Ancylostoma braziliense, Ancylostoma ceylanicum, Ancylostoma caninum and Uncinaria stenocephala are not only capable of producing morbidity and mortality in dogs but are also neglected tropical zoonoses. Each hookworm species differs considerably in its geographical distribution, life cycle, biology, pathogenic impacts on both canine and human hosts, zoonotic potential, and response to treatment with anthelminthics. Here we describe the development and validation of two Taq-Man based multiplex PCR assays capable of detecting and differentiating all four canine hookworm species in faeces of naturally infected dogs. The analytical sensitivity of both assays was assessed using 10-fold serial dilutions of synthetic gene block fragments containing individual sequence targets of each hookworm species. The sensitivity of the assays and ability to detect mixed species infections were compared to a conventional PCR-Restriction Fragment Length Polymorphism based-approach when applied to laboratory and field samples from endemic areas.The canine hookworms A. caninum (47%), U. stenocephala (33%) and A. ceylanicum (0.02%) that were missed by PCR-RFLP. These multiplex qPCR assays will assist field based epidemiological surveillance studies towards an accurate and sensitive monitoring of canine hookworm infections in dogs, to inform their species-specific zoonotic risks to populations living in endemic areas, globally.The qPCRs detected at least one species of hookworms in 82.4% of PCR-RFLP-negative but microscopy-positive samples. The qPCRs detected an additional 68% mixed infections with different species of canine hookworms, and additional single species infection with  Ancylostoma braziliense, Ancylostoma ceylanicum, Ancylostoma caninum and Uncinaria stenocephala. Each hookworm species differs in its geographical distribution, impact on canine health and more importantly its ability to infect and produce disease in humans. For example, dogs are the primary reservoir for A. ceylanicum, the second most common hookworm species infecting humans in the Asia Pacific. Ancylostoma braziliense is more geographically confined and responsible for chronic skin eruptions in humans. Accurate diagnosis of hookworm species in dogs is therefore essential for understanding the relative risk each species pose to human populations living in close association with dogs. As microscopic methods are unable to distinguish between species of hookworms in stool, highly efficient real time PCR assays capable of detecting and differentiating all four canine hookworm species in canine faeces were developed. These new assays are superior to previously published gel-based PCR assay displaying higher throughput, and ability to correctly identify single and mixed hookworm species infections with higher accuracy.There are four species of blood-sucking hookworms that infect dogs, namely  Ancylostoma braziliense, Ancylostoma ceylanicum, Ancylostoma caninum and Uncinaria stenocephala are widely distributed soil-transmitted helminths causing morbidity in dogs and are agents of zoonoses [A. caninum, A. braziliense and U. stenocephala transmission by dogs preying on paratenic hosts is also possible [A. caninum [A. caninum, is a common cause of haemorrhagic diarrhoea and death in pups and chronic iron deficiency anaemia in adult animals [A. braziliense is by far the most frequently implicated, as it is the only species capable of causing classical ‘creeping eruptions’, a prolonged highly pruritic and serpiginous eruption within the dermis that may persist for over 100 days, if untreated [A. caninum is a well-recognized agent of eosinophilic enteritis and aphthous ileitis. Although most infections are asymptomatic, a single immature adult worm residing in the small intestine is capable of eliciting abdominal pain, intestinal bleeding, diarrhoea and weigh loss [A. caninum eggs in the faeces of human patients highlights its potential to complete its life cycle in humans [The canine hookworms zoonoses –8. Each zoonoses ,9–15. Dopossible –8,16. Tr caninum ,18. Infe animals . All can animals . In humantreated . In humaigh loss ,21,22. Rn humans .A. ceylanicum on the other hand, is the only canine (and feline) hookworm species known to produce patent, long-lived infection within the intestinal tract of humans both in both natural and experimental infections [A. ceylanicum is the second most common hookworm infecting humans after Necator americanus and is responsible for between 5.5–51.6% of human infections in Laos, Malaysia, Thailand, Cambodia and the Solomon Islands [Ancylostoma spp. are mostly found in subtropical and tropical regions and may co-exist as sympatric species and/or be featured by specific ecological requirements with implications for their distribution. For instance, A. caninum is endemic in wet and dry regions of the tropics and subtropics globally, while A. ceylanicum thrives in the wet tropics and subtropics of Africa, Asia and Oceania. Ancylostoma braziliense appears limited in distribution to a narrow latitudinal areas spanning approximately 5°N to 10°S of Brazil, Kenya, Tanzania, northern Australia, Indonesia and Malaysia [U. stenocephala, inhabits predominantly temperate and colder climate such as those of Europe, Canada, southern Australia and the United States of America [fections –30. In t Islands . Ancylos America ,36.Ancylostoma-infected dogs in these tropical climates is exacerbated by the co-existence of several risk factors that allow the parasite to thrive in dogs and significantly increase their risk of infecting humans i.e. free ranging community dogs, a lack of veterinary care, irregular deworming, roaming behaviour and poor environmental hygiene [In semi-domesticated dogs residing in urban and rural areas of Asia, Africa and South America, between 40–100% of dogs may be infected with hookworms contributing to a high environmental dispersal of potentially zoonotic infectious larvae , 29–32.  hygiene ,37–39.Diagnosis of canine hookworm infections has traditionally relied on the identification of eggs isolated in faecal floatation and species-based identification by examining adult worms following necropsies or anthelmintic treatment ,40. For A. ceylanicum, A. caninum and U. stenocephala by PCR-RFLP were selected [A. caninum, 60 A. ceylanicum and 4 U. stenocephala by PCR-RFLP [A. braziliense. This negative faecal sample was collected from a fully-dewormed pet dog residing in Melbourne, Victoria, and demonstrated free of hookworm eggs using sodium nitrate faecal floatation (SG 1.20). The hookworm-free faecal sample was divided into equal aliquots and spiked with serial dilution of genomic A. braziliense DNA belonging to an existing collection of hookworm genomic DNA of Prof. Rebecca Traub (University of Melbourne). DNA concentrations were quantified using a Qubit 4 Fluorometer  as per manufacturer’s instructions. Ten-fold serial dilutions of 0.219 ng/μl genomic DNA were prepared and spiked into seven tubes containing 150 mg of faeces each and 150 mg faeces with nuclease free water was used as negative control.A total of 191 faecal samples previously demonstrated positive for selected . Of thesselected  and fromselected . Of thesPCR-RFLP . Twelve DNA was extracted from faeces using the ISOLATE Fecal DNA Kit  according to the manufacturer’s instructions. Final DNA elution was made in 100 μl of elution buffer.A. braziliense, A. ceylanicum, A. caninum and U. stenocephala. Reference nucleotide sequences for the ITS-1 region of A. caninum , A. ceylanicum , U. stenocephala  and A. braziliense  were obtained from NCBI and aligned with GenBank sequences sourced from the available range of geographical isolates for each hookworm species, as well as other closely related canine helminths using Clustal Omega   and their genus or species-based specificity assessed using NCBI Blast (http://www.ncbi.nlm.nih.gov/blast). The primers were manufactured by Integrated DNA Technologies, USA. The probes were designed and manufactured using LNA (locked nucleic acid) technology  to increase probe specificity. A four-channel Magnetic Induction Cycler  was used for the amplification, detection, and data analysis (micPCR software).The multiplex qPCR assays were designed to detect the ITS-1 rRNA region of ustalo/) . PrimersA. caninum and A. ceylanicum ITS-1 rRNA. The second qPCR used a common primer pair UncbrazF and UncbrazR to amplify a 119 bp and 118bp region of the ITS-1 rRNA of A. brazilienze and U. stenocephala, respectively. Equine herpes virus (EHV4) primers (EHVF and EHVR), probe (EHV probe) and genomic DNA were used as reaction internal control, and mammalian primers (Mam F and Mam R) and probe (MAM PROBE) designed to target a 92 bp region of the 16S mitochondrial gene of mammals was used as a DNA extraction control for both qPCR assays.Two qPCR multiplex assays were developed. The first reaction used a common primer pair designed (AcanceyF and AcanceyR) to amplify a 103 bp region of Optimisation of each singleplex and multiplex assays were carried out using synthetic double stranded DNA fragments  containing individual sequence targets of each hookworm species. Progressive concentrations of primer pairs and probes were tested to select the concentrations that guaranteed the highest efficiency and the greatest sensitivity.A. caninum and A. ceylanicum and 0.1 unit for A. braziliense and U. stenocephala. The fluorescent threshold was set to 5% for all targets. The cycling conditions were identical for both reactions: 2 min at 95°C, followed by 40 cycles of 15 sec at 95°C and 1 min at 60°C. gBlocks Gene Fragments were used as positive controls and nuclease-free water was used as negative control in all runs. The efficiency and the intra-assay reproducibility of multiplex reactions were directly compared to each singleplex reaction by plotting Ct versus 10-fold serial dilutions of target gBlock Gene Fragments of known DNA concentration. The correlation coefficients (R2) of the standard curves were produced automatically by micPCR software .The qPCR assays were conducted in 20 μL reactions containing 10 μL of GoTaq Probe qPCR Mastermix , 1 μL of known quantity of EHV4 gDNA, and 2 μL of template DNA. Nuclease free-water was added to reach the final reaction volume. The cycling threshold was set at 0.2 units for A. caninum, 7,17 ng/μl for A. ceylanicum, 13.2 ng/μl for A. braziliense and 7.22 ng/ μl for U. braziliense). In addition, the analytical sensitivity of the qPCRs and PCR-RFLP was assessed and compared using 10-fold dilutions of known concentration of genomic DNA  sourced from an existing collection of hookworm genomic DNA of Prof. Rebecca Traub (University of Melbourne).The analytical sensitivity  of the Echinococcus granulosus sensu lato, Dipylidium caninum, Taenia spp., Spirometra erinacei, Strongyloides stercoralis, Giardia duodenalis, Trichuris vulpis, Toxocara spp., and Neospora caninum were used. Additionally, we tested the newly developed qPCRs against the common human hookworm Necator americanus.To investigate the analytical specificity of the assay (i.e. the ability of an assay to exclusively identify a particular target substance or organism rather than similar but different substances or organisms ) genomicThe diagnostic sensitivity  was assAssessment of the overall diagnostic specificity  of the qPCRs was not possible due to truly negative samples being unavailable. Given that microscopy negative samples cannot be considered as truly negative for hookworm infection , microsck) <0.00, slight if 0.00≤ k ≤0.20, fair if 0.21≤ k ≤0.40; moderate if 0.41≤ k ≤0.60, substantial if 0.61≤ k ≤0.80 and almost perfect if k >0.80. The 95% confidence intervals (95% CI) were calculated using the Wald method.Comparison of the PCR-RFLP and qPCRs was therefore performed relying on Kappa statistics  using Ex2 from 0.929 to 0.999 and Slope from -2.904 to -3.716  for U. stenocephala, 38.27 (S.D.