Comparison of transcriptomes of an orthotospovirus vector and non-vector thrips species

Summary

Thrips transmit one of the most devastating plant viruses worldwide–tomato spotted wilt tospovirus (TSWV). Tomato spotted wilt tospovirus is a type species in the genus Orthotospovirus and family Tospoviridae. Although there are more than 7,000 thrips species, only nine thrips species are known to transmit TSWV. In this study, we investigated the molecular factors that could affect thrips ability to transmit TSWV. We assembled transcriptomes of a vector, Frankliniella fusca [Hinds], and a non-vector, Frankliniella tritici [Fitch], and performed qualitative comparisons of contigs associated with virus reception, virus infection, and innate immunity. Annotations of F. fusca and F. tritici contigs revealed slight differences across biological process and molecular functional groups. Comparison of virus cell surface receptors revealed that homologs of integrin were present in both species. However, homologs of another receptor, heperan sulfate, were present in F. fusca alone. Contigs associated with virus replication were identified in both species, but a contig involved in inhibition of virus replication (radical s-adenosylmethionine) was only present in the non-vector, F. tritici. Additionally, some differences in immune signaling pathways were identified between vector and non-vector thrips. Detailed investigations are necessary to functionally characterize these differences between vector and non-vector thrips and assess their relevance in orthotospovirus transmission.

Keywords:

DNA-binding proteins – Phylogenetic analysis – sulfates – Sequence databases – Transcriptome analysis – Gene ontologies – Tomato spotted wilt virus – Pattern recognition receptors

Introduction

Thrips-transmitted tomato spotted wilt tospovirus (TSWV) ranks among the ten most detrimental plant viruses worldwide [1]. Tomato spotted wilt tospovirus is the type species of the genus Orthotospovirus in the family Tospoviridae and order Bunyavirales. TSWV is an enveloped single-stranded ambisense RNA virus that is transmitted exclusively by thrips in a persistent and propagative manner [2–5]. Of the thousands of thrips species known worldwide, nine species alone are known to transmit TSWV, and only fourteen species in total are documented to transmit all known orthotospoviruses [6–8]. Also, all known vector thrips species are confined to the suborder Terebrantia and family Thripidae [9]. Several speculations have been made as to why some thrips species are vectors and others are not, but conclusive explanation or evidence is still lacking [10–11].

The interactions between thrips vectors and TSWV are complex. For instance, thrips exhibit stage-specific acquisition and inoculation of the virus. The virus must be acquired at the first or the second instar larval stage for successful inoculation at the adult stage. If thrips acquire the virus for the first time as adults, they will not be able to inoculate the virus. Once ingested, TSWV travels through the foregut to the midgut where it replicates and is subsequently translocated into salivary glands for further replication. During this process, TSWV crosses several membrane barriers [12, 13]. The complexity of TSWV-thrips interactions is further enhanced by the fact that the exact route of the virus from the midgut to salivary glands is unknown. Three hypotheses explaining the mechanism of TSWV translocation into the salivary glands have been proposed, including movement through a temporary ligament connecting the midgut and salivary glands formed during the early larval stages, through hemocoel, and through direct virus movement facilitated by proximity between the salivary glands and midgut tissues during the early larval stages [11–15]. Of the three hypotheses, the one suggesting virus movement through the ligament is supported, as studies have demonstrated salivary gland infection following TSWV infection in the ligament structure [11, 16]. The temporary ligament connecting the midgut to salivary glands is formed during the larval stages. However, as larvae develop, the connection is believed to be lost [11, 14]. Thus, only thrips that acquire TSWV during the early larval stages can serve as TSWV vectors. It is unknown whether the ligament connecting midgut tissues to salivary glands is present in all thrips species. Studies indicate that very closely related thrips species within the same genus, and presumably with a similar anatomy, function as vectors and non-vectors [10, 15,16]. Therefore, it would be reasonable to assume that ligament may not be the ultimate determinant of orthotospovirus transmission by thrips.

