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De novo transcriptome sequencing and analysis of Anisakis pegreffii (Nematoda: Anisakidae) third-stage and fourth stage larvae

U-Hwa Nam
U-Hwa Nam
, 
Jong-Oh Kim
Jong-Oh Kim
 and 
Jeong-Ho Kim
Jeong-Ho Kim
   | Apr 16, 2020
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Published Online: Apr 16, 2020
Page range: 1 - 16
Received: Nov 26, 2019
DOI: https://doi.org/10.21307/jofnem-2020-041
Keywords
© 2020 U-Hwa Nam et al., published by Sciendo.
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Figure 1:

GO analysis of DEGs in A. pegreffii transcriptomes (A. pegreffii L3 vs A. pegreffii L4).
GO analysis of DEGs in A. pegreffii transcriptomes (A. pegreffii L3 vs A. pegreffii L4).

Figure 2:

The expression level of selected DEGs either in APL3 or in APL4 by real-time PCR. A: the real-time PCR results of five genes with significantly higher expression level in APL3 compared to APL4; B: the real-time PCR results of five genes with significantly higher expression level in APL4 compared to APL3; C and D are 14 additional genes selected to compare the expression levels of ASL3 and APL4 (C: eight gene for APL3 compared to APL4, D: six gene for APL4 compared to APL3). GAPDH genes were used as the reference genes. The results are given as mean of samples (n = 3) and expressed as fold change in mRNA expression. *P < 0.05.
The expression level of selected DEGs either in APL3 or in APL4 by real-time PCR. A: the real-time PCR results of five genes with significantly higher expression level in APL3 compared to APL4; B: the real-time PCR results of five genes with significantly higher expression level in APL4 compared to APL3; C and D are 14 additional genes selected to compare the expression levels of ASL3 and APL4 (C: eight gene for APL3 compared to APL4, D: six gene for APL4 compared to APL3). GAPDH genes were used as the reference genes. The results are given as mean of samples (n = 3) and expressed as fold change in mRNA expression. *P < 0.05.

Top 20 most differentially expressed genes in A. pegreffi L3.

No. Name Description Log FC P-value Q-value
1 ergic2 Endoplasmic reticulum-Golgi intermediate compartment protein 2 16.8 0.0001179 0.247384
2 mgat4b Alpha-1,3-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase B 16.7 0.0001274 0.247384
3 Acin1 Apoptotic chromatin condensation inducer in the nucleus 16.3 0.0002229 0.255429
4 SFXN2 Sideroflexin-2 16.1 0.0003069 0.265269
5 ANKHD1 Ankyrin repeat and KH domain-containing protein 1 16.1 0.0002901 0.265269
6 RALGAPA1 Ral GTPase-activating protein subunit alpha-1 15.9 0.0003816 0.278843
7 SI Sucrase-isomaltase, intestinal 15.9 0.0004035 0.278843
8 B0361.11 Putative transporter B0361.11 15.9 0.0004042 0.278843
9 lact-2 Beta-lactamase domain-containing protein 2 15.8 0.0004392 0.291452
10 sptl-2 Serine palmitoyltransferase 2 15.8 0.0004306 0.291452
11 hyd E3 ubiquitin-protein ligase hyd 15.7 0.0005016 0.292532
12 Macf1 Microtubule-actin cross-linking factor 1 15.7 0.0005216 0.292532
13 ZK370.4 Uncharacterized NTE family protein ZK370.4 15.7 0.000554 0.292532
14 LOAG_00120 Hypothetical protein 15.7 0.0005268 0.292532
15 FZD10 Frizzled-10 15.6 0.0005786 0.292532
16 cdgs-1 Phosphatidate cytidylyltransferase 15.6 0.0005679 0.292532
17 Pcsk1 Neuroendocrine convertase 1 15.6 0.000601 0.292532
18 Bm1_49625 Hypothetical protein 15.6 0.0006287 0.292532
19 GLSN_MEDSA Glutamate synthase [NADH], amyloplastic 15.6 0.0006022 0.292532
20 Moe Moesin/ezrin/radixin homolog 1 15.6 0.0005762 0.292532

Gene expression overview of A. pegreffii L3 and L4 transcriptomes.

Gene (fpkm < 1.0)
Name Expressed Known Novel Unexpressed
APL3 47,243 18,753 28,490 3,030
APL4 43,660 18,996 24,664 6,613

Preprocessing statistics of Anisakis transcriptome.

