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Identification of Balanus amphitrite larvae from field zooplankton using species-specific primers

Published online by Cambridge University Press:  06 November 2014

Chetan C. Gaonkar ,
Lidita Khandeparker ,
Dattesh V. Desai  and
Arga Chandrashekar Anil
Chetan C. Gaonkar
Affiliation:
National Institute of Oceanography, Council of Scientific and Industrial Research, Dona Paula, Goa 403 004, India
Lidita Khandeparker*
Affiliation:
National Institute of Oceanography, Council of Scientific and Industrial Research, Dona Paula, Goa 403 004, India
Dattesh V. Desai
Affiliation:
National Institute of Oceanography, Council of Scientific and Industrial Research, Dona Paula, Goa 403 004, India
Arga Chandrashekar Anil
Affiliation:
National Institute of Oceanography, Council of Scientific and Industrial Research, Dona Paula, Goa 403 004, India
*
Correspondence should be addressed to:L. Khandeparker, National Institute of Oceanography, Council of Scientific and Industrial Research, Dona Paula, Goa 403 004, India email: klidita@nio.org
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Abstract

Identification of marine invertebrate larvae using morphological characters is laborious and complicated by phenotypic plasticity. Balanus amphitrite is a dominant barnacle, important in the context of intertidal ecology and biofouling of manmade structures. Morphological identification of barnacle larval forms in a mixed population is difficult because of their intricacy and similarity in size, shape and developmental stages. We report the development and application of a nucleic acid-based Polymerase Chain Reaction (PCR) method for the specific identification of the barnacle, B. amphitrite, from the heterogeneous zooplankton sample. This method is reliable and accurate thereby overcoming taxonomic ambiguity. Sequence alignment of the 18S rRNA gene region of selected species of barnacles allowed the design of B. amphitrite-specific PCR primers. Assay specificity was evaluated by screening DNA obtained from selected species of barnacles. The oligonucleotide primers used in the study flanked a 1600 bp region within the 18S rRNA gene. The primer is specific and can detect as few as 10 individuals of B. amphitrite larvae spiked in a background of ~186 mg of zooplankton. This technique facilitates accurate identification and the primer can be used as a marker for enumeration of B. amphitrite larvae in the plankton.

Keywords

BarnacleBalanus amphitriteLarvaeZooplankton18S rRNA
Type
Research Article
Information
Journal of the Marine Biological Association of the United Kingdom , Volume 95 , Issue 3 , May 2015 , pp. 497 - 502
Copyright
Copyright © Marine Biological Association of the United Kingdom 2014 

INTRODUCTION

Larval ecology studies help in understanding the population dynamics, community patterns, ecosystem structure and biodiversity of native and invasive species (Webb et al., Reference Webb, Barnes, Clark and Bowden2006). Identification of marine invertebrate larvae is a tedious, labour intensive task by expert taxonomists. Traditionally, planktonic larval identification is difficult because of larval intricacy and similarity in size, shape and developmental stages (Chanley & Andrews, Reference Chanley and Andrews1971; Branscomb & Vedder, Reference Branscomb and Vedder1982; Shanks, Reference Shanks1986; Nichols & Black, Reference Nichols and Black1994). Due to their small size, shape and similar developmental stages, it is difficult to identify these larvae morphologically, although they play a pivotal role in taxonomic identification (Levin, Reference Levin1990). Sometimes larval identification becomes extremely difficult due to phenotypic plasticity (Hebert, Reference Hebert2002).

