Differential gene expression in hypobiosis-induced and non-induced third-stage larvae of the bovine lungworm Dictyocaulus viviparus

International Journal for Parasitology 37 (2007) 221–231 www.elsevier.com/locate/ijpara

Differential gene expression in hypobiosis-induced and non-induced third-stage larvae of the bovine lungworm Dictyocaulus viviparus Christina Strube *, Thomas Schnieder, Georg von Samson-Himmelstjerna Institute for Parasitology, Centre for Infectious Diseases, University of Veterinary Medicine Hannover, Buenteweg 17, 30559 Hannover, Germany Received 28 June 2006; received in revised form 15 September 2006; accepted 19 September 2006

Abstract Hypobiosis is of particular importance in overwintering of the bovine lungworm Dictyocaulus viviparus. However, in parasitic nematodes there is no information available on the genetic mechanisms of hypobiosis. Suppression subtractive hybridisation was performed to identify upregulated transcripts of hypobiosis-induced and non-induced third-stage D. viviparus larvae, respectively. Subtracted libraries containing 105 clones of the hypobiosis-induced and 104 clones of the non-induced larvae were generated. By differential screening and Southern dot blot, 26 clones of the hypobiosis-induced and 22 clones of the non-induced larvae were confirmed to be differentially expressed. Sequencing of rapid amplification of cDNA ends (RACE) and spliced-leader-1 PCR products was performed to further characterise selection of the differentially regulated gene transcripts. The genes encoding an N-methyltransferase and a superoxide dismutase were upregulated in the hypobiosis-induced and non-induced larvae, respectively. The expression patterns of these genes were validated by quantitative real-time PCR. This revealed differential gene expression, particularly for the N-methyltransferase.  2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Dictyocaulus; Parasitic nematode; Hypobiosis; Inhibited development; Arrested development; Differential gene expression; Suppression subtractive hybridisation

1. Introduction The nematode Dictyocaulus viviparus is the causative organism of parasitic bronchitis, which leads to severe economic losses or even death in grazing calves. The phenomenon of hypobiosis, also termed arrested or inhibited development, represents an interruption or postponement of larval development inside the host with the consequence of reduced parasite metabolism. This enhances parasite survival during times of adverse environmental conditions (Blitz and Gibbs, 1972a,b; Gibbs, 1982). In temperate areas, where the bovine lungworm occurs, most infective L3 die on pasture during winter, whereas hypobiotic larvae persist in the lungs of infected cattle until the *

Corresponding author. Tel.: +49 511 953 8796; fax: +49 0511 953 8555. E-mail address: [email protected] (C. Strube).

following spring and then mature to egg-laying adults. Thus, hypobiosis is a significant aspect in the epidemiology of lungworm infections. Winter inhibition of parasitic nematodes is induced by changing environmental conditions in the autumn, particularly low temperatures (Blitz and Gibbs, 1972a; Armour and Bruce, 1974; Eysker, 1981). In D. viviparus, hypobiosis can be experimentally induced by chilling L3 for several weeks at 4–7 C (Gupta and Gibbs, 1970; Eysker et al., 1992). Although hypobiosis occurs in many parasitic nematodes (Gibbs, 1986), regulating mechanisms on a molecular level are still unknown. To analyze transcripts of L3 we performed suppression subtractive hybridisation (SSH) followed by differential screening and Southern dot blot using cDNA of hypobiosis-induced (L3i) and non-induced (L3ni) D. viviparus L3. Differentially expressed sequences were compared with parasite-expressed sequence tags (ESTs) and gene sequences of

0020-7519/$30.00  2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2006.09.014

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other organisms to determine the genes they may represent. A selection of upregulated transcripts was characterised and their predicted protein functions via gene ontology (GO) searches in inhibited and uninhibited development are discussed.

Dictyocaulus viviparus L1 were isolated from the feces of experimentally infected calves using the Baermann method (Baermann, 1917). Circa 100,000 larvae were incubated in 20–40 ml of tap water at room temperature for about 14 days, until development to L3 was completed. These larvae represented L3ni. To induce hypobiosis, L3 were incubated in tap water for 6 weeks at 4 C. After this incubation period, they were designated as L3i. Different incubation times of hypobiosis-induced and non-induced larvae were unavoidable, since in contrast to trichostrongyle parasites, lungworm L3 will die if they are kept for longer than about 14–20 days at room temperature. To ensure sufficient poly(A)+ RNA isolation, the L3i and L3ni were exsheathed in 0.6% v/v sodium hypochlorite (NaOCl) at 37 C with continuous shaking. Following exsheathment, the larvae were washed three times in diethylpyrocarbonate (DEPC)-treated water and stored in guanidinium isothiocyanate buffer at 80 C until use.

berg, Germany). SSH was performed with the PCRSelect cDNA Subtraction Kit (BD Biosciences Clontech) according to the manufacturer’s protocol. Briefly, following restriction-enzyme digestion of the cDNAs and adaptor ligation of the tester cDNA populations, two rounds of subtraction were performed. In the forward subtraction, L3i cDNA served as tester containing upregulated transcripts. This tester was hybridised with driver cDNA of the L3ni to remove common gene sequences. In a reverse subtraction, the L3ni cDNA served as the tester and was hybridised with L3i cDNA as driver. In a further experiment, the 4-fold amount of driver cDNA was used for the second round of the subtractions, as previous subtraction efficiencies were not sufficient. The subtracted, differentially expressed sequences of both larval populations were amplified using 27 primary and 12 secondary PCR cycles. Subtracted and unsubtracted secondary PCR products of the L3i and L3ni were amplified to examine the efficiency of subtraction. For this purpose, primers for an unspecified gene sequence known to be expressed in both larval populations were used. If subtraction was efficient, this PCR product should be detected 5–15 cycles later in the subtracted than in the unsubtracted cDNA. The sequences of these primers were 5 0 -ACG CGT TTA GCA CTA CTG GTT GT-3 0 and 5 0 -GGA CGG AAG CTG CTA CAA-3 0 . PCR cycling was performed using the following temperature profile: denaturation at 94 C for 30 s, annealing at 56 C for 30 s and extension at 68 C for 2 min.