Name Raw reads Clean reads
Reads Base pairs Reads (%) Base pairs (%)
APL3 57,831,158 4,395,168,008 53,790,942 (93.0%) 4,079,160,122 (92.8%)
APL4 61,963,202 4,709,203,352 56,726,910 (91.5%) 4,301,151,997 (91.3%)

Top 20 most abundant unigenes in A. pegreffii L4.

No. ID Name Description
1 TBIU007721 PI-3 Major pepsin inhibitor 3
2 TBIU033076 col-145 Putative cuticle collagen 145
3 TBIU014455 col-34 Cuticle collagen 34
4 TBIU001741 col-34 Cuticle collagen 34
5 TBIU018580 ART2 Putative uncharacterized protein ART2
6 TBIU006603 COIII Cytochrome c oxidase subunit 3
7 TBIU020343 col-138 Protein COL-138
8 TBIU020344 Bm1_54705 Nematode cuticle collagen N-terminal domain containing protein
9 TBIU013810 COI Cytochrome c oxidase subunit 1
10 TBIU000598 col-40 Cuticle collagen 40
11 TBIU008478 COII Cytochrome c oxidase subunit 2
12 TBIU003870 Y51F10.7 Protein Y51F10.7
13 TBIU015232 IXCI_RHIMP Chymotrypsin-elastase inhibitor ixodidin
14 TBIU017350 ZK970.7 Protein ZK970.7
15 TBIU001757 Bm1_23600 hypothetical protein
16 TBIU015233 IXCI_RHIMP Chymotrypsin-elastase inhibitor ixodidin
17 TBIU006107 col-182 Protein COL-182
18 TBIU029263 CRVP Venom allergen 3
19 TBIU004632 col-34 Cuticle collagen 34
20 TBIU030958 LGALS8 Galectin-8

Top 20 most abundant unigenes in A. pegreffii L3.

No. ID Name Description
1 TBIU006603 COIII Cytochrome c oxidase subunit 3
2 TBIU013810 COI Cytochrome c oxidase subunit 1
3 TBIU015558 UBB Polyubiquitin-B
4 TBIU017350 ZK970.7 Protein ZK970.7
5 TBIU017923 act-2b Actin-2
6 TBIU015560 UBB Polyubiquitin-B
7 TBIU017925 act-2b Actin-2
8 TBIU008478 COII Cytochrome c oxidase subunit 2
9 TBIU015561 UBIQP_XENLA Polyubiquitin
10 TBIU002664 UBIQP_XENLA Polyubiquitin
11 TBIU022916 HSP70 Heat shock 70 kDa protein
12 TBIU007173 NAP1L4 Nucleosome assembly protein 1-like 4
13 TBIU018580 ART2 Putative uncharacterized protein ART2
14 TBIU001952 R09H10.3 Probable 5-hydroxyisourate hydrolase R09H10.3
15 TBIU003351 PCCB Propionyl-CoA carboxylase beta chain, mitochondrial
16 TBIU022918 HSP70 Heat shock 70 kDa protein
17 TBIU030005 ASP2_ANISI Serine protease inhibitor 2
18 TBIU005085 Mmadhc Methylmalonic aciduria and homocystinuria type D homolog, mitochondrial
19 TBIU032617 CLPH_ONCVO Calponin homolog OV9M
20 TBIU030096 Mcee Methylmalonyl-CoA epimerase, mitochondrial

Primer information for real-time PCR analysis.

