Introduction

The bovine lungworm Dictyocaulus viviparus is an important parasitic nematode in grazing cattle occurring worldwide in temperate areas. It causes parasitic bronchopneumonia, which leads to economic losses due to illness or even death of affected animals (Coles 2001; Panuska 2006). The ability to arrest development inside the host, also referred to as hypobiosis, is one of the key elements of the biology of the parasite, enabling survival under harsh environmental conditions (Blitz and Gibbs 1972a, 1972b; Gibbs 1982). In temperate areas, hypobiosis of parasitic D. viviparus larvae occurs in winter, induced by changing environmental conditions, particularly low temperature (Blitz and Gibbs 1972a; Armour and Duncan 1987). Hypobiotic lungworm larvae outlast the winter months in the lungs of cattle, continuing their development to egg-laying adults in spring, whereas most D. viviparus larvae on pasture die during winter. Therefore, hypobiotic larvae are considered to be crucial for the epidemiology of lungworm infections enabling the parasite’s survival from year to year (Michel 1955; Michel and Shand 1955). Experimentally, hypobiosis can be induced by exposing infective L3 of D. viviparus to low temperatures of 4–7°C for 6 weeks (Gupta and Gibbs 1970; Eysker et al. 1992; Strube et al. 2007). Even though hypobiosis is a well-known phenomenon in parasitic nematodes (Eysker 1993), only few studies about the regulating mechanisms on a molecular level have been performed. One study analyzing differentially transcribed genes in hypobiosis-induced and non-induced L3 was performed by Strube et al. (2007) and resulted in the first description of genes found to be differentially transcribed in the bovine lungworm D. viviparus. However, the authors used infective L3 in which hypobiosis was induced by chilling. Nevertheless, data from actually developmentally inhibited parasitic larvae are still missing. Therefore, the present study aimed to investigate differential gene transcription in hypobiotic and non-hypobiotic preadult larvae (L5) of D. viviparus to identify genes associated with the parasites’ development, thereby focusing on the process of hypobiosis. Subtracted complementary DNA (cDNA) libraries of hypobiotic and non-hypobiotic L5 were constructed by suppression subtractive hybridization (SSH), selected by differential screening followed by verification by Northern blot. Verified differentially transcribed sequences were subjected to bioinformatic analysis. The obtained data represent a basis for further stage specific as well as hypobiosis specific transcriptional investigations, possibly providing molecular targets for diagnosis as well as nematocidal compounds or vaccine development.

Materials and methods

Parasite material

First stage larvae of D. viviparus (isolate HannoverDv2000) were isolated from the feces of experimentally infected calves using the Baermann technique (Baermann 1917). Isolated larvae were incubated for 10 days at room temperature in 20–50 ml tap water to allow development to L3. For production of normally developing L5, two calves were infected with 20,000 L3 each. Necropsy was performed 15 days post-infection (dpi), followed by perfusion of the lung. To obtain hypobiotic L5 (L5hyp), five calves were infected with 35,000 L3 induced for hypobiosis by chilling at 4°C for 10 weeks. Furthermore, two calves were infected with 50,000 hypobiosis-induced L3. Necropsy was done 35 dpi. Larvae collected by subsequent lung perfusion were examined microscopically, and those smaller than 5 mm were considered to be hypobiotic (Inderbitzin 1976; Pfeiffer 1976). Recovered L5 and L5hyp, respectively, were washed several times in 0.9% sodium chloride and stored in 5.5 M guanidinium isothiocyanate buffer at −75°C until further use.

Poly(A)+ RNA isolation

To destroy the cuticle, larvae were homogenized with a TissueRuptor (Qiagen). Afterwards, poly(A)+ RNA was extracted using the QuickPrep™ Micro messenger RNA (mRNA) Purification Kit (GE Healthcare) following the manufacturer’s protocol. The poly(A)+ RNA yield was determined by measuring the absorbance at 260 nm with the NanoDrop™ 1000 spectrophotometer (PEQLAB Biotechnologie GmbH). For the construction of the L5 and L5hyp library, 1,450 L5 larvae and 124 L5hyp were used, respectively. The subsequent cDNA synthesis was performed with 1,000 ng mRNA of each population.

Suppression subtractive hybridization

The poly(A)+ RNA obtained from the two larval populations was reverse transcribed to cDNA using the SMART™ PCR cDNA Synthesis Kit (BD Biosciences Clontech). Afterwards, SSH was carried out using the PCR-Select™ cDNA Subtraction Kit (BD Biosciences Clontech) according to manufacturer’s instructions, with the exception that the fourfold amount of driver cDNA was used for the second round of hybridization as described by Strube et al. (2007).