± 0.06) for A. braziliense, 36.89 (S.D.± 0.368) for A. ceylanicum and 37.62 (S.D. 0.30) for A. caninum corresponding to 1.4 x 10−3 fg of target gBlock Gene DNA for A. ceylanicum, 2.6 x 10−3 fg of target gBlock Gene DNA for A. braziliense, 1.4 x 10−3 fg for U. stenocephala and 3.8 x 10−4 fg for A. caninum.The analytical sensitivity of the assay was 0.0146 ng genomic DNA of A. ceylanicum, 0.00033 ng and 0.033 ng for A. caninum, 0.00058 ng and 0.0058 ng for A. braziliense, and 0.0078 ng and 0.78 ng for U. stenocephala.The limit of detection of the qPCRs and PCR-RFLP were respectively 0.00028 ng and 0.0028 ng for The multiplex qPCRs displayed a diagnostic sensitivity using microscopy as the gold standard of 97.3% . The diagnostic sensitivity of the PCR-RFLP against microscopy was 84.7% .Neither multiplex qPCR assays cross reacted with genomic DNA of the tested parasites. No cross reactions with other target hookworm species was observed, thereby showing high analytical specificity.A. caninum . Substantial agreement was shown for A. ceylanicum  and U. stenocephala .Performance of the canine hookworm multiplex qPCRs were determined using 191 DNA samples sourced from previous canine hookworm surveys in Vietnam and Queensland. Field samples, positive controls, EHV4 and mammalian DNA amplified within the expected Cq values. The detection of the signal derived from the amplification of mammalian DNA in all the samples confirmed the success of the extraction step. The multiplex qPCRs showed greater diagnostic sensitivity for the detection of hookworms in comparison to the PCR-RFLP . The results for the multiplex quantitative qPCR and the PCR-RFLP are summarized in Here we propose and validate two Taq-Man probe-based multiplex qPCRs for the detection of zoonotic species of canine hookworms. These diagnostic tools will assist large epidemiological field studies toward a timely surveillance and monitoring of canine hookworm infections in dogs and provide information on species-specific zoonotic risks to populations living in endemic areas, globally.A. ceylanicum alone estimated to infect up to 73 million people in the Asia Pacific [While several qPCR assays have been developed for high-throughput screening of human hookworm species ,53–55, t Pacific –35. SimiA. ceylaniucm and A. braziliense and 100-fold more sensitive for A. caninum and U. stenocephala.The qPCRs herein described demonstrated higher diagnostic sensitivity (97%) in comparison to previously developed PCR-RFLP (84%) by Ng-Nguyen et al. (2015). For instance, our qPCRs successfully detected at least one species of hookworms in the 82.4% of PCR-RFLP-negative but microscopy-positive samples. The absolute sensitivity of the qPCRs was 10-fold more sensitive than the PCR-RFLP for A. caninum single infections (47%), two U. stenocephala (33%) and one A. ceylanicum (0.02%) missed by PCR-RFLP. A small number of samples were qPCR-negative and PCR-RFLP positive however, it is likely that these samples were PCR-RFLP false-positives associated with the subjective and potentially inaccurate interpretation of gel electrophoresis results. These assays have proven specific as it detected all the four canine hookworms tested without cross reacting when tested against a range of 10 different intestinal parasites of dogs and the human hookworm N. americanus.One of the main advantages of our qPCRs was the capability of detecting additional mixed hookworm species infections (68%) compared to PCR-RFLP. In addition, the qPCRs detected an additional 48 A. caninum from A. duodenale DNA, given the high degree of nucleotide identity between DNA sequences of these two closely related hookworm species. The ability to differentiate canine from human hookworm species becomes apparent in resource-poor communities that practice outdoor defaecation, as dogs are known to be coprophagic [A. duodenale has been demonstrated to produce self-limiting infections in experimentally infected puppies in a non-controlled study [A. caninum-endemic settings as the transmammary route of infection in pups is likely to provide cross-protective immunity against its closely related human-hookworm counterpart. Despite this limitation, the likelihood of false positive A. caninum infection in dogs as a result of the coprophagy or patent infections with A. duodenale appears negligible. DNA of Ancylostoma duodenale was detected in a single canine faecal sample in Turkana, Kenya at an overall prevalence of <0.062%, even in the presence of widespread canine coprophagic behaviour [The primary disadvantage of the qPCR is its inability to differentiate rophagic . Moreoveed study , a phenoehaviour .A. caninum infections. The primary reason for this was the qPCRs assays’ increased sensitivity for the detection of A. caninum and mixed hookworm-species infections. Good agreement (0.61 ≤|κ|≤ 0.80) was instead shown for A. ceylanicum and U. stenocephala.Kappa statistics showed only fair agreement (0.41 ≤|κ|≤ 0.6) between the qPCRs and the PCR-RFLP for mixed and single The primary limitation of the study is that only microscopy positive samples were subjected to the PCR-RFLP assays and therefore diagnostic specificity was not assessed. Further studies testing the relationship between Cq values/copy numbers to egg numbers are needed before these qPCRs can be used as quantitative tools to assess infection intensity levels. The deployment of more sensitive techniques such as the qPCR assays herein validated, acquires pivotal importance especially in areas where preventive chemotherapy has been employed for many years and where conventional techniques may fail to detect light-intensity infections (i.e. shedding low number of eggs) ,60. The These newly developed qPCRs will facilitate large-scale detection and proper identification of canine hookworm species, globally, by researcher dealing with the assessment of STHs in endemic areas. Further, this tool will aid in promptly diagnosing and treating affected animals by veterinary clinicians whom can refer cases to diagnostic laboratories. Further, faecal samples can be easily stored and transported at room temperatures to laboratories with qPCR capabilities without the need of trained technicians or special equipment, making it an ideal tool for large survey especially when sampling animals living in remote areas. These new tools provide an alternative to the labour-intensive microscopic examination of faecal samples and to low-throughput gel-based PCR-RFLP for the diagnosis of canine hookworm infections.A. ceylanicum, A. caninum and A. braziliense are sympatric. Its greater accuracy in comparison with PRC-RFLP may be explained owing to outputs being based on an objective automated quantitation of fluorescent signals instead of a visual-based size discrimination of the bands in an electrophoresis gel.These qPCRs will enable a comprehensive investigation of the epidemiology of canine hookworm infections in dogs and their risk posed to humans especially in areas where A. caninum, the well-known cause of eosinophilic enteritis in humans, have demonstrated high-level of pyrantel resistance [U. stenocephala has also been demonstrated [Distinguishing between the four species of canine hookworm is also important to inform One Health based chemotherapeutic intervention strategies in dogs, as species-specific differences in anthelmintic efficacy have been reported ,9–15. Fosistance . Poor efnstrated .A. ceylanicum is the only canine hookworm known to produce patent infection in humans in both natural and experimental infections and is the second most common hookworm infecting humans in the Asia Pacific [A. caninum and A. braziliense cause accidental infections in humans although preliminary evidence on the ability of the former to produce patent infections in human patients have recently been reported [Furthermore, although hookworm eggs are morphologically indistinguishable, the zoonotic impact of each species differs considerably. For example,  Pacific ,19,53. Creported .A. ceylanicum [A. ceylanicum and the subsequent requirement for accurate high throughput diagnostic techniques, will become increasingly important [WHO recommends different targeted treatments in endemic populations based on the prevalence detected in people from these geographical areas . Howeverylanicum ,33,55. Amportant .These novel multiplex qPCRs for the four species of canine hookworms showed higher diagnostic sensitivity compared to the PCR-RFLP and have greater ability to detect mixed infections compared to previously published assays. These qPCRs will assist large epidemiological surveillance investigations, the assessment of the role of dogs in the transmission of hookworms to humans and the efficacy of anthelminthic treatments in canine populations.S1 TextIn grey primer pairs designed and in red probes.(DOCX)Click here for additional data file.