Few studies have investigated non-vector thrips species to elucidate factors determining thrips inability to serve as TSWV vectors [9, 10]. Assis Filho et al. (2005) demonstrated that in a non-vector thrips species, eastern flower thrips, Frankliniella tritici [Fitch], TSWV replicated successfully in the midgut epithelial cells. However, TSWV infection in the salivary glands was completely absent. Translocation of TSWV from the midgut to the salivary glands is a requisite for TSWV transmission. It is likely that the lack of TSWV in salivary glands despite successful accumulation in midgut cells is due to the midgut escape barriers, or suppression of TSWV replication caused by rapid degradation of virions in the midgut [17]. Insects possess highly efficient innate immunity that degrades and inhibits pathogen replication [18–21]. Insects’ innate immunity includes immune genes that recognize conserved motifs of invading pathogens termed pathogen associated molecular patterns (PAMP) [22, 23]. Once pathogens such as viruses are recognized, extracellular cascades are activated to amplify different signals, which lead to systemic production of pathogen suppressing molecules like antimicrobial peptides that ultimately degrade pathogens [24–26]. Until now, differences between vector and non-vector thrips species in terms of their innate immunity have not been explored. Comparative studies investigating the immune genes present in thrips species could help understand whether vector and non-vector thrips species vary in their innate immunity.

For successful virus-vector interactions, virus receptors must facilitate recognition and entry of viruses into host cells [27]. TSWV entry into thrips cells requires binding of TSWV glycoproteins to receptors located in the epithelial cells of thrips midgut. A 50-kDa protein has been identified as a putative receptor of TSWV in thrips [28]. However, that protein has not been characterized. TSWV is one of the few plant-infecting viruses in bunyavirales. The glycoprotein envelope of TSWV shares a similar structure and motif as that of other animal-infecting members of bunyavirales [29]. Several receptors that interact with the glycoproteins of animal-infecting members of bunyavirales have been identified. For instance, receptors including integrin, nucleolin, heparan sulfate, and dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) that facilitate entry of hantaviruses, nairoviruses, and phleboviruses have been identified [30–33]. Identifying similar receptors in thrips transcriptomes could provide insights into whether the presence of homologs of such receptors vary between vector and non-vector thrips species.

Comparative studies between vector and non-vector species at a molecular level could help identify species-specific constitutive factors that function as determinants of TSWV transmission. Recently, transcriptomes of different life stages of two main vector species: Frankliniella fusca (Hinds) and Frankliniella occidentalis (Pergande) were examined [34, 35]. However, transcriptomes of non-vector thrips species have neither been examined nor compared with the transcriptomes of vector thrips species. The main objective of this study was to compare transcriptomes of a vector (F. fusca) and a non-vector (F. tritici), and to investigate contigs (contiguous sequences) associated with virus-vector interactions including virus reception, virus infection, and innate immunity.

Results

Processing of the RNA-Seq reads and transcriptome assembly

Trimmomatic was used to process the sequencing reads. Trimming of the adapter sequences and filtering of low quality reads resulted in 36.6 million quality reads in F. fusca and 22.7 million high quality reads in F. Tritici. The clean reads were de novo assembled using Trinity into 27,025 and 23,605 contigs for F. fusca and F. tritici, respectively. N50 of the assembled contigs in F. fusca were 2,633 bases long, while the N50 of the assembled contigs in F. tritici was 2,975 bases “Table 1”. Further, evaluation of completeness of the de novo assemblies in F. fusca and F. tritici through the Core Eukaryotic Genes Mapping Approach (CEGMA) revealed that 99 and 100% of core proteins that are conserved within eukaryotes were present in F. fusca and F. tritici, respectively. BUSCO analysis revealed that 95.1 and 96.2% of 1658 single-copy gene orthologs from 42 insect species were present in F. fusca and F. tritici transcriptomes, respectively.