2.2. Hypobiosis of the D. viviparus strain

2.5. Construction of subtracted libraries

To confirm the occurrence of hypobiosis of the lungworm strain used in these experiments, three male calves 3 months of age were experimentally infected. Two calves were infected with 20,000 L3i. Necropsy was performed 27 days p.i. and 42 days p.i., followed by perfusion of the lungs as previously described (Eysker et al., 1990). As a control, the lungs of another calf infected with 3000 L3ni were perfused 27 days p.i.

The secondary PCR products of the L3i and L3ni generated by SSH were directly cloned into the vector pCR 4-TOPO and transformed into Escherichia coli strain One Shot TOP10 by the TOPO TA Cloning Kit for Sequencing (Invitrogen, Karlsruhe, Germany). Following the manufacturer’s instructions, about 100 clones of each larval population were randomly selected and checked for inserts using the nested primers of the secondary PCR. These selected clones were designated as the subtracted libraries of the L3ni and L3i.

2. Materials and methods 2.1. Parasite material

2.3. Poly(A)+ RNA isolation To destroy the cuticle, the exsheathed larvae were homogenised with a mortar and pestle in liquid nitrogen. Poly(A)+ RNA was extracted using oligo(dT) cellulose beads from the Quick Prep Micro mRNA Purification Kit (Amersham Biosciences, Freiburg, Germany). The poly(A)+ RNA yield was determined by measuring the absorbance at 260 nm. 2.4. Suppression subtractive hybridisation (SSH) Poly(A)+ RNA of the two larval populations was transcribed to cDNA by using the SMART PCR cDNA Synthesis Kit (BD Biosciences Clontech, Heidel-

2.6. Labeling of differential screening probes Subtracted and unsubtracted primary PCR products of the L3i and L3ni were labelled with digoxigenin (DIG)dUTP (deoxy-uridinetriphosphate) to serve as hybridisation probes in differential screening. Briefly, the cDNAs were labelled by secondary PCR with a DIG-dUTP:dTTP (deoxy-thymidinetriphosphate) ratio of 1:6 using the DIG DNA Labeling Mix (Roche, Mannheim, Germany), purified with CHROMA SPIN-100 Columns (BD Biosciences Clontech), digested with RsaI, SmaI, and EaeI (Roche) to remove adaptors and purified again with CHROMA SPIN-100 Columns.

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2.7. Differential screening

2.9. Sequencing of differentially expressed cDNAs

To eliminate false positive clones present in the subtracted libraries, we performed differential screening using the DIG-labelled subtracted and unsubtracted L3i and L3ni cDNAs as probes (see above). First, the inserts of the subtracted libraries were amplified and arrayed in duplicate onto four positively charged nylon membranes (Roche) according to the protocol of the PCR-Select Differential Screening Kit (BD Biosciences Clontech). The membranes were enclosed in a hybridisation bag (Roche) and prehybridised with DIG Easy Hyb solution (Roche) containing 50 ll blocking solution (PCR-Select Differential Screening Kit, BD Biosciences Clontech) and denatured at 99 C for 10 min before quenching on ice. Prehybridisation was performed at 42 C for 90 min in a water bath with gentle shaking. Each DIG-labelled cDNA probe (495 ng) was denatured together with 50 ll blocking solution and added to 30 ml DIG Easy Hyb (corresponding to 16.5 ng probe/ml). This hybridisation solution was added to the membranes. Hybridisation was done at 42 C for 22 h in a water bath with gentle shaking followed by washing twice at room temperature (10 min/wash) in 2· sodium chloride– sodium citrate (SSC), 1% sodium dodecyl sulfate (SDS) and twice at 68 C in 0.5· SSC, 0.1% SDS (20 min/wash). The chemiluminescent detection was performed using the DIG High Prime Labeling and Detection Starter Kit II (Roche) according to the manufacturer’s recommendations. Following exposure of the membranes to KODAK BioMax Light films (Amersham Biosciences) at room temperature for 40 min, the films were developed using KODAK GBX Developer and KODAK GBX Replenisher (Integra, Fernwald, Germany).

Plasmid DNA of the 48 clones containing differentially expressed cDNAs was obtained using the Plasmid Midi Kit (Qiagen, Hilden, Germany) and sequenced at the SEQLAB Sequence laboratories (Go¨ttingen, Germany). The sequence data were analyzed for identities to genes and proteins using the WU-Blast2 parasite genome database of the European Bioinformatics Institute (EBI) (http:// www.ebi.ac.uk/blast2/parasites.html) and BLAST for searching the non-redundant National Center for Biotechnology Information (NCBI) database (http://www.ncbi. nlm.nih.gov/BLAST/).

2.8. Verification by Southern dot blotting To confirm the results of the differential screening procedure, a further cDNA hybridisation was performed. Briefly, the amplified inserts of the subtracted libraries were arrayed again in duplicate onto two positively charged nylon membranes (Roche). Freshly isolated poly(A)+ RNA (Quick Prep Micro mRNA Purification Kit, Amersham Biosciences) of the L3i and L3ni was used to generate DIG-labelled cDNA using the SMART PCR cDNA Synthesis Kit (BD Biosciences Clontech) according to the manufacturer’s instructions. The probes were then labelled with a DIG-dUTP:dTTP ratio of 1:6 during PCR. To remove the SMART II oligonucleotides, the probes were purified with CHROMA SPIN-100 Columns (BD Biosciences Clontech) according to the manufacturer’s recommendations, digested with RsaI (Roche), and purified again with CHROMA SPIN-100 Columns. Preparation of the membranes, hybridisation and detection were carried out as described above (Section 2.7) with the following modifications: 25 ng probe/ml DIG Easy Hyb (Roche) were used and membranes were exposed for 90 min to the chemiluminescent films.

