Accepted Manuscript Title: Identification of putative genes involved in parasitism in the anchor worm, Lernaea cyprinacea by de novo Transcriptome analysis. Author: Pallavi B., Shankar K.M., Abhiman P.B., Iqlas Ahmed PII: DOI: Reference:
S0014-4894(15)00076-4 http://dx.doi.org/doi:10.1016/j.exppara.2015.03.014 YEXPR 7020
To appear in:
Experimental Parasitology
Received date: Revised date: Accepted date:
31-7-2014 19-3-2015 20-3-2015
Please cite this article as: Pallavi B., Shankar K.M., Abhiman P.B., Iqlas Ahmed, Identification of putative genes involved in parasitism in the anchor worm, Lernaea cyprinacea by de novo Transcriptome analysis., Experimental Parasitology (2015), http://dx.doi.org/doi:10.1016/j.exppara.2015.03.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 1
Identification of putative genes involved in parasitism in the anchor worm, Lernaea
2
cyprinacea by de novo Transcriptome analysis.
3
Pallavi B1,2, Shankar KM2*, Abhiman PB1,2 & Iqlas Ahmed1,2
4
1
5
Mangalore 575002, India.
6
2
7
Sciences University
8
*Corresponding author: Dean, College of Fisheries Mangalore, Tel: +91-824-2248936;
9
Fax: +91-824-2248366
Aquatic Animal Health Laboratory, Department of Aquaculture, College of Fisheries,
Department of Aquaculture,College of Fisheries,Karnataka Veterinary, Animal and Fisheries
10
E-mail:[email protected]
11
Highlights:
12 13 14 15 16
De novo transcriptome sequencing of adult and free living stages of L. cyprinacea.
36,054 unigenes found defined annotations in the pfam database.
Differences in transcription between free-living and parasitic stages established.
17
This would shorten route towards vaccine/control strategies against the parasite.
18 19
Graphical Abstract
20
Page 1 of 18
2 21
ABSTRACT
22
There is little information on the genome sequence of Lernaea cyprinacea a major
23
ectoparasite of freshwater fish throughout the world. We subjected the L. cyprinacea
24
transcriptome (adult and free living stages) to Illumina HiSeq 2000 sequencing. We obtained
25
a total of 31671751 (31.67 millions) reads for the adult parasitic stage and 33840446 (33.84
26
millions) for the free living stage. The reads were assembled into 50,792 contigs for the adult
27
stage and 69,378 for the free living stage. Using the pfam database, 41.91 % of the
28
transcriptome was annotated. The transcriptome was mined for genes associated with
29
parasitism. To examine gene expression changes associated with the parasitism of
30
L. cyprinacea during the transit from the free living to parasitic stage, we studied the
31
differentially expressed transcripts between the two stages. The microsatellite markers were
32
also identified (9,843 for adult stage; 16,813 for free living stages) and this would facilitate
33
population genetic studies in various geographical isolates of Lernaea. Our data provides the
34
most comprehensive sequence resource available for L. cyprinacea and demonstrates that
35
Illumina sequencing allows de novo transcriptome assembly and gene expression analysis in
36
a species lacking genome information. The data could open new avenues for a wide array of
37
genetic, evolutionary, biological, ecological, epidemiological studies, and a solid foundation
38
for the development of novel interventions against L. cyprinacea.
39
Keywords:
40
Lernaea cyprinacea; Transcriptome; Parasitism
41
1. Introduction
42
Lernaea infection is a major disease problem encountered in carp culture in the Indian
43
subcontinent and has been reported from Indian major carps Catla (Catla catla), Rohu (Labeo
44
rohita), Mrigal (Cirrhinus mrigala), exotic carps silver carp (Hypophthalmichthys molitrix),
Page 2 of 18
3 45
grass carp (Ctenopharyngodon idella) and indigenous carps Labeo fimbriatus (Nandeesha et
46
al., 1984, 1985; Tamuli and Shanbhouge, 1996; Zafar et al., 2001). The three Indian Major
47
Carps viz., Catla, Rohu and Mrigal, contribute significantly to Indian aquaculture production
48
with an output of over two million tonnes (Kurva et al., 2013). Ectoparasitic diseases in
49
freshwater fish farms of India result in an annual loss of 300 crores INR due to disease-
50
induced mortality and impaired growth (Sahoo and Kar, 2012; Lakra et al., 2006). Lernaea
51
infections have been reported from Africa, Asia, Europe, North America (Hoffman, 1999)
52
and South America (Plaul et al., 2010). Lernaea cyprinacea, Linnaeus, 1758, is the only
53
cosmopolitan species in the genus Lernaea (Piasecki et al., 2004).
