Lactobacillus is a diverse group with a combined species count of over 200. They are the largest group within the lactic acid bacteria and one of the most important bacterial groups involved in food microbiology and human nutrition because of their fermentative and probiotic properties. Lactobacillus salivarius, a species commonly isolated from the gastrointestinal tract of humans and animals, has been described as having potential probiotic properties and results of previous studies have revealed considerable functional diversity existing on both the chromosomes and plasmids. Our study consists of comparative genomic analyses of the functional and phylogenomic diversity of 42 genomes of strains of L. salivarius using bioinformatic techniques. The main aim of the study was to describe intra-species diversity and to determine how this diversity is spread across the replicons. We found that multiple phylogenomic and non-phylogenomic methods used for reconstructing trees all converge on similar tree topologies, showing that different metrics largely agree on the evolutionary history of the species. The greatest genomic variation lies on the small plasmids, followed by the repA-type circular megaplasmid, with the chromosome varying least of all. Additionally, the presence of extra linear and circular megaplasmids is noted in several strains, while small plasmids are not always present. Glycosyl hydrolases, bacteriocins and proteases vary considerably on all replicons while two exopolysaccharide clusters and several clustered regularly interspaced short palindromic repeats-associated systems show a lot of variation on the chromosome. Overall, despite its reputation as a mammalian gastrointestinal tract specialist, the intra-specific variation of L. salivarius reveals potential strain-dependant effects on human health.The genus  AH, alimentary health; ANI, average nucleotide identity; BLAST, Basic Local Alignment Search Tool; CCuG, Culture Collection, University of Gothenburg; CECT, Colección Española de Cultivos Tipo; CRISPR, clustered regularly interspaced short palindromic repeats; COG, clusters of orthologous groups; DSM, Deutsche Sammlung von Mikroorganismen; EPS, exopolysaccharide; GIT, gastro-intestinal tract; GH, glycosyl hydrolase; GT, glycosyl transferase; GTR, general time reversible; HGT, horizontal gene transfer; JCM, Japan Collection of Microorgansims; KEGG, Kyoto Encyclopaedia of Genes and Genomes; LAB, lactic acid bacteria; LMG, Laboratorium voor Microbiologie, Universiteit Gent; NCBI, National Center for Biotechnology Information; NCIMB, National Collection of Industrial, Food and Marine Bacteria; POCP, percentage of conserved proteins; RBB, reciprocal best BLAST; SNP, single nucleotide polymorphism; UCC, University College Cork.GenBank accession number for the whole-genome shotgun sequence of 01M14315: MSCR00000000.GenBank accession number for the whole-genome shotgun sequence of AH4231: NBEY00000000.GenBank accession number for the whole-genome shotgun sequence of AH4331: NBEX00000000.GenBank accession number for the whole-genome shotgun sequence of AH43324: NBEW00000000.GenBank accession number for the whole-genome shotgun sequence of AH43348: NBEV00000000.GenBank accession number for the whole-genome shotgun sequence of CCUG 2753OB: NBEU00000000.GenBank accession number for the whole-genome shotgun sequence of CCUG 38008: NBET00000000.GenBank accession number for the whole-genome shotgun sequence of CCUG 44481: NBES00000000.GenBank accession number for the whole-genome shotgun sequence of CCUG 45735: NBER00000000.GenBank accession number for the whole-genome shotgun sequence of CCUG 47171: NBEQ00000000.GenBank accession number for the whole-genome shotgun sequence of CCUG 47825: NBEP00000000.GenBank accession number for the whole-genome shotgun sequence of CCUG 47826: NBEO00000000.GenBank accession number for the whole-genome shotgun sequence of DSM 20554: NBEM00000000.GenBank accession number for the whole-genome shotgun sequence of DSM 20492: NBEN00000000.GenBank accession number for the whole-genome shotgun sequence of gul1: NBEL00000000.GenBank accession number for the whole-genome shotgun sequence of gul2: NBEK00000000.GenBank accession number for the whole-genome shotgun sequence of JCM 1040: NBEJ00000000.GenBank accession number for the whole-genome shotgun sequence of JCM 1042: NBEI00000000.GenBank accession number for the whole-genome shotgun sequence of JCM 1044: NBEH00000000.GenBank accession number for the whole-genome shotgun sequence of JCM 1045: NBEG00000000.GenBank accession number for the whole-genome shotgun sequence of JCM 1047: NBEF00000000.GenBank accession number for the whole-genome shotgun sequence of JCM 1230: NBEE00000000.GenBank accession number for the whole-genome shotgun sequence of L21: NBED00000000.GenBank accession number for the whole-genome shotgun sequence of LMG 14476: NBEC00000000.GenBank accession number for the whole-genome shotgun sequence of LMG 14477: NBEB00000000.GenBank accession number for the whole-genome shotgun sequence of NCIMB 702343: NBEA00000000.GenBank accession number for the whole-genome shotgun sequence of NCIMB 8816: NBDZ00000000.GenBank accession number for the whole-genome shotgun sequence of NCIMB 8817: NBDY00000000.GenBank accession number for the whole-genome shotgun sequence of NCIMB 8818: NBDX00000000Data file 1 has been deposited in figshare; DOI: dx.doi.org/10.6084/m9.figshare.4577917.v1Data file 2 has been deposited in figshare; DOI: dx.doi.org/10.6084/m9.figshare.4577947.v1Data file 3 has been deposited in figshare; DOI: dx.doi.org/10.6084/m9.figshare.4577950.v1Data file 4 has been deposited in figshare; DOI: dx.doi.org/10.6084/m9.figshare.4577953.v1Data file 5 has been deposited in figshare; DOI: dx.doi.org/10.6084/m9.figshare.4577956.v1Data file 6 has been deposited in figshare; DOI: dx.doi.org/10.6084/m9.figshare.4577965.v1Data file 7 has been deposited in figshare; DOI: dx.doi.org/10.6084/m9.figshare.4577971.v1Data file 8 has been deposited in figshare; DOI: dx.doi.org/10.6084/m9.figshare.4577977.v1.Lactobacillus is an important group of bacteria that is used in food preservation, food preparation and probiotics. The group is unusually diverse, with individual species showing considerable variation in their functional properties; results of numerous studies have indicated that this variation is also present across strains within species of the genus Lactobacillus, leading to strain-dependent health benefits. Lactobacillus salivarius is one of about 200 species (including subspecies) within this group and results reported in previous literature have revealed its role as a potential probiotic. Our study uses a dataset of 42 strains of L. salivarius and a comparative genomic approach to define genes that each strain has in common and genes that show presence–absence distributions across the strains. Bacteria are subject to selective gene loss as well as gene acquisition. Several mechanisms, collectively known as horizontal gene transfer (HGT), contribute to the process of gene acquisition. These processes lead to considerable variation in gene content within a species, which is further promoted by the presence of independently replicating sequences (replicons) called plasmids that are often present in bacterial cells and can be transferred between cells by one of several mechanisms. A description of functional diversity across L. salivarius and its distribution over the replicons will increase current knowledge and possible exploitation of the variation found in members of the genus Lactobacillus.Lactobacillus is a diverse, paraphyletic group with a combined species and subspecies count of over 200 [The genus over 200 . Lactobaover 200 . They arLactobacillus genus [et al. recently conducted an international genome sequencing initiative of the lactobacilli that revealed that the genus was more diverse than a typical taxonomic family and that confirmed that Leuconostoc, Oenococcus, Weissella, Pediococcus and Fructobacillus all branch from within the Lactobacillus phylogenetic tree [Several pivotal studies have called for a reclassification of the us genus  while otus genus . Sun et tic tree .Lactobacillus, highlighting considerable intraspecific genomic diversity among strains [Lactobacillus salivarius [Numerous studies have also focused on the comparative genomics of individual species of the genus  strains . One spelivarius .L. salivarius UCC118 was first characterised by Claesson et al. and shown to have a multi-replicon organisation with a single repA-type megaplasmid and two smaller plasmids. The megaplasmid harboured genes with an array of functions including bile salt hydrolysis, carbohydrate metabolism and genes that complete the pentose phosphate pathway. It was concluded that the megaplasmid increased the metabolic flexibility and competitiveness of the species [L. salivarius [Lactobacillus rhamnosus [The genome of  species . A previ species . Two exohamnosus .L. salivarius share a similar multi-replicon organisation to that of UCC118, each having a homologous repA-type megaplasmid and a varying number of smaller plasmids from none to two [repA-type megaplasmid while JCM1046 also has an additional circular megaplasmid [L. salivarius as well as the variation in size of the megaplasmids [et al. used the two chromosomal EPS clusters of UCC118 as a reference in a comparative genome hybridisation (CGH) experiment that revealed considerable divergence in gene synteny and gene presence among 33 strains of L. salivarius [Results of two studies indicated that other strains of e to two . Severale to two  as well aplasmid . The varplasmids   that CGH demands is not a limiting factor of the present study, and strain-specific as well as clade-specific genes and functions can be identified by comparative genomics that would otherwise be excluded. We focussed on the analysis of numerous functional traits and we also provide an overall whole-genome view of the relatedness of the strains and the extent of their diversity.The previous study by Raftis ferences . The preL. salivarius were sequenced by Macrogen  using the HiSeq platform and 100 bp paired-end reads. This dataset was supplemented by 13 genomes (5 complete and 8 draft) of strains of L. salivarius that were available in NCBI databases. Lactobacillus hayakitensis DSM18933T was also included in the study as a related outgroup. The dataset included both genome sequences for the type strain of L. salivarius from two different culture collections (DSM20555T and ATCC11741T) to test the robustness of the methods.The genomes of a panel of 29 strains of Reads for the 29 sequenced genomes were assembled using Velvet (v1.2.10)  with a kGenes were predicted using three different gene prediction software: Glimmer3 (v3.02) , GeneMarThe issue of multi-copy genes such as the 16S rRNA gene is not addressed in this study. Our dataset contains a majority of draft genome sequences where assembly software often fails to assemble multiple copies of identical or almost identical genes due to ambiguous placement of reads. Similar genes that posed no problem for assembly software were included in gene counts analysis.www.ncbi.nlm.nih.gov) to assign functional annotation. blast thresholds for assigning the function of a reference sequence to a query gene were 40 % identity, 50 % alignment length to the query gene and a blast bit score of 60. Prediction and annotation of specific functional groups in this study are described in the Supplementary Methods.The amino acid sequences of predicted genes were BLASTed (blastp) against the Kyoto Encyclopaedia of Genes and Genomes database (KEGG) , the Clublast hits (RBBs) between the genes of each pair of genomes and performing two-stage clustering (single-linkage and Markov clustering) on the RBBs. blast thresholds were 40 % identity, 50 % alignment length of the query gene and a blast bit score of 50. For clustering the RBBs, an MCL inflation value of 3 and a minimum cluster size of 2 were used.QuartetS  was usedL. salivarius genomes and the L. hayakitensis DSM18933T genome