Deterioration of traits essential for biological control has been recognized in diverse biological control agents including the insect pathogenic nematode Hb
]. These traits can deteriorate rapidly and substantially when bio-control agents are isolated from nature and reared in the laboratory, or mass-produced for commercial purposes. Genetic and non-genetic processes may be responsible for trait deterioration in laboratory-cultured bio-control agents and their symbionts. However, the specific mechanisms behind these genetic processes remain unclear. To identify genes associated with trait deterioration in the entomopathogenic nematode Hb
, we undertook an expression profiling study using custom Roche NimbleGen expression arrays that screened over 15,220 transcripts. To identify the DEGs, expression was compared between two experimental lines of Hb
; L5M and OHB. Our results showed that trait deterioration of Hb
induces substantial overall changes in the nematode transcriptome (Figure ). We observed a few general patterns suggesting that trait deterioration via inbreeding depression, taking place over a short period of time (under 20 passages), can result in massive changes in metabolic processes, cellular transportation and gene translation. In addition, the massive reprogramming of primary and secondary metabolic processes as part of trait-deterioration involved changes in signalling and other regulatory processes. The present study represents the first transcriptional analysis of degradation of beneficial traits in EPNs and highlights several key components of trait deterioration that may be common among biological control agents.
Experimental design and analysis
The advent of microarrays has enabled the screening of thousands of genes in parallel to assist in candidate gene identification. In this study we used a set of over 15,220 ESTs to construct a cDNA microarray. The main source of ESTs for this array was derived from Hb TT01 that interacts symbiotically with Photorhabdus luminescens TT01 bacterium. The microarray experiments were conducted in a reference design, where tissue samples from the original parental line, OHB, acted as reference against the inbred line, L5M. The results show 1,185 genes were DE, encompassing diverse functions. We validated our microarray observations by qRT-PCR for several genes that were chosen based on their biological interest as well as spectrum of significance in fold change expression. In general, the results revealed the evolution of altered transcript levels concomitant with trait deterioration, including major changes in the metabolism category, especially in energy, carbohydrate and lipid metabolism.
It is common practice to use an arbitrary transcription differential cut-off (such as twofold) in order to identify changes that may be biologically significant, but our results showed that the majority of DEGs exhibit a small fold change in expression level ranging from 0.38 to 2.45. A number of past studies have shown ineffectiveness of a two-fold cut-off as the basis for filtering out DEGs [24
] and low-magnitude transcriptional changes have been found to produce functionally significant changes [27
]. The low amplitude modulation of gene expression (less than two-fold changes) is suggestive of low-magnitude remodelling of the transcriptome, which may be an integral component of an organism's adaptive response to selection on physiological traits. It is possible that the overall evolutionary response of the nematode, even over very small time scales (a few generations) requires coordinated changes among a wide array of genes, and those changes in turn may require reinforcing changes in an even wider array of functionally connected genetic components. We speculate that numerous genes that function in Hb
are co-ordinately modulated to support the many physiological changes manifested in the evolution of trait deterioration.
Biological gene regulatory networks are highly interconnected systems. Non-linear, synergistic interactions are common. Large number of genes with low-magnitude transcriptional modulation could potentially be just as important in conferring phenotypes and mediating physiological adaptation as the small numbers of genes that show large-magnitude modulations. Our findings suggest that widespread, low-magnitude transcriptional remodelling may be a normal process during physiological adaptation in trait deteriorated nematodes. However, understanding the role of pervasive low-magnitude remodelling may require using computational modelling approaches at a system level, as well as improved technologies for accurately measuring those changes.
