Quantifying larvae of the coralivorous seastar Acanthaster cf. solaris using qPCR

Supplementary Information

Authors: Jason R Doyle*, A David McKinnon, Sven Uthicke

Australian Institute of Marine Science, PMB No. 3, Townsville MC, Qld, 4810, Australia

Keywords: Acanthaster planci, qPCR, larvae, plankton

*Corresponding author: Jason Doyle

Australian Institute of Marine Science, PMB No. 3, Townsville MC, Qld, 4810, Australia

Fax +61 7 47785852


Table S1. Details of primer sets designed using PrimerBlast(Ye et al. 2012) for the Acanthaster cf. solaris mitochondrial cytochrome oxidase subunit 1 gene. Parameters for primer design were; 1) non target sequences must have 6 or more mismatches to primer sequence including at least 3 mis-matches within 5 base pairs of the 3’ end of the primer, 2) primer length between 20 – 25 bp, 3) GC% between 40 – 80%, 4) Tm between 60 – 70 degrees and 5) amplicon length must be less than 150 bp

Primer set number / Amplicon length (bp) / Annealing temp (°C) / Forward/Reverse primer name / Sequence
1 / 132 / 60 / COTS-F-1321 / TCCGACTACCCGGACGCCTATAC
COTS-R-1452 / TCGAACAGTGGTTCGCTGGGAAG
2 / 78 / 60 / COTS-F-1369 / GGCAGCACCATATCCCTCGTAGC
COTS-R-1446 / AGTGGTTCGCTGGGAAGTGAAGG
3 / 84 / 60 / COTS-F-1369 / GGCAGCACCATATCCCTCGTAGC
COTS-R-1452 / TCGAACAGTGGTTCGCTGGGAAG
4 / 126 / 60 / COTS-F-1321 / TCCGACTACCCGGACGCCTATAC
COTS-R-1446 / AGTGGTTCGCTGGGAAGTGAAGG
5 / 71 / 60 / COTS-F-1321 / TCCGACTACCCGGACGCCTATAC
COTS-R-1391 / GCTACGAGGGATATGGTGCTGCC

Table S2. Tissue samples used to determine in-vitro specificity of primers. Labels in source column are, A = Uthicke et al (2015), B = Vogler et al (2008) and C = this study. The exact collection date of samples from source B is not known, nor is the tissue type. Genbank accession numbers refer to the mtCOI partial gene sequence with the exception of HE608395 which is a partial sequence of the mitochondrial control region. PC = pyloric caeca

Sample number / Source / Date / Class / GenBank No. / Species / Tissue / Location
3 / A / 21/2/14 / Asteroidea / KT271482 / Nardoanovaecaledoniae / skin / Pelorus Is.
5 / A / 20/2/14 / Asteroidea / KT271483 / Echinastersp. / skin / Picnic Bay
14 / A / 24/2/14 / Asteroidea / KT271484 / Cryptasterinasp. / skin / Orpheus Is.
18 / A / 26/2/14 / Asteroidea / KT271485 / Fromiaindica / skin / Rib Rf.
20 / A / 26/2/14 / Asteroidea / KT271486 / Echinastercallosus / skin / Rib Rf.
22 / A / 26/2/14 / Asteroidea / KT271487 / Echinastersp. / skin / Rib Rf.
28 / A / 26/2/14 / Asteroidea / KT271488 / Linckia multifora / skin / Rib Rf.
31 / A / 1/3/14 / Asteroidea / KT271489 / Choriastersp. / skin / Davies Rf.
32 / A / 16/3/14 / Asteroidea / KT271490 / Neoferdinacummingii / skin / Lizard Is.
33 / A / 16-/3/14 / Asteroidea / KT271491 / Fromiamonilis / skin / Lizard Is.
38 / A / 13/2/14 / Asteroidea / KT271492 / Linckia laevigata / PC / Davies Rf.
41 / A / 13/2/14 / Holothurioidea / KT271493 / Stichopuschloronotus / PC / Davies Rf.
59 / B / 1987-2008 / Asteroidea / HE608395# / Acanthaster planci / unknown / Mayotte (South Indian Ocean)
62 / B / 1987-2008 / Asteroidea / FM174564 / Acanthaster planci / unknown / Maldives (North Indian Ocean)
67 / B / 1987-2008 / Asteroidea / FM174654 / Acanthaster planci / unknown / Wajha Bank (Red Sea)
72 / A / 8/11/13 / Asteroidea / KT271494 / Acanthaster cf. solaris / gonads / Arlington Rf.
78 / A / 8/11/13 / Asteroidea / KT271498 / Acanthaster cf. solaris / gonads / Arlington Rf.
87 / C / 30/5/16 / Asteroidea / KX766031 / Acanthaster brevispinus / Tube feet / Trawler collection, east of Townsville

Table S3. Mitochondrial cytochrome oxidase subunit 1 (mtCOI) copy number and total DNA (ng) within pre-settlement CoTS life stages.