Control of subtraction efficiency via Southern blot analysis

Subtraction efficiency of cDNA hybridizations was examined by Southern blot analysis. Briefly, 1 μg subtracted and unsubtracted cDNA, respectively, was electrophoretically resolved in two agarose gels (1%) and subsequently transferred to positively charged nylon membranes (Roche Diagnostics GmbH). Fixation was done by baking for 30 min at 120°C. Membrane prehybridization to block unspecific binding sites was carried out at 42°C for 90 min with 10 ml DIG Easy Hyb solution (Roche Diagnostics GmbH) containing 100 μl of herring sperm DNA (10 mg/ml). Afterwards, one membrane was probed (at 42°C for 20 h) with 100 ng of RsaI-digested, digoxigenin (DIG)-dUTP-labeled subtracted cDNA of the L5hyp, the other membrane with the respective cDNA from normal L5. The blots were then washed twice at room temperature (5 min/wash) with a low stringency buffer [2× saline-sodium citrate (SSC), 0.5% sodium dodecyl sulfate (SDS)] and 2× 15 min at 68°C with high stringency buffer (0.5× SSC, 0.1% SDS). Chemiluminescence detection during 30 min exposition was conducted with the Bio-Imaging system MF-ChemiBIS 3.2 (Biostep GmbH).

Construction of subtracted libraries and differential screening

Subtracted L5hyp and L5 cDNAs were cloned into the pCR®4-TOPO® vector (Invitrogen) and transformed into Escherichia coli strain One Shot® Mach1™ (Invitrogen). Randomly picked clones were checked for inserts by PCR, and 2,016 clones containing a single insert were selected to represent the L5hyp and L5 subtracted libraries.

To verify stage-specific upregulated transcripts enriched by SSH, differential screening was performed. For this purpose, high-density arrays containing quadruplicates of each clone as well as positive and negative controls were spotted by imaGenes GmbH and fixed by UV cross-linking (120 μJ × 100, 40 s). For hybridization, subtracted and unsubtracted cDNA probes of both populations were PCR-labeled with DIG-dUTP/dTTP (ratio 1:6; DIG DNA Labeling Mix; Roche Diagnostics GmbH). To prevent false positive hybridization signal detection due to possible incomplete restriction enzyme digestion, a blocking solution (PCR-Select™ Differential Screening Kit, BD Bioscience Clontech), containing oligonucleotides complementary to the adaptor sequences, was used besides herring sperm DNA (10 mg/ml) in the prehybridization step (42°C, 90 min). During hybridization (42°C, 18 h), high-density arrays were probed with subtracted and unsubtracted cDNA probes (350 ng each). Washing and chemiluminescence detection were carried out as described in the section before.

Chemiluminescence signals were analyzed using the Phoretix™ Array software (version 2003.01, Nonlinear Dynamics). Thereby, a background correction was achieved by Spot Edge Plus correction. This method calculates the mean of the lowest intensity band of pixels around the outline of a spot, which was then implemented as the background for the spot. Hence, the background is calculated independently for each spot. The band width of the pixels around the spot was defined as 1 mm. The cutoff intensity for a signal considered to be “present” was a spot volume of 74,000,000 when using the corresponding subtracted cDNA as probe. The signal intensity differences of each clone with the four different probes were calculated. Only clones showing signals with the corresponding subtracted and non-subtracted cDNA probes alone or at least a fivefold higher intensity than with the subtracted and non-subtracted cDNA probes of the contrary population were considered to be differentially transcribed, whereas the other clones were discarded as false positives.

Verification of differential gene transcription by virtual Northern blot

To verify clones with inserts identified as differentially transcribed by the differential screening procedure, a virtual Northern blot based on freshly isolated L5 and L5hyp poly(A)+ RNA was performed. For poly(A)+ RNA isolation, 1,000 L5 and 300 L5hyp were used and 1,000 ng per population were utilized for conversion into cDNA. After PCR labeling as hybridization probes, hybridization, detection and software analysis of the signal intensities were done as described above. Clones showing less than fivefold increased signal differences between the corresponding and the contrary population were eliminated. The remaining verified clones containing stage-enriched transcript stretches were sequenced in forward and reverse directions.

EST analysis

Primer and vector sequences were clipped from the obtained differentially transcribed sequence data. The remaining pure D. viviparus expressed sequence tags (ESTs) were processed using the ESTExplorer (http://estexplorer.biolinfo.org), a semi-automated bioinformatic pipeline for clustering and comparative analysis of ESTs (Ranganathan et al. 2007). Resulting contigs and singletons were further analyzed with the Blast2GO program (version 2.4.4, http://www.blast2go.org). This bioinformatic tool supports BLAST searches as well as gene ontology annotation, KEGG mapping and InterPro motif scan. Additionally, a secretome analysis using SignalP (version 3.0, http://www.cbs.dtu.dk/services/SignalP/) and prediction of transmembrane helices with the TMHMM server (version 2.0, http://www.cbs.dtu.dk/services/TMHMM/) were performed.

