recognized roles in the transmission of the respectively associated parasites, L. (L.) amazonensis and L. (V.) guyanensis [Leishmania sp. DNA was detected in Trichophoromyia ininii, considered a low-anthropophilic phlebotomine species, in Sabajo Heuvels, Suriname [E. infraspinosa had previously been found in Oiapoque carrying promastigotes that were subsequently characterized as L. (V.) guyanensis from the residual material present on the slide [Interestingly, yanensis ,4. LeishSuriname . E. infrhe slide .L. (V.) braziliensis in four different phlebotomine species suggests concomitant contact of these insects with potential reservoirs of the parasite. Considering that rodents are the most likely animals to be involved in maintaining the enzootic cycle of L. (V.) braziliensis [B. flaviscutellata [N. umbratilis has been found alternatively feeding on blood from rodents in an environmentally-impacted area close to Manaus, Amazonian Brazil [E. infraspinosa, which infrequently bites man in the bordering region of Jari, between the Brazilian states of Pará and Amapá, was relatively frequent species in Disney-trapped rodent trap catches [Dasyprocta leporina DNA was recently detected in E. infraspinosa in Saint Georges de l'Oyapock, Guiana [E. infraspinosa, in our past experience during captures in area I, this species has drawn medical attention for having been observed biting professionals during Shannon trap captures in same circumstances of having been found naturally infected by L. (V.) guyanensis [E. infraspinosa with respect to L. (V.) braziliensis-associated ACL transmission; particularly as these phlebotomine populations may also be permissive to other Leishmania spp. and have suggestive anthropophilic behavior.The detection of iliensis –51, it iutellata  and N. un Brazil . Additio catches . Further, Guiana . Still iyanensis . Conversyanensis . These fN. umbratilis was concomitantly positive for L. (V.) braziliensis and L. (V.) guyanensis DNA; however, it cannot be ascertained whether these two parasites were within the same phlebotomine or in different specimens of that pool. In this study and in the literature [N. umbratilis feed on different animals that act as potential reservoirs of Leishmania spp.One pooled sample of ten females of terature –56 it isL. (V.) braziliensis DNA in different phlebotomine species captured in ecologically distinct environments and strata in the study area. It should be noted, however, that these findings alone are not sufficient to conclude that there is a transmission cycle involving these phlebotomines and L. (V.) braziliensis. First, absence of visible blood cannot be interpreted as the possible survival of the parasite. Second, and more importantly, studies focused to this topic demonstrated that parasite DNA is detectable few days after Leishmania are killed in the non-natural vectors [L. (V.) braziliensis in this region. Nonetheless, the known capacity of these species to harbor Leishmania spp. combined with suggestive anthropophilic behavior present a need for prioritized entomological surveillance.The results of the present study provide additional information on ACL ecology in Oiapoque, highlighting the presence  vectors ,58, rein


Spodoptera littoralis, is a phytophagous pest and current focal species for insect chemical ecology and neuroethology.Deciphering the molecular mechanisms mediating the chemical senses, taste, and smell has been of vital importance for understanding the nature of how insects interact with their chemical environment. Several gene families are implicated in the uptake, recognition, and termination of chemical signaling, including binding proteins, chemosensory receptors and degrading enzymes. The cotton leafworm, S. littoralis, including the antennae, proboscis, brain and body carcass. We have annotated 306 gene transcripts from eight gene families with known chemosensory function, including 114 novel candidate genes. Odorant receptors responsive to floral compounds are expressed in the proboscis and may play a role in guiding proboscis probing behavior. In both males and females, expression of gene transcripts with known chemosensory function, including odorant receptors and pheromone-binding proteins, has been observed in brain tissue, suggesting internal, non-sensory function for these genes.We produced male and female Illumina-based transcriptomes from chemosensory and non-chemosensory tissues of S. littoralis and closely related species. Collectively, our results expand current understanding of the expression patterns of genes with putative chemosensory function in insect sensory and non-sensory tissues. When coupled with functional data, such as the deorphanization of odorant receptors, the gene expression data can facilitate hypothesis generation, serving as a substrate for future studies.A well-curated set of annotated gene transcripts with putative chemosensory function is provided. This will serve as a resource for future chemosensory and transcriptomic studies in The online version of this article (10.1186/s12864-019-5815-x) contains supplementary material, which is available to authorized users. The chemical senses, including the olfactory and gustatory modalities of smell and taste, play a critical role throughout the life history of most insects . For phyDrosophila melanogaster GR in the brain has been linked to internal sugar monitoring [At the molecular level the processes of chemosensory detection, which include uptake, reception, and inactivation of stimulus molecules , are mednitoring , and thenitoring  resultedD. melanogaster. However, a recent report demonstrating polycistronic co-expression of four to six ORs in individual olfactory sensory neuron subtypes in a mosquito [Spodoptera littoralis  is a phytophagous pest insect indigenous to Africa and the Middle East [S littoralis has obtained status as an invasive species of continental Europe [S. littoralis has lately been the subject of intensive focused research, specifically with regards to questions