Total mtCOI copies per individual
Unfertilised egg (oocyte) / Fertilised egg / 2 cell stage / Gastrula / Early Bipinnaria / Bipinnaria / Early Brachiolaria / Competent Brachiolaria
rep 1 / 4888300 / 3705547 / 3198460 / 7036701 / 6176092 / 8401763 / 11947312 / 16048754
rep 2 / 5838151 / 2126193 / 5203152 / 5689472 / 3774027 / 5755634 / 18751621 / 18072466
rep 3 / 5632109 / 1609393 / 3240937 / 8831718 / 8471872 / 8338732 / 15809096 / 9480971
rep 4 / 6146490 / 2951565 / 5445964 / 4879080 / 3456978 / 8198645 / 12229294 / 18476682
rep 5 / 5490112 / 5728330 / 4272515 / 7761274 / 4990977 / 7334597 / 11727286 / 11547119
rep 6 / 4884946 / 4760295 / 5521615 / 8856486 / 9152863 / 8528845 / 14893898 / 17667524
rep 7 / 4254823 / 6384522 / 6263854 / 6643618 / 4241933 / 9899340 / 11488422 / 13250143
rep 8 / 3981966 / 4851690 / 5378909 / 6937508 / 7381468 / 10811290 / 10244969 / 9853393
Mean / 5139612 / 4014692 / 4815676 / 7079482 / 5955776 / 8408606 / 13386487 / 14299632
SD / 717475.9 / 1596357 / 1051809 / 1307611 / 2046502 / 1425237 / 2658972 / 3495541
CV% / 14 / 40 / 22 / 18 / 34 / 17 / 20 / 24
Total DNA per individual (ng)
Unfertilised egg (oocyte) / Fertilised egg / 2 cell stage / Gastrula / Early Bipinnaria / Bipinnaria / Early Brachiolaria / Competent Brachiolaria
rep 1 / 0.156 / 0.096 / 0.078 / 2.7 / 8.16 / 12.84 / 44.52 / 58.32
rep 2 / 0.138 / 0.09 / 0.078 / 2.64 / 6.3 / 15.24 / 59.76 / 55.2
rep 3 / 0.204 / 0.096 / 0.078 / 4.38 / 6.66 / 18.84 / 48.6 / 49.08
rep 4 / 0.156 / 0.084 / 0.078 / 2.04 / 7.56 / 13.32 / 64.8 / 39.12
rep 5 / 0.156 / 0.096 / 0.096 / 3.12 / 7.68 / 9.78 / 41.4 / 35.88
rep 6 / 0.252 / 0.09 / 0.072 / 2.52 / 7.92 / 13.44 / 45.96 / 39.84
rep 7 / 0.198 / 0.078 / 0.078 / 3 / 5.34 / 14.76 / 44.52 / 40.68
Mean / 0.18 / 0.09 / 0.079714 / 2.914286 / 7.088571 / 14.03143 / 49.93714 / 45.44571
SD / 0.036986 / 0.006414 / 0.006963 / 0.680228 / 0.946745 / 2.548498 / 8.164141 / 8.104276
CV% / 21 / 7 / 9 / 23 / 13 / 18 / 16 / 18

Table S4. Field collection details for samples used to in CoTS qPCR assay.