54
The life cycle of L. cyprinacea is composed of nine stages; following three free living
55
naupliar stages are five copepodite stages and one adult stage. The parasite feeds on fish
56
mucus, blood and tissue debris. Invasion destroys scales, skin and muscles of fish. Heavy
57
parasitosis can be the cause of mass death of fish and secondary bacterial or fungal infections
58
(Bednarska et al., 2009).
59
Unfortunately, the only effective treatment against Lernaea spp is the application of
60
organophosphate and organochlorine pesticides. Their chemical stability, lipophilic nature
61
and toxicity, has led researchers to be concerned with their presence in the environment
62
(Amaraneni, 2002). The persistence of therapeutic agents in the aquatic environment causes
63
adverse effects on the ecosystem (Anon 1988, Choo 1994). Lernaea spp has been reported to
64
develop resistance to certain pesticides (Hoole et al., 2001; Sandra, 2004). Thus,
65
immunological protection of fishes against Lernaea spp infestation is presently the practically
66
sustainable alternative control method to the current use of pesticides that is riddled with
67
serious limitations. Owing to the complex nature of parasites, search for vaccine targets has
68
proven difficult. Currently no EST records are available for Lernaea cyprinacea in the
Page 3 of 18
4 69
National Center for Biotechnology Information (NCBI) database. Lack of genomic data has
70
hampered the use of molecular tools in developing control strategies for L. cyprinacea.
71
In order to predict and prioritize novel antigenic targets expressed across different
72
developmental stages of L. cyprinacea, we employed the Illumina sequencing and predictive
73
algorithms to explore similarities and differences in the transcriptomes of the free living and
74
adult parasitic stage of L. cyprinacea. This study involves the first de novo transcriptome
75
analysis of L. cyprinacea. Bioinformatic analyses of the transcriptomic data allowed a
76
detailed exploration of molecular changes associated with the transition from the free-living
77
to the parasitic stage and a prediction of the roles that key transcripts play in the metabolic
78
pathways linked to parasitism. Overall, this study provides the first insight into the molecular
79
biology of the important parasite L. cyprinacea.
80
2. Materials and Methods
81
2.1. Parasite Isolation
82
Catla heavily infected with L. cyprinacea were brought to the laboratory from the farm of the
83
Fisheries College, Mangalore, India. The L. cyprinacea was carefully pulled out with forceps
84
to avoid contamination with the host tissues, snap frozen in liquid nitrogen and stored in it
85
until used. Intact egg sacs were removed and incubated in flasks containing filtered tap water.
86
The resulting nauplii were maintained until a majority moulted to the first copepodite stage.
87
The free living stages were recovered onto 47-mm cellulose acetate filter membranes with a
88
pore size of 0.22 µm (Millipore). The membranes were flash frozen in liquid nitrogen and
89
stored in it till the next use (Sutherland et al., 2012). The phylogenetic status of the parasite
90
was also checked by partial sequencing of the rDNA of 18S and 28S regions (communicated
91
for publication elsewhere) and it was confirmed as L. cyprinacea.
92
2.2. RNA isolation
Page 4 of 18
5 93
The pooled adult (50) and free living stages (500) of the L. cyprinacea sample were
94
separately homogenized using TOMY Homogenizer with steel beads. Total RNA was
95
extracted with the TriZOL (Invitrogen) according to the manufacturer’s instructions.
96
2.3. Library preparation and sequencing
97
Transcriptome library for sequencing was constructed using TruSeq RNA sample preparation
98
kit according to the manufacturer’s instructions. Total RNA (1µg) was subjected to Poly A
99
purification of mRNA. Purified mRNA was fragmented for 4 min at elevated temperature
100
(94oC) in the presence of divalent cations and reverse transcribed with Superscript III
101
Reverse transcriptase (Invitrogen) by priming with random hexamers. Second strand cDNA
102
was synthesized in the presence of DNA polymerase I and RnaseH. The cDNA was cleaned
103
up using Agencourt Ampure XP SPRI beads (Beckman Coulter). Illumina Adapters were
104
ligated to the cDNA molecules after end repair and addition of A base. On completion of
105
ligation, SPRI was cleaned. The library was amplified using 11 cycles of PCR for enrichment
106
of adapter ligated fragments. The prepared library was quantified using a Nanodrop
107
spectrophotometer (Thermo Scientific, DE, USA) and validated for quality by running an
108
aliquot on a High Sensitivity Bioanalyzer Chip (Agilent). The prepared library was
109
sequenced on Illumina HiSeq 2000 (Illumina) to generate reads of 2x100 bp. The sequencing
110
reactions for the parasitic and free living stages were run at the same time to prevent
111
confounding error profiles with real differences in transcription.