2.10. Generating full-length cDNA sequences Based on the sequence identities obtained by parasite WU-Blast2 and NCBI BLAST, six clones of the L3ni (L3ni 16, L3ni 22, L3ni 51, L3ni 56, L3ni 67, L3ni 69/92) and six clones of the L3i (L3i 2/10, L3i 53, L3i 75, L3i 82/101, L3i 88, L3i 100) were selected to generate full-length sequences. Two additional clones were chosen for further analysis: the clone L3i 33 because of its high E-value and the sequence encoded by L3i 18/86/94/99 because of its frequent presence in the subtracted library (see Section 3.3). To obtain full-length cDNA, gene specific forward and reverse primers were designed with the Lasergene software (DNASTAR, Version 5.06). Each primer combination was checked by PCR using double-stranded cDNA and four different single-strand cDNA-preparations of the L3ni and L3i. The amplification products were analyzed by gel electrophoresis, cut out of the gel and sequenced. Primer sequences can be obtained from the authors on request. Two methods were used to obtain full-length sequences: rapid amplification of cDNA ends (RACE) and splicedleader-1 (SL1-) primer PCR. RACE was carried out using the SMART RACE cDNA Amplification Kit (BD Biosciences Clontech). Briefly, poly(A)+ RNA of the two larval populations was converted into first-strand cDNA and subsequently amplified using touchdown PCR. SL1primer PCR was used to obtain the 5 0 -cDNA end in the case of five insert sequences. The SL1 primer sequence was 5 0 GGT TTA ATT ATC CCA AGT TTG AG-3 0 (Takacs et al., 1988). This primer was used in combination with a gene-specific reverse primer. SL1-primer PCR was performed for 40 cycles using the following temperature profile: initial denaturing at 94 C for 1 min, denaturing at 94 C for 20 s, annealing primers at 62 C for 20 s, extending primers at 72 C for 3 min and final extension at 72 C for 7 min. Candidate bands were cut out and ligated into pCR 4-TOPO vector (TOPO TA Cloning Kit for Sequencing, Invitrogen) followed by sequencing. Fulllength cDNA sequences were obtained by overlapping fragments. Additionally, for a number of clones full-length PCR products were amplified by using primer pairs complementary to nucleotide sequences near the 5 0 - and 3 0 -end.

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2.11. Bioinformatic analysis

3. Results

The resulting full-length sequence data were analyzed for sequence identities via BLAST searching the ESTs at Nematode Net (http://www.nematode.net/BLAST/) and the NCBI database (http://www.ncbi.nlm.nih.gov/ BLAST/). To obtain information about the predicted protein functions, gene ontology searches were performed using Joined Assembly of Function Annotations (http:// jafa.burnham.org/newSession.html; Friedberg et al., 2006), GO Figure Gene Ontology Annotator (http://udgenome. ags.udel.edu/gofigure/index.html; Khan et al., 2003), and Goblet (http://goblet.molgen.mpg.de/cgi-bin/goblet-batch. cgi; Groth et al., 2004).

3.1. Hypobiosis capacity of the D. viviparus experimental strain

2.12. Quantitative real-time PCR Quantitative real-time PCR (qPCR) was performed to verify differential gene expression results obtained by differential screening and Southern dot blot. Selected genes were superoxide dismutase (SOD) and N-methyltransferase (NMT). First-strand cDNA preparations generated from five different mRNA-isolations of both larval populations were used as template. The housekeeping gene elongation factor-1a (EF-1a, GenBank Accession No. AY534333) was chosen to correct for variations of mRNA amounts and cDNA synthesis efficiency. Primers and TaqManMGB-probes were designed using the Primer Express software (Applied Biosystems, Darmstadt, Germany). Probes were purchased from Applied Biosystems and primers from Invitrogen, respectively. Primer sequences were: NMT for 5 0 -CCACAAAATCGCCAAGAGCTA-3 0 , NMT rev 5 0 -T TCGAGTTTTGATTGGTCCCA-3 0 , SOD for 5 0 -TGGAC GAGCCGTTGTCATT-3 0 , SOD rev 5 0 -TTAGGCCTTG GCAGATCGG-3 0 , EF-1a for 5 0 -GGTGGGATTGACAA AAGAACCA-3 0 , and EF-1a rev 5 0 -CAAGAGATGGG TAAGGGTTCTTTC-3 0 . The corresponding TaqManMGB-probe sequences were: NMT 5 0 -FAM-TGCGGTG GTATGAGGGA-MGBNFQ-3 0 , SOD 5-FAM-ATGCCG ATGCAGATGA-MGBNFQ-3 0 , and EF-1a 5 0 -FAM-TG AAAAATTTGAGAAGGAAGC-MGBNFQ-3 0 . Five different single-stranded cDNA-preparations per gene (SOD or NMT) and larval population (L3i and L3ni) were used as undiluted, 1:10 and 1:100 diluted template. In addition, 10-fold serial dilutions of cloned NMT, SOD and EF-1a cDNA samples ranging from 107 to 101 copies per sample were used as templates to generate standard curves for estimation of copy numbers on each plate. The plates contained duplicates of each cDNA sample, serially diluted control samples for the standard curves and a no-template control. qPCR was set up using the Brilliant Quantitative PCR Core Reagent Kit (Stratagene, Heidelberg, Germany). Thermal cycling conditions were: 10 min at 95 C followed by 40 cycles of 30 s at 94 C, 30 s at 56 C and 2 min at 68 C. Experiments and data analysis were performed using the Mx4000 Multiplex Quantitative PCR System (Stratagene, Heidelberg, Germany).