Comparative analysis of differentially expressed genes
We obtained 1,185 genes that were differentially expressed in a deteriorated line of Hb. Comparative analysis of these DEGs with those available in various public databases showed that 59% (n = 698) matched C. elegans and C. briggsae proteins, and 26% (n = 313) matched parasitic nematodes. When these 313 DEGs were compared with a subset of parasitic nematodes, 7% (n = 82) matched animal and human parasitic nematode (AHPN) proteins, suggesting that these genes may participate in parasitism-related activities. Of the remaining DEGs, 19% (n = 231) did not match AHPN sequences that we designated as parasitic nematode specific. A small portion of DEGs 10% (n = 122) did not appear to match any available sequences, indicative of novel Hb genes. These findings suggest the potential of discovering new genes and gene functions, genetic networks, and metabolic pathways specific to Hb and other EPNs. Similarly, the identification of putatively secreted proteins and expression profiling of the DEGs shared between Hb and other parasitic nematodes could be a valuable resource for conducting in-depth research on gene functions that will ultimately elucidate parasitic nematode-specific biological processes.
We found several Hb
DEGs that are associated with RNA interference (RNAi) phenotypes of C. elegans
. Our analysis shows differential expression of genes like egl-8
(complete list in Additional file 1
), each of which exhibit RNAi phenotypes in C. elegans
. These genes may prove useful candidates in the ongoing RNAi endeavors for functional genomics studies of EPNs. Interestingly, we found 9 DEGs that matched proteins from various prokaryotic organisms. These transcripts encode ATP synthase (GenBank: FF679373
) and a DNA-J class molecular chaperone (GenBank: ES409751
). The presence of these transcripts could be the result of horizontal gene transfer (HGT) from bacteria encountered by Hb
during its life cycle. The presence of sequences of putative prokaryotic origin has already been reported in Hb
] as well as in plant parasitic nematodes [31
]. Given the similarity of these sequences to prokaryotic sequences, and presence of poly(A) RNA in the transcript, the possibility that these sequences are bacterial contaminants is low. Our findings of Hb
DEGs with similarity to prokaryotic sequences identified here do not imply that all these genes have been acquired by HGT, as the null hypothesis remains convergent evolution. However, their presence serves as a first step in identifying a pool of candidates from which parasitism and mutualism-related genes can be explored in the future.
Functional analysis of differentially expressed genes
Gene Ontology (GO) [22
] has been used widely to predict gene function and classification. GO provides a dynamic vocabulary and hierarchy that unifies descriptions of biological, cellular and molecular functions across genomes. We used Blast2GO [23
], a sequence-based tool, to assign GO terms, extracting them for each BLAST hit obtained by mapping to extant annotation associations. Though GO analysis showed only one third of the DEGs can be assigned to different functional categories, we observed a clear pattern of changes exhibited by deteriorated nematodes. High numbers of DEGs were assigned to biological processes, including metabolic, developmental, cellular processes and cellular stress. We observed a pattern of changes in primary as well as secondary metabolic processes, indicating that our trait-deteriorated nematodes evolved massive metabolic changes. The molecular function category was dominated by binding, catalytic, transporter, transcriptional regulator and enzyme regulator activities. Such a representation of diverse functional areas is suggestive of coordinated modulation of genes from different functional areas to support the changes undergone during the evolution of trait deterioration.
Biochemical functionality was predicted by mapping all DEGs to pathways using KEGG within Blast2GO. Molecules involved in metabolism (energy, amino acid, carbohydrate and lipid metabolism), neurodegenerative diseases and signal transduction had the highest representation amongst the sequences mapped to KEGG pathways. The enzyme cytochrome c oxidase had the highest mapping to both energy metabolism and neurodegenerative disease categories. Similarly, other enzymes well represented in KEGG pathways are vacuolar ATP synthase (GenBank: EX011485), protein disulfide isomerase (GenBank: EX012905), transglutaminase (Tgase) (GenBank: EX012170), phosphoglycerate dehydrogenase (GenBank: EX013716), NADH dehydrogenase (ubiquinone) (GenBank: ES411557, EX010284), aldehyde dehydrogenase (GenBank: ES411128) and aconitate hydratase (GenBank: EX014674, ES741155). We identified predicted proteins with potential roles in host-parasite interactions, MAPK and T-cell receptor signaling pathway and apoptosis. Although at this stage the precise role of such molecules in the nematode-bacteria-insect host interplay is unclear, they could be involved in manipulating the host's immune response or associated with nematode's innate immune response. Furthermore, we identified families of proteins representing serine, cysteine and metallo-proteinases as well as proteinase inhibitors. While these enzymes are inferred to mediate or modulate proteolytic functions, they may in turn, facilitate the nematode's interaction with its host and symbiont, as the proteinase inhibitors may protect the nematodes against its host's immune system.