Molecular techniques have the potential to accurately identify the organism to its species level, thereby overcoming taxonomic ambiguity. Identification and quantification of marine invertebrate larvae is far easier using molecular techniques (Baldwin et al., Reference Baldwin, Black, Sanjur, Gustafson, Lutz and Vrijenhoek1996; Bilodeau et al., Reference Bilodeau, Lankford, Kim, Felder and Neigel1999; Makinster et al., 1999; Morgan & Rogers, Reference Morgan and Rogers2001; Deagle et al., Reference Deagle, Bax, Hewitt and Patil2003; Larsen et al., Reference Larsen, Frischer, Rasmussen and Hansen2005; Vadopalas et al., Reference Vadopalas, Bouma, Jackels and Friedman2006; Jones et al., Reference Jones, Preston, Marin, Scholin and Vrijenhoek2008; Chen et al., Reference Chen, Hoeg and Chan2013). Polymerase Chain Reaction (PCR) along with sequencing has led to accurate identification of any organism to its species level. Appropriate use of specific primers can facilitate rapid, sensitive and accurate detection of any individual species in a population. Some molecular techniques which assist in identification or characterization of organisms are DNA barcoding (Hebert et al., Reference Hebert, Cywinska, Ball and deWaard2003a, Reference Hebert, Ratnasingham and deWaardb); Random amplified polymorphic DNA (Coffroth & Mulawka, Reference Coffroth and Mulawka1995); multiplex PCR (Hare et al., Reference Hare, Palumbi and Butman2000); Middle repetitive sequence analysis (MaKinster et al., Reference MaKinster, Felder, Chlan, Boudreaux and Neigel1999); Amplified fragment length polymorphism (Bucklin, Reference Bucklin, Harris, Wiebe, Lenz, Skjoldal and Huntley2000; Rogers, Reference Rogers, Atkinson and Thorndyke2001); Restriction fragment length polymorphism and Single strand conformation polymorphism analysis (Hillis et al., Reference Hillis, Mable, Moritz, Hillis, Moritz and Mable1996). Oligonucleotide probes used for specific detection of individual larvae in a mixed population are either concise to family level (Bell & Grassle, Reference Bell and Grassle1998), genus level (Frischer et al., Reference Frischer, Danforth, Tyner, Leverone, Marelli, Arnold and Blake2000) or species level (Frischer et al., Reference Frischer, Danforth, Tyner, Leverone, Marelli, Arnold and Blake2000; Hare et al., Reference Hare, Palumbi and Butman2000). Molecular tools with respect to PCR-based approaches are more reliable and frequently used in larval identification (Hare et al., Reference Hare, Palumbi and Butman2000; Wood et al., Reference Wood, Krogran, Dover, Schneider, Heidt, Boateng, Dean, Golshani, Zhang, Greenblatt, Johnston and Shilatifard2003; Webb et al., Reference Webb, Barnes, Clark and Bowden2006; Chen et al., Reference Chen, Hoeg and Chan2013).

Barnacles are of major concern in biofouling studies around the world. They have drawn the attention of many investigators in marine plankton ecology owing to their easy accessibility on the rocky intertidal regions and also because some species are dominant in marine fouling (Strathmann et al., Reference Strathmann, Branscomb and Vedder1981; Crisp, Reference Crisp, Costlow and Tipper1984; Connell, Reference Connell1985; Holm, Reference Holm1990; Sutherland, Reference Sutherland1990; Bertness et al., Reference Bertness, Gaines, Bermudez and Sandford1991; Raimondi, Reference Raimondi1991; Thiyagarajan et al., Reference Thiyagarajan, Venugopalan, Nair and Subramoniam1997a, Reference Thiyagarajan, Venugopalan, Subramoniam and Nairb). Barnacles possess both a planktotrophic and a lecithotropic larval stage, which settle and metamorphose on hard substratum resulting in macrofouling. Morphological identification of barnacle larval forms in a population is difficult because of their intricacy and similarity in size, shape and developmental stages, and requires extensive microscopy and taxonomic expertise.

Balanus amphitrite, an acorn barnacle, has wide distribution, can be easily maintained in the laboratory, and possesses six planktonic naupliar stages followed by a pre-settlement cypris stage. This species has been extensively used in different studies related to larval development, metamorphosis, influence of different chemical cues and antifouling assays (Rittschof et al., Reference Rittschof, Branscomb and Costlow1984; Maki et al., Reference Maki, Rittschof, Costlow and Mitchell1988; Clare et al., Reference Clare, Freet and McClary1994; Anil et al., Reference Anil, Chiba, Okamoto and Kurokura1995; Khandeparker et al., Reference Khandeparker, Anil and Raghukumar2003, Reference Khandeparker, Anil and Raghukumar2006; Khandeparker & Anil, Reference Khandeparker and Anil2011). Since B. amphitrite larvae are the primary target of investigations related to biofouling and plankton ecology, their fast enumeration and identification is crucial. In the present study, a PCR-based approach was used for detection of a dominant fouling barnacle, B. amphitrite (syn. Amphibalanus amphitrite; Pitombo, Reference Pitombo2004) larvae from the mixed population.