combined had a predicted core genome of 938 genes. For each genome, these 938 genes were concatenated and the resulting sequences were aligned across the genome set using Muscle (v3.8.31) [L. hayakitensis DSM18933T. The root branch was artificially shortened to provide greater visual discrimination across L. salivarius sub-clades so all other branches are informative relative to each other.The 42 v3.8.31) . Gap regv3.8.31)  where eav3.8.31)  was usedgroEL, rpsB, parB and rpoA, which were identified in each genome using reference sequences from UCC118.To supplement the core-gene phylogeny, four single-gene phylogenies were also generated based on nucleotide sequences using the above methods and a generalised time reversible CAT model. These four genes are L. hayakitensis DSM18933T was excluded from this analysis. Unique genes that were excluded by QuartetS (due to a minimum cluster size of 2) were also added to the matrix at this point. The numbers of core, pan and new genes were calculated by starting with two genomes and sequentially adding genomes, one at a time, until all 42 genomes were included. This procedure was repeated 1000 times, each time the order of the matrix being permuted to randomise the order of addition of genomes. Median values along with the variation from each permutation were recorded and plotted using R. In order to assess the open or closed nature of a pan-genome, the log10 median values for the new-gene curve were also plotted, where a slope of less than 1 is interpreted as indicating an open pan-genome (α<1) [A binary gene matrix modified from the QuartetS output was used to generate core-, pan- and new-gene curves in R. me (α<1) . The R chttps://github.com/chjp/ANI/blob/master/ANI.pl) and implemented. Qin et al. [blast thresholds listed in their paper. The script used for POCP calculation is on figshare .Two whole-genome comparative metrics were used to supplement the core-gene and single-gene phylogenies. Average Nucleotide Identity (ANI)  and Percn et al.  did not Additional descriptions of Methods can be found in Supplementary Methods. These have the sub-headings, ‘Quality assessment of genomes’, ‘Assigning contigs to replicons’ and ‘Specific functional groups’.L. salivarius consisted of 1236 genes. Applying a leave-one-out-strategy to the 42 L. salivarius genomes and re-computing the core genome indicated that it varies from 1236 to 1246 with 1281 as an outlier when JCM1230 is excluded. Table S1 shows that the JCM1230 strain sequenced in this study possesses no plasmids, which explains why the core genome increased so much when the strain was excluded – the absence of a megaplasmid excludes all extrachromosomal genes from being part of the core genome. Li et al. [repA-type megaplasmid in JCM1230 and predicted its size to be approximately 100 kb. It is difficult to explain the absence of plasmid sequences in JCM1230 in the current study: the megaplasmid might have been artificially excluded by a procedural artefact during the DNA extraction/preparation procedure or, alternatively, since 100 kb is the smallest repA-type megaplasmid in the Li et al. [in vitro during laboratory passage.The core genome of i et al.  identifii et al.  dataset,L. salivarius genomes. The curve starts to plateau after the addition of only a few genomes and has substantially levelled out by genome number 42. This suggests that a dataset of 42 genomes is sufficient to define the core genome of L. salivarius. Hutchison et al. [L. salivarius, with approximately 1200 genes, indicates that most of the core genes of a specific group of bacteria are necessary for processes outside of basic cell viability such as niche adaptation and interaction with competitors and pathogens.n et al.  recentlyL. salivarius (excluding unique genes) consists of 3057 gene clusters ranging from 802 genes present in only two genomes to 109 genes present in 41 genomes . L. salivarius. The steep slope indicates that the current dataset is not large enough to define the accessory genome of L. salivarius and that the addition of more genomes from other strains would continue to increase the size of the accessory gene set. The accessory genomes of the 42 strains of L. salivarius genomes will continue to acquire novel genes. Variation in the presence of genes within species is brought about by two main processes, HGT and gene decay, both of which apparently began to act upon all L. salivarius strains after they diverged from their common ancestor, leading to the intra-specific variation observed in this dataset.Overall, the data presented in This intra-specific variation can be summarised in a very general sense using the median number of genes per replicon with the first and third quartiles representing inter-genome variation: chromosome = 1737 ; megaplasmid=249  and small plasmid=47.5 .L. salivarius, rooted on L. hayakitensis DSM18933T. The bootstrap values are high, indicating a robust tree topology and the length of most of the branches leading to the nodes indicates that some divergence has occurred even in more closely related strains. Note that the outgroup branch (DSM18933T) has been shortened for this analysis (see Methods), but the scale indicating 0.003 substitutions per amino acid position can still be applied to all branches corresponding to strains of L. salivarius. A few sub-clades have little to no outer branch lengths, reflecting a lack of phylogenetic divergence. LMG14476 and LMG14477 have a difference of only eight SNPs in the predicted core of 938 genes even though they were isolated from different sources (Table S1). Three strains isolated from the oral cavity, gul1 and gul2 (isolated in the same study), and DSM20555T (independent isolate), also show limited phylogenetic divergence (8–19 SNPs). ATCC11741T is the same type strain of L. salivarius as DSM20555T from another culture collection and they have a difference of zero SNPs in the predicted core of 938 genes, highlighting the limited accrual of variation over short periods of time during vertical gene transfer. A similar case can be observed for three strains, AH4231, AH4331 and AH43348 (17–48 SNPs), all isolated from the human ileocecal region in the same study, and between UCC118 and AH43324 (54 SNPs), also isolated from the human ileocecal region. In contrast to these sub-clades, CCUG 44481  and CCUG 38008  have the most divergent core genome across all 42 strains of L. salivarius (3643 SNPs). Average Nucleotide Identity (ANI) [L. salivarius strains. L. hayakitensis was excluded from the heatmap so an unrooted clustering is presented. ANI was designed as a method to identify whether a particular strain belongs within a species, using a cut-off value of 95 % as the species boundary [L. salivarius is 96.8 % between JCM1047 (isolated from swine intestine) and CECT5713 (isolated from human breast milk), indicating that all strains represent members of the same species.groEL, rpsB, parB and rpoA. When sub-clades had sufficient phylogenetic signal, bootstrap values were high and agreed with the tree topology of the core-gene phylogeny in parB is included as Fig. S1 since it shows the most phylogenetically informative signal of the four genes.Single-gene phylogenies were also constructed using four marker genes, et al. have already shown that there is considerable size variation in L. salivarius repA-type megaplasmids ranging from 100 kb (JCM 1230) to 380 kb (DSM 20555T) [repA-type megaplasmids that we predicted ranged from 165 genes in NIAS840 to 408 genes in cp400. NIAS840 has a complete genome sequence while that of strain cp400 is a draft, indicating that closed genomes are not a factor for bias when predicting the number of genes on megaplasmids. The lack of plasmids in L. hayakitensis DSM 18933T was not discussed when the description of the strain was published [repA-type megaplasmid was acquired early in the evolution of L. salivarius [blast results of three repA-type marker genes  against the contigs of each genome. If contigs were assigned to replicons accurately, it is expected that blast hits for each gene would lie on predicted repA-type megaplasmid contigs. This is indeed the case, with all three genes having between 93% and 100 % identities over their full length aligned to a repA-type megaplasmid contig, usually all three genes align to the same contig. Exceptions include JCM1230, which had no blast hits due to its missing megaplasmid, AH43348, which had an extra parA gene on a predicted repA-type megaplasmid contig and L. hayakitensis DSM 18933T, which has a repA gene and a parA gene on a predicted chromosomal contig. The repA and parA genes of DSM 18933T have a lower identity than the other hits  and it is possible that these genes belong to an unidentified megaplasmid, although there was no mention of extrachromosomal sequences in the original species/strain description [Li  20555T) . This inublished  and plaslivarius . The poscription .repA-type megaplasmid, a finding that was first documented by Li et al. [Lactobacillus plasmids (see Supplementary methods). The contigs of AH43348 had very little homology to pLMP1046; however, several contigs do cover most of a second megaplasmid present in NIAS840 aside from the contigs that align to repA-type megaplasmids. The second megaplasmid of NIAS840 was not described as being circular or linear [repA-type megaplasmid; its draft genome is made up of ten contigs, six belonging to the chromosome and the remaining four described as representing a 143 kb megaplasmid, an 85 kb large plasmid and two small plasmids (31 and 9 kb) [repA-type megaplasmid of UCC118 (pMP118), together adding up to over 94 % of its length. This indicates that these two sequences do not represent separate plasmids, but together make up the repA-type megaplasmid of SMXD51, a finding made more probable by the fact that the available SMXD51 genome is a draft genome.Several strains in the dataset also possess linear megaplasmids that have little homology to the i et al. . These snd 9 kb) . We founet al. in UCC118 [et al. [The smaller plasmids show even greater variation. Table S1 shows that 15 strains have no small plasmids, 20 strains have a single small plasmid and 8 strains have two small plasmids. The number of predicted genes on the small plasmids ranged from 11 in a GJ24 plasmid to 144 in an AH4231 plasmid. Many of these plasmids show high-level homology to the two endogenous plasmids described by Fang n UCC118 . The sma [et al. .L. salivarius, this includes host–bacterium interactions since most strains have been isolated from human or animal sources. Sortases are important enzymes for recognising and anchoring surface proteins containing an LPXTG motif, and sortase-anchored surface proteins are often involved in the interaction of a bacterium with its surrounding environment . In L. srepA-type megaplasmid  where three were isolated from the oral cavity and ATCC 11741T is a reference strain from the Human Microbiome Project (www.hmpdacc.org). Pili are commonly involved in adhesion and their production in this sub-clade might reflect an adaptation to the oral environment by allowing the bacterial cell to adhere to the tooth surface or underlying dentine. JCM 1047 is a swine intestinal isolate and it is not clear why it is the only other strain with a predicted pilus operon, except that the presence of pili surely has an adaptive role in the intestine as well as the oral cavity.All 43 genomes have at least one sortase A gene, the ‘housekeeping’ sortase, that typically acts on many protein targets and is considered to be essential for the survival of most Gram-positive bacteria. Additionally, seven genomes have an extra sortase A and five of these have a sortase C gene. All five strains with a sortase C have a putative pilus operon, confirming the results of previous studies that describe the role of sortase C in pilus construction . The exti et al. ) of AH43L. hayakitensis DSM 18933T has the most genes containing an LPXTG motif (n=18). This indicates that there might have been selective pressure leading to a reduced number of cell-surface and