of chemical ecology and for providing a foundation for novel pest control strategies.The molecular mechanisms of insect olfaction are mostly understood through research in mosquito  highlighdle East . In recel Europe . AccordiS. littoralis respond to a broad range of ecologically relevant pheromonal as well as host plant volatiles [S. littoralis have recently been investigated, culminating in the identification of a repertoire of ORs that are activated by many of the same odorants shown previously to activate olfactory sensory neurons in this species [S. littoralis [It has been demonstrated that the antennae of olatiles –16. The  species . Modulatttoralis . Interesttoralis –23. Furtttoralis .S. littoralis, initial reports have characterized chemosensory gene expression in male [In  in male  and fema in male  antennal in male . These t in male  and CYP  in male  gene famS. littoralis antennae. Furthermore, we provide a comprehensive first-look at chemosensory gene expression in the proboscis of S. littoralis. We have also aimed to enhance the availability of adult S. littoralis transcriptomics resources in general and have thus sequenced transcripts from the brain and whole body minus head (henceforth body carcass) tissues. In sum, our study includes the analyses of male and female RNA-Seq transcriptomes, both derived from antennae, proboscis, brain and body carcass tissues. We have expanded the existing scientific knowledge on the number of putative chemosensory genes from most of the gene families with chemosensory function, as well as the completeness of coding sequence information for many of the previously identified but incomplete chemosensory transcripts. The expression of putative chemosensory receptor genes in brain tissue of both male and female moths suggests a potential novel function for these genes as monitors of internal chemical signaling. Finally, we report olfactory receptor genes that may mediate proboscis probing behaviors in moths.In this report, we have expanded on these studies by performing in-depth qualitative as well as preliminary quantitative analyses of chemosensory gene expression in male and female De novo transcriptomes were derived from nine tissue samples each for both male and female S. littoralis. After quality control processing of the raw sequencing reads, input for the male and female transcriptomes consisted of 240.8 and 236.4 million read pairs, respectively  have previously been reported [S. frugiperda genome [For reported –27. OR1-reported . For thea genome , the preS. littoralis ORs and a selection of those from other Lepidoptera with sequenced genomes is shown ), OR16 (58.6 FPKM) and OR11 (34.4 FPKM) were the most abundantly expressed tuning ORs, of which the latter two cluster within the Lepidoptera pheromone receptor (PR) subfamily . In virgIn other tissues, limited relatively low-level OR transcript expression was observed in both male and female samples. In the proboscis of both male and female moths, SlitOrco and SlitOR14 showed consistent expression patterns. In virgin female and male brain, SlitOrco, SlitOR14, and SlitOR25 displayed consistent expression patterns with FPKM values higher than one.  receptors, SlitGR2 and SlitGR3, represent the most highly expressed GRs  were among the most highly expressed GRs in the proboscis. Notably, a single putative bitter-compound receptor, SlitGR230, was observed to be expressed across all tissue types. Expression of this gene was confirmed in antennae, proboscis, and brain via RT-PCR assay  and relatively lower expression in proboscis  and brain . SlitSNMP2 displayed relatively moderate to high expression in antenna , proboscis  and brain . RT-PCR assays of SlitSNMP1 and SlitSNMP2 expression in antennae, proboscis, and brain are consistent with these observations . Likewise, complete ORFs are predicted for 86% (n=264) of all of the gene transcripts characterized in this study. Nearly all of the annotated transcripts were identified in the male (n=290) and female (n=293) transcriptomes, providing high confidence in the accuracy of the sequence information for the transcripts being studied.We used Illumina-based RNA Sequencing methodology to provide a expanded picture of the expression patterns of several S. littoralis. Recently, several reports on genomic studies of the odorant receptors in lepidopteran species have provided a better range approximation on the number of ORs per species within this insect order: 95 ORs in P. xylostella [E. postvittana [B. mori [M. sexta [D. plexippus [H. melpomene [Sixty ORs are now annotated for lostella , 38, 70 tvittana , 71 ORs [B. mori , 41 , 73M. sexta , 43, 64 lexippus  and 74 Oelpomene . These vS. littoralis PRs [E. postvitanna male antennae (namely EposOR30/OR34) [B. mori (BmorOR30)[C. pomonella (CpomOR30/OR31/OR41) [A novel OR, SlitOR56, has been identified that clusters phylogenetically with other alis PRs , 47. In alis PRs . However30/OR34) . InteresBmorOR30) and C. p31/OR41) . High exS. littoralis, with RNA-Seq and PCR confirmation of expression of SlitOrco, SlitOR14, and SlitOR25. SlitOR14 has been determined to be strongly responsive to phenylacetaldehyde [Manduca sexta [Consistent with a previous report , we havealdehyde , which ialdehyde , 50. Basca sexta ; SlitOR1ca sexta , and theca sexta , 52.Aedes aegypti [We additionally observed expression of several SlitORs in male and female brain, including SlitOrco, SlitOR14, and SlitOR25. Similar observations in this species were previously reported . As with. It has  aegypti , suggestS. littoralis, including the first description of putative bitter-compound GRs in this species. This number is far lower than what has been described in other Lepidoptera from genome analyses. Typically, 45-70 GRs are predicted . The deB. mori [2 GRs to be expressed in both males and females, namely, SlitGR2 and SlitGR3. In the antennae of both males and females, SlitGR2 and SlitGR3 are the most highly expressed GRs; this expression pattern is consistent with a previous report