Results

Parasite material

Perfusion of the lung to obtain normally developed L5 resulted in approximately 16,500 L5 from the first calf and 4,000 L5 from the second calf. After necropsy, the number of L5hyp obtained was 49, 57, 75, 75, and 106, respectively, after infection with 35,000 larvae and 74 and 226 after infection with 50,000 larvae.

The microscopic examination revealed that both male and female L5s exhibited apparent genital anlages (Fig. 1a, b). In the L5hyp population, males showed a bursa with bursal rays and spicules (Fig. 1c), whereas the development of the vulva anlage of females was still at the beginning (Fig. 1d).

Fig. 1
figure 1

a Male L5 of D. viviparus (note distinct bursa anlage). b Female L5 of D. viviparus (note distinct vulva anlage). c Hypobiotic male L5 of D. viviparus (note small size and distinct bursa anlage). d Hypobiotic female L5 of D. viviparus (note small size and initial vulva development)

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SSH subtraction efficiency control

The Southern blot analysis to evaluate SSH subtraction efficiency showed that the subtracted L5hyp probe exhibited a strong hybridization signal to both L5hyp cDNAs. However, the hybridization signal to the subtracted L5hyp cDNA in direct comparison to the unsubtracted L5hyp cDNA signal is more intense and stretches over a wider range, representing sufficient subtraction efficiency. With the unsubtracted non-hypobiotic L5 cDNA, the signal was significantly weaker and no signal was observed with the subtracted L5 cDNA. The blot probed with the subtracted non-hypobiotic cDNA showed the reciprocal result. The Southern blot analyses are displayed in Fig. 2a, b.

Fig. 2
figure 2

a L5hyp Southern blot subtraction efficiency control. b “Normal” L5 Southern blot subtraction efficiency control. Equal cDNA amounts of subtracted L5 (L5s), subtracted hypobiotic L5 (L5hyps), unsubtracted L5 (L5u), and unsubtracted hypobiotic L5 (L5hypu) were transferred to positively charged nylon membranes followed by hybridization with DIG-labeled subtracted cDNA of hypobiotic and “normal” L5 cDNA as probes, respectively

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Clustering and assembly of differentially transcribed ESTs

Of the 2,016 clones of each subtracted library, 500 L5hyp clones (25%) and 100 L5 clones (5%) were confirmed to contain differentially transcribed ESTs by differential screening and finally verified by virtual Northern blot. Insert lengths of the clones varied between 250 and 900 bp. Sequencing of the 500 L5hyp clones in both directions resulted in 950 sequences, and 192 sequences were obtained from the sequencing of 100 L5 clones. After manual revision, 849 and 161 sequences remained, respectively. By further processing, using the ESTExplorer pipeline, 833 L5hyp high quality ESTs remained and were clustered into 54 contigs and 21 singletons (equivalent to 75 representative ESTs). This corresponds to 499 clones (99.8%) of the originally sequenced 500 clones. The contigs were ranked depending on the number of containing ESTs. Thereby, 148 ESTs (18%) were included in the top 5 contigs and 236 (28%) in the top 10 contigs. The L5 population contained 150 high quality ESTs assigned to 15 contigs and 43 singletons (58 representative ESTs), representing 83 (83%) of the originally sequenced 100 clones. The top 5 contigs included 21 ESTs (14%) and the top 10 contigs represented 31 sequences (21%). The high-quality ESTs of both larval populations were submitted to GenBank (accession numbers GW915699-GW916698 and GW992802-GW992804).

Bioinformatic characterization of differentially transcribed ESTs

Of the 75 representative ESTs (rESTs) of the L5hyp population 12 rEST were found to be homologous to either Caenorhabditis elegans or Caenorhabditis briggsae sequences, four rESTs were homologous only to other organisms than C. elegans or C. briggsae, namely, with Angiostrongylus cantonensis, Brugia malayi, and Schistosoma mansoni, whereas 47 rESTs of the L5hyp population revealed no sequence homologies. Thereby, the criteria for homology were an at least 55% amino acid similarity on a sequence segment of at least 40 amino acids with a corresponding e value of ≤1.00E−05. From the 58 rESTs of the L5 population, six were found to be homologous to C. elegans and C. briggsae transcripts. In contrast, four rESTs showed no homologies with C. elegans or C. briggsae but with hypothetical proteins of A. cantonensis. Again, 47 rESTs showed no homologies with published sequences.