Mitochondrial DNA and nuclear DNA have been the major targets for species identification due to their high conservation and high copy numbers per cell (Stach & Tubeville, Reference Stach and Tubeville2002). Application of mtDNA (12S and 16S) has been useful for species identification, because sequences from various kinds of species have been deposited in the database. Identification of barnacles, based on analysis of 12S and 16S rRNA genes, has been reported by Begum et al. (Reference Begum, Yamaguchi and Watabe2004) and Simon-Blecher et al. (Reference Simon-Blecher, Huchon and Achituv2007). Recently, a species-specific primer for quantitative real-time PCR (qPCR) was evaluated for specific detection and quantification of B. amphitrite using the 12S rRNA gene (Endo et al., Reference Endo, Sato, Matsumura, Yoshimura, Odaka and Nogata2010). Nucleic acid-based sandwich hybridization assays using an rRNA target probe was used for barnacle detection of the group (order Thoracica) and species (Balanus glandula) which could detect even a single barnacle larva in a water column (Goffredi et al., Reference Goffredi, Jones, Scholin, Marin and Vrijenhoek2006). Designing of species-specific primers within the 18S rRNA gene region helps in detecting individual species, since 18S rRNA gene regions have slowly evolved among different orders and families, including invertebrates (Winnepenninckx et al., Reference Winnepenninckx, Backeljau and de Wachter1995; Bleidorn et al., Reference Bleidorn, Vogt and Bartolomaeus2003; Pradillon et al., Reference Pradillon, Schmidt, Peplies and Dubilier2007). In the present study an attempt was made to develop species-specific primers within the 18S rRNA gene region which has not been attempted earlier for B. amphitrite and this provides an additional dimension to this field, especially with reference to identification of B. amphitrite larvae in a mixed zooplankton sample. The primers were designed by comparing the 18S rRNA sequences of closely related Balanus sp. and evaluating the conserved region within the 18S rRNA gene sequence. This approach for planktonic larval detection is less time-consuming compared with morphological microscopic examination and less expensive than other DNA-based approaches.

MATERIALS AND METHODS

Sample collection

Adult B. amphitrite were collected from the intertidal region of Goa, West Coast of India. Adults obtained from field samples were brought to the laboratory and exposed to air for 1–2 h and then immersed in filtered seawater, which triggered the release of larvae. The Instar II nauplius larvae obtained from the adults were used as a positive control and for internal spiking in the present investigation. Horizontal hauls were taken for collection of zooplankton using a 100 µm mesh Heron-Tranter (HT) zooplankton net with a calibrated flow meter attached to it in the viscinity of Dona Paula Bay (15°27.5′N 73°48′E), west coast of India. The plankton samples were either preserved in 95% ethanol or directly processed for DNA isolation and PCR analysis. The preserved samples were quantified for the presence of cirripede larvae. The number of larvae present in different samples varied from 50 to 4000 ind m−3. Four other barnacle species (Chthamalus malayensis, Megabalanus tintinnabulum, Lepas sp. and Ibla sp.) were also collected from the study area. The adult barnacles were identified based on previously described morphological features (Karande, Reference Karande1967; Wagh & Bal, Reference Wagh and Bal1970; Henry & McLaughlin, Reference Henry and McLaughlin1975; Flowerdew, Reference Flowerdew1985; Pitombo, Reference Pitombo2004; Fernando, Reference Fernando2006). They were collected for extraction of genomic DNA and verification of primer specificity.

Extraction of genomic DNA

The adults of B. amphitrite were starved overnight prior to DNA extraction. Genomic DNA was extracted from adult barnacles namely B. amphitrite, Chthamalus malayensis, Megabalanus tintinnabulum, Lepas sp. and Ibla sp. Whole adult muscle tissue was used for genomic DNA isolation. DNA extracted from newly hatched Artemia sp. nauplii was used as control. The zooplankton samples were weighed and then subjected to DNA extraction using DNA Extraction solution (GeneI, India). The extracted DNA was visualized on a 0.8% Agarose gel stained with ethidium bromide and observed under UV illumination.