showing their expression in the antennae of males and females [Spodoptera exigua, comparing expression levels of putative CO2 receptors in antennae, proboscis and labial palps [S. littoralis, and indeed, other Lepidoptera.Similar to B. mori , three pB. mori , with COB. mori . While w females . In lighal palps , furtherB. mori [P. xylostella [H. melpomene [D. plexippus [Similar to other moths, a total of five putative sugar-compound receptors have been identified; in this highly conserved GR sub-family, five receptors were identified in B. mori , and P. lostella , 38 and elpomene , while 1lexippus , 44.S. littoralis. A role for the insect antenna in contact chemoreception is well understood [S. littoralis antennal sensitivity to sugars sucrose, glucose and fructose [S. littoralis.Putative sugar-detecting GR transcripts were detected in male and female antennae and proboscis of derstood . A recenfructose ; here weB. mori orthologue (BmorGR9), which is 63% identical to SlitGR9, has been shown to be responsive to fructose [D. melanogaster fructose-clade receptor DmelGR43a [While we did not detect the previously reported fructose sub-family receptor, SlitGR10 (formerly SlitGR4) in our transcriptomes, we did identify a novel gene transcript that clusters with other receptors in this clade, SlitGR9. Expression of this receptor was detected in male and female antennae and brain tissues, but not in proboscis and body carcass. A fructose , suggestfructose . SimilarS. littoralis ovipositors [A recent report characterized contact chemosensory sensilla on positors , demonstS. littoralis.A total of seven putative bitter-compound receptor GRs have been identified in this report, representing the first identification of putative GRs from this clade in S. littoralis is highly polyphagous, one would hypothesize a large number of so-called bitter-compound GRs to be expressed in this species, though it remains to be seen if that will be true.However, recent studies have suggested an expansion of the bitter-compound GR clade in related polyphagous moths , 54, supH. armigera, described novel classes of intronless insect GRs, including functionally characterized receptors with shorter ORFs (ca. 200-350 amino acids) [The putative bitter-compound GRs identified here displayed different expression patterns across all tissues examined in both male and females, with a single receptor candidate, SlitGR230, having been detected in antennal, proboscis and brain tissues. A recent report on GRs in the noctuid moth, o acids) . SimilarS. littoralis, however, identification of insufficient numbers of putative bitter-compound GRs precludes characterization of this phenomenon.Five of these seven SlitGR candidates clustered in different bitter-compound receptor sub-families, with only one set of two genes clustering together as potential paralogues. Species-specific expansions of different putative bitter-compound receptor lineages have been noted as a more prominent feature of GRs in Lepidoptera compared to most OR sub-families , 41; in S. exigua [, C. pomonella [M. sexta [A total of 17 IRs were identified in this study, building on the gene transcripts previously identified , 32, and. exigua , C. pomoomonella  and M. sM. sexta . SimilarM. sexta , 32 all M. sexta , 48, 58 M. sexta , 64. In S. littoralis, both OBPs and CSPs display broad and diverse expression patterns in all tissues examined here, suggesting diverse roles for these proteins related to chemosensory and non-chemosensory processes. Indeed, these findings are consistent with other reports on expression patterns of both OBPs and CSPs [In and CSPs –67. Furtand CSPs ).Among all of the gene families examined in this report, annotated OBPs claimed the highest percentage share of total summed FPKM expression values in both male and female antennae, at 17.7% in males and 11.0% in females. Previous reports have similarly shown that OBPs are among the most abundantly expressed genes in insect antennal tissues from moth  to mosquB. mori can bind and interact with non-pheromonal compounds [The pheromone-binding protein (PBP) sub-family of OBPs contains proteins known to bind moth pheromone components and facilitate PR activation , 71. Conompounds .Vanessa gonerilla comprise greater than 50% of the total content of the larval mandibular gland proteome [Compared to OBPs, CSPs claimed the highest percentage share of total summed FPKM expression values, among the gene families studied here, in the proboscis, with 9.1% in females and 5.2% in males. It has recently been reported that two CSPs from the butterfly, proteome . SimilarS. littoralis [Similar to the OBPs and CSPs, the CXE/CCE and CYPs displayed diverse expression patterns in all tissues examined in both males and females. Among possible ODE families, we focused on these gene families to provide an extension of previous studies on antennal expressed genes that may function as ODEs; indeed, it has been reported that some SlitCXEs bind to and modify odorants , 76; Slittoralis .To that end, all previously identified antennal expressed CXEs were found to be expressed in both male and female antennae in this report. Of the 26 novel SlitCCEs identified, 12 displayed antennal expression in males, females or both, bringing the total number of antennal expressed esterases to 42.S. littoralis will serve as a useful resource for future transcriptome and genome annotation efforts in S. littoralis and closely related insects. A thorough analysis of the expression patterns of known and putative chemosensory genes in male and female chemosensory tissues provides a framework for a better understanding of the molecular mechanisms of olfaction and gustation in S. littoralis. Furthermore, the data presented here, when compared to gene expression studies of other insects, may provide evolutionary insights with regards to conserved and divergent molecular function and physiological/behavioral processes. The observation of gene expression of known chemosensory genes in non-sensory tissues, such