Station Name / Latitude (S) / Longitude (E) / Date / Location / Water Depth (m) / Volume water filtered (m3) / Total Plankton concentrate volume (ml) / Sub sampled volume (ml) / Sample lysis volume (ml) / Lysis sub-sample (ml) / DNA extraction volume (ul) / qPCR test volume (ul) / Concentration factor / Stock DNA concentr-ation (ng/ul) / DNA dilution tested / CoTS COI copies per m3
COT002 / 15.69583 / 145.729 / 18/12/2014 / S of Osterlund Rf. / 52 / 9.8 / 35 / 5 / 6 / 0.6 / 150 / 5 / 21000 / 39.3 / 10 / 6.42E+07
COT003 / 15.66383 / 145.6607 / 18/12/2014 / S of Osterlund Rf. / 40 / 7.4 / 36 / 4 / 6 / 0.6 / 150 / 5 / 27000 / 58.5 / 10 / 6.76E+07
COT004 / 15.625 / 145.595 / 18/12/2014 / S of Osterlund Rf. / 35 / 6.4 / 35 / 5 / 6 / 0.6 / 150 / 5 / 21000 / 24.7 / 10 / 6.25E+07
COT005 / 15.58667 / 145.5447 / 18/12/2014 / S of Osterlund Rf. / 35 / 6.4 / 40 / 5 / 6 / 0.6 / 150 / 5 / 24000 / 22.2 / 10 / 5.38E+07
COT006 / 15.5455 / 145.456 / 18/12/2014 / S of Osterlund Rf. / 32 / 5.8 / 45 / 5 / 6 / 0.6 / 150 / 5 / 27000 / 37 / 10 / 9.73E+07
COT012 / 15.6155 / 145.77 / 19/12/2014 / Irene Rf./Ribbon Rf. 1 / 48 / 9 / 40 / 7 / 6 / 0.6 / 150 / 5 / 17143 / 47.2 / 10 / 8.02E+07
COT013 / 15.58617 / 145.7993 / 19/12/2014 / Ribbon Rf. 1/2 / 44 / 8.2 / 25 / 6 / 6 / 0.6 / 150 / 5 / 12500 / 18.6 / 10 / 3.87E+06
COT014 / 15.57117 / 145.8838 / 19/12/2014 / Coral Sea / 1400* / 20 / 30 / 7.5 / 6 / 0.6 / 150 / 5 / 12000 / 15.8 / 10 / 0.00E+00
COT015 / 15.55583 / 145.97 / 19/12/2014 / Coral Sea / 2000* / 20 / 24 / 6 / 6 / 0.6 / 150 / 5 / 12000 / 15.7 / 10 / 0.00E+00
COT022 / 15.647 / 145.5763 / 20/12/2014 / E of Ribbon Rf. 1 / 38 / 7 / 40 / 5 / 6 / 0.6 / 150 / 5 / 24000 / 53.8 / 10 / 1.50E+07
COT023 / 15.74217 / 145.571 / 20/12/2014 / Cairns Rf./Endeavour Rf. / 31 / 5.6 / 50 / 7 / 6 / 0.6 / 150 / 5 / 21429 / 28.5 / 10 / 2.09E+07
COT024 / 15.8035 / 145.5847 / 20/12/2014 / Endeavour Rf. /Pickersgill Rf. / 33 / 6 / 35 / 5 / 6 / 0.6 / 150 / 5 / 21000 / 22.8 / 10 / 5.04E+07
COT025 / 15.84967 / 145.5883 / 20/12/2014 / N of Pickersgill Rf. / 34 / 6.2 / 36 / 5 / 6 / 0.6 / 150 / 5 / 21600 / 14.5 / 10 / 2.83E+06
COT026 / 15.90917 / 145.575 / 20/12/2014 / S of Pickersgill Rf. / 32 / 5.8 / 48 / 6 / 6 / 0.6 / 150 / 5 / 24000 / 16.8 / 10 / 9.00E+07
Station Name / Latitude (S) / Longitude (E) / Date / Location / Water Depth (m) / Volume water filtered (m3) / Total plankton concentrate volume (ml) / Sub sampled volume (ml) / Sample lysis volume (ml) / Lysis sub-sample (ml) / DNA extraction volume (ul) / qPCR test volume (ul) / Concentration factor / Stock DNA concentration (ng/ul) / DNA dilution tested / CoTS COI copies per m3
COT027 / 15.9385 / 145.6527 / 20/12/2014 / Evening Rf. / 39 / 7.2 / 48 / 6 / 6 / 0.6 / 150 / 5 / 24000 / 21.4 / 10 / 1.89E+08