Designing of B. amphitrite-specific primers

18S rRNA gene sequences of barnacles (Table 1) were obtained from the NCBI GenBank (http://www.ncbi.nim.nih.gov/) and compiled. These 18S rRNA gene sequences were aligned using Clustal X 1.8 (Thompson et al., Reference Thompson, Gibson, Plewnaik, Jeanmougin and Higgins1997). Since the sequence of B. amphitrite 18S rRNA gene was not available in any of the databases, primers were designed by selecting a short highly conserved region within the 5′ end region between barnacles with few mismatches in the last five nucleotides in the 3′ region of the primer (Table 1). Target primers were designed in the gene region where there were mismatch pairs in relation to other barnacle species. Primers were manually designed using BioEdit, with standard priming conditions such as primer length, self annealing, possible loops, GC content and melting temperature (T m), which were evaluated every time during each primer design. During our analysis, some of the 3′ end nucleotides of the forward primer were changed in order to eliminate strong loop, self annealing or primer dimer formation.

Table 1. Balanus amphitrite specific primers aligned with the corresponding sequence from other available barnacles.

A dot represents similar nucleotides within the primer sequence.

Polymerase chain reaction

DNA isolated from the adult samples was used for PCR amplification of the 18S rRNA gene. DNA amplification of the 18S rRNA gene region was performed using primers which annealed only with the B. amphitrite species. 5 µl of extracted DNA was used for PCR amplification using a PTC 200 Thermal cycler (MJ Research). PCR reaction was carried out with a 50 µl reaction mixture containing 10 mM of each dNTPs, 20 pmoles of each primer, 1 U of Taq polymerase, 1× PCR reaction buffer containing 10 mM MgCl2. Amplification of the 18S rRNA gene region was carried out using B. amphitrite specific primers (Table 2). The thermal cycler was programmed using a touchdown PCR protocol. One cycle of 94 °C for 2 min, followed by 35 cycles of 1 min at 94 °C, 45 s at 58 °C, 1 min at 72 °C and final cycle of 10 min at 72 °C. The resulting fragments were resolved electrophoretically on 1% agarose gel for 1 h at 90 V. The resulting amplicons were compared with a commercial 1 Kb DNA ladder (Genetix, India). Amplification was carried out in replicates and batches, to determine the specificity, sensitivity and reproducibility of the designed primers.

Table 2. Sequences of oligonucleotide primers used for species-specific detection of Balanus amphitrite.

Verification of primer's specificity

Primer specificity was evaluated by PCR amplification of the 18S rRNA gene region using extracted genomic DNA from four other barnacle species (Chthamalus malayensis, Megabalanus tintinnabulum, Lepas sp. and Ibla sp.). The designed primers amplified ~1600 bp amplicons only from the B. amphitrite (Figure 1). However, other species of barnacles did not show amplification with similar primer and PCR conditions. PCR amplification using gDNA from Artemia sp. also showed no amplicon, resulting as negative control for the designed primers.

Fig. 1. Agarose gel electrophoresis of PCR amplified 1600 bp product of the 18S rRNA gene from Balanus amphitrite using specific primers. Left lane represents standard size markers (1 Kb DNA ladder, Chromous). Lane 1 is control using Artemia sp., Lane 2 is the PCR product of 18S rRNA gene region using B. amphitrite genomic DNA as template. Lanes 3–6 are genomic DNA from other known available barnacles as template: Megabalanus tintinnabulum (3); Chthamalus malayensis (4); Lepas sp. (5); Ibla sp. (6).

Evaluation of primers for B. amphitrite specificity

DNA isolated from mixed zooplankton samples were subjected to PCR amplification using the above protocol. The zooplankton samples were screened under a microscope and all cirripede larvae were eliminated which were used as control, in order to check the specificity of our designed primers. In order to check the presence or absence of any PCR inhibitors in extracted DNA, the zooplankton samples without cirripede larvae were spiked with 10 and 100 larvae (Instar II) of B. amphitrite. Owing to small size and low DNA content in the case of Instar II barnacle nauplii one larva could not be amplified. DNA isolated from Artemia sp. was used as control.