secreted proteins with an LPXTG motif in L. salivarius.The range of values for gene products with an LPXTG motif is partly explained by the number of sortase genes and the presence of pilus operons, with more genes being present in strains with multiple sortases and a pilus operon. L. salivarius and L. hayakitensis DSM 18933T, separated into their respective replicons.Glycosyl hydrolases (GHs) and glycosyl transferases (GTs) are two large and important groups of genes that are responsible for the hydrolysis (or modification) and synthesis, respectively, of the glycosidic bonds of carbohydrates. P=0.67), showing the independence of a strain’s ability to synthesise carbohydrates compared with its ability to break them down. This is not surprising since the selective pressures acting on genes that break down particular carbohydrates are largely determined by the availability of that substrate in the environment while carbohydrate synthesis can lead to complex interactive traits such as EPS, which vary in structure, composition and function depending on the biotic and abiotic environmental factors and the species of bacteria in question [There is no correlation between the number of GHs and the number of GTs per strain in this dataset , but there is considerably more extrachromosomal diversity for GHs than GTs and no GTs are located on the smaller plasmids. These results indicate that GHs are horizontally acquired more frequently than GTs in L. salivarius but absent from L. hayakitensis DSM 18933T or, alternatively, absent from all 42 strains of L. salivarius but present in DSM 18933T. These families are GH2 and GT32 , and GH68 (present in L. hayakitensis only). GH68 is a levansucrase and present in DSM 18933T only while GH2 and GT32 are quite general and act on multiple substrates. Levansucrase enzymes, unlike sucrases, are localised almost entirely extracellularly and they contribute to 60 % of extracellular sucrase activity [T indicates that this strain is more adapted to the breakdown of sucrose, an ability that may compensate for the fact that this strain has the lowest number of GH genes (n=12) in this dataset and the lowest number of GH families (n=9) along with 01M14315, DSM 20492 and SMXD51.Numerous gene families for both GHs and GTs are present in all 43 genomes and found on the chromosomes only. For GHs, these are GH13, GH32 and GH73; for GTs, these are GT26, GT28, GT41 and GT51. All these families have numerous predicted substrates and functional properties and their absence from extrachromosomal replicons indicates that these genes are important for cell processes independent of particular niches. More interesting are the families that are present in the genomes of all 42 strains of repA-type megaplasmid of JCM 1046. Peptidoglycan lyase, an enzyme that can hydrolyse the cell walls of bacteria, is found on the smaller plasmids of JCM 1046 and JCM 1047, both isolates from the swine intestine. GT27 and GT92 are limited to the chromosomes of five strains: the sub-clade of four strains containing DSM 20555T and the singleton, NCIMB 8817.A few other GH and GT families have very limited distributions. GH70, a dextransucrase, is limited to CCUG 44481 and GJ24, a branch pair isolated from different sources. A gene for GH119, an α-amylase, is found only on the The distribution of genes across the strains in these two major functional groups indicates considerable gene loss and HGT with very limited association of GH and GT families with isolation source.L. salivarius UCC118 EPS cluster 1 is located on the chromosome and is composed of 21 genes spread across 23 kb. Of the strains of L. salivarius studied 29 harbour at least 18 genes from UCC118 EPS cluster 1 and the other 13 strains do not have the cluster in their genomes . In contrast to this, the majority of strains harbouring EPS cluster 1 have a human origin, indicating that EPS cluster 1 is not essential for the survival of L. salivarius UCC118 EPS cluster 2 is also located on the chromosome and is composed of 28 genes spread across 33 kb. The two physical extremities of EPS cluster 2 are shared by all the strains . In all 22 cases, this bacteriocin is found on the repA-type circular megaplasmid and appears to have no strong association with a particular isolation source, but its distribution on the core-gene tree (n=3), the AH43348 branch (n=6) and a few small sub-branches (of n=2) and singletons. It is interesting that some of the strains lack this bacteriocin; the size and functional variation of the repA-type megaplasmid highlights the fast evolutionary rate that these replicons display, perhaps losing Abp118 if bacteria co-inhabiting the same environment did not compete strongly with L. salivarius for limiting resources.Flynn activity . This baactivity . Homologene tree  is assocL. salivarius in this dataset. All 43 strains possess between one and four enterolysin genes. The N-termini of these bacteriocins have considerable sequence homology to a bacteriophage lysin and they act to degrade the bacterial cell wall in a range of genera including enterococci, pediococci, lactococci and lactobacilli [n=4) and shows homology to bacteriocins in several oral streptococci. The two-strain sub-clade consisting of CCUG 44481 and GJ24 is the only branch to harbour a plantaricin S while MR10B is present on the small plasmid of three strains, JCM 1046, JCM 1047 and DSM 20554. A cluster of three bacteriocins is present on two divergent strains, CCUG 44481 and CCUG 47171, harbouring plantaricin NC8, lactacin F and acidocin LF221B. The distribution of bacteriocins in this dataset gives an indication of HGT: LS2 is confined to a single sub-clade and was probably transferred into the megaplasmid of the ancestor of these four strains; MR10B is present on the only small plasmid in three divergent strains.A number of other bacteriocins are also present in the strains of obacilli . LS2, anobacilli , is confThe production of bacteriocins gives a strain an obvious competitive advantage since it inhibits similar strains and species that may compete strongly for limited resources. Specific environments impose different biotic and abiotic factors and the details of microbial competition and horizontal transfer of genes (including bacteriocin genes) are dependent on a complicated interplay among these factors, potentially explaining the scattered distribution of bacteriocin genes in this dataset.L. salivarius and a closely related outgroup, L. hayakitenis DSM 18933T. Results from previous comparative studies indicate that there is considerable functional and phylogenetic diversity across species of the genus Lactobacillus species. Smaller scale intra-specific studies focusing on single species of the genus Lactobacillus highlight the continuation of this trend across strains.We conducted a comparative genomic study of 42 strains of L. salivarius has an open pan-genome and that all major functional groups described show considerable functional variation across strains, often displaying greater similarity within sub-clusters as opposed to niche-specific trends. Variation in gene function is greater across the megaplasmids than across the chromosomes and greater across the smaller plasmids than across the megaplasmids. The level of functional variation revealed in L. salivarius indicates that strain-specific properties might be applied to commercial areas of human health and nutrition such as probiotics and food preservation.We demonstrate that Harris HMB. Genbank. BioProject ID: PRJNA357984; BioSample accession number: SAMN06163248; GenBank accession number: MSCR00000000 (01M14315)Harris HMB. Genbank. BioProject ID: PRJNA357984; BioSample accession number: SAMN06163249. GenBank accession number: NBEY00000000 (AH4231).Harris HMB. Genbank. BioProject ID: PRJNA357984; BioSample accession number: SAMN06163250. GenBank accession number: NBEX00000000 (AH4331).Harris HMB. Genbank. BioProject ID: PRJNA357984; BioSample accession number: SAMN06163251. GenBank accession number: NBEW00000000 (AH43324).Harris HMB. Genbank. BioProject ID: PRJNA357984; BioSample accession number: SAMN06163252. GenBank accession number: NBEV00000000 (AH43348).Harris HMB. Genbank. BioProject ID: PRJNA357984; BioSample accession number: SAMN06163253. GenBank accession number: NBEU00000000 (CCUG 2753OB).Harris HMB. Genbank. BioProject ID: PRJNA357984; BioSample accession number: SAMN06163254. GenBank accession number: NBET00000000 (CCUG 38008).Harris HMB. Genbank. BioProject ID: PRJNA357984; BioSample accession number: SAMN06163255. GenBank accession number: NBES00000000 (CCUG 44481).Harris HMB. Genbank. BioProject ID: PRJNA357984; BioSample accession number: SAMN06163256. GenBank accession number: NBER00000000 (CCUG 45735).Harris HMB. Genbank. BioProject ID: PRJNA357984; BioSample accession number: SAMN06163257. GenBank accession number: NBEQ00000000 (CCUG 47171).Harris HMB. Genbank. BioProject ID: PRJNA357984; BioSample accession number: SAMN06163258. GenBank accession number: NBEP00000000 (CCUG 47825).Harris HMB. Genbank. BioProject ID: PRJNA357984; BioSample accession number: SAMN06163259. GenBank accession number: NBEO00000000 (CCuG47826).Harris HMB. Genbank. BioProject ID: PRJNA357984; BioSample accession number: SAMN06163260. GenBank accession number: NBEN00000000 (DSM 20492).Harris HMB. Genbank. BioProject ID: PRJNA357984; BioSample accession number: SAMN06163261. GenBank accession number: NBEM00000000 (DSM 20554).Harris HMB. Genbank. BioProject ID: PRJNA357984; BioSample accession number: SAMN06163262. GenBank accession number: NBEL00000000 (gul1).Harris HMB. Genbank. BioProject ID: PRJNA357984; BioSample accession number: SAMN06163263. GenBank accession number: NBEK00000000 (gul2).Harris HMB. Genbank. BioProject ID: PRJNA357984; BioSample accession number: SAMN06163264. GenBank accession number: NBEJ00000000 (JCM 1040).Harris HMB. Genbank. BioProject ID: PRJNA357984; BioSample accession number: SAMN06163265. GenBank accession number: NBEI00000000 (JCM 1042).Harris HMB. Genbank. BioProject ID: PRJNA357984; BioSample accession number: SAMN06163266. GenBank accession number: NBEH00000000 (JCM 1044).Harris HMB. Genbank. BioProject ID: PRJNA357984; BioSample accession number: SAMN06163267. GenBank accession number: NBEG00000000 (JCM 1045).Harris HMB. Genbank. BioProject ID: PRJNA357984; BioSample accession number: SAMN06163268. GenBank accession number: NBEF00000000 (JCM 1047).Harris HMB. Genbank. BioProject ID: PRJNA357984; BioSample accession number: SAMN06163269. GenBank accession number: NBEE00000000 (JCM 1230).Harris HMB. Genbank. BioProject ID: PRJNA357984; BioSample accession number: SAMN06163270. GenBank accession number: NBED00000000 (L21).Harris HMB. Genbank. BioProject ID: PRJNA357984; BioSample accession number: SAMN06163271. GenBank accession number: NBEC00000000 (LMG 14476).Harris HMB. Genbank. BioProject ID: PRJNA357984; BioSample accession number: SAMN06163272. GenBank accession number: NBEB00000000 (LMG 14477).Harris HMB. Genbank. BioProject ID: PRJNA357984; BioSample accession number: SAMN06163273. GenBank accession number: NBEA00000000 (NCIMB 702343).Harris HMB. Genbank. BioProject ID: PRJNA357984; BioSample accession number: SAMN06163274. GenBank accession number: NBDZ00000000 (NCIMB 8816).Harris HMB. Genbank. BioProject ID: PRJNA357984; BioSample accession number: SAMN06163275. GenBank accession number: NBDY00000000 (NCIMB 8817).Harris HMB. Genbank. BioProject ID: PRJNA357984; BioSample accession number: SAMN06163276. GenBank accession number: NBDX00000000 (NCIMB 8818).Harris HMB. dx.doi.org/10.6084/m9.figshare.4577917.v1 (data file 1).Harris HMB. dx.doi.org/10.6084/m9.figshare.4577947.v1 (data file 2).Harris HMB. dx.doi.org/10.6084/m9.figshare.4577950.v1 (data file 3).Harris HMB. dx.doi.org/10.6084/m9.figshare.4577953.v1 (data file 4).Harris HMB. dx.doi.org/10.6084/m9.figshare.4577956.v1 (data file 5).Harris HMB. dx.doi.org/10.6084/m9.figshare.4577965.v1 (data file 6).Harris HMB. dx.doi.org/10.6084/m9.figshare.4577971.v1 (data file 7).Harris HMB. dx.doi.org/10.6084/m9.figshare.4577977.v1 (data file 8).