Genes of general and secondary metabolism
Results obtained from our analysis showed that trait deteriorated nematodes undergo massive changes of the transcripts encoding metabolic enzymes and processes. We observed a pattern emerging from our studies suggesting that the trait-deteriorated nematodes down-regulate their primary metabolic processes, which at the same time activate secondary metabolic processes. We also identified significant changes in the dynamics of the genes responsible for energy, amino acid, carbohydrate and lipid metabolism. Enzymes involved in xenobiotic biodegradation, glycan biosynthesis and metabolism and biosynthesis of secondary metabolites were also changed (Additional file 4
). These results show that the evolution of trait deterioration can result in metabolic upheavals that could be responsible for reduced pathogenicity. The biggest change was observed in energy metabolism, involving the up-regulation of cytochrome c oxidases (CCO) (GenBank: EX012198
) and down-regulation of vacuolar ATPases (V-ATPases) and NADPH-cytochrome P450 (GenBank: ES411356
). Cytochrome c oxidase encodes an important enzyme involved in oxidation phosphorylation pathways and thus energy production. In Cryptococcus neoformans
, the up-regulation of CCOI was shown to be related to stress response of the yeast, which is vital for survival in its hostile host [32
]. It is possible that the up-regulation of this mitochondrial gene might be linked to an increased energy production critically important to the survival of Hb
in a deteriorated condition. The V-ATPases are ATP-dependent proton pumps present in both intracellular and plasma membranes, and function in processes such as receptor recycling, protein processing and degradation [33
]. In C. elegans
H+-V-ATPases are required for development and osmoregulation in animal excretory systems [34
] and act as potent lifespan regulators [35
]. The down-regulation of V-ATPases is indicative of the deterioration in cellular homeostasis, and general reduction in cellular transportation activities associated with the trait-deteriorated Hb
We observed an interesting transcriptional pattern of genes involved in amino acid, lipid and carbohydrate metabolism, with the majority of the genes being down-regulated. There was up-regulation of sterol metabolism and down-regulation of enzymes in the category of synthases and hydrolases, suggestive of huge shifts in metabolism. Similarly, we observed down-regulation in the category of a dehydrogenase-like aldehyde dehydrogenase, glutamate dehydrogenase, suggesting repression of fermentative pathways. Carbohydrate metabolism was mostly down-regulated with the exception of pyruvate dehydrogenase (GenBank: NP_500340
) and phosphoglycolate phosphatase (GenBank: EX910617
). Similarly, amino acid metabolism was also mostly down-regulated, except for sorbitol dehydrogenase (SDH) (GenBank: XP_790483
), glutathione peroxidase (GenBank: NP_497078
) and a few other enzymes (Additional file 4
). During anhydrobiosis, nematodes reportedly accumulate polyols like sorbitol and glycerol, which are known to protect animal tissues and cells from injuries caused by freezing or dehydration [36
]. As anhydrobiosis is an ametabolic stage, the induction of SDH suggests a general reduction in metabolic activities, and nematodes might be using SDH as a stress survival mechanism. We also observed the differential regulation of enzymes involved in the tricarboxylic acid (TCA) cycle, including the up-regulation of pyruvate dehydrogenase and down-regulation of citrate synthase (GenBank: ES412521
), aconitate hydratase (GenBank: ES741155
), and dihydrolipoyl dehydrogenase (GenBank: EX010778
). Two DEGs encoding fructose-bisphosphate aldolase (FBPA) (GenBank: EG025510
) were down-regulated in the deteriorated line relative to original line. FBPA is an early step in the glycolysis pathway. The products of this pathway are ATP and pyruvic acid (PVA).