In order to further elucidate biological, molecular and cellular functions of the differentially transcribed sequences, Gene Ontology (GO) terms were assigned. It was found that 23 of the 75 L5hyp rESTs and seven of the 58 L5 rESTs could be assigned to GO terms. Since individual sequences can be assigned to more than one GO term, all in all, 108 annotations were possible for the hypobiotic and 23 for the normal L5 population.

The top hits of the BlastX search with corresponding percentage of identity and similarity as well as GO annotation results are displayed in Table 1 (L5hyp) and Table 2 (L5).

Table 1 Top hits of BlastX and gene ontology annotation of Dictyocaulus viviparus hypobiotic L5 upregulated rESTs
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Table 2 Top hits of BlastX and gene ontology annotation of Dictyocaulus viviparus L5 upregulated rESTs
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To gain knowledge about biological pathways, in which the differentially transcribed genes are involved, rESTs of both populations were mapped using KOBAS, which was implemented within the Blast2GO program. In the hypobiotic population, five rESTs were assigned to 14 pathways. One rEST was assigned to four different pathways (metabolic pathways, purine, pyrimidine, nicotinate, and nicotinamide metabolism as well as biosynthesis of alkaloids derived from histidine and purine) and a further rEST was assigned to nine pathways (metabolic pathways, glycolysis and gluconeogenesis, biosynthesis of alkaloids derived from histidine and purine, of phenylpropanoids, terpenoids, and steroids, plant hormones and alkaloids derived from shikimate pathway, ornithine pathway, and terpenoid pathway). Furthermore, one rEST was assigned to metabolic pathways and pyrimidine metabolism and two rESTs to methane metabolism, tropane, piperidine, and pyridine alkaloid biosynthesis. In the L5 population, one rEST could be assigned to the pathway of aminoacyl-t-RNA biosynthesis. However, it has to be kept in mind that some of the pathways covered by KEGG do not (for example plant pathways) or may not exist in worms (for example mammalian pathways).

SignalP predicted that 15 of the rESTs in the L5hyp population had signal peptide cleavage sides. Regarding the L5 population, 14 rESTs were predicted to have signal peptide cleavage sides. The presence of transmembrane helices could not be predicted with TMHMM in any of the rESTs of the L5hyp and L5 population.

Discussion

Hypobiosis or arrested development ensures the survival of D. viviparus during adverse environments (Blitz and Gibbs 1972a,b; Gibbs 1982). Thereby, hypobiotic larvae outlast in the lungs of cattle without being eliminated by the immune system of the host. Since knowledge about regulatory mechanisms of hypobiosis is extremely limited, a subtractive hybridization approach was used to identify transcripts either associated with hypobiotic (L5hyp) or normally developed (L5) preadult lungworm larvae. Thereby, 500 L5hyp sequences, later on assigned to 75 rESTs and 100 L5 sequences, assigned to 58 rESTs were identified as differentially transcribed and subjected to further analysis. The small number of confirmed differentially transcribed sequences from the subtracted library (25% in the L5hyp and 5% in the L5 population) was due to inserts whose chemiluminescence signals did not exceed the cutoff value as well as due to the claim of an at least fivefold difference in signal intensities between both populations. If the threshold had been set to a twofold difference, as applied in a study with Oesophagostomum dentatum (Cottee et al. 2006), 220 additional clones in the L5hyp population and 47 in the L5 population would have matched the definition for differential transcription. However, to minimize the possibility of picking false positive clones, the difference was set this high. Furthermore, only a limited number of differences may be expected (Stubbs et al. 1999). This was the case in the present study in which two populations of preadult larvae were compared.

BlastX sequence alignment revealed that a high number of differential transcripts showed homologies with either C. elegans or C. briggsae rather than with parasitic nematodes. This can be ascribed to the fact that those two free living nematode species belong to the same clade as D. viviparus (Blaxter 1998). Furthermore, their genome is completely sequenced, whereas most parasitic nematodes are not.