Caenorhabditis elegans is an important model species for understanding genetic variation of complex traits. So far, most studies involve axenic laboratory settings using Escherichia coli as the sole bacterial species. Over the past decade, however, investigations into the genetic variation of responses to pathogenic microbiota have increasingly received attention. Quantitative genetic analyses have revealed detailed insight into loci, genetic variants, and pathways in C. elegans underlying interactions with bacteria, microsporidia, and viruses. As various quantitative genetic platforms and resources like C. elegans Natural Diversity Resource (CeNDR) and Worm Quantitative Trait Loci (WormQTL) have been developed, we anticipate that expanding C. elegans research along the lines of genetic variation will be a treasure trove for opening up new insights into genetic pathways and gene functionality of microbiota interactions.The bacterivorous nematode  Caenorhabditis elegans  is a cosmopolitan hermaphroditic nematode that thrives on bacteria in rotting plant material [C. elegans populations can easily be reared in the lab. Research using C. elegans has mainly investigated nematodes kept under axenic laboratory conditions  that are devoid of interactions with natural pathogens. Microbiota provide C. elegans with nutrients, but they may also be pathogenic, leading to decreased growth and fitness of nematodes [C. elegans and its pathogenic microbiota, like microsporidia, bacteria, and viruses, with the aim to better understand C. elegans ecology away from E. coli [C. elegans microbiota community normally inhabits or is closely associated with the nematode [C. elegans and that the microbiota assembly is highly dynamic in time and species composition [C. elegans and microbiota may help further in gaining a deeper insight of microbiota interactions in other species. This comes at an important moment since it is increasingly realized that microbiota play in an important role in metazoan health [material ,2. It wamaterial , and sinematodes . Over th E. coli ,6. The Cnematode . Recentlposition . Understn health .C. elegans microbiota studies are host-pathogen interaction studies and have been limited to elucidating the molecular mechanism of host-pathogen interaction using a single C. elegans wild type strain, the canonical strain Bristol N2 [C. elegans strains will provide a broader view on the genetic architecture of host-pathogen interactions  was established to collect all genetic and genomic studies based on wild strains, thus facilitating genome-wide association studies (GWAS) [C. elegans worldwide and facilitating genome-wide association analyses. Worm Quantitative Trait Loci (WormQTL) was recently launched, a platform for systems genetics in C. elegans [C. elegans eight parental experimental evolution (CeMEE) contains more than 500 RIL. CeMEE has already been applied in C. elegans research to understand the genetic basis of complex phenotypes, like fertility and the hermaphrodite body size during reproduction [C. elegans–microbiota interactions  . It is a elegans . Next to elegans . For insoduction ,13,17. Aractions . These sC. elegans microbiota groups, bacteria are the most well studied. Bacteria may serve as food and/or may be pathogenic to C. elegans. The most abundant bacteria studied include Gammaproteobacteria  and Bacteroidetes  complemented with a few Acetobacteriaceae [Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria are bacterial phyla found living in the same habitat as C. elegans [C. elegans populations thrive well on these bacteria, and some of these bacteria may even increase resistance to abiotic and biotic stress factors, including pathogen infection [C. elegans is able to avoid the pathogenic bacteria, Pseudomonas aeruginosa, through aversive olfactory learning. This ability could be transmitted to next generations under the function of P-element Induced WImpy testis (Piwi) Argonaute homolog Piwi Related Gene (PRG-1), thus improving the fitness of the progeny [Pseudomonas vranovensis is a natural pathogen of C. elegans, parental exposure of C. elegans to P. vranovensis upregulates the cysteine synthases CYSL-1 and CYSL-2 and the regulator of hypoxia inducible factor RHY-1, which promotes the resistance of progeny to infection [C. elegans also have impact on the resistance to Bacillus thuringiensis [B. thuringiensis, males of C. elegans have lower resistance and survival than hermaphrodites. Next to this, proteome levels in C. elegans differ depending on the type of bacteria that they feed on [Ochrobactrum spp.  or E. coli OP50.Of all eriaceae ,26. Prot elegans . C. elegnfection ,28. C. e progeny . Besides progeny . Pseudomnfection . Besidesngiensis . Under i feed on , like OcC. elegans by a parasitic strain of Gram-positive B. thuringiensis using ten wild strains [C. elegans. Here, the behavioral trait comprised evasion and reduced parasite ingestion. As for the gene level, different quantitative genetic studies have identified the neuropeptide Y receptor gene, npr-1, as a major pleiotropic determinant regarding bacterial food intake. Natural variation in npr-1 was first reported to modify social behavior and responses to food [npr-1 affected behavioral responses to the Gram-negative pathogen Pseudomonas aeruginosa by means of oxygen-dependent behavioral avoidance rather than direct regulation of innate immunity [npr-1 in C. elegans exposed to B. thuringiensis and P. aeruginosa using QTL analyses of behavioral immune defense and RNAseq-based transcriptomics using recombinant inbred lines and introgression lines [npr-1. The wild type N2 allele was found to be associated with reduced defense against B. thuringiensis and had an opposite phenotype to that previously mapped for the N2 npr-1 allele against P. aeruginosa. Global gene expression profiling suggested that npr-1 plays an important role against both pathogenic bacteria through p38 Mitogen-Activated Protein kinases (MAPK) signaling, insulin-like signaling, and C-type lectins.Schulenburg and Muller (2004) showed that genetic variation affects infection of  strains . Their a to food . Later, immunity . Nakad eon lines . SeveralP. aeruginosa was associated with the induction of oxidative stress genes and activation of GATA transcription factors. In contrast, oxidative stress gene suppression combined with the activation of Ebox transcription factors determined B. thuringiensis effects. Like npr-1, genetic variation in other genes related to neuronal functions was found to play a key role in detecting food related cues, like tyramine receptor gene tyra-1, encoding a G protein-coupled catecholamine receptor [npr-1 was found to control a range of phenotypic differences through behavioral avoidance of ambient oxygen concentrations to growth and physiology. Together, these results show that genetic variation in npr-1 gives rise to pleiotropic effects that are associated with bacterial food [The reduced sensitivity to receptor . Interesial food .npr-1, Chang et al. (2011) found polymorphisms in a Homologous to the E6–AP Carboxyl Terminus (HECT) domain-containing E3 ubiquitin ligase, HECW-1. Two polymorphisms close to residues of HECW-1 each affected C. elegans behavioral avoidance exposed to P. aeruginosa [C. elegans to P. aeruginosa is achieved by avoidance instead of physiological stress pathways. Neuronal analysis pointed out that HECW-1 functions in two of sensory neurons against P. aeruginosa through inhibition of the neuropeptide receptor NPR-110.Next to ruginosa . The resC. elegans–bacteria interaction studies, QTL analysis was not only used to study npr-1, but also to detect other genetic variants. For example, it was found that the ability of C. elegans to sense bacteria also differed among the different strains of C. elegans. The odor of Serratia marcescens is more attractive to Bristol N2 than Hawaii CB4856. In this case, QTL analysis helped to define the region that interacts and affects S. marcescens preference based on RILs [C. elegans–microbiota systems were mostly researched in bacteria until now. The results varied from gene level to behavior level, expanding the insight into different host mechanisms in C. elegans against bacterial pathogen.In  on RILs . In summNematocida parisii was the first microsporidian species found to infect C. elegans and causes lethal infections. The mechanism of microsporidia to invade host tissue is mainly achieved by restructuring multicellular host tissues into syncytia and proliferating cells across the host tissue cells before forming spores [N. parisii has been isolated from wild C. elegans, C. elegans–N. parisii interactions have become an important model for studying both the infection process of pathogen and response of the host. The response of C. elegans to N. parisii involves a transcriptional response named intracellular pathogen response (IPR) that can be triggered by proteotoxic stress to improve the resistance to the stress [pals-22 and pals-25 [C. elegans including microsporidia, it was found that IPR involves gene pals-22 and pals-25 which play an essential role in combatting pathogens. Mutation of pals-22 showed increased resistance to microsporidia infection as well as virus infection. However, mutants of pals-25 showed suppressed phenotypes caused by mutation in pals-22, including increased IPR gene expression, thermotolerance, and immunity against natural pathogens. Gene pals-22 was proved to repress expression of some ubiquitin ligase complex components and other IPR genes [Microsporidia are a group of eukaryotic intracellular pathogens comprising more than a thousand species that are associated with a wide host range . Nematocg spores . As N. pe stress . This re pals-25 . Whilst PR genes .C. elegans. However, among different species of microsporidia, host range [N. parisii only invades and replicates in the intestinal cells of C. elegans, while Nematocida displodere is able to infect multiple host tissues [N. parisii in C. elegans were hardly induced by Nematocida ausubeli, suggesting it may evolve to counteract with the host response [C. elegans and C. briggsae, 50% of the worms were infected, while for Oscheius tipulae, no infectious behavior was found [Microsporidia are transmitted horizontally and replicate predominantly in intestinal cells in st range , virulenst range , and tisst range . For exa tissues . Besidesresponse . In the as found .C. elegans after infection of the intestines by Nematocida ironsii [C. elegans is a complex trait associated with higher reproductive output later in life, thus enhancing overall fitness. Currently, only a few genetic variation studies of microsporidia infections in C. elegans have been conducted. More research into the genetic variants affecting epithelial tolerance is required since the epithelium plays a major role in host–pathogen interactions.Genetic variation in epithelial immunity was found in  ironsii ,22,23. WC. elegans–virus interactions were hampered by the lack of a naturally infectious virus until 2011 when Orsay virus was found [C. elegans intestinal cells [Studies on as found ,44. Orsaas found . It is tal cells ,46. Orsaal cells . ORF δ ial cells ,49. Contal cells .C. elegans share the same target tissue, the intestine [C. elegans consists of 20 cells, holding a proportion of one third of somatic mass and is essential for absorbing nutrient and producing food derived macromolecule [C. elegans and mammals [C. elegans to be translated to higher species.Many pathogens of ntestine . Orsay vmolecule . It was molecule . Orsay v mammals , which aC. elegans–Orsay virus and C. elegans–microsporidia interaction pathways both belong to IPR, there is a difference between the two pathogens. The C. elegans retinoic acid-inducible gene (RIG)-I-like receptor DRH-1 was proved to be necessary for inducing IPR when infected by Orsay virus, but not for microsporidia infection or proteotoxic stress [C. elegans–microsporidia and C. elegans–Orsay virus interactions were also not the same.Orsay virus infection also triggers the IPR pathway. The exposure to Orsay virus led to upregulation of the Skp, Cullin, F-box (SCF) ubiquitin ligase components, such as the cullin ortholog CUL-6. The ubiquitylation components, the proteasome, and the autophagy pathway are all important for defense against viral infection . Howeverc stress . DiffereC. elegans is highly variant [C. elegans strains have distinct susceptibilities when faced with Orsay virus infection. Studies into the genetic variation to Orsay virus infection revealed that DRH-1, encoding a RIG-I-like helicase, is required for the initiation of an antiviral RNAi pathway and the generation of virus-derived siRNAs (viRNAs) [C. elegans [drh-1 polymorphism is involved in viral susceptibility [C. elegans genetic variation studies involving Orsay virus, insight into antiviral immunity in C. elegans was developed and more detail in viral infection and host response was revealed.The intraspecies susceptibility to Orsay virus of  variant ,54, as d(viRNAs) . Wild ty(viRNAs) . The dif elegans . For instibility . In summC. elegans genotypes of pathogenic microbiota interactions can be complementary to studying interactions in a single genotype. Studying microbiota interactions across multiple genotypes allows for unraveling the genetic architecture of these interactions.Quantitative genetic analyses into different C. elegans and its associated microbiota. Resources like CeNDR, in combination with QTL studies, could provide more insight of gene function and evolutionary studies. To overcome the limitations of RILs that were derived from a combination of two parental strains , multiparent populations like CeMEE and the four parental population panel can improve the power of detecting natural genetic variants [Mapping of genetic variation is an essential tool in investigating the genetic basis of complex traits that underlie the interaction between variants .C. elegans could be explored more to reveal the genetic architecture of variants relating to complex microbiota related traits. This may promote the study of the underlying molecular genetic mechanisms at, for instance, the metabolic and protein levels. Further, it may aid to understand how microbiota influence the fitness of C. elegans and how functional genes affect the microbiota interactions. For example, a recent study on nematode trapping fungi (NTF) demonstrated the trap induction, involving G protein β–subunit Gpb 1, plays as fitness character in NTF family [In the future, with the application of advanced methods of quantitative genetics study, genetic variation of F family . A next


Bartonella spp. are increasingly implicated in association with a spectrum of zoonotic infectious diseases. One hundred sanitary workers in La Rioja, Spain, completed a questionnaire and provided blood specimens for Bartonella spp. serology and Bartonella Alpha-Proteobacteria growth medium (BAPGM) enrichment blood culture/PCR. Six immunofluorescence assays (IFA) were performed and aseptically obtained blood specimens were inoculated into liquid BAPGM and subcultured onto blood agar plates. Bartonella DNA was amplified using conventional and real-time PCR assays. The Bartonella spp., strain, or genotype was determined by DNA sequencing. Bartonella seroreactivity was documented in 83.1% and bloodstream infection in 21.6% of participants. Bartonella henselae, B. vinsonii subsp. berkhoffii genotypes I and III, and B. quintana were identified. IFA seroreactivity and PCR positivity were not statistically associated with self-reported symptoms. Our results suggest that exposure to and non-clinical infection with Bartonella spp. may occur more often than previously suspected in the La Rioja region. Bartonella comprises fastidious Gram-negative, slow growing and facultative intracellular bacteria belonging to the Alpha-2 subgroup of the class Proteobacteria, Order Rhizobiales. These microorganisms are most often transmitted to humans through animal bites or scratches , or by scratch inoculation of infected flea and body louse feces [Bartonella bacilliformis in Peru during the last century [Bartonella spp. infections, but their role as competent vectors requires further confirmation [Bartonella spp. can survive within different hosts and reservoirs  for months to years. This genus has been increasingly associated with a wide spectrum of zoonotic emerging and reemerging infectious diseases. The number of validated species (more than 35), potential species (at least 17) and subspecies (at least three) continues to increase <http://www.bacterio.net/bartonella.html>. Some species, such as B. bacilliformis, cause potentially life threatening illness, but are limited geographically by the transmitting vector, whereas other species such as B. quintana are transmitted under poor hygienic conditions throughout the world. Flea-transmitted B. henselae is the most frequent etiological agent of sub-acute and chronic lymphadenopathy named cat-scratch disease (CSD) in children and teenagers and occurs throughout the world. A subset of CSD patients develop severe or systemic disease manifestations, including endocarditis, osteomyelitis, granulomatous hepatitis and hepatosplenic abscess [Bartonella spp. have been associated with human diseases  or, at least, have been detected in humans (e.g. B. vinsonii subsp. vinsonii).The genus se feces . In addiirmation ,4,5,6,7. abscess ,10,11,12Bartonella spp. may represent potential opportunistic pathogens for infections in animals and human patients [Bartonella epidemiology and pathogenesis continues to change [Bartonella infection (positive PCR/DNA sequencing results) in healthy blood donors [Bartonella spp. infection in sanitary workers in a localized region of Spain. Previously, a high prevalence of Bartonella spp. antibodies, as well as DNA of these bacteria, were reported in blood samples collected from veterinary workers in Spain [Bartonella spp. In addition to microbiological testing, the potential relationship of Bartonella spp. antibodies or blood stream infection (as assessed by DNA amplification and sequencing) with minor or nonspecific, self-reported symptoms, such as fatigue or insomnia was examined.Seemingly, all patients ,14. Undeo change . After rd donors ,16,17, tin Spain . CompareBartonella infections. The CIBIR houses the Center of Rickettsiosis and Arthropod-borne Diseases, where researchers support the diagnosis of arthropod  associated diseases. Clinical specimens are tested using serological and molecular assays for Bartonella, Borrelia, Coxiella, Rickettsia spp. and other pathogens. Bartonella spp. are not routinely cultured and are infrequently isolated. Isolates of B. quintana and B. vinsonii subsp. berkhoffii genotypes have not been cultured in the research laboratories.Study participants were recruited from among physicians, nurses, researchers, medical students, and administrative personnel who worked at the Center of Biomedical Research (CIBIR) or San Pedro’s University Hospital at La Rioja (Northern Spain). The buildings housing these facilities are adjacent to each other and personnel share a common café. Physicians, medical students and nurses have contact with patients diagnosed with CSD or other After excluding three individuals who had recently taken antimicrobials, 97 sanitary workers were included in the study. A total of 194 participant samples (97 EDTA-blood and 97 sera) and the accompanying questionnaires were used to generate data reported in the results. Questionnaire demographic and risk exposure characteristics are provided in Bartonella spp. or genotypes ranged from 16.5% to 62.9% with the lowest percentage reactivity to B. quintana  testing  enrichment blood culture/PCR result  of Bartonella vinsonii subsp. berkhoffii genotype II, B. quintana or B. koehlerae was found among participant blood DNA extractions. Following BAPGM enrichment blood culture, sixteen participants were positive for Bartonella DNA amplification or bacterial isolation. Four of nine participants, who were PCR positive following blood DNA extraction , were again PCR/DNA sequence positive for the same organism following BAPGM enrichment blood culture. The species identified in blood cultures were B. henselae (six participants), B. vinsonii subsp. berkhoffii genotype I (six participants), B. vinsonii subsp. berkhoffii genotype III (four participants), and B. quintana (one participant)  had a positive R result . Bartoneectively . An addiicipant) . Co-infeectively . PCR seqBartonella sp. antigen . Interestingly, two B. henselae bacteremic patients were not B. henselae seroreactive, but one each was seroreactive to B. vinsonii subsp. berkhoffii genotypes II and III, or B. vinsonii subsp. berkhoffii genotype II and B. koehlerae  IFA serology panel, as well as the high prevalence of occult bloodstream infection among individuals from the Rioja region of Spain [B. henselae seroreactor (antibody titer 64); however, 16 (50%) individuals were B. vinsonii subsp. berkhoffii genotype II seroreactive [B. vinsonii subsp. berkhoffii genotype I or III, B. koehlerae, or B. quintana seroreactive and Bartonella spp. DNA was not PCR amplified from any blood or BAPGM enrichment blood culture using the same diagnostic platform used in this study [Bartonella spp. DNA results (Maggi R. unpublished data). Technically, the same personnel (Bradley and Maggi) performed the serology and BAPGM enrichment blood culture testing in the current and previous North Carolina study. Based upon the results of this study, Bartonella spp. occupational exposure risk in sanitary workers should be further investigated.In this study, conducted among ‘healthy volunteers’ working in a sanitary setting in Spain, 83.1% participants were of Spain ,16. Usinreactive . Among tis study ,20. TestB. vinsonii subsp. berkhoffii genotype II seroreactivity was unexpectedly high among healthy sanitary workers in both studies . In the only other study involving healthy individuals and the BAPGM enrichment blood culture platform, Brazilian medical personnel screened 500 blood donors [B. henselae and B. quintana (IFA antigen slides provided by the Centers for Disease Control and Prevention), and blood stream infection was found in 16 (3.2%) of the 500 donors, infected with either B. henselae (n = 15) or B. clarridgeiae (n = 1). It is important to point out that similar to the current study, IFA serology was not consistently associated with bloodstream infection. Recent documentation of bloodstream infection among healthy individuals has further increased the diagnostic complexity associated with the genus Bartonella. As fastidious Gram-negative bacteria, Bartonella spp. require several weeks to grow axenically. Despite efforts to improve yield, obtaining agar plate isolates remains difficult and successful isolation has only been reported in a small number of human cases, often requiring microbiological techniques limited to specialty laboratories. Isolation is more likely to occur when culturing immunocompromised individuals, who presumably maintain higher levels of bacteremia [Bartonella infection in animals and humans, although sensitivity has been considered poor [Coxiella burnetii, Chlamydia spp., Rickettsia spp. (spotted fever group), Treponema pallidum, Orientia tsutsugamushi, Francisella tularensis, Ehrlichia chaffeensis, Mycoplasma pneumoniae, and Escherichia coli have been reported in human patients [Bartonella spp. IFA has a high degree of specificity (97% or greater depending upon IFA antigen) when testing dogs [Rickettsia rickettsii do not induce fluorescence (cross reactivity) to Bartonella spp. antigens [Bartonella IFA assays have poor sensitivity when testing sera from PCR/culture positive dogs [Bartonella spp. or the frequency of Bartonella intergenus IFA specificity contributes to serology results in dogs and humans, when using a broad panel of antigens that differ from the classical B. henselae and B. quintana diagnostic assays, awaits additional clarification. Dogs also developed Bartonella species-specific IFA antibodies following experimental infection with B. henselae or B. vinsonii subsp. berkhoffii [B. vinsonii subsp. berkhoffii genotype-specific antibodies after an inadvertent needle stick transmission [Bartonella