112
2.4. De novo assembly and sequence clustering
113
Raw reads were processed using the perl script. Raw read processing step involved Adapter
114
trimming, B-block removal and low quality base filtering. Read Quality was assessed using
115
read quality check tool, SeqQC. Contamination at reads level was checked by aligning the
116
processed reads with the RNA sequences of the host fish using the Bowtie-0.12.8 tool and
117
reads aligning to the host fish were removed. The Velvet_1.2.10 tool (Zerbino and Birney,
Page 5 of 18
6 118
2008) was used for the de-novo assembly of high quality reads to get contigs. The
119
Oases_0.2.08 (Schulz et al., 2012) tool was used for transcript generation from de novo
120
assembled contigs.
121
combined transcripts to form unigenes with minimum similarity cut-off of 95%.
122
2.5. Ontology and annotation
123
Assembled transcripts were mapped against UniProt and associated GO, pfam databases and
124
COG database using the ncbi-BLAST-2.2.28 tool (Altschul et al., 1990).
125
2.6. SSRs identification
126
MISA tool was used for identification and localization of perfect microsatellites as well as
127
compound microsatellites, which are interrupted by a certain number of bases.
128
2.7. Differential Gene Expression Analysis
129
The transcripts from the adult and larval stages were combined and clustered using the CD
130
Hit tool with a minimum similarity cutoff of 95%. The reads were aligned using the Bowtie
131
version (0.12.8) tool and the read count profile generated. The Differential Gene Expression
132
analysis was carried out with the DESeq tool (Anders and Huber, 2010). The P-value given
133
by DESeq package was calculated based on significance test incorporated within DESeq.
134
Transcripts having corrected P-value as
S0014-4894(15)00076-4 http://dx.doi.org/doi:10.1016/j.exppara.2015.03.014 YEXPR 7020
To appear in:
Experimental Parasitology
Received date: Revised date: Accepted date:
31-7-2014 19-3-2015 20-3-2015
Please cite this article as: Pallavi B., Shankar K.M., Abhiman P.B., Iqlas Ahmed, Identification of putative genes involved in parasitism in the anchor worm, Lernaea cyprinacea by de novo Transcriptome analysis., Experimental Parasitology (2015), http://dx.doi.org/doi:10.1016/j.exppara.2015.03.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 1
Identification of putative genes involved in parasitism in the anchor worm, Lernaea
2
cyprinacea by de novo Transcriptome analysis.
3
Pallavi B1,2, Shankar KM2*, Abhiman PB1,2 & Iqlas Ahmed1,2
4
1
5
Mangalore 575002, India.
6
2
7
Sciences University
8
*Corresponding author: Dean, College of Fisheries Mangalore, Tel: +91-824-2248936;
9
Fax: +91-824-2248366
Aquatic Animal Health Laboratory, Department of Aquaculture, College of Fisheries,
Department of Aquaculture,College of Fisheries,Karnataka Veterinary, Animal and Fisheries
10
E-mail:[email protected]
11
Highlights:
12 13 14 15 16
De novo transcriptome sequencing of adult and free living stages of L. cyprinacea.
36,054 unigenes found defined annotations in the pfam database.
Differences in transcription between free-living and parasitic stages established.
17
This would shorten route towards vaccine/control strategies against the parasite.