as the brain, suggests novel functions for these genes in non-chemosensory contexts. When coupled with functional data, such as the deorphanization of ORs, the gene expression data can facilitate hypothesis generation, serving as a substrate for future studies. For example, the expression in the proboscis of an OR that detects a floral odorant suggests that this OR may underlie floral/nectar feeding behavior as observed in a closely related moth.The curation of a broad set of putative chemosensory genes in S. littoralis) was obtained from cotton fields  in 2010 and reared on a standard semi-artificial potato-based diet; the colony has been refreshed with new wild-collected individuals approximately every 6 months.Cotton leafworm  on ice. Dissections were made from 3-4 hours into the scotophase in a dark environment with dimmed background lighting. Antennae and proboscis were separated from the head of living organisms. For brain dissections, after removal of antennae and proboscis, the head was removed into 1X Phosphate Buffer Saline (PBS) and brain tissue was removed from the head capsule. For each body carcass sample, three individuals were used directly after removal of the head. After dissections, all samples were snap frozen on dry ice or liquid nitrogen and stored at -80°C until all samples were ready for processing.Total RNA was extracted and purified with a combined approach of Trizol-based extraction followed by RNeasy Mini spin column purification , as previously described . RNA wasPure total RNA samples from both males and females  were prepared and sequenced commercially at the Beijing Genomics Institute (BGI) sequencing facility in Hong Kong (BGI Hong Kong Co.) using standard protocol  [Initial quality control measures for obtained raw reads were undertaken prior to assembly  . The Tri3-02-05) . The malhttps://gvolante.riken.jp/) was utilized with the following parameters: min_length_of_seq_stats: 1, assembly_type: trans, Program: BUSCO_v2/v3, selected reference_gene_set: Arthropoda.To assess the completeness of the transcriptomes, an Arthropoda BUSCO database, consisting of 1066 core genes that are highly conserved single-copy orthologues , 83, wasS. littoralis genes. OR, GR, OBP, and CSP sequences were taken from the supplementary materials of previous reports [Text files were compiled in fasta format for the different chemosensory gene families, with protein sequences included for previously characterized  reports , 27. IR  reports , CXE [28 reports  sequencehttp://www.ebi.ac.uk/Tools/msa/clustalo/) [Blast nucleotide databases were created from The Trinity.fasta files and were queried by the protein sequence fasta files for each of the chemosensory gene families. For this procedure, Blast version 2.2.24+ was used to perform a tblastn query and a minimum e-score threshold of 1e-05 was required for hits; additional parameters included num_descriptions 50 and -num_threads 8; blast output files were generated with output format six . For eacustalo/) .Novel chemosensory genes were identified and annotated in an iterative search process. First, all sequences that were reported in the original blast searches that did not correlate to previously annotated genes were examined. These sequences were used as input for a web-based blastx query in order to verify homology to putative chemosensory genes and identify ORF orientation. In order to reduce the possibility of mis-annotating two uncoupled fragments of the same gene as distinct genes, only sequences with ORFs greater the 50% of the average length of a complete ORF in a given gene family (OR = 406 amino acids (aa), GR= 447 aa, IR= 676 aa, OBP = 178 aa, CSP= 132 aa, CXE= 563 aa, CYP= 510 aa) were included for further analysis. The protein sequences of novel gene candidates identified as described here were incorporated into fasta files for each gene family, and an additional tblastn query was performed against the Trinity.fasta nucleotide databases in order to determine if any further gene candidates would be identified.de novo generated transcriptomes and subsequent expression level abundance estimations were carried out, as described [Read mapping of individual sample reads to the escribed  with theescribed , versionescribed  and versescribed . Subsequescribed  of each S. littoralis ORs were compared to sequences from Bombyx mori [Epiphyias postvitanna [Heliconius melpomene [S. littoralis GRs were compared to sequences from B. mori [Helicoverpa armigera [. S. littoralis IRs were compared to sequences from B. mori and D. melanogaster [Manduca sexta [Cydia pomonella [Danaus plexipus [S. littoralis OBPs were compared to sequences from B. mori [H. melpomene [S. littoralis CSPs were compared to sequences from B. mori [H. melpomene [S. littoralis CXEs were compared to sequences from B. mori [E. postvitanna [S. littoralis P450s were compared to sequences from B. mori [D. melanogaster [For the qualitative report of gene family transcripts, published sets of genes from different species were used for comparison with our data. byx mori , 41, Epitvitanna  and Helielpomene . S. litt B. mori , and Helarmigera . S. littnogaster , Manducaca sexta , Cydia pomonella  and Danaplexipus . S. litt B. mori  and H. melpomene . S. litt B. mori  and H. melpomene . S. litt B. mori  and E. ptvitanna . S. litt B. mori  and D. mnogaster .https://mafft.cbrc.jp/alignment/server/) through the FFT-NS-i iterative refinement method, with JTT200 scoring matrix, “leave gappy regions” set, and other default parameters [Amino acid sequences for each gene family were aligned using MAFFT online version 7.220  according to the manufacturer's protocol. PCR assays were performed, with the Dream Taq Green master mix system (Thermo Fisher Scientific), on cDNA from single biological samples of virgin male and female antennae, proboscis and brain. Specific primer pairs Additional file 2:Bioinformatic Information for Male and Female Transcriptomes. Contains basic information on the transcriptomes at various stages of