COT028 / 16.03283 / 145.6317 / 20/12/2014 / Mackay Rf. / 33 / 6 / 24 / 4 / 6 / 0.6 / 150 / 5 / 18000 / 13.1 / 10 / 1.90E+08
COT029 / 16.153 / 145.6743 / 20/12/2014 / N of Rudder Rf / 42 / 7.8 / 36 / 6 / 6 / 0.6 / 150 / 5 / 18000 / 9.32 / 10 / 1.00E+07
COT036 / 16.2325 / 145.643 / 21/12/2014 / Rudder Rf./Tongue Rf. / 42 / 7.8 / 36 / 4 / 6 / 0.6 / 150 / 5 / 27000 / 32.8 / 10 / 3.38E+08
COT037 / 16.39383 / 145.6245 / 21/12/2014 / E of Batt Rf / 31 / 5.6 / 36 / 4 / 6 / 0.6 / 150 / 5 / 27000 / 13.9 / 10 / 2.63E+06
COT038 / 16.48783 / 145.8273 / 21/12/2014 / S of Batt Rf. / 39 / 7.2 / 36 / 4 / 6 / 0.6 / 150 / 5 / 27000 / 23.4 / 10 / 5.78E+07
COT039 / 16.59733 / 145.8703 / 21/12/2014 / E of Oyster Rf. / 39 / 7.2 / 40 / 4 / 6 / 0.6 / 150 / 5 / 30000 / 14.1 / 10 / 4.78E+06
COT040 / 16.73033 / 145.9717 / 21/12/2014 / Arlington Rf./Green Isl. / 43 / 8 / 32 / 4 / 6 / 0.6 / 150 / 5 / 24000 / 24.9 / 10 / 2.22E+07
COT041 / 16.90417 / 146.1355 / 22/12/2014 / E of Sudbury Rf. / 39 / 7.2 / 30 / 5 / 6 / 0.6 / 150 / 5 / 18000 / 17 / 10 / 3.22E+07
COT042 / 17.22017 / 146.2545 / 22/12/2014 / S of Flora Rf. / 38 / 7 / 36 / 6 / 6 / 0.6 / 150 / 5 / 18000 / 80.4 / 10 / 7.71E+07
COT043 / 17.49183 / 146.3413 / 22/12/2014 / E of Peart Rf. / 38 / 7 / 30 / 5 / 6 / 0.6 / 150 / 5 / 18000 / 29.4 / 10 / 8.97E+06
COT044 / 17.63203 / 146.3516 / 22/12/2014 / N of Ellison Rf. / 42 / 7.8 / 32 / 4.5 / 6 / 0.6 / 150 / 5 / 21333 / 40.4 / 10 / 0.00E+00
COT045 / 17.94017 / 146.4468 / 22/12/2014 / S of Beaver Rf. / 36 / 6.6 / 36 / 6 / 6 / 0.6 / 150 / 5 / 18000 / 35.6 / 10 / 1.51E+07
COT046 / 18.08009 / 146.4744 / 22/12/2014 / E of Otter Rf. / 32 / 5.8 / 36 / 6 / 6 / 0.6 / 150 / 5 / 18000 / 27.6 / 10 / 3.76E+06
COT047 / 18.214 / 146.5213 / 22/12/2014 / E of Britomart Rf. / 35 / 6.4 / 36 / 6 / 6 / 0.6 / 150 / 5 / 18000 / 34.7 / 10 / 1.57E+07
COT048 / 19.101 / 146.863 / 23/12/2014 / Magnetic Isl. / 15 / 2.4 / 36 / 6 / 6 / 0.6 / 150 / 5 / 18000 / 8.48 / 10 / 0.00E+00
COT055 / 16.77304 / 145.9529 / 17/06/2015 / Green Isl. / 38 / 7 / 30 / 5 / 6 / 0.6 / 150 / 5 / 18000 / 49.6 / 10 / 0.00E+00
COT056 / 16.92076 / 145.9941 / 17/06/2015 / Fitzroy Isl. / 18 / 3 / 10 / 5 / 6 / 0.6 / 150 / 5 / 6000 / 44.8 / 10 / 0.00E+00

Fig. S1. PCR using the primers COTS-F-1321 and COTS-R-1446 which amplified a 126 bp fragment from all four CoTS lineages. Sample 59 is from the South Indian Ocean (Mayotte), sample 62 is from the North Indian Ocean (Maldives), sample 67 is from the Red Sea (Wajha Bank) and samples 72 and 78 are from the Greta Barrier Reef. NTC = no template control

References

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Doi 10.1186/1471-2105-13-134