spp. IFA cross-reactivity in dogs to closely related alpha-Proteobacteria or other more distantly related bacteria is lacking and the extent to which co-exposures, rather than cross-reactivity, contributes to serology findings in humans remains unclear. For people who live in places like La Rioja where free time is often spent in rural environments, co-exposures may be more frequent than previously suspected.Interestingly, d donors ,16. Serocteremia . Serologred poor ,23. In tpatients ,25. In cing dogs ,27,28. Santigens . Howeverive dogs . The extrkhoffii ,27. Simismission . OverallBartonella spp. DNA in blood or tissues using PCR assays can be technically limiting due to low level infection , a relapsing bacteremia, or Bartonella PCR primer designs that differ from the numerous Bartonella spp. genotype and strain targets . Thus, negative PCR results do not exclude the presence of authentic Bartonella infections, as previously described [Bartonella infection since other infectious diseases  cannot be readily distinguished. Although immunohistochemical analyses are less sensitive than PCR amplification techniques, their usefulness to establish the etiologic diagnosis of Bartonella infections has been reported, but these assays are available in only a few laboratories [Demonstration of escribed . Histoloratories .Bartonella infection [Bartonella spp. bloodstream infection in dogs was originally reported in the Journal of Microbiological Methods [Recently, specialized culture techniques based on growth enrichment in modified media combined with PCR assays and subculture bacterial isolation (BAPGM platform) have been developed with the aim of enhancing documentation of nfection ,34. Vali Methods ,36. Subs Methods ,38 and i Methods ,39,40,41 Methods ,43,44.Bartonella. Increased detection sensitivity can be a benefit to the patient and physician, but as illustrated by the results of this study, increased sensitivity can also complicate clinical decision-making, as well as microbiological and pathological interpretations. In this study, statistically significant associations were not detected between serology or the detection of Bartonella bloodstream infection and self-reported symptoms, such as fatigue and insomnia.At times, these more sensitive techniques detect organisms in healthy individuals that were previously associated with pathology in sick individuals, which is seemingly historically applicable to the genus B. vinsonii subsp. berkhoffii genotype III DNA from four participants. In contrast to the results of this study, human studies from the United States using the BAPGM platform most often result in amplification of B. vinsonii subsp. berkhoffii genotype II and less frequently genotype I DNA [B. vinsonii genotype III has not been reported from North America, whereas this genotype has been identified in dogs and humans in several Mediterranean countries, including Spain [B. vinsonii subsp. berkhoffii genotype II, in conjunction with the detection of genotype III provides further support for a lack of laboratory contamination with PCR products or organisms. In addition, the B. henselae and B. quintana sequences obtained in this study were most consistent with sequences reported by European, as compared to North American investigators , include Bartonella spp. PCR positive or an IFA antibody titer of ≥800 against B. henselae or B. quintana antigens [B. henselae or B. quintana DNA from excised heart valves also establishes a diagnosis of Bartonella endocarditis in BCNE cases [Bartonella infection using serological techniques is also a cause of concern. In the present study, Bartonella seroprevalence in healthy participants (>83%) was substantially higher than the prevalence’s reported from blood donors from the same area nearly 20 years before (<6%), and even exceeded previous seroprevalence values from risk groups, such as cat owners (28.9%) and HIV-infected people (17.3%) [Bartonella spp. antigens and variable sensitivity among individual IFA antigens used at these two times are likely contributors, but it is also possible that the epidemiology of Bartonella spp. transmission has changed in the region. In support of this possibility, using the same antigens, the seroprevalence of sanitary workers was more than twice the seroprevalence found among veterinary sanitary workers from different regions of Spain according to a study performed during the same year [Bartonella DNA is amplified, but antibody reactivity is low or not detected. Obviously, a single serological result  does not confirm an acute or chronic infection, since IgG antibodies may be due to a prior exposure to the microorganism.The results of this study have created additional diagnostic challenges for clinicians attempting to confirm a diagnosis of bartonellosis in an individual patient. For example, microbiological criteria for a clinical diagnosis of antigens . AmplifiNE cases ,48. The  (17.3%) ,50. Cleaame year . The resBartonella spp. among mammalian reservoir hosts located throughout much of the world represents an amazing and evolving area of research [Bartonella spp. have been transmitted by needle stick, animal bites and scratches and potentially by blood transfusion [Recent documentation of the historical co-evolutionary efficiency of research ,52. In ansfusion ,53. ThusBartonella species/genotypes. Bacteremia was concurrently assessed by means of Bartonella alpha-Proteobacteria growth medium (BAPGM) enrichment blood culture platform. Institutional review board approval for this study was obtained from the Ethical Committee of Clinical Research from La Rioja (CEICLAR) in January 29, 2016 (Ref. CEICLAR PI-209). A cross-sectional study was performed to determine the seroprevalence to six A standardized questionnaire including demographic information, symptoms experienced, domestic and wild animal bites, scratches or exposures, and travel history, was completed. Exposure to, or bites by different arthropods  was recorded. Approximately 10–12 mL of blood  was collected at the time of enrollment. Aseptic blood collection, including chlorhexidine decontamination of the skin, was performed by an experienced nurse. Three participants, who reported antimicrobial use within the last 2 months on the questionnaire, were subsequently excluded from the study.Refrigerated EDTA-anticoagulated blood and serum samples were processed in less than two hours at the Center of Rickettsiosis and Arthropod-Borne Diseases (CRETAV), located at the Center for Biomedical Research from La Rioja , where blood was centrifuged and sera stored at −80 °C until prepared for shipping to Galaxy Diagnostics, Inc., Research Triangle Park, North Carolina, USA.Bartonella vinsonii subsp. berkhoffii, B. henselae, B. koehlerae and B. quintana antibodies were determined in the Intracellular Pathogens Research Laboratory (IPRL) at North Carolina State University  using cell culture grown bacteria as antigens and following standard immunofluorescent antibody assay (IFA) techniques [B. vinsonii subsp. berkhoffii genotype I , B. vinsonii subsp. berkhoffii genotype II  and B. vinsonii subsp. berkhoffii genotype III (NCSU 06CO-01 Klara), feline isolates of B. henselae SA2 strain  and B. koehlerae  and B. quintana (NCSU11-MO-01 Monkey origin) were passed from agar plate grown cultures into Bartonella-permissive cell lines, i.e., the DH82 (a canine monocytoid) cell line for strains B. henselae SA2, B. quintana, B. vinsonii subsp. berkhoffii I and B. koehlerae and Vero cells  for B. vinsonii subsp. berkhoffii II and III to obtain antigens for IFA testing. For each antigen, heavily infected cell cultures were spotted onto 30-well Teflon-coated slides (Cell-Line/Thermo Scientific), air-dried, acetone-fixed, and stored frozen. Fluorescein conjugated goat anti-human IgG  was used to detect bacteria within cells using a fluorescent microscope . Serum samples diluted in a phosphate-buffered saline (PBS) solution containing normal goat serum, Tween-20, and powdered nonfat dry milk to block nonspecific antigen binding sites were first screened at dilutions of 1:16 to 1:64. All sera that were reactive at a reciprocal titer of 64 were further tested with two-fold dilutions out to 1:8192. To avoid confusion with possible nonspecific binding found at low dilutions, a cutoff of 64 was selected as a seroreactive titer.chniques ,19. Cani2, water-saturated atmosphere. After 7, 14, and 21-day incubation periods, PCR was performed on each inoculated liquid culture and a 1 mL aliquot of the enrichment culture was inoculated onto blood agar plates and incubated at 35 °C. Plates were checked for colony formation at 7, 14, and 21 days after plating. An un-inoculated BAPGM culture (negative control) was processed in an identical manner with each group of study participant specimens.Enrichment blood culture was performed at Galaxy Diagnostics Inc., Research Triangle Park, North Carolina, USA, as previously described ,19,20. ABartonella spp. DNA was amplified using primers designed to amplify two distinct consensus sequences in the Bartonella 16S-23S intergenic spacer (ITS) region as described previously with minor modifications [B. koehlerae was performed using species-specific oligonucleotides Bkoehl-1s and Bkoehl-1125as as forward and reverse primers, respectively (B. koehlerae) levels were performed in a 25 μL final volume reaction containing 12.5 μL of either MyTaq HS Red Mix 2X (Bioline) for B. koehlerae conventional PCR or Sso Advanced Universal Probe Supermix (BioRad) for Bartonella genus real-time PCR; 0.2 μL of 100 μM of each forward and reverse primer (IDT-DNA Technology), 7.3 μL of molecular-grade water, and 5 μL of DNA from each sample tested. PCR negative controls were prepared using 5 μL of dH2O (when testing isolates from plates), 5 μL of DNA from blood of a healthy dog, or 5 μL of DNA extracted from un-inoculated BAPGM-negative controls (when testing BAPGM enrichment cultures). Positive controls for PCR were prepared by serial dilution (using dog blood DNA) of genomic DNA from B. henselae (Houston I strain type) down to 0.001 pg/μL . Conventional PCR was performed in an Eppendorf Mastercycler EP gradient under the following conditions—a single hot-start cycle at 95 °C for 3 min followed by 55 cycles of denaturing at 94 °C for 15 seconds (s), annealing at 66 °C for 15 s, and extension at 72 °C for 18 s. Amplification was completed by an additional cycle at 72 °C for 1 min, and products were analyzed by 2% agarose gel electrophoresis with detection using ethidium bromide under ultraviolet light. Amplicon products were sequenced to determine the Bartonella species and ITS strain type. Real-time PCR was performed in an CFX96 Real-time System (Bio-Rad) under the following conditions: a single hot-start cycle at 95 °C for 3 min followed by 44 cycles of denaturing at 94 °C for 10 s, annealing at 66 °C for 10 s, and extension at 72 °C for 10 s. Fluorescence at channel 1 was detected during the extension cycle. As in conventional PCR, all amplicon products were sequenced to determine the Bartonella species and ITS strain type. All PCR and uninoculated BAPGM enrichment controls remained negative throughout the study period. PCR assays targeting GroEl, rpoB and 16S rRNA genes were also performed to confirm identification of agar plate isolates (DNA was extracted using standard operating procedures from EDTA anticoagulated blood, enrichment liquid blood cultures incubated for 7, 14, and 21 days, and from blood agar plate colony isolates, if obtained after subculture from BAPGM enriched blood specimens . Bartoneications . Two setications , conventectively . Amplifiisolates .Questionnaire data for the study were collected on paper forms and entered into a Microsoft Excel database. Data entry was validated by comparison of the electronic records with the information on the forms. Associations of demographic, risk factor, symptoms and exposure variables were assessed with means and medians for continuous variables, and with counts and rates in contingency tables for categorical data. Group comparisons were performed. The Fisher exact test for categorical variables and the Mann–Whitney U test for non-categorical variables were used. Data processing was carried out with the R software , version