Information about sequence homologies combined with assignment to GO terms, KEGG maps, or protein domains and motifs provide a first functional bioinformatic characterization of stage-specific upregulated genes in hypobiotic and normally developed L5. In the present study, a high number of transcription factors was found to be upregulated in the hypobiotic compared to the normal L5. Transcription factors are known not only to activate but also to repress the transcription of target genes (Beato et al. 1995). Hypobiotic larvae are in a state of retarded development (von Samson-Himmelstjerna and Schnieder 1999), this perhaps being initiated and maintained by a high number of transcription factors, repressing genes needed for physiological functions and growth. Nevertheless, stage-specific proteins involved in the metabolism like the steroidogenic acute regulatory protein (StAR) were found. Being the top 5 contig with 22 high-quality ESTs included, it may be of special importance for the hypobiotic stage. It is known that StAR regulates the delivery of cholesterol from the outer to the inner mitochondrial membrane, which is considered as the rate-limiting step in steroid biosynthesis (Manna et al. 2009). Cholesterol is the precursor for steroid hormones, which can act as ligands for nuclear hormone receptors like daf-12 (Rottiers and Antebi 2006). These receptors can function as transcription factors and mediate the activation or repression of target genes (Beato et al. 1995). Subsequently, it could be assumed that StAR might play a major role in cholesterol trafficking in hypobiotic larvae, perhaps being the precursor for transcription factors promoting the hypobiotic state.

Another group of proteins, which was found to be upregulated in hypobiotic larvae, were H2B and H4 histones. Histones are essential for the packing of DNA into nucleoprotein complexes, which are essential for the control of gene expression (Keall et al. 2007). Gene expression control is also achieved by various posttranslational modifications of histones, referred to as “language” or “histone code,” which is read by other proteins (Strahl and Allis 2000; Sun and Allis 2002). Therefore, it can be speculated that the upregulation of histones in hypobiotic D. viviparus larvae might be necessary for the control of gene expression to promote prolonged development by repressing genes promoting development. Thus, hypobiosis might not be induced and maintained by the upregulation of certain genes but rather caused by silencing of certain genes. Besides regulation of gene expression, histones may play an important role in sexual development, since in adult C. elegans, inhibited H4 protein synthesis results in sterility (Keall et al. 2007). Interestingly, in hypobiotic lungworm larvae sexual development seems to be less inhibited by hypobiosis than growth. Strube et al. (2009) examined the transcription rate of the main sperm component, the major sperm protein (MSP), in ten developmental lungworm stages and found a significantly higher transcription level in hypobiotic than in normally developed male L5. Furthermore, the present study provides clues of hypobiosis decelerating growth, whereas sexual development seems to be less affected. Thus, both hypobiotic and non-hypobiotic male L5 showed a fully formed bursa with bursa rays and spicules. In contrast, while the hypobiotic female L5 showed an indistinct vulva opening, the non-hypobiotic one already possessed undifferentiated organs of the reproductive system.

Indeed, one of the genes upregulated in non-hypobiotic L5 encodes a vitellogenin-like protein, which functions not only as a cholesterol transporter (Entchev and Kurzchalia 2005) but also in maintaining reproductive capacity (Manna et al. 2009). Schneider (1996) describes its function as vital for oozyte maturation. Furthermore, a homologue of the anterior pharynx defective family member APH-1 was found to be associated with normal L5 development. Mutations of this protein lead to embryonic lethality as well as defects in vulva development. Consequently, upregulated transcription of a vitellogenin-like protein and APH-1 in combination with the morphological findings in female larvae leads to the conclusion that female sexual development is inhibited compared to normally developed L5. This contrasts with male sexual development, which seems to be more advanced in hypobiotic larvae. However, further studies such as real-time PCR experiments are necessary to prove this hypothesis. Nevertheless, drawing these conclusions on the basis of single genes is speculative and needs to be functionally proved by further investigations.

From a total of 133 rESTs, the large number of 94 rESTs (47 from each population) showed no homologies to sequences in the current databases. These genes might represent parasite or even Dictyocaulus-specific genes and thus could be possible targets for anthelmintic and/or vaccine development.

The fact that, in the present study, no genes involved in C. elegans dauer larvae maintenance were found to be upregulated in the hypobiotic larvae seems to confirm the assumption that the equivalent of the C. elegans dauer larvae is the free-living infective third stage larva (Burglin et al. 1998) rather than the hypobiotic larva. Another observation within this context is that genes described to be upregulated in hypobiosis-induced D. viviparus L3 (Strube et al. 2007) were not recognized in this study. This could be explained by stage differences of L5 in the present and L3 in the previous study. While hypobiosis-induced L3 neither feed nor develop in terms of completely arrested longitudinal growth, hypobiotic L5 do feed and show a slow but measurable growth in length.

In conclusion, this is the first study dealing with transcripts differentially transcribed in hypobiotic nematode larvae and the corresponding normally developed stage. It delivers two sets of stage-specific ESTs, which function as the basis for further studies on the genetic mechanisms of hypobiosis in lungworms and other parasitic nematodes. Furthermore, identified genes and encoded proteins may serve as targets for vaccine and drug development.