Stage Gene Primer sequences (5´ − 3´) Length (bp)
APL3 Acin1 F: 5´-TGAATGACGAAGAAGGGAGATGA-3´ 108
R: 5´-CAGTGTCAAATGAGAATAGCGTTTC-3´
SI F: 5´-GAGGCGATTGCTGGAAACAT-3´ 111
R: 5´-CTTCGTTGGTTCTTTTTGTCGTT-3´
lact-2 F: 5´-CACCCCACCATCCTCTCCTT-3´ 114
R: 5´-CCGAGTGTATCTGCGAGGAAA-3´
sptl-2 F: 5´-GGCAACCAAGAACGAGTGATG-3´ 108
R: 5´-TCAGCATGGGATTTGCAACA-3´
Macf1 F: 5´-ATGCTTGCTCCAGTTCTCCTAAA-3´ 125
R: 5´-ATGGACAGGACGCTCTTCTTG-3´
ergic2 F: 5´-CTGTTGAGTTGATGGCTGGAAA-3´ 106
R: 5´-ATTTTGGCACCACTGCTTTTATTAG-3´
mgat4b F: 5´-AATGCCAGCGATTGAAGAGTTT-3´ 115
R: 5´-CGCTGGTGTACGCAAATCATA-3´
B036.11 F: 5´-TAATGATTGCTGAATGCGTCTGT-3´ 120
R: 5´-ACACCGAAAGATAACAACGAATACG-3´
nas-13 F: 5´-AGCAATAGCAGCAGCGATGA-3´ 131
R: 5´-GTAGCCAATGCTTTT-3´
EF-TSMT F: 5´-TTCTTCTACTCCTCATCGCATCTG-3´ 100
R: 5´-TTATGCTCCTCCAGTGCTTTTG-3´
SFXN2 F: 5´-TTAGAATGGCGTTGAAGCAGTAGTAG-3´ 130
R: 5´-AGTATCGGTTCTGACCAGTTTTTTG-3´
hyd F: 5´-CCATCCAGTGAAGAAGGATTCC-3´ 116
R: 5´-GAATAGAGCGGTAAGTAGAGCCTTGA-3´
dhs-27 F: 5´-ACGATGAACGATTCGGGTAAGT-3´ 140
R: 5´-GCGTTATCCCCACATTTTCAA-3´
APL4 vps-11 F: 5´-CTCAACACACCACAATACTCATCAAT 105
R: 5´-CAGCAACATCAACATCACAACTCA
EGF1 F: 5´-CACATCAGCCCAGAAACATACG 106
R: 5´-ATTTAGACCGTCGCCAGGAA
CLINT1 F: 5´-AACGCAAGAATAGGCAAACACA-3´ 113
R: 5´-AGGAGCATCAACAGAAAACAATGAG-3´
let-805 F: 5´-CACCGAGCACCGCTATCAA-3´ 120
R: 5´-GAAGGAGGCATCAAGGAAAGTG-3´
VIT F: 5´-AGGCTTATCATTACTTCGTTCACTCA-3´ 105
R: 5´-GTTTGGTTGTCGGTTCAGGTGTA-3´
F09E10.7 F: 5´-CCACGCTCAAAAGCCCATT-3´ 116
R: 5´-GGACCAGCGGAAGTTCAGAA-3´
ATP8B4 F: 5´-CGACACATTCTTCGTCAGCATT-3´ 135
R: 5´-CCGAACCCAAGCATGAAAA-3´
pept-1 F: 5´-TGCTCAAGGAGAAGGAAGTTTACA-3´ 113
R: 5´-TACCCCACTTCACCGATTCG-3´
col-34 F: 5´-GCCGAGTAAGCCACAAAACG-3´ 121
R: 5´-TACACCCCGTCCACCTTCA-3´
col-40 F: TTCAGCAGGTGGGCAGTCA 98
R: ATGGAACTCCGGGTGAGGAT
SAM-MT F: 5´-CCATCAAGGCGGCGTTAC-3´ 93
R: 5´-CAGCATGCCATAGATCCAGTGT-3´

Top 20 most differentially expressed genes in A. pegreffi L4.

No Name Description Log FC p-value q-value
1 F09E10.7 Protein F09E10.7 20.7 0.0000115 0.171197
2 ATP8B4 Probable phospholipid-transporting ATPase IM 18.7 0.0000172 0.171197
3 pept-1 Peptide transporter family 1 18.5 0.0000214 0.171197
4 vps-11 Vacuolar protein sorting-associated protein 11 homolog 18.5 0.0000214 0.171197
5 col-34 Cuticle collagen 34 18.4 0.0000229 0.171197
6 EGF1 Fibropellin-1 17.5 0.000063 0.206261
7 CLINT1 Clathrin interactor 1 17.4 0.0000751 0.218706
8 let-805 Protein LET-805 17.2 0.0000989 0.247384
9 Bm1_33530 Hypothetical protein 17 0.0001257 0.247384
10 VIT Vitrin 17 0.0001194 0.247384
11 col-40 Cuticle collagen 40 16.9 0.0001524 0.255429
12 VIT Vitrin 16.7 0.0002081 0.255429
13 unc-104 Kinesin-like protein unc-104 16.7 0.0001838 0.255429
14 SAM-MT Probable fatty acid methyltransferase 16.7 0.000202 0.255429
15 sulp-3 Sulfate permease family protein 3 16.6 0.0002432 0.258155
16 CLINT1 Clathrin interactor 1 16.6 0.0002241 0.255429
17 Oplah 5-oxoprolinase 16.5 0.0002634 0.265269
18 VIT Vitrin 16.4 0.0003171 0.265269
19 LOAG_09410 Hypothetical protein 16.4 0.0003238 0.265269
20 ZAN Zonadhesin 16.4 0.0003204 0.265269
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