Halyomorpha halys (Stål), the brown marmorated stink bug, is a highly invasive insect species due in part to its exceptionally high levels of polyphagy. This species is also a nuisance due to overwintering in human-made structures. It has caused significant agricultural losses in recent years along the Atlantic seaboard of North America and in continental Europe. Genomic resources will assist with determining the molecular basis for this species’ feeding and habitat traits, defining potential targets for pest management strategies.H. halys’ capacity for invasiveness. Many of the genes identified herein have potential for biomolecular pesticide applications.Analysis of the 1.15-Gb draft genome assembly has identified a wide variety of genetic elements underpinning the biological characteristics of this formidable pest species, encompassing the roles of sensory functions, digestion, immunity, detoxification and development, all of which likely support H. halys genome sequence will be useful for the development of environmentally friendly biomolecular pesticides to be applied in concert with more traditional, synthetic chemical-based controls.Availability of the  Halyomorpha halys (Stål) (Heteroptera: Pentatomidae), the brown marmorated stink bug (BMSB), is native to Asia  and has emerged in recent decades as a major insect pest of worldwide importance due to its exceptional capacity to colonize new habitats . Accidentally introduced outside its native range, H. halys has become established in North America , Europe  and South America  [H. halys’ high dispersal capacity, polyphagy (at least 170 plant species) and ability to compete with endemic species have assisted its spread , grapes, ornamental plants, vegetables, seed crops, as well as staple crops [H. halys continues to expand its range, it poses major threats to agriculture, especially to such staple crops as corn and soybean grown in the primary agricultural production regions of the American Midwest [H. halys is also a nuisance pest, well known for its invasion of human structures such as houses, schools and other indoor spaces in large numbers when it overwinters [iewed in ). In comle crops . As H. h Midwest . H. halyrwinters .H. halys is a member of the insect order Hemiptera, which contains approximately 82,000 described species and constitutes the most speciose order of hemimetabolous insects [H. halys belongs to the sub-order Heteroptera, and to the family Pentatomidae, which encompasses all stink bugs  [Oncopeltus fasciatus [ insects . All hem insects . As a “tprolixus ; the peaon pisum ; the wats buenoi ; the broomorpha) ; and theasciatus ; among oH. halys genome was conducted as a community annotation project under the “i5K” initiative to sequence the genomes of 5,000 insects and other arthropods with important biological significance or economic value [H. halys as a worldwide invasive pest, top priority was given to the annotation and analysis of gene families related to sensory functions, digestion, immunity, detoxification and development. These efforts revealed informative genome features potentially related to broad phytophagy , xenobiotic detoxification  and digestion.Analysis of the ic value . Given tH. halys [H. halys is attracted to and infests new host plants, of its potential to develop insecticide resistance and possibly of its biological vulnerabilities, thereby assisting in the development of environmentally sustainable biomolecular pesticides for controlling this important pest.Numerous integrated pest management and biological control measures, as well as monitoring and targeted chemical control tactics, have been explored for H. halys , 18. TheThe genome sequencing and assembly yielded an assembly of 1.15 Gb (1.00 Gb in gap-free scaffolds) with a contig N50 of 17.7 kb and scaffold N50 of 802 kb. The overall genome size was estimated to be 1.143 +/- 0.019 Gb (n= 4) and 1.095 +/- 0.023 Gb (n=4) for the female and male, respectively, using flow cytometry  and the annotated gene set (98.7%), missing only 29 and 13 of the 1,658 Insecta BUSCO genes, respectively  LGT event with the milkweed bug, Oncopeltus fasciatus, of a cell wall degradation enzyme, endo-1,4-beta-mannosidase [H. halys through a series of species-specific tandem duplications  does support a potential tandem expansion of mannosidase genes in this polyphagous lineage  from microbes into arthropod genomes were once thought rare or non-existent, but are now know to be relatively common . H. halynosidase . StrikinH. halys  , we iden9 , we id species , 28 and  species . AdditioH. halys : one froH. halys with those from other hemipterans with available genome sequences to detect whether any potential expansions or contractions may have occurred along the hemipteran lineage leading to H. halys . Most remarkable, however, is a major potential expansion of the GR family, both in number of genes and the prevalence of alternative splicing yielding different isoforms from 63 of the 198 genes. This is among the largest GR families known in insects, even taking into account that 37 of them are pseudogenic, leaving 330 apparently functional GR proteins. This total exceeds the 215 genes encoding 245 proteins (219 of them intact) in the flour beetle Tribolium castaneum [Helicoverpa armigera and Spodoptera frugiperda [Periplaneta americana [Blattella germanica [Tetranychus urticae has 689 genes [Insects depend on the members of three large families of chemoreceptors for the specificity and sensitivity of most of their senses of smell and taste , 31. Theastaneum , and theugiperda –38. The mericana  and Blat89 genes . This ma89 genes , 37 and H. halys, which are expected to encode 58 proteins due to isoforms , Chinavia ubica (25) and Dichelops melacanthus (9) [Forty-eight odorant-binding protein (OBP) genes were identified in the genome of  species , 44. The with 51 –47 and B[B. mori , with 44 gambiae –52, Aede with 21 , and in thus (9) , 55.Halyomorpha halys has a ratio of OR to OBP genes of 148:48, approximately 3:1. The ratio of OR:OBP genes has been quite variable among insects, but always with more OR genes than OBP genes. For example, for D. melanogaster and An. gambiae the ratio is 70:50 [A. mellifera it is 170:21 [is 70:50 , 50, ands 170:21 .D. melanogaster, An. gambiae, A. mellifera, Ae. aegypti, B. mori and T. castaneum [The HhalOBP genes and pseudogenes are distributed across 25 scaffolds. Forty are organized into nine clusters of 2-14 genes  to 37,085 (obp21) bases, which is longer than that found in, for example, the hemipteran aphids A. pisum [Aphis gossypii [Drosophila melanogaster [H. halys, compared to zero to eight introns ranging from 58 to 12 kb in A. pisum, one to eight introns ranging from 0.6 to 2 kb for A. gossypii and zero to two introns ranging from zero to 638 bases in D. melanogaster.The length of the HhalOBP genes ranged from 2,130  uncovered varying representations of retinal opsin subfamilies , Metatropis rufescens (Berytidae) and Nezara viridula (Pentatomidae). Combined, these findings date the expansion of the H. halys LW opsin gene cluster to the early evolution of the Pentatomorpha. These LW opsin gene clusters are therefore referred to as the “Pentatomomorpha LW” clades 1-3.In the oot Fig. . In addiH. halys LW opsin subfamily expansion ranks as one of many examples in the unexpectedly dynamic diversification of LW-opsins in the Heteroptera.The hemipteran LW opsin gene tree further revealed that the four-member water strider LW-opsin cluster is of independent origin Fig. . In addiH. halys and O. fasciatus LW opsin paralogs are characterized by the ancestral green-sensitivity associated residues at the sites of strongest comparative tuning substitution evidence  [Lygus hesperus: JAG03839, JAG63746; Bemisia tabaci: XP_018896152.1, XP_018897455; and Diuraphis noxia: XP_015365906.1, XP_015372008.1). Moreover, all three non-retinal opsins, including Rh7 opsin, have been found in the water strider genome [H. halys and O. fasciatus are due to genuine gene losses or insufficient genome sequence coverage.Three ancient opsin subfamilies that are expressed in non-retinal tissues have been discovered in insects: ciliary (C) opsins , arthropeed bug) , 26. A Hr genome . Taken tT. castaneum larvae became important components of adaptive responses in overcoming the effect of cereal protease inhibitors [T. castaneum and the related tenebrionid, Tenebrio molitor, large expansions of genes encoding cysteine cathepsins were driven not only by protection against inhibitors, but also by more efficient digestion of complex proteins in grains [Cysteine peptidases from the papain C1 family  are typhibitors . In T. cn grains –76.Euschistus euschistoides [Brachynema germari [Perillus bioculatus [Podisus maculiventris [Apodiphus amygdali [N. viridula [For insects from the family Pentatomidae, evidence suggests that cysteine cathepsins also participate as digestive enzymes in intraoral digestion. There were early reports of cathepsin B activity in the posterior midgut of the brown stink bug, istoides , and lat germari . Digestioculatus ; spined iventris ; shield amygdali  and the viridula . In factviridula .H. halys, we found 41 genes and gene fragments encoding cysteine cathepsins of the C1 family  and O (Hh CatO); and orthologs of insect cathepsins I (Hh CatI) and Ll , as well as cathepsin B-like subfamily gene ).In ily Fig. . Thirty-H. halys encode digestive peptidases to enable specific functions, such as expanded dietary choices. This hypothesis is supported by similar species-specific clades of cysteine peptidases in T. castaneum [T. molitor [Leptinotarsa decemlineata [The most numerous group is the species-specific category, containing species-specific cathepsin L-like genes (shaded yellow) with 30 members clustered in three phylogenetic clades derived from the cathepsin L subfamily enzymes (Hh Cat.ss.uLx.x). These species-specific genes are localized in the draft genome assembly either as separate genes or as tandem arrays of up to seven copies. Species-specific cathepsin B-like peptidases (shaded pink) are represented by one phylogenetic clade of six genes (Hh Cat.ss.uBx.x). We propose that these species-specific genes in astaneum , 75, T.  molitor , 84 and mlineata , 86, whiH. halys genome assembly. If in fact they are not encoded in the genome, this divergence would have significant biological and evolutionary consequences.Surprisingly, we did not find complete gene sequences of one of the major conserved cathepsins–“true” cathepsin L (ortholog of human CatL1 or L2), as well as a conserved and presumably catalytically inactive TINAL-like protein from the cathepsin B-like group . These sH. halys, like other hemipteran species, injects saliva into plants using highly evolved, needle-like and flexible mouthparts  [H. halys genome  as salivary effector genes. Further, we annotated three genes for disulfide isomerases, thought to aid in the gelling nature of sheath saliva by catalyzing the formation of disulfide bridges in proteins [H. halys overcome a plant’s physical and chemical defenses and/or perform extra-oral digestion. The discovery of salivary effector genes in the H. halys genome is significant because these genes were recently found to play a key role in generalist herbivory behavior [H. halys across North America.stylets) . Hemiptestylets) . Salivarffectors . Severalns Table . For exaA. pisum ), Ejaculpersicae , 93), AnA. pisum ), and Mu. lugens , 96). Th. lugens –96. Herbn plants . Accordiproteins . We idenproteins . We alsobehavior , which htoll, toll-interacting isoform X1, toll family protein 10, toll-6 and toll-7 were present on four different scaffolds. Single copies of spaetzle, myeloid differentiation primary response protein (MyD88), pelle kinase, dorsal, tube, cactus, cactin, traf, pellino, persephone and various serine proteases (serpins) were also confirmed by manual annotation and BLAST alignments. However, the dif  gene was not found. In Drosophila, dif is a second cactus-bound NfK-B transcription factor  