18 19
Graphical Abstract
20
Page 1 of 18
2 21
ABSTRACT
22
There is little information on the genome sequence of Lernaea cyprinacea a major
23
ectoparasite of freshwater fish throughout the world. We subjected the L. cyprinacea
24
transcriptome (adult and free living stages) to Illumina HiSeq 2000 sequencing. We obtained
25
a total of 31671751 (31.67 millions) reads for the adult parasitic stage and 33840446 (33.84
26
millions) for the free living stage. The reads were assembled into 50,792 contigs for the adult
27
stage and 69,378 for the free living stage. Using the pfam database, 41.91 % of the
28
transcriptome was annotated. The transcriptome was mined for genes associated with
29
parasitism. To examine gene expression changes associated with the parasitism of
30
L. cyprinacea during the transit from the free living to parasitic stage, we studied the
31
differentially expressed transcripts between the two stages. The microsatellite markers were
32
also identified (9,843 for adult stage; 16,813 for free living stages) and this would facilitate
33
population genetic studies in various geographical isolates of Lernaea. Our data provides the
34
most comprehensive sequence resource available for L. cyprinacea and demonstrates that
35
Illumina sequencing allows de novo transcriptome assembly and gene expression analysis in
36
a species lacking genome information. The data could open new avenues for a wide array of
37
genetic, evolutionary, biological, ecological, epidemiological studies, and a solid foundation
38
for the development of novel interventions against L. cyprinacea.
39
Keywords:
40
Lernaea cyprinacea; Transcriptome; Parasitism
41
1. Introduction
42
Lernaea infection is a major disease problem encountered in carp culture in the Indian
43
subcontinent and has been reported from Indian major carps Catla (Catla catla), Rohu (Labeo
44
rohita), Mrigal (Cirrhinus mrigala), exotic carps silver carp (Hypophthalmichthys molitrix),
Page 2 of 18
3 45
grass carp (Ctenopharyngodon idella) and indigenous carps Labeo fimbriatus (Nandeesha et
46
al., 1984, 1985; Tamuli and Shanbhouge, 1996; Zafar et al., 2001). The three Indian Major
47
Carps viz., Catla, Rohu and Mrigal, contribute significantly to Indian aquaculture production
48
with an output of over two million tonnes (Kurva et al., 2013). Ectoparasitic diseases in
49
freshwater fish farms of India result in an annual loss of 300 crores INR due to disease-
50
induced mortality and impaired growth (Sahoo and Kar, 2012; Lakra et al., 2006). Lernaea
51
infections have been reported from Africa, Asia, Europe, North America (Hoffman, 1999)
52
and South America (Plaul et al., 2010). Lernaea cyprinacea, Linnaeus, 1758, is the only
53
cosmopolitan species in the genus Lernaea (Piasecki et al., 2004).
54
The life cycle of L. cyprinacea is composed of nine stages; following three free living
55
naupliar stages are five copepodite stages and one adult stage. The parasite feeds on fish
56
mucus, blood and tissue debris. Invasion destroys scales, skin and muscles of fish. Heavy
57
parasitosis can be the cause of mass death of fish and secondary bacterial or fungal infections
58
(Bednarska et al., 2009).
59
Unfortunately, the only effective treatment against Lernaea spp is the application of
60
organophosphate and organochlorine pesticides. Their chemical stability, lipophilic nature
61
and toxicity, has led researchers to be concerned with their presence in the environment
62
(Amaraneni, 2002). The persistence of therapeutic agents in the aquatic environment causes
63
adverse effects on the ecosystem (Anon 1988, Choo 1994). Lernaea spp has been reported to
64
develop resistance to certain pesticides (Hoole et al., 2001; Sandra, 2004). Thus,
65
immunological protection of fishes against Lernaea spp infestation is presently the practically
66
sustainable alternative control method to the current use of pesticides that is riddled with
67
serious limitations. Owing to the complex nature of parasites, search for vaccine targets has
68
proven difficult. Currently no EST records are available for Lernaea cyprinacea in the
Page 3 of 18
4 69
National Center for Biotechnology Information (NCBI) database. Lack of genomic data has
70
hampered the use of molecular tools in developing control strategies for L. cyprinacea.
71
In order to predict and prioritize novel antigenic targets expressed across different
72
developmental stages of L. cyprinacea, we employed the Illumina sequencing and predictive
73
algorithms to explore similarities and differences in the transcriptomes of the free living and
74
adult parasitic stage of L. cyprinacea. This study involves the first de novo transcriptome
75
analysis of L. cyprinacea. Bioinformatic analyses of the transcriptomic data allowed a
76
detailed exploration of molecular changes associated with the transition from the free-living
77
to the parasitic stage and a prediction of the roles that key transcripts play in the metabolic
78
pathways linked to parasitism. Overall, this study provides the first insight into the molecular
79
biology of the important parasite L. cyprinacea.