The capacity for hypobiosis in the D. viviparus strain was examined experimentally in calves. Hypobiosis was induced by incubating third-stage larvae for 6 weeks at 4 C, followed by infection of two calves and perfusion of the lungs at 27 and 42 days p.i. All 418 parasitic stages isolated were pre-adult, ranging in length from 0.45 to 3.0 mm and lacking complete sex differentiation. More precisely, only five individuals showed a developing bursa. In contrast, only fully sexually developed adult parasites (3–7 cm) were obtained from the lungs of a calf infected with non-induced larvae and necropsied at 27 days p.i. 3.2. Construction and analysis of subtracted libraries Suppression subtractive hybridisation was performed to enrich gene sequences differentially expressed in the tester populations L3i and L3ni. The second round of subtractive hybridisation was performed using the recommended amount of driver in a first experiment as well as a 4-fold amount of driver in a second experiment. The subtraction efficiency was tested by amplification of a gene sequence known to be expressed in both larval populations (see Section 2.4). Subtraction efficiency test of the first experiment revealed that subtraction efficiency was insufficient. By contrast, the subtraction efficiency test of the second experiment resulted in PCR products observable 10 cycles earlier when using the unsubtracted cDNAs than the subtracted cDNAs of the two larval populations. This result indicated efficient subtraction of common sequences in both larval populations when using the 4-fold amount of driver instead of the recommended tester:driver ratio. The cDNAs subtracted by SSH were cloned to create subtracted libraries. According to the manufacturer’s recommendation, 105 clones of the L3i and 104 clones of the L3ni were randomly selected. These clones were termed subtracted libraries. The inserts of the clones varied from 250 to 2200 bp in length. To identify differentially expressed sequences, we carried out differential screening. Hybridisation of the inserts dotted on nylon membranes with subtracted tester and driver probes resulted in strong signal differences between the clones of the two subtracted libraries. In particular, the clones of the L3i-subtracted library showed high signal intensity to the subtracted tester probe (L3i, Fig. 1A), whereas the signal intensity using the subtracted driver probe (L3ni, Fig. 1C) was weak. Using unsubtracted probes of the two larval populations, fewer differences in signal intensity were observed (Figs. 1B and D). Fifty-eight clones (55.2%) of the L3i and 44 clones (42.3%) of the L3ni were found to show signals only with the tester cDNA probes or at least 4-fold more intense compared with the driver cDNA probes. Following the manufacturer’s recommendation, these differences in signal

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probes. In the L3ni-subtracted library, 22 of 104 clones (21.2%) were differentially expressed whereas 78.8% of the clones did not meet the threshold to be considered as differentially expressed. 3.3. Sequencing and analysis of differentially expressed sequences The 48 differentially expressed gene fragments of the L3ni and L3i (26 of the L3i and 22 of the L3ni) populations were sequenced. Poly(A)+ tails representing 3 0 ends were found in the clones L3ni 7, L3ni 80, L3i 51, and L3i 69. A comparison of all sequences among themselves revealed that some clones of the subtracted libraries encoded the same gene. These were the clones L3ni 69 and L3ni 92, L3i 2 and L3i 10, L3i 82 and the L3i 101 as well as the clones L3i 18, L3i 86, L3i 94 and L3i 99. The search for identities to published sequences was done using WU-Blast2 at EBI and NCBI BLAST. The results of the WU-tBlastx searching parasite ESTs are summarised in Table 1. There were 28 clones (11 L3ni and 17 L3i clones) showing no identity or identities with an E-value > 0.0001 at the amino acid level with parasite ESTs, but E-values < 0.0001 at the nucleic acid level to parasite ESTs (data not shown). Notably, there was no sequence identity at all of the sequence L3ni 75 to nematode ESTs. 3.4. Characterisation of differentially expressed sequences and variants of transcripts

Fig. 1. Differential screening results of the L3i-subtracted library. Each clone is spotted on the membranes in duplicate. A selection of three differentially expressed clones is indicated by circles (black = L3i 25, white = L3i 64, grey = L3i 94). These clones show higher signal intensities when hybridised with subtracted (A) than with unsubtracted L3i-cDNA (B) as a probe. No or only very weak signals were obtained when hybridisation was done with subtracted (C) and unsubtracted (D) L3nicDNA, respectively.

intensity observed by macroscopic estimation of the density were determined as threshold for differential expression. Confirmation of the differential expression of these clones was performed by Southern dot blotting using cDNA probes generated from freshly isolated poly(A)+ RNA. Finally, 26 of 105 clones (24.8%) of the L3i-subtracted library were confirmed to obtain differentially expressed inserts, whereas the remaining clones (75.2%) were considered as false positive clones. These showed either low or no differences in signal intensities across the four cDNA

The gene-specific primers designed to obtain full-length cDNA of six L3ni and eight L3i clones were tested by PCR using cDNA of both larval populations. When the amplification products were analyzed, there were two notable observations. First, no or faint bands were observed for the clones L3i 2/10, L3i 33, L3ni 51, L3i 75, L3i 82/101, L3i 88, L3i 94/99, L3i 100, L3ni 16 and L3ni 69/92 when using driver cDNA. Thus, for these clones differential gene expression was reinforced. Secondly, amplification of L3i 33, L3i 53, L3i 75, L3i 82/101, L3i 88, L3i 100 and L3ni 56 resulted in two or three bands in the gel instead of the expected single band. Sequence analysis showed that the multiple bands were due to deletions. Further transcript variants were obtained by sequencing RACE products resulting in isoforms of seven genes (see Table 1). The lengths of the similar proteins obtained by BLASTP search indicate that the L3i 88, L3i 94/99 and L3i 67 sequences were incomplete at their 5 0 end. The sequence similarities to parasite ESTs and proteins deposited in the NCBI database are summarised in Table 2. The results of the gene ontology search via Goblet, performed to obtain information about the gene function, are listed in Table 3. By using RPSBLAST, a number of conserved domains were found within the deduced amino acid sequences. The percentage of aligned amino acid residues to conserved domains is shown in Table 4.