Vibrio parahaemolyticus is a Gram-negative bacterium that widely inhabits the marine and estuarine environments worldwide  and originated from a fishery market in Malaysia. This strain exhibited multidrug resistance profiles toward 5/14 antibiotics tested. Based on the antibiotic susceptibility phenotype, the strain exhibited multiple-antibiotic resistance toward ampicillin, 3rd generation cephalosporins (cefotaxime and ceftazidime), and aminoglycosides (amikacin and kanamycin) .Genomic DNA of VP103 was extracted using Masterpure™ DNA purification kit  followed by RNase  treatment (Ser et al., Gene prediction was carried out using Prodigal 2.6, while rRNA and tRNA were analyzed using RNAmmer and tRNAscan SE version 1.21 (Lowe and Eddy, V. parahaemolyticus VP103 consists of 4,988,425 bp with a mean genome coverage of 177.8-fold and with an average G + C content of 53.37% (Table LBDB00000000. The version described in this paper is the first version, LBDB01000000. The genome sequences data are available in FASTA, annotated GenBank flat file, graphical, and ASN.1 formats.The genome of V. parahaemolyticus VP103 strain genome. The genome analysis on ARG-ANNOT noted the presences of β-lactam resistant gene, bla gene within the genome at 99% similarities when compared to other V. parahaemolyticus strains. The phenotypic resistance shown by V. parahaemolyticus VP103 toward ampicillin, cefotaxime, and ceftazidime is closely related to the gene coding Beta-lactamase in the genome. The gene coding aminoglycosides adenylyltransferase of V. parahaemolyticus VP103 confers resistance phenotypic observed toward amikacin and kanamycin.The analysis obtained from RAST server revealed 573 subsystems Figure . The annV. parahaemolyticus VP103 genome illustrates how extensive antibiotics have been used in the aquaculture. Although antibiotics namely oxytetracycline, tetracycline, quinolone, sulphonamides, and trimethoprim are allowed in the Asian aquaculture industry (Rico et al., V. parahaemolyticus VP103 will be useful in future studies to determine antimicrobial resistance and virulence attributes as well as mechanisms that enhance its environmental or host fitness.Multidrug resistance profile seen in the phenotype and genes of The experiments, data analysis and manuscript writing were performed by VL and HS, while KC, BG, and LL provided vital guidance and technical support. LL founded the research project.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


RNA interference (RNAi) is a powerful tool to silence endogenous mosquito and mosquito-borne pathogen genes in vivo. As the number of studies utilizing RNAi in basic research grows, so too does the arsenal of physiological targets that can be developed into products that interrupt mosquito life cycles and behaviors and, thereby, relieve the burden of mosquitoes on human health and well-being. As this technology becomes more viable for use in beneficial and pest insect management in agricultural settings, it is exciting to consider its role in public health entomology. Existing and burgeoning strategies for insecticide delivery could be adapted to function as RNAi trigger delivery systems and thereby expedite transformation of RNAi from the lab to the field for mosquito control. Taken together, development of RNAi-based vector and pathogen management techniques & strategies are within reach. That said, tools for successful RNAi design, studies exploring RNAi in the context of vector control, and studies demonstrating field efficacy of RNAi trigger delivery have yet to be honed and/or developed for mosquito control. Aedes, Culex, and Anopheles target species less than a decade after the introduction of DDT for public health campaigns in Europe and the USA [Insecticide resistance to DDT was originally documented in  the USA ,2,3,4. T the USA ,6,7. To  the USA .Aedes, Anopheles, Culex, and Armigeres disease vector species have resulted in disruption of processes including: morphogenesis, olfaction for host seeking and oviposition, blood feeding, fertility, fecundity, and survival [RNA interference (RNAi) may lend itself to the cause through the suppression of gene products involved in key physiologies that impact mosquito survival, fecundity, behavior, or vector status. The RNAi pathway functions as a powerful subcellular anti-viral mechanism that post-transcriptionally suppresses mRNA transcripts based on sequence complementarity to double-stranded RNAs (dsRNAs) . The patsurvival ,17,18,19per os or contact applications for aquatic stages . Interestingly, a similar ubiquitin gene was found to be strongly linked to apoptosis and cell survival in a genome wide screen of D. melanogaster [D. melanogaster screen revealed 438 dsRNAs that induced apoptosis in vitro. This screen both validated known knockout lethal phenotype genes and identified novel essential genes including the ubiquitin-like Ubi-p63E and CG11700 genes as well as DNA binding proteins bss and CG15455 [D. melanogaster genes have orthologs in An. gambiae, there likely is a cache of highly potent, mortality-inducing target genes in mosquito species [There is great interest in developing RNAi to act as direct alternative to chemical insecticides by virtue of suppressing essential genes leading to RNAi-induced mortality. The most direct pathway to inducing mortality is via manipulation of programmed cell death. Apoptosis in insects is regulated by the Inhibitor of Apoptosis Protein 1 (IAP1) . IAP1 prdal RNAi ,53,54,55ppressed . In a scin vitro . The othnogaster . In tota species .Ae. aegypti, Drosophila species, Blattella germanica, Diabrotica virgifera virgifera, and Rhodnius prolixus [Ae. aegypti, mortality is induced by soaking larvae with a dsRNA targeting β-tubulin without transfection [actin, in Ae. aegypti larvae has also led to increased Cry11Aa toxin sensitivity [Beyond apoptosis, other essential ubiquitously and constitutively expressed genes are targeted for insecticidal activity such as tubulins and Vacuolar ATPase proton pumps (V-ATPases). Tubulin suppression by RNAi induces high levels of mortality in a diverse array of insects including prolixus ,72,73,74sfection . RNAi-basitivity .Drosophila and as such are considered vital to maintaining membrane proton translocation [Drosophila, M. sexta, T. castaneum, A. pisum, Bactericerca cockerelli, Helicoverpa armigera, and D. virgifera virgifera all result in death of the insect [Ae. aegypti midguts, and noted a dramatic reduction in virus titer following suppression of V-ATPase subunits by RNAi [V-ATPase subunit V0B by injection resulted in 80.5% knockdown (72 h post exposure) leading to reduced longevity (measured over 50 days), fecundity (98% reduction), and fertility (19% reduction). Therefore the reduction in virus may be due to an overall impact on mosquito health following V-ATPase silencing. Conversely, Coy et al. (2012) showed that dsRNA delivered orally suppresses V-ATPase subunit C by 60% (168 h post exposure) without any noticeable death [V-ATPase subunit β suppression increased Cry11Aa toxin hypersensitivity in Ae. aegypti larvae [ATPase subunit A significantly reduced the lifespan of Ae. albopictus larvae [Drosophila identified 33 distinct V-ATPase genes, with many subunits having multiple copies or splice variants [V-ATPase mutations have long been associated with lethal phenotypes in location ,77. RNAie insect ,73,78,79 by RNAi . In addile death . Death mle death ,80. RNAii larvae . Additios larvae . VariatiAn. gambiae Serpin2 (SRPN2), which functions in processing prophenoloxidase, proves lethal to older mosquitoes and could be useful as a late life insecticide [Ar. subalbatus induced significant mortality and gross deformities in eclosing adults. COatamer Proteins (COPs) are required for vesicle formation and nutrient trafficking in the midgut. RNAi suppression of COPs induces death in H. armigera and Ae. aegypti [Ae. aegypti larvae is lethal during development [Finally, death can also be a by-product of suppression of genes with roles in processes beyond cell death and cytoskeletal structure. For instance, suppression of ecticide . An RNAi aegypti ,82,83,84elopment ,49,85. Uelopment .An. gambiae, Ae. aegypti, and Cx. pipiens [2 detection required for host-seeking [Ae. aegypti [Ae. albopictus, RNAi knockdown of OR7 results in reduced blood feeding success and loss of human host preference in a human or mouse choice experiment [Cx. pipiens, RNAi suppression of OR37 & OR99 results in reduced egg raft production and ability to sense the stimulant 4-ethylphenol [Olfaction is essential for hematophagy and associated fundamental mosquito behaviors including host-seeking and oviposition. Disruption of olfactory processes is a particularly useful approach to repelling a host-seeking mosquito—witness DEET, the most widely used active ingredient in repellent formulations on the market ,88. Ther pipiens ,91,92,93-seeking . Beyond  aegypti . In Ae. periment . Olfactiylphenol .orco gene can alter the repellency effect of DEET in Ae. aegypti demonstrating the potential loss of efficacy that can occur through odorant receptor mutations [Suppressing olfaction genes may offer non-chemical approaches to altering blood-feeding behavior and oviposition with clear public health benefits. However achieving this goal requires better understanding of the sequence diversity and evolution rates of olfaction genes in wild mosquito populations. As mentioned above, mutations in the utations . Anotherutations .An. gambiae [Ae. aegypti, suppression of the aegyptin gene results in drastically reduced blood meal uptake and egg output [timeless, chryptochrome 1, takeout 1–3) odorant receptor genes have been linked to reduced blood feeding success in An. gambiae and Ae. aegypti respectively [gbf1, gap1/gap2, late trypsin, AaSPVI, and AaSPVII in Ae. aegypti result in impaired oogenesis but not mortality [COPI and ARF1/ARF4 induce rapid mortality following a blood meal indicating their importance in digestion [Blood feeding stimulates differential expression in 50% (2388 upregulated transcripts) of all genes in  gambiae ,99. Disrg output . Additioectively ,97. Afteortality ,100. Othigestion ,83.arginase and urate oxidase delays vitellogenesis and thereby stalls oogenesis following a blood meal in Ae. aegypti [Anopheles and Drosophila are likely to be essential for survival” [aquaporin suppression increases fluid retention resulting in mosquito tolerance to desiccation improving survival under stress [In addition to digestion, impairing diuresis via suppression of  aegypti . Overendurvival” . Indeed,r stress ,104.kir in An. gambiae, Catalase 2 in Ae. aegypti, and Cx. pipiens (Insulin like peptides) adult females can have no impact on survival but significantly reduce egg output or egg length [Beyond inducing rapid mortality, the next logical course to reduce vector populations is to interfere with sexual development and reproduction. Preventing oogenesis, diminishing the number of female adults through sexual dependency genes, and producing sterile males are viable approaches to population control. Numerous genes limit egg batch size and nutrient uptake through disruption of nutrient regulation and developmental processes in the oocyte ,108,109.g length ,110,111.zero population growth or transglutaminase genes, which function in sperm development and storage respectively [E. coli expressing dsRNAs targeting multiple male testes genes resulted in sterility in 92% of emerging male adults. This study also suppressed the female splice variant of the mosquito sex dependency gene doublesex in larvae, with the result that 97% of emerging adults were male. Suppression of other sex dependency genes such as transformer 2 using an inducible RNAi-plasmid transformation was also found to result in 70% male progeny following mating of transformed adult males and females [Fecundity and sex bias also have been heavily investigated as a means of vector control. Sterile males can be generated via suppression of testis specific genes such as ectively ,112. In ectively , feeding females .Ae. aegypti, 8400 genes have altered transcript levels, and in An. gambiae the embryo contains more differently transcribed sequences (624–1009) than any other life-stage [semaphorin A, frazzled, and commissureless2 [+ methionine symporter (AeNat5) in Ae. aegypti embryos resulted in larval mortality but no impact on embryos was measured [An. gambiae [Embryogenesis is a transcriptionally intensive process. In fe-stage ,115. Thefe-stage ,116,117.ureless2 ,116,117.measured . In the measured . In thismeasured . Knockdomeasured ,120. Her gambiae . Therefo gambiae . Howeverper os, soaking, or rehydration in aqueous solutions [Ae. aegypti larvae soaked in an RNAi trigger for β-tubulin showed enhanced mortality [per os, through the cuticle, via the anal papillae or by another route. Many different delivery approaches have been used for per os RNAi trigger delivery in larvae, including naked dsRNA in buffer or water [® (Qiagen) liposomes, chitosan nanoparticles, E. coli expression systems, and Pichia pastoris expression systems have been explored [en masse with knockdown measured at fourth instar. Chitosan is currently the most economical and environmentally safe nanoparticle delivery system for larvae. Chitosan has the added benefit of having anti-microbial activity when complexed in particles which could limit dsRNA degradation by microbes [Ae. aegypti and An. gambiae [E. coli and P. pastoris expression systems facilitate even cheaper, scalable dsRNA delivery which protect dsRNA until consumption [Management of larval stage insects is a key component of any pest or vector control strategy. In larvae, RNAi is functional throughout the course of development with knockdown being inducible either by direct exposure or via inheritance from exposed parents ,122,123.olutions ,125. Ae.or water ,127. Altor water ,22. To pexplored ,129. Curexplored ,131. In microbes . Numerou gambiae ,128,129.Aedes, Armigeres, and Culex species pupae produces knockdown of targets up to 90% [per os, RNAi trigger exposure in larvae, or even adult female F0s could provide sufficient long term persistence to suppress pupal genes [single-minded gene (AAEL011013) [For pupae, RNAi trigger exposure requires injection because pupae are a non-feeding stage. Nevertheless, injection of RNAi triggers into p to 90% ,19,96. Pp to 90% . Althougal genes ,120. IndL011013) .Ae. aegypti larvae, exposure to the oganophosphate temephos increases expression of a ABC type transporter P-glycoprotein eightfold [P-glycoprotein resulted in no mortality but increased larval sensitivity to temephos by ~25%.Because RNAi can suppress detoxification and pesticide resistance genes, RNAi triggers could act as synergists to enhance insecticidal activity. Using RNAi to increase insect sensitivity to toxins was an early concept in agricultural pest control. First demonstrated in cotton bollworm, RNAi-silencing of cytochrome p450 resulted in larval susceptibility to toxic plant metabolites . The useightfold . RNAi siAe. aegypti larval midguts. Here disruption of the peritrophic matrix facilitates uptake of diflubenzuron, calcofluor white, and dithiothreitol leading to mortality [Synergistic action of RNAi trigger knockdown is not limited to insecticide resistance genes such as noted in suppression of chitin synthase 1 in ortality .Ae. aegypti-Sindbis virus interactions [RNAi is highly attractive as a technology to parallel chemical insecticides, counter basic mosquito physiology and impact populations. Unlike chemical approaches, RNAi could also be harnessed to impact vector competence, or the inherent capacity of a mosquito to support the development and transmission of a pathogen. This concept originated in a body of mosquito-arbovirus studies that constituted the first evidence that RNAi is an antiviral innate immune response. In mosquito cells, expression of an antisense segment of the La Crosse Virus was noted as a form of “intracellular immunization” because these cells had far lower LACV titers than control cells . The samractions ,143.Plasmodium parasite control in the vector abound because the innate immune response to these parasites has been studied extensively. For example, RNAi suppression of a number of proteins involved in complement, signal modulation  and melanization pathways decreases numbers of midgut-stage parasites as reviewed by Blandin et al. (2008) [Although RNAi cannot be used to directly target malaria parasites developing in the mosquito host, leads for RNAi approaches to . (2008) . Further. (2008) ,146.Brugia malayi, proved amenable to RNAi by injection of an RNAi trigger for a B. malayi cathepsin gene, with a suspected role in molting, into the mosquito host body cavity. Parasites exposed to this trigger at key life stages proved to be significantly less motile than those from control groups, and thereby are highly unlikely to be successfully transmitted [The mosquito-borne filarial worm parasites present yet another potential scenario for RNAi-based interventions for mosquito-borne disease control. Animal parasitic nematodes are often considered recalcitrant to RNAi-based gene suppression . Howevernsmitted .Schistocerca gregaria and the German cockroach B. germanica [Ae. aegypti where vATPase C dsRNA was found partially degraded in tissues after 24 h [Plutella xylostella [P. xylostella when sprayed onto cabbage leaves demonstrating potential for modified siRNAs to be used in ATSB or other per os exposure applications targeting mosquito species. The ability for modified nucleotides to be delivered by per os exposure has been demonstrated in Anopheles stephensi. In this case a single stranded antisense morpholino reduced transcript of the target anti-mitogen-activated protein kinase by 60% following administration via a synthetic blood-meal [A variety of effective oral delivery systems using unmodified dsRNAs have been developed for larvae. In some cases larvae are soaked in large quantities of naked RNAi triggers resulting in suppression of the target after consumption ,126,127.ermanica ,150. Theter 24 h . The tralostella ,152. Theood-meal . These oood-meal .jmtA resulted in suppression and 47% reduction in egg production in Ae. aegypti [Leptinotarsa decemlineata) potato plant leaves were coated with an aqueous solution of dsRNA targeting actin and left to dry [In adults, oral exposure of naked dsRNA has also proven effective when exposed via a nuclease free medium. Feeding a 3%–5% sucrose solution via capillary tubes containing 3–16 mg/mL dsRNA targeting  aegypti . In termt to dry . dsRNA dper os [Plasmid expression systems offer major advantages over in vitro production of dsRNA. Firstly, expression of dsRNAs using bacterial systems have long been utilized to greatly reduce the cost of synthesis for nucleic acids and proteins. These plasmids contain a promoter followed by the target sequence of interest, a loop region, and the reverse complement of the sequence of interest, which when transcribed form a long hairpin dsRNA. Bacterial production of dsRNA still requires the dsRNA to be delivered to the mosquito following production, which has proven effective per os . Alternaper os ,155,156.per os ,120. Theper os . The nexAe. aegypti larvae [Spodoptera frugiperda Sf9 cells, CPPs can facilitate delivery of plasmids [Ae. aegypti Aag2 cells, CPPs increase the potency of IAP1 dsRNA without increasing cytotoxicity [Nanoparticle complexes can provide protection of RNAi triggers from degradation in the environment and the midgut. The composition of nanoparticles varies greatly from biomacromolecular material like chitosan or peptides to chemically produced liposomes and polyethylene glycol (PEG) ,72,157. i larvae . Cell Pei larvae . RNAi tri larvae ,160. In toxicity ). These B. germanica, naked dsRNA is degraded in the midgut following ingestion but protected when encapsulated in liposomes , which subsequently facilitated uptake and knockdown of β-tubulin with 100% mortality [Drosophila species where neonate larvae soaked in Lipofectamine 2000 (Invitrogen) reduced RNAi target transcriptinon by 50% while soaking without lipofectamine failed to induce silencing [Ae. aegypti and M. sexta naked dsRNA and E. coli expressed dsRNA failed to induce RNAi silencing, but Effectene® (Qiagen) liposomes achieved 90% knockdown of target genes [® liposome feeding assays 77% knockdown was achieved on average, with up to 97% knockdown across 11 genes in Ae. aegypti [Ae. aegypti with or without liposomes induced mortality when using β-tubulin, Chitin synthase I, or Heat shock protein 83 RNAi triggers [Chemically produced liposome nanoparticles have also been explored extensively as RNAi delivery systems. In the cockroach, ortality . The samilencing . In Ae. et genes . Of all  aegypti ,130,131.triggers . RegardlAn. gambiae [An alternative to both chitosan and liposome systems is the utilization of PRINT particles. Here numerous different reagents, such as PEG, can be molded to form complexes with defined shapes, sizes, and charges in a highly scalable production system. Uptake and bio-distribution without toxicity have been demonstrated in larval and adult  gambiae ,51.Ae. aegypti adults [Considering the variety of entomopathogenic viruses already safely used as biocontrol agents mosquito viral systems offer a sophisticated means of delivering and expressing RNAi triggers directly in the mosquito cell cytosol . The spei adults .Ae. aegypti densovirus (AaDNV) genome was expressed in a plasmid containing a Pol III promoter-driven expression cassettes containing short hairpin RNA (shRNA) targeting Ae. aegypti V-ATPase subunit A [Ae. albopictus C6/36 cells produced viral stocks that when exposed to larvae resulted in silencing of V-ATPase A and significant reduction in larval longevity.Densoviruses have also been utilized as expression and delivery vectors of RNAs in larvae. Here the ubunit A . ExpressE. coli strain can be transformed with dsRNA plasmid expression vectors and grown in bulk worry of RNA degradation. In one study, a Pet17B plasmid containing an ampicillin resistance cassette, origin of replication, and cloned inverted repeats targeting 3 genes of interest, was mass produced in HT115 E. coli [Ae. aegypti larvae in water (soaking method) resulting in 81%–97% gene suppression of 3 target genes in larval midguts. Others take this one step further and expose mosquitoes directly to the organism producing the dsRNA. There are several lines of thought for taking on such an approach. For one, biologically produced dsRNA does not need to be expressed in the mosquito and as such does not require transfection or a mosquito specific promoter. Also dsRNA is rapidly degraded in aqueous environments [E. coli to C. elegans [E. coli expressing Ae. aegypti sexual dependency genes (embedded in agar pellets) were fed to larvae, resulting in sterility in up to 90% of adults [A major alternative to nanoparticle complexes are direct expression systems. The main advantage of such a system is scalability and cost compared to in silico production methods. For instance, the RNase III deficient HT115  E. coli . Purifieronments . This co elegans . The metf adults . Direct Pichia pastoris yeast have also been successfully transformed with pPicZB plasmid containing a long hairpin RNA sequence encoding an Ae. aegypti juvenile hormone acid methyl transferase gene [Ae. aegypti fed on fermented yeasts resulted in >90% knockdown and death of larvae up to 144 h post exposure.ase gene . Ae. aegTo combat the proliferation of pesticide resistant mosquito vectors and control associated mosquito-borne diseases, Integrated Vector Management and Integrated Vector Borne Disease Management programs will require alternatives to chemical pesticides ,8. Herei