Functional annotations of F. fusca and F. tritici contigs

Frankliniella fusca and F. tritici contigs were annotated using Blastx search against NCBI non-redundant database. Blastx retrieved sequence match for 14,081 contigs in F. fusca and 12,984 contigs in F. tritici. Annotated contigs were further assigned functional groups under three classification systems using Blast2go analysis: biological process, molecular function, and cellular component. Blast2go assigned 23 Gene Ontology (GO) terms under the biological process category “Fig 1” and 13 GO terms under the molecular function category “Fig 2”. Most of the contigs under the biological process category consisted of functional annotations associated with cellular macromolecule metabolic process (12%), protein metabolic process (10%), nucleobase-containing compound metabolic process (10%), signal transduction (7%), and gene expression (6%). Under the molecular function category, nucleotide binding was the most dominant GO term (32%) followed by DNA binding (12%), kinase activity (12%), and phosphotransferase activity (7%). In both categories, all the GO terms that were assigned to F. fusca were also present in F. tritici. In the cellular component category, five and three GO terms were assigned to F. fusca and F. tritici, respectively “Fig 3”. Two GO terms specific to F. fusca included cell envelope and external encapsulating structure.

Pathway analysis mapped 127 and 125 biochemical pathways to F. fusca and F. tritici, respectively “S1 Table”. Most of the pathways were associated with metabolism including carbohydrate metabolism, biosynthesis of secondary metabolites, glycan biosynthesis and metabolism, lipid metabolism, and amino acid metabolism. Five pathways including toluene degradation, biotin metabolism, flavone and flavonol biosynthesis, PI3K-Akt signaling pathways, and nitrotoluene degradation were unique to F. fusca. However, three pathways associated with sesquiterpenoid/triterpenoid biosynthesis, vitamin B6 metabolism, and D-arginine and D-ornithine metabolism were unique to F. tritici.

Following the overview of functional annotation and pathway analyses of F. fusca and F. tritici contigs, contigs that could influence virus-vector interactions were examined.

Virus receptors in F. fusca and F. tritici

Frankliniella fusca and Frankliniella tritici transcriptomes were examined for Known receptors of animal-infecting bunyavirales’ members. Using OrthoMCL and phylogenetic analysis, homologs of integrin were identified in F. fusca and F. tritici “Fig 4A”. However, the homologs of heparan sulfate were only identified in F. fusca “Fig 4B”. Homologs of other bunyavirales receptors such as DC-SIGN and nucleolin were not present in either thrips species in this study.

Viral infection related contigs in F. fusca and F. tritici

Blast2go annotated several contigs under the GO term “viral process” in F. fusca and F. tritici. The GO term viral process consisted of contigs associated with various processes of virus infection such as viral attachment, viral replication, and virus assembly. In F. fusca, 41 contigs were assigned to viral process “Table 2A”, while 31 contigs were assigned to viral process in F. tritici “Table 2B”. Some of the homologs of proteins present in both thrips species included ankyrin repeat domain-containing protein 17, host cell factors, heat shock protein 70, transcription elongation factor, and serine arginine-rich protein specific kinase 1b. The contigs of common homologs between two species were 88.1± 0.62% (Mean ± SE) similar. There were substantial number of polymorphisms that exist between the two species, it is not clear what these polymorphisms represent at this juncture. Further, homologs of several proteins specific to each thrips species also were identified. Out of 41 viral process contigs, 19 contigs including creb-binding protein, transcription initiation factor, transcriptional activator, and mitogen-activated protein kinase were only present in F. fusca. Eight homologs of proteins including nucleoporin seh1, radical s-adenosyl methionine domain containing protein, Ras-specific GTPase-activating protein, and Ras-related c3 botulinum toxin substrate were specific to F. tritici.