processing, including total number of sequences, mean sequence length, N50 and other parameters. (XLSX 35 kb)Additional file 3:Novel Candidate Chemosensory Gene Informatics. Includes annotation name, ORF size, best blast hit and other parameters. (DOCX 178 kb)Additional file 4:Estimated Transcript Abundance Peak Values by Gene Family, Tissue and Sex, Fragments per Kilobase per Million (FPKM). (DOCX 15 kb)Additional file 5:S. littoralis OR protein sequences. Contains updated amino acid sequences for all annotated S. littoralis odorant receptors. (TXT 24 kb)Additional file 6:Candidate Chemosensory Gene Abundance Estimates. Contains “FPKM” and “estimated mapped reads” values for candidate chemosensory gene transcripts, sorted by chemosensory gene family, sex and tissue type. (XLSX 47 kb)Additional file 7:S. littoralis GR protein sequences. Contains updated amino acid sequences for all annotated S. littoralis gustatory receptors. (TXT 6 kb)Additional file 8:S. littoralis GRs. Estimation of abundance values determined by read mapping. Black indicates low/no expression, dark colors indicate low/moderate expression, bright colors indicate moderate/high expression. Color plots represent binary log of FPKM plus one for each gene Additional file 10:S. littoralis OBP protein sequences. Contains updated amino acid sequences for all annotated S. littoralis odorant binding proteins. (TXT 9 kb)Additional file 11:S. littoralis (Slit), Heliconius melpomene (Hmel) and Bombyx mori (Bmor). Branches containing “Plus-C” subfamily OBPs are colored green; branches containing “Minus-C” subfamily OBPs are colored blue; branches containing putative pheromone binding proteins (PBPs) and general odorant binding proteins (GOBPs) are colored orange; S. littoralis OBPs are indicated with a larger bold font, and novel S. littoralis OBPs are marked with a “•”. Node support was assessed with 600 bootstrap replicates and values greater than 70% are shown. (PDF 368 kb)Maximum likelihood cladogram of candidate SlitOBP sequences with other lepidopteran OBP sequences. Unrooted. Includes sequences from Additional file 12:S. littoralis CSP protein sequences. Contains updated amino acid sequences for all annotated S. littoralis chemosensory proteins. (TXT 2 kb)Additional file 13:S. littoralis (Slit), Heliconius melpomene (Hmel) and Bombyx mori (Bmor). S. littoralis CSPs are indicated with a larger bold font, and novel S. littoralis CSP is marked with a “•”. Node support was assessed with 600 bootstrap replicates and values greater than 70% are shown. (PDF 283 kb)Maximum likelihood cladogram of candidate SlitCSP sequences with other lepidopteran CSP sequences. Unrooted. Includes sequences from Additional file 14:S. littoralis CXE/CCE protein sequences. Contains updated amino acid sequences for all annotated S. littoralis carboxy/cholinesterase proteins. (TXT 29 kb)Additional file 15:S. littoralis (Sl), E. postvittana (Epos) and Bombyx mori (Bm). S. littoralis CXE/CCEs are indicated with a larger bold font, and novel S. littoralis CXE/CCEs is marked with a “•”. Node support was assessed with 600 bootstrap replicates and values greater than 70% are shown. (PDF 386 kb)Maximum likelihood cladogram of candidate SlitCXE sequences with other lepidopteran CXE/CCE sequences. Unrooted. Includes sequences from Additional file 16:S. littoralis CXE/CCEs. Estimation of abundance values determined by read mapping. Black indicates low/no expression, dark colors indicate low/moderate expression, bright colors indicate moderate/high expression. Color plots represent binary log of FPKM plus one for each gene Additional file 18S. littoralis (Sl), D. melanogaster (Dmel) and B. mori (Bmor). Branches containing mitochondrial clan CYPs are colored red. S. littoralis CYPs are indicated with a larger bold font, and novel S. littoralis CYPs are marked with a “•”. Node support was assessed with 600 bootstrap replicates and values greater than 70% are shown. (PDF 532 kb)Maximum likelihood phylogenetic tree of candidate SlitCYPs sequences with other insect CYP sequences. Unrooted. Includes sequences from Additional file 19:Heat-plot of relative expression values for SlitCYPs. Estimation of abundance values determined by read mapping. Black indicates low/no expression, dark colors indicate low/moderate expression, bright colors indicate moderate/high expression. Color plots represent binary log of FPKM plus one for each gene Additional file 21:supplemental materials and methods. (DOCX 22 kb)Additional file 22:PCR primer sequences and expected fragment sizes. (XLSX 10 kb)Additional file 23:uncropped PCR gels, including all experimental assays and no template-controls. For all gels, 1kb Gene Ruler ladder (Thermo Fisher Scientific) was used . MVA – male virgin antennae, FVA – female virgin antennae, MVPR – male virgin proboscis, FVPR, female virgin proboscis, MVBR – male virgin brain, FVBR – female virgin brain. On some gels, part of the gel space was used for experiments unrelated to this manuscript; those sections are indicated as “not applicable.” (PDF 5006 kb)Additional file 24:uncropped PCR gels, including all no-RT controls. For all gels, 1kb Gene Ruler ladder (Thermo Fisher Scientific) was used. MVA – male virgin antennae, FVA – female virgin antennae, MVPR – male virgin proboscis, FVPR, female virgin proboscis, MVBR – male virgin brain, FVBR – female virgin brain, N/A – not applicable. On some gels, part of the gel space was used for experiments unrelated to this manuscript; those sections are indicated as “not applicable.” (PDF 3777 kb)