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Table 1 Parasite expressed sequence tag (EST) top hits to differentially expressed bovine lungworm sequences using WU-tBlastx (http://www.ebi.ac.uk/blast2/ parasites.html) Clone

EST

DB:ID

Identity %

Positives %

E-value

L3ni 7 L3ni 16a

Ancylostoma caninum genomic DNA Haemonchus contortus cDNA similar to Caenorhabditis elegans unc-15 paramyosin A. caninum cDNA similar to C. elegans troponin Nippostrongylus brasiliensis cDNA similar to C. elegans C05D11.10 Pristionchus pacificus genomic DNA A. caninum cDNA A. caninum cDNA similar to C. elegans Y53H1B.2 n/a Meloidogyne incognita cDNA similar to C. elegans tth-1 n/a Teladorsagia circumcincta cDNA similar to C. elegans ccg-1 Ascaris suum cDNA similar to C. elegans mrp-1 Parastrongyloides trichosuri cDNA similar to C. elegans mdt-18 n/a Globodera pallida cDNA n/a Necator americanus cDNA similar to C. elegans anc-1 A. caninum cDNA similar to H. contortus extracellular SOD (Cu-Zn) precursor Brugia malayi cDNA A. suum cDNA n/a n/a

EMBL:CW699514 EMBL:CB016244

81 97

88 97

1.7e13 4.2e86

EMBL:BQ667338 EMBL:BM279145 EMBL:CL660478 EMBL:BM077715 EMBL:BQ666776

85 83 51 68 74

89 88 72 82 87

1.9e39 8.2e24 1.5 4.0e26 5.8e47

EMBL:CK984877

48

55

0.0097

EMBL:CB037140 EMBL:CB013947 EMBL:BM513244

68 46 68

84 66 86

3.2e82 4.0e37 2.5e51

EMBL:BM415502

34

50

4.2

EMBL:BU87981 EMBL:BF250403

69 74

76 87

1.3e27 7.5e39

EMBL:AI856874 EMBL:BU606069

30 32

47 61

0.00012 2.7

EMBL:CB189921 EMBL:BQ090615

77 32

87 54

4.9e29 4.3

EMBL:AI436816

70

75

0.0039

EMBL:CW698256 EMBL:BI502329

58 44

76 58

6.7 4.9

EMBL:CB175662 EMBL:CG755720 EMBL:AI087660 EMBL:CB175491

68 40 45 61

74 61 65 73

2.3e18 5.5 4.0 4.7e09

EMBL:AI261187 EMBL:BM033581

34 53

67 80

1.8 0.91

EMBL:BU86980 EMBL:CA304096 EMBL:BI48470

85 57 54

90 73 70

9.6e37 6.7e65 6.0e05

EMBL:BQ625687

57

70

1.3e37

L3ni L3ni L3ni L3ni L3ni L3ni L3ni L3ni L3ni L3ni L3ni L3ni L3ni L3ni L3ni L3ni

17 18 19 20 22a 28 32 41 51a 54 56a 57 63 75 67a 69/92a

L3ni L3ni L3ni L3ni

80 83 91 103

L3i 2/10a L3i 8 L3i 14 L3i 18/86/94/ 99a L3i 19 L3i 25 L3i 26 L3i 29 L3i 33a L3i 46 L3i 51 L3i 53a L3i 64 L3i 68 L3i 69 L3i 71 L3i 75a L3i 82/101a L3i 88a L3i 90 L3i 100a a

Ancylostoma ceylanicum cDNA similar to C. elegans F45D3.2 Strongyloides ratti cDNA n/a Onchocerca volvolus cDNA n/a A. caninum genomic DNA S. ratti cDNA n/a A. ceylanicum cDNA P. pacificus genomic DNA B. malayi cDNA A. ceylanicum cDNA similar to C. elegans probable glycerol kinase n/a Onchocerca volvolus cDNA A. suum cDNA n/a N. americanus cDNA similar to C. elegans Y73B6BL.4 A. suum cDNA similar to C. elegans N-methyltransferase Heterodera glycines cDNA n/a Ostertagia ostertagi cDNA similar to C. elegans E02A10.3

Sequences selected for further characterisation.