Potential signal transduction related genes
We identified a set of signal transduction components which likely orchestrate a rapid and general response to a wide range of changes, but also a set of signalling components that may mediate responses more specific to nematode trait deterioration (examples are highlighted in Table ). Transcriptome patterns associated with signalling during trait deterioration of insect parasitic nematodes have not been well established. We observed differential expression of signalling components like stress-induced-phosphoprotein 1(GenBank: ES740228), phospholipase c beta (GenBank: EX009150), cyclophilin-1 (GenBank: ES411663) and sodium/potassium transporting ATPase (GenBank: ES741918), which are involved generally in stress response and transduction.
The stress-induced-phosphoprotein 1 (sip-1
) or the Hsc70/Hsp90-organizing protein belongs to a group of co-chaperones, which regulate and assist the major chaperones. sip-1
modulates the chaperone activities of linked proteins and also interacts with other chaperones. The loss of sip-1
function in C. elegans
results in embryonic lethality and reduction in life span [37
]. Among our DEGs are several transcripts encoding phospholipase c beta of the gene encoded by C. elegans egl-8
. PLCβ in conjunction egl-30
acts in motor neuronswith to directly or indirectly regulate acetylcholine release, thereby modulating locomotion rate and behavior [38
]. Also included in our DEGs were two transcripts encoding C. elegans
), a class of peptidyl-prolyl cis-trans
isomerase (PPIase) enzymes which play an important role in protein folding [39
]. These cyclophilins are predicted to be secreted proteins in nematodes that are constitutively expressed [40
]. Induced signalling of these genes is likely an attempt on the part of stressed nematodes to combat reduction in life span and maintain the proper functioning of proteins in a changing environment.
A number of transcripts encoding protein tyrosine kinase (GenBank: EX009882
) and NADH-cytochrome P450 (GenBank: EX009598
) were down-regulated and nitric oxide synthase interacting protein (NOSIP) (GenBank: ES412752
) was up-regulated in L5M compared to OHB. Protein tyrosine kinases (PTKs) are important for intra- and inter-cellular communication as well as for survival in eukaryotes and play a major role in signal transduction processes. These proteins are also known to be involved in developmental and differentiation processes of cells [41
]. In C. elegans, a
nother signalling molecule, NADH-cytochrome P450 (NADH-CYP), has been shown to be involved in the detoxification of environmental pollutants and synthesis and degradation of signalling molecules [42
]. One of the C. elegans
CYP isoforms plays an important role in regulating lifecycle progression and the adult life span [43
]. The down-regulation of CYP in the deteriorated Hb
indicates an impaired ability of these nematodes to detoxify environmental pollutants and use of alternative pathways.
NOSIP is an enzyme that regulates nitric oxide (NO) production through binding with high affinity to the carboxyl-terminus of the endothelial nitric oxide synthase (eNOS) oxygenase domain and preventing NO synthesis. NO, a signalling molecule produced by nitric oxide synthase (NOS), is part of the immune response and acts as a neurotransmitter and proliferation signal. It is produced by both parasitic nematodes and their host. The excretory/secretory products from filarial nematodes, which include NO, have been shown to inhibit the proliferation of host cells mediating innate or acquired immunity [44
]. Although NO from Hb
has not been characterized yet, it could play a significant role in invasion and/or suppression of the host immune system. Up-regulation of NOSIP, which negatively regulates NO production, could result in reduced virulence, one of the observed characteristics in the trait-deteriorated Hb
. In addition to the signalling molecules described here, the discovery of more signalling transcripts from Hb
adds to the existing knowledge base of dauer-related genes in C. elegans
, furthering exploration of the importance of signalling components on trait improvement, increased longevity and stress resistance in nematodes.