Oedaleus asiaticus grasshoppers under four different single-plant diets to examine the relationships among diet, performance, stress, and transcription patterns. Grasshoppers fed only Artemisia frigida (Asteraceae) were stressed, as indicated by their lower growth, size, development, and survival, in comparison to grasshoppers fed on any of three grasses, Cleistogenes squarrosa, Leymus chinensis, or Stipa krylovii . We then used transcriptome analysis to examine how gene expression levels in O. asiaticus were altered by feeding on these diets. Nymphs fed A. frigida had the largest variation in gene expression profiles with a total of 299 genes significantly up- or down-regulated compared to those feeding on the three grasses: down-regulated genes included those involved in cuticle biosynthesis, DNA replication, biosynthesis and metabolism of nutrition. The up-regulated genes included stress-resistant and detoxifying enzymes. GO and KEGG enrichment analysis also showed that feeding on A. frigida could down-regulate biosynthesis and metabolism related pathways, and up-regulate stress-resistant and detoxification terms and pathways. Our results show that diet significantly altered gene-expression, and that unfavorable, stressful diets induce more transcriptional changes than favorable diets. Altered gene-expression represents phenotypic plasticity, and many such changes appear to be evolved, adaptive responses. The ease and regularity by which individuals shift phenotypes via altered transcription suggests that populations consist not of similar, fixed phenotypes, but of a collection of ever-changing, divergent phenotypes.We reared  Phenotypic plasticity refers to the ability of individuals to alter their phenotypes in response to changing environments . PhenotyAn interesting and important category of phenotypic plasticity relates to how animals alter their phenotypes in response to different foods. Diet resources vary in space and time, and it is not surprising that animals have evolved the ability to rapidly change their phenotypes in response to these challenges. Many animals alter their biochemical phenotypes to better optimize favorable food items, or, conversely, to ameliorate unfavorable food items. An example of the former is the rapid production of lipases, carbohydrases, or proteases after consuming lipids, sugars, or proteins –6. ExampIn addition to diet’s direct influence on nutritional biochemistry, altered diets also induce numerous broader, derivative (down-stream) changes to the phenotype, including to general homeostasis, physiology, growth, development, fecundity, and survival. Such down-stream effects of diet change also represent phenotypic plasticity –2.Oedaleus asiaticus (Orthoptera: Acrididae). Our ultimate goal is to use knowledge about nutrition and diet-induced gene-regulation to control insect pests [Although, the biochemical, metabolic, and performance responses to changing diets are well documented, the gene-regulation that often underlies these downstream responses is not well-studied. In this paper, we seek to understand how diet influences gene-regulation and, subsequently, performance. Specifically, we examine the transcriptomics that underlie the manifold changes resulting from altered diets. Altered gene-regulation lies at the basis of much phenotypic plasticity , 3, 14. ct pests .Controlling pests by manipulating their nutritional/feeding biology is reasonable –16. MostSpodoptera spp. have been studied by RNA-Seq analysis [O. asiaticus specializes on grasses [O. asiaticus often cause grassland damage and economic disruption [O. asiaticus on four different single-plant diets, and measured resulting performance. We then sequenced and compared the transcriptomes from grasshoppers fed on each diet. Bioinformatics and differential gene expression analysis revealed that different food plants induced different gene expression profiles in O. asiaticus. These results provide new insights into the alteration of transcription by host plants and enhance our understanding of the gene expression variation underlying phenotypic plasticity in phytophagous insects.Previous biological and ecological research has examined the adaptations of herbivores to their host plants. For example, diet-dependent metabolic responses of insect herbivores such as analysis . However grasses –28, and  grasses , 29. Outsruption –31. To sOedaleus asiaticus B. Bienko (Orthoptera: Acrididae), were field-collected at Xilin Gol grassland in 2015. Grasshoppers are common agricultural pests and are not included in the “List of Protected Animals in China”. No specific permits were required for the described field studies.Grasshoppers, °C in December and reach 35°C in July. The mean annual precipitation is 346 mm, more than 80% of which occurs during the growing season from May to September [Cleistogenes squarrosa (Trin.) Keng, Leymus chinensis (Trin.) and Stipa krylovii Roshev , Artemisia frigida Willd (Asteraceae), and, Caragana microphylla Lam. (Leguminosea). However, in this ecosystem, plant composition is highly variable in space, and the abundance of any one plant species can change dramatically over fairly short distances. For example, A. frigida can comprise 0.3% to 29.6% of plant individuals at different sites [O. asiaticus grasshoppers must somehow adapt to this plant diversity. Three grasshoppers, O. asiaticus B. Bienko, Calliptamus abbreviatus Ikonn and Dasyhippus barbipes  are widely distributed in this region [The research site  was located in the Xilin Gol League, Inner Mongolia, northeastern China, a region representative of Eurasian steppe grassland . The meaeptember , 32–33. nt sites , 34. O. O. asiaticus performance when reared on different host plant species during late June, 2015. A 200-m2 flat area of steppe was prepared by removing all vegetation using a mower. We then installed 20 gauze-covered cages (each measuring 1 m × 1 m × 1 m) in five rows with four cages in each row. The distance between cages was 1 m. We placed field-collected soil on the floor of each cage to a depth of 10 cm, and removed spiders and other natural enemies from the cages before adding O. asiaticus nymphs. The mesh covering stifled wind flow, and the cage placement gave equal exposure to sunlight, reducing microclimate differences amongst cages. Each cage was assigned to one of four treatments, consisting of one of four single plants; C. squarrosa, L. chinensis, S. krylovii or A. frigida. To start the experiment, we collected 3rd-instar O. asiaticus of mixed sex from the field. Thirty were immediately euthanized, dried at 90°C for 24 h, and individually weighed to establish the dry starting mass of our experimental animals. We then randomly assigned 16 individuals to each cage. Hence, each of the four treatments contained 80 individuals, divided among five replicates (cages)/per treatment, in a randomized block design.We investigated th instar nymphs. The experiment ran for ~30 d. Each morning, fresh plants were cut at ground level and each species was placed in a separate plastic container and returned to the laboratory. The wet weight of each plant was determined  and 50 g of a single plant species was placed into a rectangular plastic container (20 cm × 10 cm × 2 cm) containing sterile water. The top of the container was perforated, through which the plant stems were inserted. One container was placed into each cage, and embedded in the soil so that the top of the container was flush with the soil surface. Fresh vegetation was replaced every 24 h, thus providing surplus food in a semi-natural environment. Morning feeding provided the freshest food for the grasshoppers, which fed heavily in the morning. We surveyed field cages daily to monitor survival until all surviving individuals became 5th instar nymphs. They were then euthanized and weighed to obtain their dry mass using the method described above.Insects remained in their cages and received the specified feeding treatment until all individuals developed into 5rd to 5th instar nymphs. Survival rate was calculated by the number surviving to 5th instar/the number of initial 3rd instar nymphs (n = 16) [Development time was calculated from 3n = 16) . Growth  . Growthth instar, we analyzed their RNA. We collected 2 samples from each of our 4 treatments. Each sample consisted of 5 newly molted 5th instar female nymphs (1 chosen randomly from each of the 5 replicates). Hence, in total, we analyzed 8 samples (2 from each diet). Each sample consisted of 5 female nymphs (combined) from a single diet. The collected samples were named by abbreviating the insect name and food plant followed by the sample number; OA_Sk_1, OA_Sk_2, OA_Cs_1, OA_Cs _2, OA_Lc_1, OA_Lc_2, OA_Af_1, and OA_Af_2.When our treatment insects reached 5Total RNA was extracted from each of the 8 samples, using the TRIzol reagent  following manufacturer instructions. The RNA sample quality was examined in 4 steps: RNA degradation and contamination was monitored on 1% agarose gels; RNA purity was checked using the NanoPhotometer spectrophotometer ; RNA concentration was measured using the Qubit RNA Assay Kit in the Qubit 2.0 Fluorometer ; and RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system , respectively. The OD260/280 ratios of extracted RNA were between 1.9 and 2.1, which were deemed high quality. All samples had RNA integrity number (RIN) >8.0.-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptors with a hairpin loop structure were ligated to prepare for hybridization. To select cDNA fragments that were 150~200 bp in length, the library fragments were purified with AMPure XP system . Then, 3 μl USER Enzyme  was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR. Then, PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) primer. Finally, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.A total of 1.5 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext Ultra RNA Library Prep Kit for Illumina  following manufacturer recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primers and M-MuLV Reverse Transcriptase (RNase HClustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer instructions. After cluster generation, the libraries were sequenced on an Illumina HiSeq 2000 platform and 125 paired-end reads were generated.Raw data (raw reads) in fastq format were first processed through an in-house perl script . In this step, clean reads were obtained by removing adapters and reads containing ploy-N and low quality reads. At the same time, Q20, Q30, GC-content and sequence duplication level of the clean data were used for data filtering. All downstream analyses were based on clean, high quality data.https://github.com/trinityrnaseq/trinityrnaseq/wiki).The left files (read1 files) from all libraries/samples were pooled into one big left.fq file, and right files (read2 files) into one big right.fq file. Transcriptome assembly was accomplished based on left.fq and right.fq using Trinity (version: r20140413p1) with min_kmer_cov set to 2, K set to 25 by default and all other parameters set at default. Trinity  partitioGene function was annotated based on the following databases: Nr (NCBI non-redundant protein sequences); Nt (NCBI non-redundant nucleotide sequences); Pfam (Protein family); KOG/COG (Clusters of Orthologous Groups of proteins); Swiss-Prot ; KO (KEGG Ortholog database) and GO (Gene Ontology).Gene expression levels for each sample were estimated by RSEM . First, P values were adjusted using the Benjamini and Hochberg approach to control for false discovery rates. Genes with an adjusted P-value < 0.05 were assigned as differentially expressed.Differential gene expression analysis was performed using the DESeq R package (1.10.1) to compare two feeding conditions/group . DESeq pGene Ontology (GO) enrichment analysis of the differentially expressed genes (DEGs) was performed using the GOseq R packages based on Wallenius non-central hyper-geometric distribution , which chttp://www.genome.jp/kegg/). We used KOBAS [KEGG  is a dated KOBAS  softwareCHS , CUP2 , LCP , ESG , CUP1 , CYP , CAT , HSP , SBD  and NSO . Gene-specific primers of those ten genes were designed using Primer Express Software v2.0 . All primers used are listed in −ΔΔCT Method [O. asiaticus feeding on S. krylovii to be 1 for each gene. All qRT-PCRs for each gene was performed in three technical repeats. Statistical analysis of qRT-PCR data was conducted using the ANOVA procedure of SAS 8.0.Ten candidate DEGs involved in insect cuticle biosynthesis, stress-resistant or detoxifying enzymes were chosen for validation using quantitative real-time PCR (qRT-PCR), including O. asiaticus phenotypic plasticity, in response to four different diets . This indicates that feeding on A. frigida provided less benefit for O. asiaticus growth and development, presumably because of poor adaptability to this plant compared to the other three grass species. By comparison, among the three grasses, survival rate, developmental time, adult dry mass, growth rate and overall performance, were not