Immune genes in F. fusca and F. tritici

Using OrthoMCL, homologs of immune genes associated with pathogen pattern recognition, signal modulation, signaling pathways, and pathogen-suppressing molecules were identified in F. fusca and F. tritici. To confirm the presence of immune genes in F. fusca and F. tritici, homologs of immune genes from thrips species were aligned with best matched immune gene sequences from other arthropods and phylogenetic trees were constructed. Phylogenetic analysis confirmed the presence of 95 and 89 immune genes in F. fusca and F. tritici, respectively, and the data are presented in “Table 3”. Phylogenetic trees constructed on homologs of multigene families associated with immune pathways are presented as supplementary information (S1 File). Under pathogen pattern recognition, thirty-one homologs of immune genes associated with pathogen pattern recognition encoding peptidoglycan recognition protein (PGRP) “Figure A in S1 File”, scavenger receptor (SCR) “Figure B in S1 File”, and C-type lectin (CTL) “Figure C in S1 File” were identified in F. fusca, while 29 homologs of pattern recognizing proteins were identified in F. tritici. Immune genes such as clip domain serine proteases (CLIP) and serine protease inhibitors (Serpins) that are associated with signal modulation were also examined. The number of homologs of CLIP “Figure D in S1 File” was more in F. fusca (24) than in F. tritici (20). Four homologs of serpin “Figure E in S1 File” were identified in both F. fusca and F. tritici.

Most of the immune genes under the Toll pathway that were identified in F. fusca were also present in F. tritici “Figure F in S1 File”. However, homologs of Tollip were absent in F. tritici. Under IMD pathway, six and eight homologs of immune genes were identified in F. fusca and F. tritici, respectively. Several IMD pathway related immune genes such as Dredd, transforming growth factor b activated kinase (TAK), and Tak1-binding protein 2 (Tab2) were present in F. tritici but absent in F. fusca. In F. tritici, homologs of UbC13 were not present. Three homologs of JNK pathways related immune genes were identified in F. fusca, while in F. tritici two homologs were present. All the immune genes under JAK/STAT pathway: Domeless, STAT, and SOCs were present in both thrips species. Also, homologs of RNA interference (RNAi) associated immune genes including dicer and argonaute were present in F. fusca and F. tritici.

The presence of pathogen suppressing molecules that are activated by signaling pathways such as Toll, IMD, and JNK was examined. Pathogen suppressing molecules (antimicrobial peptides and enzymes) including prophenoloxidase “Figure G in S1 File”, Nitric oxide synthase (NOS), lysozyme, and caspase were identified in both thrips species. In F. fusca, nine homologs of pathogen suppressing molecules were identified, while in F. tritici, 10 homologs of pathogen suppressing molecules were identified.

Discussion

In this study, we performed qualitative comparisons of F. fusca and F. tritici transcriptomes. The functional annotations for the contigs in both transcriptomes were assigned into three categories. In the cellular component category, analysis of functional annotations revealed that they were mostly similar except for the GO terms cell envelope and external encapsulating structure, which were unique to F. fusca. Cell envelope and the external encapsulating structure could constitute the membrane complex in thrips cells. Cell membrane structures are particularly useful for plant-infecting enveloped viruses such as tospoviruses and rhabdoviruses to acquire their own envelopes and enter the host cells through fusion [36, 37]. It is interesting to find that these host factors are abundant in a vector as opposed to a non-vector. The role of these structures in virus transmission by thrips deserves further scrutiny. Both thrips species had similar functional groups across the biological process and molecular function categories. Analysis of biochemical pathways revealed that most of the pathways were associated with metabolism of lipids, carbohydrates, and amino acids. We further examined whether molecular factors associated with virus-vector interactions including virus reception, virus infection, and innate immunity varied between vector and non-vector thrips. Frankliniella fusca and F. tritici transcriptomes included homologs of the cell surface receptor integrin, while homologs of another receptor, heparan sulfate, were only present in F. fusca. Heparan sulfate is a heavily sulfated polysaccharide that is found pervasively on the cell surface and in the extracellular matrix of animal tissues [38]. The ubiquitous presence of heparan sulfate has allowed numerous viruses, bacteria, and other parasites to use heparan sulfate as a cell surface adhesion receptor to bind and gain entry in to host cells [39, 40]. Numerous mammalian DNA and RNA viruses (enveloped or non-enveloped) that do not require arthropod vectors such as members of herpesvirales, monengavirales, ortervirales, and papillomoviridae, and arthropod-borne viruses such as members of bunyavirales and flaviviridae also use heparan sulfate to enter host cells [41–50]. Studies have revealed that viruses have established close relationships with heparan sulfate based on its polysaccharide structure thereby allowing heparan sulfate to serve as a specific receptor [39]. It is interesting to find homologs of a robust and ubiquitous receptor such as heparan sulfate in vector thrips but not in non-vector thrips. Future studies should further examine in detail the role of heparan sulfate in the transmission of TSWV by thrips. Enzymatic removal of heparan sulfate has been specifically shown to reduce the binding of an enveloped RNA virus, rabies virus [50]. Such an approach or dsRNA mediated knockdown assay will help assess the importance of heparan sulfate as a cell surface adhesion receptor of TSWV in vector thrips.