Stress and defense related genes
With the advent of microarray technology, researchers can now identify a broad range of genes that are involved in trait deterioration in EPNs. While some genes may be developmental stage-specific, others may be part of a general stress response shared across multiple nematode species. A large portion of the DEGs we found are involved with stress and defense response of nematodes, including two up-regulated transcripts encoding a homolog of C. elegans hsp-12.6
) and daf-21
). The C. elegans daf-21
gene encodes a member of the HSP90 family of molecular chaperones important for maturation of signal transduction kinases in neurons involved in odorant perception [45
]. The post-embryonic phenotype of Hb
treated with double stranded (ds) Hba-daf-21
RNA resulted in abnormal gonad morphology [46
]. Another up-regulated gene, hsp-12.6
, which encodes a small heat shock protein (sHSP) in C. elegans
, is developmentally regulated but is not up-regulated by a wide range of stressors [47
]. Despite its lack of chaperone activity, hsp-12.6
regulates the functions of other sHSPs, acting as a co-chaperone with other molecular chaperones [48
]. It is possible that hsp-12.6
is also developmentally regulated, specifically during the infective stage and plays a significant role in driving the chaperone activities of other Hsps, and conferring protection against oxidative stress. Transcripts encoding homologs of the mitochondrial cytochrome c oxidase (CCO) subunits I, II, and III were abundantly expressed and up-regulated in our trait-deteriorated line. Interestingly, the up-regulated DEGs included 4 transcripts (GenBank: ES740428
) encoding ubiquitin conjugating enzymes (UBCs) and one encoding the 26S proteasome regulatory subunit (GenBank: EX009185
). UBCs have been shown to be induced under stress conditions in nematodes [49
], and nutrient deprivation in plants has shown to induce ubiquitin degradation of proteins and lipids [50
]. It has been suggested that cellular stress results in improperly folded proteins, which are targeted for degradation by ubiquitinization. Interestingly, one of the over-expressed genes in the deteriorated line encoded a chaperonin protein (GenBank: ES743704
), which are needed for proper folding of nascent proteins.
Among the down-regulated genes, eat-6
encodes an alpha subunit of sodium/potassium ATPase, which in turn affects the NA+
-ATPase activity of membranes. It plays a significant role in the relaxation of pharyngeal muscle, fertility, and also affects body length and life span [51
]. Transcriptional profiling showed two transcripts (GenBank: ES744087
) encoding fructose-1, 6-bisphosphate (FBP) aldolase, which was down-regulated in the trait-deteriorated nematode line. FBP aldolase, a member of the class I aldolase family, is a glycolytic enzyme that catalyzes the cleavage of FBP into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate [52
]. Predicted proteins with potential roles in T-cell receptor (TCR) and transforming growth factor beta (TGF-beta) signaling were also down-regulated. In the parasitic nematode B. malayi
, a C. elegans daf-7
homolog which encodes a member of the TGF-beta superfamily was reported to be involved in manipulation of the host immune response [53
]. Although at this stage the precise role of such molecules in host-parasite interactions is not clear, they could be involved in manipulating the host's immune response.