Histomonas meleagridis are non-existent. The present study employed traditional 1D-in-gel-zymography (1D-IGZ) and micro-LC-ESI-MS/MS (shotgun proteomics), to investigate H. meleagridis exoproteomes, obtained from a clonal virulent and an attenuated strain. Both strains were maintained as mono-eukaryotic monoxenic cultures with Escherichia coli. We demonstrated active in vitro secretion kinetics of proteases by both parasite strains, with a widespread proteolytic activity ranging from 17 kDa to 120 kDa. Based on protease inhibitor susceptibility assay, the majority of proteases present in both exoproteomes belonged to the family of cysteine proteases and showed stronger activity in the exoproteome of a virulent H. meleagridis. Shotgun proteomics, aided by customized database search, identified 176 proteins including actin, potential moonlighting glycolytic enzymes, lytic molecules such as pore-forming proteins (PFPs) and proteases like cathepsin-L like cysteine protease. To quantify the exoproteomic differences between the virulent and the attenuated H. meleagridis cultures, a sequential window acquisition of all theoretical spectra mass spectrometric (SWATH-MS) approach was applied. Surprisingly, results showed most of the exoproteomic differences to be of bacterial origin, especially targeting metabolism and locomotion. By deciphering such molecular signatures, novel insights into a complex in vitro protozoan- bacteria relationship were elucidated.The exoproteome of parasitic protists constitutes extracellular proteins that play a fundamental role in host-parasite interactions. Lytic factors, especially secreted proteases, are capable of modulating tissue invasion, thereby aggravating host susceptibility. Despite the important role of exoproteins during infection, the exoproteomic data on  Histomonas meleagridis is a unicellular microaerophilic flagellate pathogen causing histomonosis (blackhead disease) in gallinaceous birds with a worldwide prevalence. Histomonosis in poultry is of significant importance, as it causes high mortality in turkeys and production losses in chickens  and proteins from the common Repository of Adventitious Proteins (cRAP) (n = 117) (ftp://ftp.thegpm.org/fasta/cRAP/crap.fasta) [downloaded: 18.10.2017]. Acquired raw data were processed with ProteinPilot Software version 5.0  for re-calibration and database searches. Mass tolerance in MS mode was set with 0.05 and 0.1 Da in MSMS mode for the rapid recalibration search, and 0.0011 Da in MS and 0.01 Da in MSMS mode for the final search. Parameters for database search were: trypsin digestion, cysteine alkylation set to iodoacetamide, search effort set to rapid ID. False discovery rate analysis (FDR) was performed using the integrated tools in ProteinPilot Software version 5.0 . Global false discovery rate was set to <1% on protein level. For quantification of proteins, IDA identification results were used to create the SWATH ion library with the MS/MS (ALL) with SWATH Acquisition MicroApp 2.0 in PeakView 2.2 . Peptides were chosen based on a FDR rate <1%, excluding shared and modified peptides. Up to 6 peptides per protein and up to 6 transitions per peptide were used. MarkerView 1.2.1  was used for calculation of peak areas of SWATH samples after retention time alignment and normalization using total area sums.The database consisted of translated amino acid sequences originating from the 3356 non-redundant contigs of the criptome  [translaStatistical evaluation was performed using R programming language. cRAP proteins and proteins quantified with just one peptide were removed from Marker View raw protein list before further processing. Raw peak areas after normalization to total area sums were log2-transformed to approach a normal distribution. On a logarithmic scale, technical replicates were aggregated by arithmetic mean before application of statistical tests. Differential expression of proteins was assessed using two-tailed t-Test for independent samples for each protein. To adjust for multiple testing, the method of Benjamini and Hochberg was used to control the FDR. Protein expression was considered differential if the adjusted p-value was below α = 0.05 and the absolute fold change was at least two (fold change < −2 or > +2).H. meleagridis transcriptome [Using pre-existing annotations from the criptome  the idenH. meleagridis exoproteins were submitted to SignalP 4.1 server [E. coli exoproteins identified by SWATH analysis to be differentially expressed were also subjected to analysis via SignalP 4.1 and SecretomeP 2.0 server.In order to detect structural features indicative for protein secretion by detecting N-terminal signal peptides predicting a possible secretion, the translated amino acid sequences of the 176 1 server  using de1 server  employinH. meleagridis exoproteomes were clustered according to their biological process. A color coded cytoscape [H. meleagridis culture.Proteins quantified to be differentially expressed between the virulent and the attenuated ytoscape  network H. meleagridis cells were monitored under serum-free conditions. Under such conditions, the cell count of virulent H. meleagridis remained constant up to 6h, which was followed by a steady decline. This indicated that upon the medium exchange, cells stopped to divide but the progressive cell death started only after 6 hours of incubation in medium without serum  of approximately 17kDa, 22 kDa, 30 kDa, 36 kDa, 68 kDa and 120kDa  in the former at any time-point except 0 hours  [H. meleagridis transcriptome. The SignalP algorithm detected 17 proteins possessing the N-terminus signal peptide, whereas SecretomeP 2.0 algorithm predicted 37 proteins to be secreted independently of signal peptides via non-classical pathways   identifypathways . In addiproteins  Tables.H. meleagridis cultures were compared by SWATH-MS to determine the difference in protein expression. The SWATH data from two injections for each replicate were analysed by ProteinPilot Software version 5.0 . Data analysis  resulted in the identification of 35 differentially expressed proteins , with two cathepsins and one legumain protease displaying a theoretical MM of 34.88 kDa, 35.65 kDa and 47.25 kDa, respectively. Correlating this data to the 1D-IGZ results, it seems that the majority if not all of the proteolytic activities up to ~ 40 kDa in the H. meleagridis exoproteome, stem from the cysteine proteases. Discrepancy of few kDa between the theoretical and actual MM as observed in here is not