3.5. Quantitative real-time PCR Two differentially expressed genes found by performing SSH and differential screening were exemplarily validated using five biological replicates as template. In this, NMT and SOD represented genes found to be specifically upregulated in the hypobiosis-induced and non-induced larvae, respectively. Relative to EF-1a as an internal standard, there was a mean of 1176 NMT copies per 1000 EF-1a copies (SD = 0.724 and SEM = 0.296) in the hypobiosisinduced larvae in contrast to 45 NMT copies per 1000

EF-1a copies (SD = 0.016, SEM = 0.006) in the non-induced larvae. This result corresponds to a statistically significant 26.13-fold upregulation (P = < 0.001) of the NMT expression in the hypobiosis-induced larvae and confirms the results of SSH and differential screening. In the case of SOD, which was clearly found to be upregulated in the non-induced larvae, qPCR revealed only minor differences between the two larval populations: 136 SOD copies per 1000 EF-1a copies (SD = 0.104, SEM = 0.046) were detected in the non-induced larvae compared to 115 SOD-copies (SD = 0.098, SEM = 0.040) in the hypobiosis-induced

Table 2 Similarities of deduced amino acid sequences of differentially expressed full-length Dictyocaulus viviparus sequences to GenBank proteins and expressed sequence tags (ESTs) of human and animal parasites in Nematode Net Organism

Gene code/description of the homologue

Partition of similarity

Identity/Positives %

AY552027

L3ni 22

AY533126

L3ni 51

AY533127

L3ni 56aa

AY533128

L3ni 56ba

AY533129

L3ni 56ca

AY533130

L3ni 56da

AY533131

L3ni 56ea

AY533132

L3ni 67

AY533133

L3ni 69/92

AY533134

L3i 2/10aa/ L3i 2/10ba

AY536871

L3i 33aa/ L3i 33ba

AY542514

L3i 53

AY570697

L3i 75aa

AY534336

Ancylostoma caninum Caenorhabditis elegans Strongyloides stercoralis C. elegans A. caninum Ostertagia ostertagi C. elegans A. ceylanicum O. ostertagi C. elegans Parastrongyloides trichosuri O. ostertagi C. elegans O. ostertagi P. trichosuri C. elegans O. ostertagi P. trichosuri C. elegans O. ostertagi P. trichosuri C. elegans O. ostertagi P. trichosuri C. elegans Ascaris suum Brugia malayi Haemonchus contortus C. elegans A. caninum C. elegans O. ostertagi Ancylostoma ceylanicum C. elegans A. ceylanicum S. stercoralis C. elegans A. ceylanicum O. ostertagi C. elegans

877 873 218 202 182 158 286 210 183 215 212 152 182 152 179 157 152 154 127 132 124 126 131 130 213 170 99 171 156 149 200 134 83 189 129 113 515 212 210 503

aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa

98/98 94/97 88/95 61/76 75/82 74/85 67/78 66/81 67/83 57/72 66/82 91/94 48/65 91/94 56/76 52/68 91/94 64/81 55/69 91/94 62/79 54/69 91/94 60/76 56/69 51/70 66/77 68/80 62/81 64/77 37/52 77/85 77/87 40/50 67/74 38/53 67/80 76/87 71/83 54/72

AY534337

A. ceylanicum S. stercoralis C. elegans

paramyosin F07A5.7/paramyosin kq40g02.y1 Y53H1B.2/unnamed protein pb47d10.y1 ph04d12.y2 Y73F8A.6/CGG-1 pl03e02.y1 ph29g03.y2 C55B7.9/MDT-18 kx74h05.y1 ph24b07.y2 C55B7.9/MDT-18 ph24b07.y2 kx74h05.y1 C55B7.9/MDT-18 ph24b07.y2 kx74h05.y1 C55B7.9/MDT-18 ph24b07.y2 kx74h05.y1 C55B7.9/MDT-18 ph24b07.y2 kx74h05.y1 ZK973.6/ANC-1 kh20g07.y1 kb57g09.y1 extracellular SOD F55H2.1/SOD-4 pb08g11.y1 F45D3.2 ph03b02.y2 pk87b01.y1 F53H2.3 pk75a12.y1 kq26a06.y1 R11F4.1/glycerol kinase pk95f04.y1 ph30g12.y2 Y73B6BL.4/intracell. membrane-bound Ca2+-iPL A2 pk86d08.y1 kp03f12.y1 Y73B6BL.4/intracell. membrane-bound Ca2+-iPL A2 pk86d08.y1

216 aa 96 aa 393 aa

57/73 31/53 55/75

216 aa

57/73 (continued on next page)

L3i 75ba

A. ceylanicum

227

Accession number

L3ni 16

C. Strube et al. / International Journal for Parasitology 37 (2007) 221–231

Sequence

228

Table 2 (continued) Accession number

Organism

Gene code/description of the homologue

Partition of similarity

Identity/Positives %

L3i 82aa/ L3i 101

AY542519 AY542518

S. stercoralis C. elegans

kp03f12.y1 T07C12.9/N-methyltransferase (PNMT/NNMT/TEMT) kj85e02.y1 T07C12.9/N-methyltransferase (PNMT/NNMT/TEMT) kj85e02.y1 T07C12.9/N-methyltransferase (PNMT/NNMT/TEMT) kj85e02.y1 T07C12.9/N-methyltransferase (PNMT/NNMT/TEMT) kj85e02.y1 M04G12.3/GCY-34 kl36a07.y1 pl11f01.y1 M04G12.3/GCY-34 kl36a07.y1 pl11f01.y1 M04G12.3/GCY-34 kl36a07.y1 pl11f01.y1 M04G12.3/GCY-34 kl36a07.y1 pl11f01.y1 C46E1.2/GCY-36 kl36a07.y1 pl11f01.y1 F13G11.1/TAG-193 pl15a04.y1 kj91b08.y1 E02A10.3/calmodulin and related proteins ph91c06.y1 kq39d10.y1 E02A10.3/calmodulin and related proteins ph91c06.y1 kx47d11.y1 E02A10.3/calmodulin and related proteins ph91c06.y1 kq39d10.y1

96 aa 254 aa

31/53 61/74

L3i 82ba/ L3i 82da

AY542520 AY542522

123 aa 190 aa

59/73 61/72

L3i 82ca

AY542521

A. suum C. elegans

123 aa 185 aa

59/73 61/72

L3i 82ea

AY542523

A. suum C. elegans

191 aa 152 aa

59/73 61/72

L3i 88aa

AY536870

158 190 52 74 146 52 74 135 52 74 121 52 74 83 52 74 135 126 107 181 195 139 180 177 61 181 160 136