In addition to virus receptors, we also identified several homologs of virus infection related proteins that were common and/or specific to F. fusca and F. tritici. In both thrips species, homologs of heat shock protein 70-kDa that is known to be involved in transcription and replication of influenza virus A [51, 52], and ankyrin repeat protein that is important for replication of myxoma virus [53] were identified. Homologs of mitogen activated protein kinase 1 that facilitates cellular entry of hepatitis C virus [54], and ribosomal protein s10 that interacts with human immunodeficiency virus [55], were present in F. fusca alone. In F. tritici, we identified homologs of GTPase- activating protein that facilitates replication of hepatitis C virus and sindbis virus [56, 57], serine arginine-rich protein specific kinase 1b that leads to phosphorylation of hepatitis B virus core protein [58], and nucleoporin protein that facilitates attachment of herpes simplex virus capsid protein to host nuclear complex [59]. Homologs of radical s-adenosylmethionine, an enzyme superfamily known to inhibit replication of several DNA and RNA viruses, were present only in F. tritici. The most well-studied example in this family is Viperin–virus inhibitory protein, endoplasmic reticulum-associated, interferon inducible [60]. Infection in humans and other animals by numerous viruses is characterized by upregulation of Viperin [61]. In influenza virus, radical s-adenosylmethionine domain containing Viperin is known to disrupt cholesterol rich lipid rafts that are used in virus budding off from the plasma membrane [62–64]. Viperin has also been known to suppress the multiplication of an enveloped rabies virus by targeting cholesterol and sphingomyelin production [65]. In addition to inhibiting virus budding, in the lentivirus, equine infectious anemia virus, Viperin is known to inhibit the release of viral group specific antigen (Gag) and envelop coding protein (Env), and disrupt virus receptors [66]. These studies suggest that virus suppression by radical s-adenosylmethionine domain containing Viperin could occur in multiple ways. The functional characterization of Viperin is accomplished either by correlating upregulation of Viperin with reduced virus multiplication or by blocking Viperin transcription and correlating it to enhanced virus multiplication [66]. The presence of an important virus multiplication inhibitor such as radical s-adenosylmethionine in the non-vector (F. tritici) and absence in the vector (F. fusca) offers substantive insights on virus-vector interactions. As mentioned earlier, TSWV is enveloped with glycolipids, and is similar to many animal infecting members of bunyavirales. Studies on the role of radical s-adenosylmethionine in TSWV transmission should be undertaken to assess if this enzyme affects thrips ability to function as vector of TSWV.

Homologs pertaining to several immune genes were found in F. fusca and F. tritici transcriptomes. Most of the immune genes associated with major antiviral pathways including RNAi, Toll, and JAK/STAT were present in both thrips species. The IMD pathway has been demonstrated to have antiviral activity in D. melanogaster [67, 68]. The loss of IMD pathway related immune genes in D. melanogaster cell lines increased cricket paralysis virus (CrPV) load and enhanced sensitivity to CrPV infection. Several IMD pathway related upstream immune genes including Dredd, TAK, Tab2 were present in F. tritici but absent in F. fusca. In Drosophila and other insects, Dredd is required to cleave Imd, leading to ubiquination by Iap2 and subsequent binding and activation of the Tab2/Tak1 complex, resulting in phosphorylation and activation of the IKK complex [69]. The absence of Dredd, TAK, and Tab2 in F. fusca suggests that the IMD pathway could be disabled in F. fusca. It is not clear whether the lack of IMD related immune genes is specific to F. fusca or in multiple vector thrips species. More studies need to be conducted to examine the importance of IMD pathway in virus suppression by vector and non-vector thrips.