significantly different  by blastx, and to the NCBI Nt database . The unigenes were named and functionally annotated based on the highest sequence similarity to the retrieved proteins/genes . Of the Zootermopsis nevadensis (21.4%), Stegodyphus mimosarum (7.8%), Tribolium castaneum (5.5%), Lasius niger (5.0%) and Acyrthosiphon pisum (4.5%). GO annotation  vs. OA_Sk (sample feeding S. krylovii) , OA_Af vs. OA_Lc (sample feeding L. chinensis) , OA_Af vs. OA_Cs (sample feeding C. squarrosa) , OA_Lc vs. OA_Sk , OA_Cs vs. OA_Sk , and OA_Lc vs. OA_Cs   between O. asiaticus fed on A. frigida compared to those fed on the respective gramineous plants , DNA replication -methyltransferase, et al.), and biosynthesis and metabolism of carbohydrate , fat  and proteins . This suggested that feeding on A. frigida could result in decreased biosynthesis activity and metabolism in O. asiaticus. Among the up-regulated genes (A. frigida could activate many stress-resistance and detoxification related genes.Our data showed that 76.15%-80.81% of the clean reads successfully mapped to the assembled transcriptome . Differegulated) , S5 Fig.ed genes , which ss plants . The ress plants  includined genes , some weO. asiaticus that fed on A. frigida and those that fed on poaceae plants. There was very little variation between nymphs that fed on poaceae plants. In the GO  and KEGG enrichment  analyses we compared DEGs from O. asiaticus that had fed A. frigida and the three poaceae plants , cellular component (CC) and molecular function (MF) , regulation of cell communication (BP), regulation of signaling (BP), inositol catabolic process (BP), alcohol catabolic process (BP), polyol catabolic process (BP) and organic hydroxy compound catabolic process (BP). This suggests that O. asiaticus fed A. frigida had increased signal transduction and metabolized other substances, such as hydroxyl compounds.From DEGs analysis, we found that the gene expression profiles varied significantly between e plants . With GOion (MF) . Down-reO. asiaticus fed A. frigida and those fed on the three grasses were assigned to 16  pathways . Moreover, ten genes showed significant correlations (P < 0.05) between the RT-qPCR data and the RNA-seq results, which indicated good reproducibility between transcript abundance assayed by RNA-seq and the expression profile revealed by qRT-PCR data.The qRT-PCR results for all ten candidate genes were tested statistically, and the cuticle biosynthesis related Oedaleus asiaticus grasshoppers. Our results demonstrate that the Artemisia frigida (family Asteraceae), is less suitable for O. asiaticus, in comparison to three grasses . Grasshoppers fed only on A. frigida had reduced size, growth, development, and survival, in comparison to those fed on any of the three grass species. These results confirm previous studies [th instar through to maturity was lowest for A. frigida [A. frigida-fed grasshoppers exhibited dramatically different transcription profiles from grass-fed insects. What does this mean? Why would feeding on an unsuitable plant dramatically alter transcription profiles, and what are the consequences of such changes? These questions are best addressed under the theory of phenotypic plasticity.In our experiment, we employed single-plant feeding trials to compare the suitability of four different food plants for  studies , 44–45.  frigida . SubsequPhenotypic plasticity occurs when an individual changes its phenotype. All living things can undergo phenotypic plasticity, which can be expressed as changes to biochemistry, metabolism, physiology, morphology, development, behavior, or life-history, etc. –2. AlterA confusing aspect of transcriptomic studies is that altered transcription in response to changed environments can range from highly evolved and beneficial responses, to non-evolved responses, whose outcome might be beneficial, neutral, or highly detrimental to the organism , 47. SorA. frigida exhibited at least 1, 138 differently expressed genes in comparison to grass-fed insects. This agrees with previous studies showing increased transcription with increased stress [A. frigida was stressful, because it significantly lowered performance. Third, we suggest that the 196 unique up- and 103 down-regulated genes in the A. frigida-fed grasshoppers produced a different animal than the grass-fed grasshoppers . This idea is supported by the significant morphological /performance differences between grass- and A. frigida-fed grasshoppers; the two groups have significantly different phenotypes. Considering that untold environmental and social factors can alter transcription [However, we can still draw some broad conclusions from our study. First is that different food plants induce different gene expression profiles. This confirms previous studies linking changed transcription to changed environments . Second d stress . In our cription , this sucription , 49.A. frigida resulted in down-regulation of some genes related to insect cuticle biosynthesis, DNA replication, and biosynthesis and metabolism of carbohydrate, fat and protein, and up-regulation of some genes related to stress-resistance or detoxification enzymes such as heat shock protein (Hsp), cytochrome P450, and carboxylesterase and apoptosis inhibitor. Likewise, GO and KEGG enrichment of A. frigida-fed insects indicated altered transcription of biological processes and biosynthesis and metabolism pathways, including down-regulation of structural constituents to cuticle production and carbohydrate metabolic process, and up-regulation of many biological processes and pathways related to stress-resistance and detoxification enzymes . The altered transcriptome offers insights into the observed biological performance of O. asiaticus. Many of these plastic changes are probably responses to stress. The fact that O. asiaticus grasshoppers up-regulated stress-resistance genes after feeding on A. frigida is not surprising, given that this plant is less preferred [A. frigida-fed insects were stressed, as indicated by significantly lower performance, and that A. frigida contains potent toxins, including terpenoids and sesquiterpene lactones [In regards to specific genes: feeding on referred , 44–45, lactones . Heat-shlactones . Up-reguLocusta migratoria manilensis (Meyen), a species where outbreaks correlate with the distribution of the host plant, Phragmites australis (Cav.) [Studies on the co-adaptation, co-evolution and co-speciation between herbivores and their host plants have provided an understanding of the behavioral, physiological, chemical, genetic, ecological and evolutionary mechanisms involved in these interactions , 28. Hers (Cav.) –55. Manys (Cav.) . In thisThe question of which selective factors have driven the evolution of host adaptability by insect herbivores is an area of research interest. Although ecological factors such as susceptibility to predation and other aspects of habitat association have been identified as important in selection, plant chemistry is critical, including nutrition, nutritional barriers, and secondary compounds , 56–58. Hyposidra infixaria (Lepidoptera: Geometridae), when reared on artificial diet compared to tea leaves, was linked to higher activity of nutrition metabolism-related enzymes [A. frigida less preferred host).Specifically, it is a widely accepted hypothesis that the evolution of diet choice and food adaptability is tightly correlated with nutrition metabolism-related enzymes . For exa enzymes . This waet al. are differentially induced for various plants in herbivorous insect [O. asiaticus fed on the less preferred plant A. frigida.In addition, the evolution of diet choice and food adaptability is tightly correlated with detoxication related enzymes . The exps insect , 51. WheIn our experiment, we analyzed 2 samples from each treatment for RNA. Although each sample contained 5 pooled insects , analyzing more samples, or individuals (instead of groups), would have allowed a statistical analysis of transcriptomic variance among treatments –64. TodaO. asiaticus. In addition, we need to examine exactly how the terpenoids and sesquiterpene lactones present in A. frigida influence physiology and biology. Analysis of quantitative relations between chemical plant traits (nutrition and secondary compounds) and metabolism/detoxication related gene-expression provide the opportunity for ongoing molecular research to decipher the biological mechanisms of herbivorous insect host choice and adaptability.Lastly, we would like to comment on the role of plant primary and secondary metabolites in mediating plant–insect interactions . Future S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigThe unigenes are grouped into 26 hierarchically structured KOG terms. The y-axis indicates the number of genes in each KOG.(TIF)Click here for additional data file.S4 FigKEGG analysis divided pathways into five groups, A-Cellular Processes, B-Environmental Information Processing, C-Genetic Information Processing, D-Metabolism, E-Organismal Systems. The x-axis indicates the number and percent of genes in each KEGG pathways.(TIF)Click here for additional data file.S5 FigO. asiaticus individuals feeding L. chinensis, S. krylovii, C. squarrosa, A. frigida, respectively.Genes were divided among three classes: red genes are up-regulated in the right sample vs. the left sample, green genes are down-regulated in the right sample vs. the left sample, and blue genes are not differentially expressed. OA_Lc, OA_Sk, OA_Cs, OA_Af were representive of (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


Sessile plants detect and ward off invading microorganisms with a robust and sophisticated innate immune system in addition to structural, physical, and chemical barriers . The fir+) to a target protein, which is termed mono(ADP-ribosyl)ation (MARylation) or poly(ADP-ribosyl)ation (PARylation), respectively  based on the conserved motifs in the catalytic domains . MAR. MAR+) t domains . In ART,g pocket ,7. ARTs g pocket . PARylatg pocket . PARylatg pocket . ADP-ribg pocket . Proteing pocket .Arabidopsis genome encodes three PARPs  with the conserved ARTD motif   . AtPARG1elopment . It is pAtPARG2 as one of the highly induced genes in multiple plant–bacterium interactions . MA. MA27]. s an ART . It appeProtein ADP-ribosylation as a versatile molecular switch has been utilized by plants as an intrinsic regulatory mechanism for immunity and by bacteria as an effective invasion strategy for pathogenicity. Meanwhile, many outstanding questions underlying these processes still remain to be addressed. Are there any additional ARTs and hydrolases in plants? What are the enzymes that remove the terminal ADP-ribose of target proteins in plants? How are PARP and PARG activities regulated in plant immunity? Human HsPARP-1 is regulated by multiple posttranslational modification processes, such as phosphorylation, ubiquitination, SUMOylation, and cleavage . It rema