59/73 51/66 48/73 39/63 54/69 48/73 39/63 49/65 48/73 39/63 52/67 48/73 39/63 48/71 48/73 39/63 47/61 32/67 33/42 82/90 90/93 39/57 82/90 90/93 61/78 61/69 57/70 39/57

L3i 88ba

AY536869

L3i 88ca

AY536867

L3i 88da

AY536868

L3i 88-2

AY536866

L3i 94/99

AY534891

L3i 100aa

AY542515

L3i 100ba

AY542516

L3i 100ca

AY542517

A. suum C. elegans

A. suum C. elegans A. suum A. ceylanicum C. elegans A. suum A. ceylanicum C. elegans A. suum A. ceylanicum C. elegans A. suum A. ceylanicum C. elegans A. suum A. ceylanicum C. elegans A. ceylanicum A. suum C. elegans O. ostertagia S. stercoralis C. elegans O. ostertagia P. trichosuri C. elegans O. ostertagia S. stercoralis

aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa

aa, amino acid residues; Ca2+-iPLA2, Ca2+-independent phospholipase A2; CCG, conserved cysteine/glycine domain protein family member; GCY, guanylyl cyclase; MDT, mediator family member; NNMT/PNMT/TEMT, nicotinamide N-methyltransferase/phenylethanolamine N-methyltransferase/thioether S-methyltransferase; SOD, superoxide dismutase; TAG, temporarily assigned gene name family member. a Sequences named with letters represent isoforms of the corresponding gene.

C. Strube et al. / International Journal for Parasitology 37 (2007) 221–231

Sequence

C. Strube et al. / International Journal for Parasitology 37 (2007) 221–231

229

Table 3 Gene function of differentially expressed Dictyocaulus viviparus transcripts based on Goblet gene ontology search Transcript

Molecular function L3ni 16 L3ni 67

Actin binding structural molecule activity

L3ni 69/92

L3i 82/101 L3i 88 L3i 94/99

Metal ion binding copper/zinc superoxide dismutase activity Serine-type endopetidase inhibitor activity Glycerol kinase activity ATP-binding Ca2+-independent phospholipase A2 activity ATP-binding nutrient reservoir activity Methyltransferases activity Guanylate cyclase activity DNA binding transcription factor activity

L3i 100

Calcium ion binding

L3i 2/10 L3i 53 L3i 75

a

E-valuea

Gene ontology Biological process

Cellular component

Striated muscle contraction muscle development

Myosin striated muscle thick filament Cytoskeleton integral to membrane

0.0 8e36

Superoxide metabolism

4e56

Glycerol-3-phosphate metabolism Fatty acid metabolism

3e30 0.0 6e69

Intracellular signaling cascade Regulation of transcription, DNA-dependent sex differentiation

Intracellular membrane fraction peroxisomal membrane

4e87 7e40 2e21

Nucleus

3e80

The E-value refers to the corresponding Goblet top hit.

Table 4 Significant alignments of differentially expressed Dictyocaulus viviparus sequences to conserved domains Sequence

Conserved domain

Accession number

Length

Aligned residues

L3ni 16 L3ni 69/92 L3i 53

Myosin-tail Copper/zinc superoxide dismutase FGGY family of carbohydrate kinases, N-terminal domain FGGY family of carbohydrate kinases, C-terminal domain Glycerol-kinase Sugar (pentulose and hexulose) kinases Ribulose-kinase Patatin, Patatin-like phospholipase NNMT/PNMT/TEMT family EF-hand, calcium-binding motif EF-hand, calcium-binding motif Eps15 homology domain FRQ1, Ca2+-binding protein EF-hand, calcium-binding motif EF-hand, calcium-binding motif Eps15 homology domain FRQ1, Ca2+-binding protein EF-hand, calcium-binding motif EF hand FRQ1, Ca2+-binding protein

pfam01576 pfam00080 pfam00370 pfam02782 COG0554 COG1070 COG1069 pfam01734 pfam01234 cd00051 cd00051 cd00052 COG5126 cd00051 cd00051 cd00052 COG5126 cd00051 pfam0036 COG5126

860 152 245 224 499 502 544 179 261 63 63 67 160 63 63 67 160 63 29 160

95.5 96.1 98.8 100 98.8 95.0 94.1 98.9 98.9 100 98.4 100 91.2 100 98.4 100 91.2 90.5 93.1 91.2

L3i 75a/75b L3i 82a/101 L3i 100a

L3i 100b

L3i 100c

aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa aa

The table shows only alignments of >90 aligned residues sorted by E-value. aa, amino acid residues; EF-hand, calcium binding motif named arbitrarily after the E and F helices of parvalbumin; Eps15, epidermal growth factor receptor pathway substrate 15; FGGY, amino acid domain; FRQ1, yeast frequenin 1; NNMT/PNMT/TEMT, nicotinamide N-methyltransferase/ phenylethanolamine N-methyltransferase/thioether S-methyltransferase.

larvae. These copy numbers correspond to a 1.18-fold upregulation in the non-induced larvae, which is not statistically significant (P = 0.543). 4. Discussion The genetic regulation of hypobiosis in parasitic nematodes is still unknown. To identify genes differentially expressed in hypobiosis-induced and non-induced L3 of the bovine lungworm D. viviparus, SSH using cDNA of

the mentioned larval populations was performed. Following creation of subtracted libraries, and differential screening, further cDNA hybridisation was used to confirm differentially expressed insert sequences due to the fact that subtracted libraries always contained false positive clones. Since a 4-fold higher signal intensity using the corresponding tester cDNA relative to the driver cDNA was postulated as the threshold for differential expression, there was a comparatively high number of background clones in both subtracted libraries (75.2% false positives in the L3i-sub-