80
2. Materials and Methods
81
2.1. Parasite Isolation
82
Catla heavily infected with L. cyprinacea were brought to the laboratory from the farm of the
83
Fisheries College, Mangalore, India. The L. cyprinacea was carefully pulled out with forceps
84
to avoid contamination with the host tissues, snap frozen in liquid nitrogen and stored in it
85
until used. Intact egg sacs were removed and incubated in flasks containing filtered tap water.
86
The resulting nauplii were maintained until a majority moulted to the first copepodite stage.
87
The free living stages were recovered onto 47-mm cellulose acetate filter membranes with a
88
pore size of 0.22 µm (Millipore). The membranes were flash frozen in liquid nitrogen and
89
stored in it till the next use (Sutherland et al., 2012). The phylogenetic status of the parasite
90
was also checked by partial sequencing of the rDNA of 18S and 28S regions (communicated
91
for publication elsewhere) and it was confirmed as L. cyprinacea.
92
2.2. RNA isolation
Page 4 of 18
5 93
The pooled adult (50) and free living stages (500) of the L. cyprinacea sample were
94
separately homogenized using TOMY Homogenizer with steel beads. Total RNA was
95
extracted with the TriZOL (Invitrogen) according to the manufacturer’s instructions.
96
2.3. Library preparation and sequencing
97
Transcriptome library for sequencing was constructed using TruSeq RNA sample preparation
98
kit according to the manufacturer’s instructions. Total RNA (1µg) was subjected to Poly A
99
purification of mRNA. Purified mRNA was fragmented for 4 min at elevated temperature
100
(94oC) in the presence of divalent cations and reverse transcribed with Superscript III
101
Reverse transcriptase (Invitrogen) by priming with random hexamers. Second strand cDNA
102
was synthesized in the presence of DNA polymerase I and RnaseH. The cDNA was cleaned
103
up using Agencourt Ampure XP SPRI beads (Beckman Coulter). Illumina Adapters were
104
ligated to the cDNA molecules after end repair and addition of A base. On completion of
105
ligation, SPRI was cleaned. The library was amplified using 11 cycles of PCR for enrichment
106
of adapter ligated fragments. The prepared library was quantified using a Nanodrop
107
spectrophotometer (Thermo Scientific, DE, USA) and validated for quality by running an
108
aliquot on a High Sensitivity Bioanalyzer Chip (Agilent). The prepared library was
109
sequenced on Illumina HiSeq 2000 (Illumina) to generate reads of 2x100 bp. The sequencing
110
reactions for the parasitic and free living stages were run at the same time to prevent
111
confounding error profiles with real differences in transcription.
112
2.4. De novo assembly and sequence clustering
113
Raw reads were processed using the perl script. Raw read processing step involved Adapter
114
trimming, B-block removal and low quality base filtering. Read Quality was assessed using
115
read quality check tool, SeqQC. Contamination at reads level was checked by aligning the
116
processed reads with the RNA sequences of the host fish using the Bowtie-0.12.8 tool and
117
reads aligning to the host fish were removed. The Velvet_1.2.10 tool (Zerbino and Birney,
Page 5 of 18
6 118
2008) was used for the de-novo assembly of high quality reads to get contigs. The
119
Oases_0.2.08 (Schulz et al., 2012) tool was used for transcript generation from de novo
120
assembled contigs.
121
combined transcripts to form unigenes with minimum similarity cut-off of 95%.
122
2.5. Ontology and annotation
123
Assembled transcripts were mapped against UniProt and associated GO, pfam databases and
124
COG database using the ncbi-BLAST-2.2.28 tool (Altschul et al., 1990).
125
2.6. SSRs identification
126
MISA tool was used for identification and localization of perfect microsatellites as well as
127
compound microsatellites, which are interrupted by a certain number of bases.
128
2.7. Differential Gene Expression Analysis
129
The transcripts from the adult and larval stages were combined and clustered using the CD
130
Hit tool with a minimum similarity cutoff of 95%. The reads were aligned using the Bowtie
131
version (0.12.8) tool and the read count profile generated. The Differential Gene Expression
132
analysis was carried out with the DESeq tool (Anders and Huber, 2010). The P-value given
133
by DESeq package was calculated based on significance test incorporated within DESeq.
134
Transcripts having corrected P-value as