Qualitative comparison between F. fusca and F. tritici transcriptomes revealed several important differences between vector and non-vector thrips species in terms of virus reception, virus infection, and innate immunity. A majority of contigs associated with virus-vector interaction was present in both thrips species. However, some contigs such as homologs of heparan sulfate associated with virus entry were only present in F. fusca, homologs of radical s-adenosylmethionine that inhibits virus replication were specific to F. tritici, and several IMD pathway related immune genes’ homologs were absent in F. fusca. The role of these genes in TSWV transmission needs further examination. Nevertheless, identification of these genes suggests that vector and non-vector thrips species could differently interact with the virus. To our knowledge, this is the first study to compare transcriptomes of vector and non-vector thrips species. Transcriptomic data generated in this study were deposited into a public (National Center for Biotechnology Information—NCBI) database with Sequence Read Archive (SRA) accession numbers SRP023246 and SRP023248. Frankliniella fusca and F. tritici transcriptomes from this study could serve as important genomic resources for future studies on other vector and non-vector thrips species.

Materials and methods

Maintenance of thrips colony

A F. fusca colony was established in 2009 with thrips collected from peanut blooms at the Belflower Farm (Coastal Plain Experimental Station, Tifton, GA, USA) and identified using published morphological keys under a dissecting microscope (Leica MZ6) at 64 X magnification [70]. The identified adults were used for initiating the colony. Thrips were maintained in Munger cages (0.11 X 0.89 X 0.18 m) in a growth chamber (Thermo Scientific, Dubuque, IA, USA) at 25–30°C, 40–50% relative humidity, and L14:D10 photoperiod [71]. Thrips were reared on non-infected leaflets of the peanut cultivar, Georgia Green. The peanut plants were maintained in thrips-proof cages (Megaview Science, Taichung, Taiwan). Frankliniella tritici used for the study were collected from peanut fields at the Coastal Plain Experimental Station.

Total RNA extraction, library preparation, and sequencing

Approximately, 35 non-virus exposed female F. fusca and field-collected F. tritici adult females were pooled separately for total RNA extraction. Total RNA was extracted using RNeasy mini kit using manufacturer’s protocol (Qiagen, Valencia, CA, USA). Subsequently, mRNAs (polyadenylated RNAs) were selected using oligo-dT and cDNA libraries were constructed at Georgia Genomic Facility of the University of Georgia. Prior to library construction, Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) was used to evaluate RNA quality and concentration of the samples. Illumina sequencing libraries were constructed using TruSeq RNA sample preparation kits using at least 1 μg of the total RNA. Messenger RNA was selected, fragmented, and then reverse transcribed into cDNA. Subsequently, second strand cDNA was prepared using Polymerase I and RNase H, and TruSeqLT adapters were ligated to the DNA fragments for PCR amplification. Finally, two libraries were sequenced on Illumina HiSeq 2000 platform using paired-end 100 cycle sequencing settings at the University of Texas Health Science Center, at San Antonio, Texas.

Processing of the RNA-seq reads and transcriptome assembly

Raw RNA-seq reads were processed using bioinformatics software available at the Georgia Advanced Computing Resource Center, UGA (https://wiki.gacrc.uga.edu/wiki/Software). Adapter sequences were trimmed using the Trimmomatic software (Version 0.36) [72]. Trimmomatic was also used to produce quality reads by removing three bases at the beginning and end of each read, setting minimum read length threshold to 50 bases, and discarding reads if the average quality of four bases fell below 20. After cleaning reads, Trinity (Version 0.36) software was used to perform de novo assembly on F. fusca and F. tritici reads individually using the following parameters “–kmer 25 –minimum-contig-length 300 bp” [73]. The assembled contigs from each thrips species were subjected to CEGMA (Core Eukaryotic Genes Mapping Approach) program (Version 2.5) to assess the completeness of the assembly [74]. The completeness of the transcriptomes was also evaluated using another software (BUSCO, Version 3.0.2) [75].