that functions primarily in the immune response rather than in playing a developmental role. Unlike dorsal, dif can be activated in both a Toll-dependent and independent manner depending on the challenge [dorsal in immunity in Hemiptera, or possibly reduced specificity and complexity of response to certain pathogens.Genes for the Toll signaling cascade, involved in both development and innate immunity, were annotated. Several sequences homologous to known Toll-pathway transmembrane receptors were encountered: hallenge . The absDrosophila is also involved in both development and immunity. It is hypothesized that induction of the JAK/STAT pathway leads to overproliferation of hemocytes and an upregulation of thiolester-containing proteins (TEPs), as well as triggering of the antiviral response [H. halys genome has homologs of all core JAK/STAT genes, including genes encoding the cytokine receptor Domeless, JAK tyrosine kinase Hopscotch, and the Signal Transducer and Activator of Transcription (Stat) transcription factor, which was found on NW_014466899.1:1005475-1044464, with highest homology to the Stat 5B isoform X1 from Bombus terrestris (XP_003401031.1). Two thiolester-containing proteins (TEPs) were located on NW_014467684.1. A putative uroporphyrinogen decarboxylase , considered a key ligand in Drosophila JAK/STAT induction, was located on NW_014466467.1. Interestingly, this ligand is missing in other insects, such as A. mellifera [Drosophila encodes three upd-like ligands; however, their sequences are highly divergent, and this degree of divergence may account for why orthologs have not been identified in other insects. In Drosophila, upd-3 is largely responsible for the activation of the Jak-STAT pathway in the context of an immune response. Additionally, in both Ae. aegypti and Drosophila, an alternate activating ligand, vago, initates a Jak-STAT response to virus infection [Vago was not found in the H. halys genome, and this absence may imply an alternate means of responding to viral challenge.The JAK/STAT pathway in response . The H. ellifera . In contnfection , 115. VaA. pisum; the body louse, Pediculus humanus corporis; or the deer tick, Ixodes scapularis. Indeed, recent evidence suggests IMD is absent among hempiterans [The IMD and JNK signaling pathways were complete with the notable exception of the immune deficiency (IMD) death domain protein itself, which initiates the IMD signaling pathway after peptidoglycan recognition protein (PGRP) attaches to the cell membrane. The lack of IMD is not necessarily unusual, as it has also not been found in the pea aphid, piterans , 117, alH. halys contained all requisite genes for the signaling pathway, including tab (1), tak (3), hep (1), basket (2), jra (2) and kayak (1). Furthermore, the signaling genes kenny, ird5 and relish were also found, but not iap2. The procaspase precursors to apoptosis, death domain protein Dredd, as well as two homologues of the aspartate-specific cysteine proteases CASP1 were annotated, indicating that the signaling pathway to apoptosis is intact, even in the absence of IMD. The absence of fadd is notable, as this may be unique to the Drosophila pathway due to its role in making flies susceptible to Gram-negative bacterial infection [The JNK pathway role in antimicrobial peptide gene expression and cellular immune responses is very well described in other insects , 119. H.nfection .A. pisum. This is consistent with the observation that Eiger serves as an IMD-like inducer for TAK in H. halys, similar to the pea aphid, whose genome also lacks IMD notwithstanding an intact JNK signaling pathway [Eiger (NW_014467110.1:500566-503446) has been proposed as an IMD-independent alternative inducer for activation of the protein kinase TAK , which then triggers the the JNK pathway . A BLAST pathway .In addition to immune signaling pathways, numerous other genes likely involved in insect immunity were identified, including PGRPs, Gram-negative binding proteins, lectins, antimicrobial peptides, RNA interference pathway components and a variety of such miscellaneous immune-related genes as putative prophenoloxidases and nitric oxide synthases , carboxylesterases (COEs) and cytochrome P450s (CYPs) are three well-documented gene families associated with xenobiotic detoxification in insects. Additionally, they have all been associated with increased insecticide tolerance and/or insecticide resistance in other insects through various mechanisms. Understanding H. halys’ xenobiotic detoxification gene repertoire is vital for successful pest control and to combat insecticide resistance that may arise in the future.Detoxification of xenobiotic compounds is an imperative cellular function that protects the organism from harmful compounds it may encounter. Glutathione S-transferases (GSTs) compose a large gene family associated with xenobiotic detoxification. Microsomal GST enzymes are typically trimeric and membrane bound, while cytosolic GSTs are typically dimeric and unbound . Based oH. halys transcribe 41 unique transcripts, which in turn translate 35 unique GST protein sequences , A. mellifera (4), N. vitripennis (8), T. castaneum (6) and B. mori (2), and may result in new endogenous functions [H. halys’ large complement of Sigma GSTs could correspond with structural roles and/or detoxification of reactive oxygen species in muscle tissue or other novel endogenous roles.The 30 glutathione S-transferase genomic loci present in the  tissues . Many spunctions . H. halyAn. gambiae (15) and D. melanogaster (11) [H. halys appears to possess only two Delta GST genes. High expression levels of Delta GSTs are a mechanism for conferring insecticide resistance, obtained by either upregulation of expression, or gene duplication. Insecticide resistance in H. halys has not yet been reported. Sparks et al. (2014) [H. halys in response to septic puncture: adult females exhibited a 9.8-fold change and adult males a 6.1-fold change. This up-regulation could be in response to foreign substances introduced during septic puncture and/or to help process a potential increase of metabolic products resulting from an immune response. The ability of H. halys to quickly alter expression levels of Delta GSTs, as seen after septic puncture, suggests the insect may utilize this mechanism in response to insecticide exposure.High counts of Delta GSTs have been reported in ter (11) , 130. H.. (2014)  noted inThe carboxylesterase (COE) family is typically divided into three clades –134. TheIn total, there are 75 genomic loci from which 90 distinct transcripts arise and which translate to 82 unique COE protein sequences. Of these, 59 are β-esterase genes, which produce 68 unique transcripts in total. A bootstrapped maximum likelihood phylogenetic tree of all unique translation products is provided in Fig. H. halys genome contains multiple scaffolds with β-esterase gene duplications in close proximity: H. halys scaffolds NW_014466677.1, NW_014469008.1, NW_014466575.1, NW_014467841.1 and NW_014466532.1 contain 11, 6, 6, 4 and 2 β-esterase genes, respectively. Within each of these scaffolds, inferred protein sequences are typically highly similar and cluster together in the phylogeny. For example, all eleven β-esterase genes on NW_014466677.1 are organized in a tandem array  [H. halys than does the single-species phylogeny, underscoring the importance of sequencing genomes from additional pentatomid taxa to enable comparative genomics studies  in the future.The paraphyletic placement of neuroligins, some of which are derived within β-esterases, as well as acetylcholinesterases derived within β-esterases, may be caused by the small size of the phylogenetic tree and the sharing of protein domains. The branch lengths of neuroligins and acetylcholinesterase in Fig. . (2017) ) places Cytochrome P450s (CYPs) have a two-fold role in gene-environment interactions, participating both in detoxification of xenobiotic compounds and in host biosynthetic pathways. P450-mediated biosynthesis of critical endogenous molecules affecting molting, hormone/pheromone synthesis and turnover, and cuticular hydrocarbon waterproofing processes can be targeted by pesticides. In response, insects have evolved modified P450s to detoxify exogenous chemicals like pesticides, leading to resistance. These two classes of P450s are easily observed in phylogenetic trees. Highly conserved one-to-one orthologs between insect species are parts of pathways to make essential biomolecules like ecdysone , juvenResistance has been associated with numerous cytochrome P450s, often members of “gene blooms,” which are large expansions of P450s in tandem duplication arrays on chromosomes. These are not highly conserved or even limited to one CYP clan. Almost any P450 family can become adapted to detoxify a pesticide –152. ResH. halys P450s sorted into the four known P450 clans: CYP2 (6 sequences), CYP3 (84 sequences), CYP4 (45 sequences) and mito (6 sequences). A maximum likelihood tree was constructed from 126 full or nearly full-length sequences, excluding 14 fragments and one pseudogene. Four additional sequences were included to stabilize the positions of single outlier sequences in the tree . CYP74 family P450s are involved in generating hexenal in plants by a hydroperoxide cleavage reaction. Although no H. halys CYP74 was found, convergent evolution could produce similar products. P450s are known to be involved in pheromone clearance in the antenna [The 141 osophila . Also foy organs . Other P antenna  and thisH. halys genomic resource has a high degree of gene content completeness, and the overall high quality of the assembly is also corroborated by well-assembled Hox and Iro-C gene clusters, in addition to two independent contamination screens  genes in this species is approximately 3:1, not unusal for an insect. Most OBP genes are organized into gene clusters, with an atypical instance of an OBP gene being embedded within the intron of an odorant receptor per the draft assembly. Although LWS and UV opsin homologs are observed in H. halys, no ortholog of the SWS-B opsin subfamily was detected, consistent with the notion that this subfamily was lost during early heteropteran diversification.H. halys include 34 genes from the cathepsin L-like subfamily and seven from the cathepsin B-like subfamily—30 and six of these, respectively, seem to represent H. halys-specific instances of cysteine peptidases, which may have been involved in the diversification of the insect’s broad dietary selections. The discovery of 64 salivary effector genes—several of which are homologous to known effectors in other herbivorous hemipterans—is significant due to the key role such genes play in the type of generalist herbivory exhibited by this species. Expansion of a cell wall degradation mannosidase, originally incorporated via an LGT event, to nine copies may also contribute to the digestive capabilities of this insect, providing a clear candidate enzyme for future functional assays and demonstrating the need for further species sampling within the Pentatominae and close relatives.Cysteine cathepsins of the C1 family observed in in vivo. This apparent lack of the IMD initiator in H. halys is consistent with findings made among other hemipteran species. A variety of additional immunity-related genes were also identified in the genome assembly, including peptidoglycan receptor proteins, gram-negative binding proteins, lectins, and the requisite molecular machinery to enable RNAi  were R&R Consensus domain-containing CPR proteins, and approximately three-fourths of the cuticular genic repertoire was arrayed in a type-specific clustering manner. Seven aquaporin genes were identified, an amount commensurate with what has been reported in other arthropods. Please see Additional file H. halys genome features reflect its broad phytophagy—in particular, its remarkable abundance of chemosensory genes—as well as the diversity of genes associated with xenobiotic detoxification and digestion. Availability of the H. halys genome sequence will undoubtedly prove useful towards the development of environmentally sustainable biomolecular