unusual and discrepancies of up to 13 kDa have been reported [H. meleagridis transcripts may not be of full length.The shotgun proteomics analysis of the reported . Proteinreported , which cH. meleagridis exoproteome remained predominantly inert to PMSF, suggesting a sparse distribution of serine proteases. In agreement with this, the shotgun proteomic approach identified only a single serine protease in the exoproteome with one confident peptide , glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and triose-phosphate isomerase (TPI), in the sminogen . The rectomonads , 41. Howaginalis ,43 Bindiaginalis . Using taginalis . The prest cells . An earlgenosome . The resH. meleagridis exoproteome particularly surfactant B/saposin-like proteins, zinc dependent phospholipase C and N-acetylmuramoyl-L-alanine amidase. Most of these lytic molecules operate by attacking target cell membrane and compromising cellular integrity [H. meleagridis to launch assaults against co-incubating bacteria, a function they might display in vivo against host cells.Data mining also revealed the presence of several lytic molecules in the ntegrity –49. In tH. meleagridis cultures. The majority of the differentially expressed proteins were of E. coli origin whose detection was not surprising due to the monoxenic co-cultivation with E. coli DH5α. The applied statistical parameters filtered only two H. meleagridis proteins as differentially expressed, a C2 domain containing protein and a RNA-binding protein, both over-expressed in the virulent exoproteome. Both proteins have a wide spectrum of functions [2+), hence sometimes referred to as a Ca2+ dependent lipid binding domains [H. meleagridis, might indicate a possible cytoadherence mechanism that involves the interaction of the parasite’s C2 motif protein and host cells.The SWATH-MS approach was applied to obtain a quantitative proteome expression between the virulent and the attenuated unctions ,51. The unctions . The maj domains . Lipid-b domains ,54. The H. meleagridis derived exRNA-binding motif in the disruption of the host cell signalling.The RNA-binding proteins engage in the formation of ribonucleoprotein complexes by binding to sequence/structural motifs in the RNA, which can alter the fate of that RNA molecule. The preH. meleagridis proteins were detected as significantly over-expressed. One possible explanation would be that the serum-free conditions provoked a stress effect and by that possibly reduced the overall metabolism of both H. meleagridis strains which lead to a minimization of excreted proteins. Additionally, since the attenuated H. meleagridis strain originated from the virulent by prolonged in vitro cultivation, both strains are genetically very similar [H. meleagridis proteome analysis study somewhat argues for the first hypothesis, since the use of identical H. meleagridis strains and the SWATH MS approach identified 42 differentially expressed H. meleagridis proteins [H. meleagridis strains in in vitro conditions is endogenousIt is intriguing to consider why only two  similar . The recproteins . HoweverE. coli origin. This demonstrates the rapid change in the prokaryote’s gene expression which is dependent on the parasite’s phenotype. At the same time, it indicates a strong interaction between H. meleagridis and bacteria. This relationship is particularly thought-provoking, as the bacteria are not only required for proliferation of H. meleagridis in vitro; but their presence in the intestines of host birds is essential for successful infection [H. meleagridis growth in vitro [H. meleagridis and bacteria [H. meleagridis -prokaryote relationship is still unresolved.The majority of differentially expressed proteins identified in the present study were of nfection . Furtherin vitro . A recenbacteria . HoweverE. coli proteins over-expressed in the H. meleagridis virulent background were involved in the flagellar motility system or carbohydrate uptake. It seems that the low glucose environment of Medium 199 induced the uptake mechanisms of carbohydrate sources, such as maltodextrin or D-allose. The MalE protein was also shown to be important in glucose transport under glucose-limited conditions [E. coli is unable to use rice starch due to the absence of endogenous amylolytic enzymes [H. meleagridis is able to hydrolyse starch molecules [H. meleagridis to severely minimize/or stop metabolizing, since even in optimal conditions, this strain of the parasite is not well adapted to in vitro growth and shows much lower metabolic rates as compared to the attenuated parasite [The nditions . Aside fnditions . The med enzymes , glucoseolecules , and couparasite .E. coli proteins up regulated during co-cultivation with attenuated H. meleagridis indicate a stress response toward the parasite. This particularly concerns ATP-dependent Clp protease (ClpX), hydrogen peroxide and cadmium resistance periplasmic protein (YgiW) and CsbD family protein, which were all described to be associated with stress response [in vitro growth, the attenuated H. meleagridis seems to cope more successfully with the serum-free conditions than its’ virulent counterpart [H. meleagridis phagocytised co-cultivating E. coli more effectively than the virulent parasite, which in turn resulted in the up-regulation of E. coli stress proteins. In addition, the up-regulation of the tryptophanase (TnaA) together with proteins involved in the formation of type -1 fimbriae (FimA and FimC), detected in the attenuated H. meleagridis exoproteome, can be related to the interaction with the parasite. Type-1 fimbriae are most likely involved in the direct contact with the parasite, since they enable the bacteria to bind specific receptor structures [E. coli towards phagocytosis by the parasite. The tryptophanase is an enzyme responsible for the inter-conversion between tryptophan and indole [Vibrio cholerae, active tryptophanase and the presence of indole are related to grazing resistance against the phagocytic eukaryote Dictyostelium discoideum [E. coli–histomonad interaction has to be investigated in more detail. However, considering that a reduction of E. coli during co-cultivation with H. meleagridis was not observed [E. coli AdhP alcohol dehydrogenase in the exoproteome of attenuated H. meleagridis is especially intriguing. Unlike the more common alcohol dehydrogenase AdhE, the AdhP is an ethanol-inducible dehydrogenase whose expression is not linked to anaerobic conditions [H. meleagridis culture indicates an increased concentration of ethanol in the media, whose likely contributor is the parasite itself. This situation resembles low-iron-dependent changes described for Tritrichomonas foetus, in which the pyruvate metabolism is shifted to the cytoplasm resulting in the production of ethanol and some glycerol [H. meleagridis, indicates the requirement for iron supplementation in order to maintain normal hydrogenosomal metabolism and energy production [in vitro conditions the serum represents an important iron source that is absent in the current experiment. Therefore, it is likely that due to the lack of iron, the majority of hydrogenosomal metabolism is inhibited and pyruvate is utilized in the cytoplasm, resulting in the production and subsequent secretion of the ethanol [E. coli AdhP enzyme, in turn, converts ethanol to acetaldehyde which can again be used by the protozoan cytoplasmic fermentive metabolism, releasing the reduced NADP+ molecules and maintaining redox balance. When compared to the virulent H. meleagridis culture, there seems to be no elevation in ethanol concentration in the media. Based on the serum-free growth and previous reports [in vitro maladaptive state, dramatically slowed down its’ metabolism just for mere survival purpose. Overall, this underlines the complexity of the bacteria-parasite interaction.In contrast to the virulent culture, response –63. As anterpart ,16. It iructures . Howeverd indole . The mord indole . In Vibrscoideum . Whetherobserved , it is pnditions . Its’ upglycerol . The exioduction . In in v ethanol . The E.  reports ,16, it iin vitro secretion kinetics assay of proteases demonstrated the significance of these lytic molecules in H. meleagridis extracellular milieu, showing a time-dependent secretion from as early as 2 hours post-incubation. Comparative 1D-IGZ analysis between the virulent and the attenuated H. meleagridis, displayed the robustness of proteases secreted by the virulent strain. The protease inhibitor susceptibility assay revealed cysteine proteases to be the predominant lytic molecules in the H. meleagridis exoproteome, with the higher potency in cultures of virulent parasites. The comprehensive analysis of the H. meleagridis exoproteome using high-resolution proteomics identified a pool of 176 proteins, providing an extensive molecular overview of the H. meleagridis extracellular milieu and indicating its’ dynamic micro-environment. The quantitative exoproteome analysis of the virulent and the attenuated H. meleagridis cultures displayed differences in proteins of co-cultivating E. coli, which in turn reflected the protozoa-bacteria interaction for the first time at the molecular level. Differences between H. meleagridis-derived exoproteins were almost non-existent, suggesting that in in vitro conditions the majority of proteomic alterations are endogenous, as previously observed in our comparative proteome studies. Overall, the present study investigating the exoproteome of H. meleagridis provides an attractive avenue for further research, hypothesis testing and comparative analysis of strains with different biological properties.The S1 TableH. meleagridis specific proteins identified in the exoproteome with ≥2 confident peptides in at least 3 replicates. Information on SignalP and SecretomeP data, number of confident peptides, the % of protein sequence coverage (>95% confidence) and the number of ambiguous accessions is given. The total ProtScore means the score with all peptides (unique and shared). The Unused ProtScore is the score using only the unique peptides. Term “ALL” represents combined data for all 6 replicates. The LP1, LP2, and LP3 are 3 replicates of the H. meleagridis virulent strain . The HP1, HP2 and HP3 are 3 replicates of the H. meleagridis attenuated strain .The list contains (XLSX)Click here for additional data file.S2 TableH. meleagridis strain. Folders named “Prot_HMx_319_A”, “Prot_HMx_319_B”, “Prot_HMx_319_C” contain lists of proteins identified in the exoproteome of each biological replicate  of the high passsage H. meleagridis strain. Folders named “Ambiguous Accessions_HMx_28 or 319_A or B or C” contain lists of all ambiguous accessions for the given protein identified in the exoproteome of each biological replicate  of either LP (28) or HP (319) H. meleagridis. The last folder named “Contigs-accessions” contains the list of all contigs in H. meleagridis transcriptome together with their corresponding accession number and the annotation.The file contains 15 folders. Folders named “Prot_HMx_all” and “Ambiguous Accessions_HMx_all” contain lists of all identified proteins and their ambiguous accessions. The total ProtScore means the score with all peptides (unique and shared). The Unused ProtScore is the score using only the unique peptides. Folders named “Prot_HMx_28_A”, “Prot_HMx_28_B”, “Prot_HMx_28_C” contain lists of proteins identified in the exoproteome of each biological replicate  of the low passsage (XLSX)Click here for additional data file.S3 TableThe file contains two folders. Folder named “Protein abundances incl. t-test” contains detailed output of the statistical analysis including absolute and relative abundances as well as p-values for all quantified proteins. Folder named “Peptide abundances” contains a list of unique peptides used for the quantification of each protein. Information on peptide sequence, peptide charge, mass (m/z), retention time and corresponding protein accession is given. All absolute peptide abundances are shown.(XLSX)Click here for additional data file.