Functional annotations of F. fusca and F. tritici contigs

Assembled contigs from F. fusca and F. tritici were annotated using a java-based Blast2go software (Version 3.2) (https://www.blast2go.com) [76]. First, Blastx was used to search sequence similarity against the NCBI non-redundant protein database with E-value threshold of 10⁻⁶. Subsequently, Blast2go assigned Gene Ontology (GO) terms to each contig with an E-value of 10⁻⁶ and annotation cutoff of 55. GO terms were categorized under biological process, molecular process, and cellular component based on a node score of 5 and level 5. Biochemical pathways were assigned to F. fusca and F. tritici contigs using the KEGG database in Blast2go.

Virus-vector interaction associated molecular factors

Following functional annotations of F. fusca and F. tritici contigs, we focused on molecular factors that could influence thrips ability to transmit TSWV including 1) virus receptors, 2) virus infection related proteins, and 3) immune genes in F. fusca and F. tritici.

Virus receptors in F. fusca and F. tritici

We investigated known receptors of animal-infecting members of bunyavirales including integrin, heparan sulfate, nucleolin, and DC-SIGN in F. fusca and F. tritici using OrthoMCL software (Version 2.0.9) [77]. First, well annotated receptor sequences from humans, Homo sapiens [L.], mouse, Mus musculus [L.], sheep, Ovis aries [L.], southern house mosquito, Culex quinquefasciatus [Say], and Asian tiger mosquito, Aedes albopictus [Skuse] were gathered and a database was created [32, 33, 78]. Sequences from other arthropods including red flour beetle, Tribolium castaneum [Herbs], pea aphids, Acyrthosiphon pisum [Harris], deer tick, Ixodes scapularis [say], and silkworm, Bombyx mori [L.] were also downloaded from the National center for biotechnology information and added to the database. The receptor sequences were used as query sequences and their homologs were identified in F. fusca and F. tritici contigs generated in this study. First, using Blastp, sequence match of all the proteins were identified with an E-value of 10⁻⁶. Blast result was then filtered by setting threshold of percent match for each pair of sequences to 50%. The homologous sequences were grouped using MCL software, and phylogenetic trees were constructed to validate grouping of the sequences. Protein sequences from each group were aligned using a Multiple alignment using Fast Fourier Transform software (http://mafft.cbrc.jp/alignment/software/), and the alignment was manually cured in Mesquite (http://mesquiteproject.wikispaces.com/installation) [79]. Subsequently, phylogenetic analyses were performed using Randomized Axelerated Maximum Likelihood (RAxML) program with CIPRES (Version 8) [80], and trees were constructed using Fig Tree software (Version 1.4.3) [81] to confirm the presence of receptors in F. fusca and F. tritici. For this analysis, protein/AA data type was chosen, and a JTT protein substitution matrix for analysis was selected. Boot strapping analysis was conducted to search for the best-scoring maximum likelihood tree. Based on software recommendations, bootstrapping was halted automatically. The RaxML generated tree was subsequently opened in FigTree software, and node and tip labels were added. Only sequences in the trees with the node score of more than 60 were considered for the study.

Immune genes in F. fusca and F. tritici

Homologs of immune genes were also identified in F. fusca and F. tritici using the OrthMCL software. A database of well-annotated immune genes from arthropods including T. castaneum [82], A. pisum [83], fruit fly Drosophila melanogaster [Fallen] [21], and B. mori [84] was created, and sequence match of the immune genes was identified in F. fusca and F. tritici as described previously using OrthoMCL. To validate the clustering of immune genes, homologs of immune genes from the thrips species along with their best matched immune gene sequences from the arthropod species identified in OrthoMCL were aligned, and phylogenetic trees were constructed with the RAxML program using CIPRES software as described previously. Phylogenetic trees of multigene immune families are provided as supplementary materials. Identified immune genes were categorized into three gene groups namely pathogen recognition, signal modulation (including signaling pathways), and pathogen suppressing molecules.

Supporting information

S1 File [tc]
Phylogenetic analysis of immune genes in and .

S1 Table [xlsx]
KEGG pathway analysis in and .