Drosophila melanogaster is an excellent model to dissect the molecular components and pathways of the innate anti-pathogen immune response. The nematode parasite Steinernema carpocapsae and its mutualistic bacterium Xenorhabdus nematophila form a complex that is highly pathogenic to insects, including D. melanogaster. We have used symbiotic (carrying X. nematophila) and axenic (lacking X. nematophila) nematodes to probe the regulation of genes belonging to different immune signaling pathways in D. melanogaster larvae and assess the survival response of certain mutants to these pathogens. We found that both types of S. carpocapsae upregulate MyD88 (Toll), but not PGRP-LE (Imd); whereas axenic S. carpocapsae strongly upregulate Wengen (Jnk), Domeless (Jak/Stat), Dawdle , and Decapentaplegic . We further found that inactivation of Wengen and Decapentaplegic confers a survival advantage to larvae infected with axenic S. carpocapsae, whereas mutating PGRP-LE promotes the survival of larvae infected with symbiotic nematodes. Steinernema carpocapsae forms an excellent tool to study the molecular interplay between insect hosts and nematode parasites [S. carpocapsae nematodes have developed mutualistic relationship with the Gram-negative bacteria Xenorhabdus nematophila, which live in the gut of the parasite and are potent pathogens of insects [Caenorhabditis dauer stage [S. carpocapsae nematodes devoid of their mutualistic bacteria (axenic worms) has permitted elegant studies for the identification of host responses directed exclusively against the nematodes [Entomopathogenic nematodes are natural parasites that infect a range of insect species ,2,3. In arasites ,7. S. ca insects ,7,8,9. Ter stage . Once ther stage ,9,11. Ther stage . When ther stage . In addiematodes ,14.Drosophila melanogaster is a well-established insect model to investigate the genetic basis of the innate immune response to a wide range of pathogens, including parasitic nematodes [D. melanogaster larvae and adult flies that are induced in response to nematode infections [S. carpocapsae as model of nematode parasitism has led to the identification of potential anti-nematode factors in insect hosts [Drosophila larval genes that could be involved in the recognition of and defense against S. carpocapsae nematodes [ematodes . Althougfections ,16,17. Ict hosts . A recenD. melanogaster larvae to S. carpocapsae symbiotic or axenic nematodes and analyzed the transcriptional regulation of PGRP-LE , MyD88 (Toll pathway), Wengen , Domeless , Dawdle  and Decapentaplegic , which belong to different immune signaling pathways. We also tested the survival ability of D. melanogaster loss-of-function mutant larvae against S. carpocapsae to assess the participation of certain signaling pathways in the D. melanogaster response to this parasitic nematode.Here, we exposed Drosophila melanogaster strains used included Oregon, 1118w, yw, PGRP-LE , c03881MyD88 [Wgn , DNUAS-Dome [Daw , and Dpp  [Wgn-RNAi and UAS-DNDome were crossed with ubiquitous Actin-Gal4 driver  and approximately 10 granules of dry baker’s yeast. All fly stocks were maintained at 25 °C with a 12:12-h light:dark cycle. Late second-early third instar larvae were used for all experiments. 88c03881 , Wgn    were reared in the larvae of the greater wax moth Galleria mellonella [S. carpocapsae nematodes (free of X. nematophila bacteria) were cultured using a previously described protocol [The entomopathogenic nematodes llonella . Axenic protocol . These nPGRP-LE, Myd88, Wgn, Dome, Daw, and Dpp genes were obtained from a recent RNA sequencing study [D. melanogaster larvae during the course of infection with 100 S. carpocapsae symbiotic or axenic nematodes. The RPKM values for genes from larvae that were infected with either symbiotic or axenic nematodes were calculated relative to the RPKM values of the uninfected controls, which were set to 1. Reads Per Kilobase Million (RPKM) values for ng study , which iS. carpocapsae symbiotic or axenic nematodes suspended in 10 µL of distilled water were added to each well containing a single larva. Addition of 10 µL of distilled water served as negative control. The plate was covered with a Masterclear real-time PCR film  and holes were poked for ventilation. Each experiment contained 10 larvae per immune mutant or background strain, per treatment. Survival experiments were monitored every 8 h for up to 64 h post-infection. Experiments were repeated at least three times.Microtiter 96-well plates  were prepared by adding 100 µL of 1.25% agarose to each well. Ten Statistical tests for gene transcript level analysis were performed using one-way analysis of variance (ANOVA) and a Tukey post-hoc test for multiple comparisons. Statistics for survival experiments were carried out using log-rank (Mantel-Cox) and Chi-square tests. All statistical tests were conducted using the GraphPad Prism 7 software .PGRP-LE (Imd), MyD88 (Toll), Wgn (Jnk), Dome (Jak/Stat), Daw , and Dpp  from D. melanogaster Oregon larvae infected for 6 or 24 h with either symbiotic or axenic S. carpocapsae. At 6 h post-infection, we found no difference in the expression of any of these genes in larvae infected with symbiotic or axenic nematodes compared to uninfected controls  or axenic (p = 0.003) nematodes compared to controls  and Dpp (p = 0.0014) in larvae infected with symbiotic nematodes , which are key components of signaling pathways that participate in innate immune processes.We plotted the RPKM values for controls . At 24 hcontrols B. Also, ematodes . In contematodes . In larvD. melanogaster PGRP-LE, Myd88, Wgn, Dome, Daw, and Dpp loss-of-function mutant larvae and individuals from their respective background strains with S. carpocapsae symbiotic or axenic nematodes and monitored their survival response every 8 h for three days. Upon symbiotic nematode infection, the survival rate for PGRP-LE mutant larvae was significantly higher compared to their controls  as read-outs of the fly humoral immune response [D. melanogaster larvae with S. carpocapsae nematodes leads to the transcriptional induction of certain genes regulated by the Toll, Jnk, Jak/Stat, and TGFβ immune signaling pathways, but not of the Imd pathway. We also show that inactivation of PGRP-LE, which plays a key role in innate immunity by activating the Imd pathway, promotes the survival of D. melanogaster against symbiotic S. carpocapsae, whereas inactivation of Wgn (Jnk pathway) or Dpp (TGFβ pathway) genes promotes larval survival in response to axenic nematode infection. A recent study investigating the immune response of response . Here, wD. melanogaster wild-type larvae infected with either S. carpocapsae symbiotic or axenic nematodes. Because RNA-seq is designed to estimate differential expression of a large number of genes, qRT-PCR can be used to validate the expression data [Myd88, Wgn, Dome, Daw, and Dpp genes in larvae responding to axenic nematodes compared to symbiotic nematode infections. These results suggest that certain parasitic nematodes can broadly activate immune signaling in D. melanogaster larvae even in the absence of their mutualistic bacteria. Interestingly, S. carpocapsae nematodes carrying their mutualistic bacteria were recently shown to upregulate antimicrobial peptide coding genes in D. melanogaster larvae [D. melanogaster adult flies showing upregulation of certain antimicrobial peptide genes in response to Heterorhabditis bacteriophora axenic nematodes [S. carpocapsae and H. bacteriophora, our current results indicate that PGRP-LE, a receptor of the Imd pathway, is not induced in response to S. carpocapsae infection. Thus, we speculate that S. carpocapsae nematodes can be detected by an unknown receptor in the Imd pathway, which leads to the induction of AMPs, as previously shown [D. melanogaster immune system will be a future focus of our research.The transcriptional regulation of immune genes in insects provides an indication for the potential involvement of specific immune signaling pathways in modulating the immune response against microbial invaders. Here, we used RNA-seq to determine the transcriptional induction of genes encoding receptors or signaling components upstream of the receptors in Imd, Toll, Jnk, Jak/Stat, and TGFβ pathways in ion data . Resultsr larvae . Our finematodes . Althougly shown ,7. FurthS. carpocapsae in the current study suggests that the mutualistic X. nematophila bacteria can interfere with the activation of immune signaling. Indeed, previous studies have shown that X. nematophila secretes a variety of toxins and virulence factors, many of which act on the insect immune system, thereby suppressing host immune genes that regulate important functions for fighting off the infection [Spodoptera exigua antimicrobial peptide genes by X. nematophila has been attributed to the activity of the metabolite benzylideneacetone, which is produced by the pathogen and acts through the eicosanoid pathway [cecropin expression in Manduca sexta is due to the presence of lrp, a global transcription factor in X. nematophila [PGRP-LE failed to show significant changes in transcript levels in response to S. carpocapsae infection. A previous study has demonstrated that the antimicrobial peptide gene diptericin is upregulated in D. melanogaster wild-type flies in response to X. nematophila infection and inactivation of diptericin confers high sensitivity to the mutant flies, suggesting that the Imd pathway is not only activated in response to X. nematophila, but it can also regulate the fly survival response to this pathogen [D. melanogaster response to S. carpocapsae nematode infection. The lower immune gene expression in response to symbiotic nfection ,27. For  pathway . Also, spathogen . The indD. melanogaster larvae to the nematode parasites. We found delayed mortality of PGRP-LE mutants compared to their background controls upon infection with symbiotic, but not axenic, S. carpocapsae nematode infection. These results indicate that upon inactivation of Imd signaling, the presence of X. nematophila bacteria in S. carpocapsae nematodes confers a pathogenicity disadvantage towards D. melanogaster. Previously, inactivation of either Toll or Imd pathways had no effect on the survival of D. melanogaster larvae upon infection with symbiotic H. bacteriophora [H. bacteriophora and S. carpocapsae, elicit different responses in D. melanogaster, and that Toll and Imd signaling might participate in modulating distinct aspects of the insect anti-nematode immune response depending on the nematode species encountered. The conflicting results showing lack of PGRP-LE transcriptional induction by S. carpocapsae and the survival advantage of PGRP-LE deficient mutant larvae to S. carpocapsae infection suggests the existence of another Imd component that participates in the interaction with these parasitic nematodes. We further tested whether inactivating any of these genes affects the survival ability of riophora . These fH. bacteriophora elicits the transcriptional activation of distinct antimicrobial peptide genes in D. melanogaster larvae and adults. For example, metchnikowin was induced in D. melanogaster larvae in response to symbiotic H. bacteriophora, but very low to no induction was observed in adult flies [Dpp mutants have increased survival upon infection with axenic S. carpocapsae is in disagreement with our recent results showing that inactivation of Dpp decreases the survival ability of the mutant flies in response to H. bacteriophora axenic nematodes [In addition, infection with symbiotic lt flies ,29. Alsoematodes . Thus, tWgn encoding the sole tumor necrosis factor receptor in D. melanogaster promotes the survival of larvae in response to infection by axenic S. carpocapsae, suggesting that Jnk signaling, which is involved in several D. melanogaster processes such as stress response, morphogenesis, and wound healing [D. melanogaster response to certain nematode parasites. The induction of the Jnk-regulated pathway gene, puckered, in D. melanogaster adult flies in response to axenic H. bacteriophora reinforces the notion that Jnk signaling, through an unknown mechanism, might participate in the interaction with entomopathogenic nematodes [Interestingly, we find that inactivation of  healing ,32,33, mematodes . D. melanogaster and regulation of the systemic immune function against entomopathogenic nematodes. It will be of particular interest to identify those genes that serve to recognize molecular patterns of entomopathogenic nematodes. The identification and characterization of the immune signaling molecular components that participate in the D. melanogaster defense against potent parasitic nematodes will potentially reveal novel anti-nematode mechanisms in other insects and perhaps even mammals.Future work will focus on understanding the connection between immune pathway activation in PGRP-LE (Imd pathway), MyD88 (Toll pathway), Wgn (Jnk pathway), Dome (Jak/Stat pathway), Daw , and Dpp  genes in D. melanogaster larvae responding to infection with S. carpocapsae symbiotic or axenic nematodes. We also measured the survival response of mutant larvae against the parasitic nematodes. We have found upregulation of MyD88, Wgn, Dome, Daw, and Dpp, but not PGRP-LE, in D. melanogaster infected with the nematode parasites. We have also shown that inactivation of PGRP-LE, Wgn, and Dpp prolongs the survival of nematode-infected larvae. This work demonstrates the modulation of the D. melanogaster immune signaling pathways during infection with S. carpocapsae entomopathogenic nematodes.In this study, we examined the regulation of