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tracted library and 78.8% in the L3ni-subtracted library). This observation is most likely based on limited differences between the cDNAs of the two larval populations of the same larval stage (Stubbs et al., 1999; Rebrikov et al., 2000). All differentially expressed transcripts were unknown gene sequences of the bovine lungworm. Generation of full-length sequences resulted in two or more isoforms of seven differentially expressed transcripts. These isoforms characterised by differences in the 5 0 -region, are most likely due to alternative splicing. Clones containing inserts encoding N-methyltransferase, a protein with serine-type endopeptidase inhibitor activity, a transcription factor, and SOD were found more than once in the subtracted libraries. This may be a reconfirmation of their differential expression and may give a hint that these are genes with high differences in their expression levels. qPCR confirmed this suggestion for the N-methyltransferase showing a 26.13-fold upregulation in the hypobiosis-induced larvae. In contrast, only a 1.18-fold upregulation was observed for the SOD in the corresponding non-induced larval population. It is unknown whether these minor changes in the SOD gene expression represent an unrecognised upregulation of the housekeeping gene EF1a in the L3i, poor performance of the qPCR, or whether they represent the real SOD expression pattern between L3i and L3ni. In the latter case, it remains unclear whether the slight upregulation is of significance for superoxide metabolism. As a result of the search for sequence identities to parasite ESTs, 20 of 48 differentially expressed inserts of the subtracted libraries showed deduced amino acid identities with an E-value <0.0001. From the clones not matching this threshold, eight sequences were found to have nucleic acid identities with an E-value <0.0001. The 20 remaining gene fragments very likely represent novel genes or 5 0 and 3 0 untranslated regions. The assumption of a novel gene is confirmed for one clone (L3ni 75) showing no identity at all currently available nematode ESTs. A poly(A)+-tail was found in three clones (L3ni 80, L3i 51 and L3i 69), indicating that these gene fragments represent 3 0 ends. Unlike all other clones (with the exception of L3ni 75), there were no sequence identities between ESTs of clade V parasites to the sequence L3i 82/101, neither nucleic acids nor deduced amino acids. This was unexpected as D. viviparus is grouped in clade V. This clade represents, amongst others, animal parasitic strongylids and Caenorhabditis elegans (Blaxter et al., 1998; Blaxter, 1998). As a consequence, NCBI BLAST searches for identities to the differentially expressed lungworm sequences resulted, in the majority of cases, in homologues of C. elegans proteins (considering relevant information available in WormBase). In combination with GO and conserved domain searches, information concerning protein function was obtained. For example, the transcript L3i 53 enriched in the hypobiosis-induced larvae was identified as glycerol kinase. This metabolic enzyme probably provides energy needed for survival since hypobiotic larvae

do not feed. They have to survive on their reserve granules where glycogen is the primary energy-storage molecule (Roberts and Fairbairn, 1965). Upregulated regulatory proteins involved in cellular processes and signaling cascades are N-methyltransferase (L3i 82/101), guanylyl cyclase (L3i 88), and a calmodulin/EF-hand (calcium binding motif named arbitrarily after the E and F helices of parvalbumin) protein (L3i 100). Further proteins found to be upregulated in hypobiosis-induced Dictyocaulus larvae are a calcium-independent phospholipase A2 (L3i 75) and a protein with DNA-dependent transcription-factor activity (L3i 94/99), which may regulate gene expression. Moreover, enriched transcripts of a protein with serine-type endopeptidase inhibitor activity (L3i 2/10) were observed in the hypobiosis-induced lungworm larvae. In the subtracted library of the non-induced larvae we found the gene L3ni 16, amongst others, to be upregulated. This gene encodes for paramyosin which assemblies with myosin into thick filaments of striated muscles of the body wall and pharyngeal muscles (Ardizzi and Epstein, 1987). Thus, paramyosin is required for locomotion and feeding. A further structural component of musculature is troponin, which is encoded by L3ni 17. Another gene found to be enriched in L3ni encodes an extracellular copper/zinc superoxide dismutase (L3ni 69/92), which detoxifies reactive oxygen species such as superoxide. The two main sources of these reactive oxygen species are mitochondrial respiration and activated phagocytes (Morel et al., 1991; Gems, 1999). SOD is then needed to metabolise free radicals released from activated phagocytes in the course of the oxidative burst and therefore SOD is designated as an immune defense protein playing an important role for parasite survival in the host (Callahan et al., 1990; Knox and Jones, 1992; Brophy et al., 1995). Possibly the differential SOD expression between the L3i and L3ni mirrors differences in the immune reaction of the bovine host towards hypobiotic and non-hypobiotic larvae in the lungs. While hypobiotic larvae persist in the lungs of infected cattle for months without any cellular infiltration, there is a strong cellular reaction around uninhibited larvae (Schnieder et al., 1991). The structural protein L3ni 67 interacts selectively with actin and contributes to the integrity of a complex or assembly within or outside a cell. The full-length sequences L3ni 22, 51, 56 as well as L3i 33 are not assigned to a GO term and conserved domain. Thus, the function of these genes as well as the differentially expressed gene fragments (with the exception of L3ni 17) not selected for further characterisation remains hypothetical or unclear. This is the first description of genes found to be differentially expressed in the bovine lungworm D. viviparus. However, further investigation, for instance with hypobiotic L4, is necessary to determine if the expressed proteins play a specific role in the process of hypobiosis.

C. Strube et al. / International Journal for Parasitology 37 (2007) 221–231

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