pesticides for use in concert with more traditional, synthetic chemical-based controls. In addition, given the presence of RNAi pathway components, these genomic resources can, for example, assist researchers in designing functional studies of gene function by dsRNA-mediated knockdown experiments.Perhaps the most striking N. lugens (Fulgoromorpha) [Anoplophora glabripennis (Asian long-horned beetle) [The genome features described here can be directly contrasted with those of other Hemiptera with sequenced genomes, such as the brown plant hopper, omorpha) , a destromorpha) . Another beetle) . These dH. halys is one of thirty arthropod species sequenced as part of a pilot project for the i5K arthropod genomes project at the Baylor College of Medicine Human Genome Sequencing Center. An enhanced Illumina-ALLPATHS-LG sequencing and assembly strategy was used, in which four libraries of nominal insert sizes  prepared from a single female insect (the homogametic sex) were sequenced, as well as one 300bp-insert library derived from a single male specimen (heterogametic sex). The amount of sequence generated from each of these libraries is noted in Additional file H. halys /Hhal_1.0), as well as the Ag Data Commons (doi: 10.15482/USDA.ADC/1504240). Detailed information for all annotation-related topics is available in the supplement.Automated gene annotation was performed both with a MAKER 2.0 annotation pipeline  tuned sp. halys  tool, v3.0.2 [A. pisum ; the bectorBase ). For alH. halys genome assembly was screened for lateral gene transfers using a DNA based homology pipeline similar to that of Wheeler et al. (2013) [O. fasciatus [The . (2013)  and alsoasciatus .O. fasciatus [C. lectularius [R. prolixus [A. pisum [P. humanus corporis [The genome assembly was searched using tBLASTn with chemoreceptors from the most closely related hemipteran genomes available, specifically those of three other heteropterans, the milkweed bug, asciatus ; the bedtularius ; and theprolixus . CompariA. pisum ; and thecorporis , are avacorporis . Most gehttp://tree.bio.ed.ac.uk/software/figtree/).The final multiple alignments for each family included the members of the three other heteropterans noted above, as well as relevant proteins from other insects, and were generated with CLUSTALX v2.1 . AlignmeH. halys genome scaffolds through BLAST in Apollo/JBrowse in The i5K Workspace. The OBP gene search used 30 putative HhalOBP transcripts mined in the antennae of females and males through RNA-Seq [H. halys Gnomon-predicted proteins and six-frame translation products of the H. halys RNA collection screened against Classic, Plus-C and Atypical OBP motif-patterns (bit score 40.0). The OBP motif-patterns were built up using a set of 6,064 OBPs as a reference retrieved from NCBI by querying for “odorant binding protein”. In addition, OBP transcripts in other closely related heteropterans were also used, such as from the Miridae [H. halys specimens from nymphs of 1st, 2nd, 3rd and 4th instars, unmated three day-old females and males that were killed in liquid nitrogen. Dissected antennae and legs were immediately immersed together in TRIzol and homogenized in FastPrep®-24 Instrument at 6.5 m/s for 60 s. Total RNA was extracted using PureLink RNA Mini kit (Ambion by Thermo Fisher Scientific) according to the manufacturer’s instructions, with DNase treatment on-column. RNA yield was verified using a Qubit RNA HS Assay (Thermo Fisher Scientific). One microgram of total RNA was used for first strand cDNA synthesis using SuperScript III First-Strand Synthesis System for RT-PCR (Thermo Fisher Scientific) and used for qPCR reactions (3-8 replicates each gene/isoform) using PowerUp SYBR Green Master Mix (Applied Biosystems) in Roche Applied Science LightCycler® 480 Real-Time PCR System. Primers were designed using version 2.62 of the PrimerPlex program  to make them unique and cross-homology-intolerant. Their sequences are presented in Additional file Odorant-binding protein (OBP) family members were searched in the  RNA-Seq , as well Miridae  and Pent Miridae . OBP genH. halys sequences were collected by tBLASTn searches against the genome sequence draft version 1.0 (GCA_000696795.1). A multiple sequence alignment was generated with Clustal Omega [H. halys sequences were collected by tBLASTn searches against the NCBI TSA database; multiple alignment, variable site clearance and bootstrapped maximum likelihood analysis were performed as described above.For preparation of the global opsin gene tree, al Omega  and varial Omega . A bootsal Omega , 186, wiThe evolutionary history among cysteine peptidases was inferred by the Maximum Likelihood method (using the JTT matrix-based model ) as implH. halys were identified using a reciprocal best BLAST hit approach as described earlier for effector identification [H. halys predicted proteins. Top hits in the H. halys genome were BLAST searched against an A. pisum protein dataset to identify false positive candidates. If H. halys genes had a top hit different from the A. pisum query used in the first step, these were excluded from further analysis. Retained H. halys candidate genes were validated based on following three criteria: 1) presence of secretion signal peptide as revealed by signalP (version 4.1) [H. halys effector was used in pairwise comparisons. Analyses were conducted using the Nei-Gojobori method [Salivary effector genes in fication , 189. Infication , 190–194ion 4.1) , 2) abseion 4.1) , and 3) ion 4.1) , which wi method  in MEGA i method . All ambManual annotation efforts were organized around a list of genes involved in the innate, humoral immune response contributing to recognition, signaling and response to bacteria and fungi in arthropods. Genes were found using a combination of approaches. Immunity genes with the same gene id names  and identified as belonging to the phylum Arthropoda (taxid:6656) were downloaded from the UniProt database and used to create an HMM profile to search against proteins identified in the genome assembly (HVIT v.1.0). Proteins with similar domains to the HMM-constructed protein families were ranked by similarity using the lowest E-values  and thhttps://i5k.nal.usda.gov/webapp/blast/). If the original search failed, default parameters were relaxed to remove the low complexity filter. Upon determination of a genomic location using one of these two methods, genes were reviewed and manually annotated. If a gene model was successfully annotated, the putative protein was compared to the NCBI NR database for arthropoda (taxid: 6656) using the BLASTp algorithm to reconfirm the annotation and gene name.When HMM-constructed protein families were not successful in finding a match to an immunity protein of interest, a consensus sequence was manually constructed using protein sequences from UniProt containing the same gene identifier restricted to Arthropoda (taxid:6656), then compared with the genome sequence using tBLASTn and the default parameters provided by The i5K Workspace’s BLAST tool  and glutathione-S-transferase (GST) enzyme inventory was performed via keyword search of Gnomon annotated H. halys inferred proteome. For each protein family, the resulting protein sequences were then used as queries in a BLASTp search against the inferred proteome to identify any sequences that may have been missed during the Gnomon annotation process. Results were then manually filtered on alignment quality and biological relevance. Both curated COE and GST protein sets were multiply aligned using MUSCLE [Identification of g MUSCLE . SeqBootg MUSCLE  was usedg MUSCLE  as impleg MUSCLE . Phylogeg MUSCLE .H. halys were mined by batch BLAST of NCBI’s NR database using 52 P450 sequences representative of insects. Results from each search were combined and filtered to remove duplicate hits. The results were 212 gene models predicted by Gnomon from the genome. Some of these were fusions of adjacent genes that had to be split. After further refinement to split fusions and remove variants of the same gene, 141 P450s remained. To look for any additional P450s, 126 of the 141 sequences were used to BLAST search the WGS section for genomic contigs. 38,000 hits distilled to just 65 contigs, indicating P450 gene linkage. The 65 contigs were BLASTx searched against a database of named insect P450s to find all exons for P450s in the genome and to determine associated start and stop coordinates.Cytochrome P450s (CYP) from H. halys sequences, as well as one Riptortus pedestris (CYP3231A2 ~ AK417387.1) and three R. prolixus  sequences used to stabilize the position of outlier branches in the tree. All positions in the multiple sequence alignment with less than 70% site coverage were purged—that is, positions with fewer than 30% alignment gaps, missing data and ambiguous bases were allowed . The final dataset contained a total of 479 positions. Evolutionary analyses were conducted in MEGA X [CYP evolutionary history was inferred by using the Maximum Likelihood method and JTT matrix-based model . Initialn MEGA X .Additional file 1:  Main Supplementary Information text file, including Tables S1-S17 and Figures S1-S18. Table S1. Sequencing, assembly, annotation statistics and accession numbers. Table S2. OrthoDB v10 comparison of five species for ortholog presence and copy-number in Hemiptera-level orthogroups. Table S3. Scaffolds present in the H. halys assembly (accession GCA_000696795.1) that may originate from contaminant sources. Table S4. Counts of repetitive DNA elements encountered in the H. halys genome assembly. Table S5.H. halys predicted protein products associated with the RNAi pathway. Table S6. Positional information for the annotated homeobox genes. Table S7. Nuclear receptors of H. halys. Table S8. Listing of candidate Y-linked genes. Table S9. Number of genes identified as putative cuticle proteins per family in the genome of H. halys. Table S10. Number of genes identified as putative cuticle proteins per species in the genomes of several insect orders. Table S11. Clusters of genes coding for cuticle proteins in the genome of H. halys. Table S12. Odorant-binding protein genes and pseudogenes (Ψ) annotated in the genome of H. halys. Table S13. Primer sequences used to validate the HhalOBP gene annotations. Table S14. Correspondences between H. halys predicted protein identifiers and cathepsin labels. Table S15. A total of 64 salivary effector proteins were identified in the H. halys genome. Table S16. A select subset of 15 H. halys salivary effector proteins having variable expression levels between nymphal and adult stages (up- or down-regulation). Table S17. Gene expression data for H. halys glutathione S-transferase genes. Figure S1. Phylogenetic organization of the Hemiptera. Figure S2. Ortholog distributions among hemipterans. Figure S3. Genome assembly quality control. Figure S4. Hox and Iro-C cluster gene loci. Figure S5.Halyomorpha mannosidase expansion. Figure S6. Maximum likelihood phylogenetic tree of selected mannosidase proteins from three bacterial outgroups and three hemipteran species. Figure S7. Phylogenetic tree of the OR family. Figure S8. Phylogenetic tree of the GR family. Figure S9. Phylogenetic tree of the IR family. Figure S10. Heteropteran global opsin gene tree. Figure S11. Array of β-esterase genes. Figure S12. Distribution of transcription factor families across insect genomes. Figure S13. Nanos amino acid sequence alignments from different species. Figure S14. Location of pair-rule gene orthologs in the H. halys genome. Figure S15.H. halys odd-family genes. Figure S16. Alignment of Wnt family domain proteins. Figure S17. Engrailed and Invected are shared among diverse insects. Figure S18. Phylogenetic analysis of hemipteran genes named “NR2E1” reveals that they are orthologous to NR2E6.Additional file 2. H. halys gene expression omnibus (per RNA-Seq data). Additional file 3. H. halys transcription factor details.Additional file 4 . Manually curated H. halys chemoreceptor protein sequences.Additional file 5. Heteropteran LW opsin sequences. Additional file 6. LW opsin tuning site comparison. Additional file 7. Y-linked gene sequences.