Dauer and nematode life span regulation
The infective juvenile stage of entomopathogenic nematodes is developmentally similar to the dauer stage in many bacterivorous nematodes, including C. elegans
and C. briggsae
. The dauer is a developmentally arrested stage triggered by food deprivation, high population density, and other harsh environmental conditions. Elucidation of this process is of specific interest in the case of entomopathogenic nematodes because the dauer juvenile is the only life stage capable of infecting insects [8
]. We observed differential expression of genes which are commonly expressed in dauer and starved adults of C. elegans
. Transcripts encoding hsp90
) were up-regulated while a GTP-binding ribosomal protein homolog (GenBank: ES410054
) and SH3-domain containing protein (GenBank: FF681443
) were down-regulated in the trait-deteriorated line compared to original. These genes are more abundantly expressed in dauer than in non-dauer (L3) larvae of C. elegans
]. One of the down-regulated DEGs that we encountered encodes a serine/threonine protein kinase (unc-51
) (GenBank: FF681332
) that is orthologous to Saccharomyces cerevisiae
autophagy protein. It is required for normal dauer morphogenesis of the C. elegans daf-2
]. The down-regulation of unc-51
may limit reallocation of nutrients in starving cells, such as those in dauer juveniles in free-living nematodes, or infective juveniles in entomopathogenic nematodes. Vacuolar H+
-ATPases (GenBank: EX011485
) were another potent lifespan regulator we found differentially expressed in trait-deteriorated nematodes. These proteins acidify intracellular compartments and act in synaptic transmission and the cell death signaling cascade [56
]. Another down-regulated daf-16
dependent gene encodes a glucose-6-phosphate isomerase (GenBank: ES740896) homolog which functions in the insulin/IGF-1 pathway to affect lifespan. In mammals, glucose-6-isomerase functions in glycolysis, which influences aging [57
]. The DEGs we recovered included 10 transcripts encoding components of the mitochondrial respiratory chain, including ATP synthase and NADH-ubiquinone oxidoreductase (ES411557
). RNAi of respiratory-chain components decreases body size and slows movement and eating behavior (pumping) of nematodes [58
]. Nematodes exposed to stress induce generation of reactive oxygen species (ROS), and therefore it is important for nematodes to have effective ROS scavenging mechanisms. A transcript which encodes a putative Ras related protein (GenBank: ES411895
), a C. elegans rab-33
homolog, was up-regulated (Figure ), suggesting that trait-deteriorated nematodes may have to elevate their ROS scavenging mechanisms.
Refined gene-specific expression using quantitative reverse transcription-PCR
The microarray observations were validated by quantitative reverse transcription-PCR (qRT-PCR) for some representative transcripts (Figure ). Sixteen genes that were differentially expressed in trait deteriorated nematodes were selected for qRT-PCR validation. For all the selected genes for which primers worked, qRT-PCR validation revealed similar expression kinetics for all the genes tested, indicating reliability of the microarray data (Figure ). The expression values obtained by qRT-PCR were generally more exaggerated than the corresponding microarray values, as reported in previous studies [59
]. Although microarray analyses showed low-magnitude change in DEGs (Figure ), we are able to verify the differential expression by means of qRT-PCR. It is possible that these levels may fall below a technical threshold and therefore do not allow a reliable transcript quantification by using only hybridization-based methods such as microarray analysis. The biological significance of such a change depends on the particular gene under consideration. Therefore, we believe that since the two-fold cut-off is somewhat artificial, its use could lead to misrepresentation of the set of up- or down-regulated genes, resulting in the loss of biologically important information. This conclusion is reinforced by the strong correlation (R2
= 0.84, P
< 0.05) for all of the transcript-concordant genes that we examined in this study irrespective of their differential regulation.
Microarrays tend to have a low dynamic range, which can lead to small yet significant under-representation of fold change in gene expression [61
]. As qRT-PCR has a greater dynamic range, it is often used to validate the observed trends rather than duplicate the fold changes obtained by chip experiments [62
]. The overall physiological response of an organism or cell to a stimulus may require coordinated changes in a wide array of genes. Those changes in turn may require compensating or reinforcing changes in an even wider array of functionally connected genetic components. Our analysis suggests that low-magnitude expression changes may be of functional significance. It is possible that some genes may show low-magnitude transcriptional modulation but still play a significant role in the resulting physiological response. We speculate that the genes involved in the evolution of EPN trait deterioration are co-ordinately modulated and thus show moderate levels of transcriptional change.