RESULTS AND DISCUSSION

Alignment of the 18S rRNA gene sequences of barnacles (Table 1) obtained from GenBank were evaluated using Clustal X. Sequences of all individuals revealed a high rate of conservation within the 18S rRNA gene region. Very low mismatch regions were present within the 18S rRNA gene region of Balanus sp. The forward primer was designed in this region because it had a conserved 5′ end with few mismatches at the 3′ region and the reverse primer was designed in a conserved region compared with other barnacles (Table 1). The designed primers amplified ~1600 bp amplicons only from the B. amphitrite species (Figure 1).

DNA extracted from the mixed planktonic population showed a positive result when amplified with the B. amphitrite specific primer, indicating the presence of B. amphitrite larvae in the mixed sample. The zooplankton samples without cirrepede larvae spiked with 10 and 100 B. amphitrite larvae produced ~1600 bp fragment stating the sensitivity of the designed primer for the specific detection of B. amphitrite and also eliminating the presence of any PCR inhibitors (Figure 2). In the present study a simple and inexpensive methodology was adopted for the specific detection of B. amphitrite larvae in a mixed population. 18S rRNA genes contain regions which are either highly conserved or variable, and specific primers can be targeted to these characteristic sites for families, genus or species (Amann et al., Reference Amann, Krumholz and Stahl1990). Mostly these ribosomal regions are best suited for probe designing (Peplies et al., Reference Peplies, Glockner, Amann and Ludwig2004). In the present study we used this information for designing species-specific primers. However, 28S and the mitochondrial rRNA (12S and 16S rRNA) gene regions can also be used for such studies if there is no single mismatch in the 18S rRNA gene, within the species level. Identification of organisms using a 12S rRNA gene sequence has also been attempted in detection and quantification of barnacle larvae in plankton samples using qPCR (Endo et al., Reference Endo, Sato, Matsumura, Yoshimura, Odaka and Nogata2010). It has a high inter-specific variability along with low intra-specific variability (Hebert et al., Reference Hebert, Cywinska, Ball and deWaard2003a, Reference Hebert, Ratnasingham and deWaardb). In an environmental sample where a mixed population of species is present, a nested PCR approach has also been used to resolve the individual species (Patil et al., Reference Patil, Gunasekera, Deagle, Bax and Blackburn2005b). That study developed species-specific PCR assays for the detection of single species of a dinoflagellate, Gymnodinium catenatum in both environmental samples and in ballast water. The specificity of the primer showed that up to five cysts of G. catenatum can be detected in mixed populations. Similar results were achieved in detecting larval forms in Pacific oysters Crassostrea gigas (Patil et al., Reference Patil, Gunasekera, Deagle and Bax2005a), Tropical oyster C. belchiri (Klinbunga et al., Reference Klinbunga, Ampayup, Tassanakajon, Jarayabhand and Yoosukh2000) and in the sea star Asterias sp. (Deagle et al., Reference Deagle, Bax, Hewitt and Patil2003) using a PCR-based approach.

Fig. 2. PCR detection of Balanus amphitrite larvae in plankton sample. Left lane contains standard size markers (1 Kb DNA ladder, Chromous). Lane 1 is control. Lane 2 is zooplankton without cirrepede larvae. Lane 3 is PCR product of zooplankton. Lane 4 is zooplankton (without cirrepede larvae) spiked with 10 B. amphitrite larvae. Lane 5 is PCR product of zooplankton (without cirrepede larvae) spiked with 100 B. amphitrite larvae.

In the present study, efficiency of the PCR assay was enhanced by increasing the number of target species. Primer sensitivity was cross-checked with all available barnacle species in the study location. Balanus amphitrite specific primers did not amplify the other barnacle species, indicating that the primers were specific only to B. amphitrite. The presence of PCR inhibitors in the zooplankton sample was ruled out by conducting a PCR with zooplankton spiked with known numbers of B. amphitrite larvae. The resulting amplicons in these samples resulted in PCR success, ruling out the presence of any PCR inhibitors.

We demonstrate that the B. amphitrite larvae can be detected with extreme sensitivity by PCR amplification using the 18S species-specific primers designed in this study. Application of this method for detection of the B. amphitrite larvae in a mixed population can facilitate accurate screening of large numbers of samples and solve significant problems associated with larval ecology. This approach also can be used to differentiate B. amphitrite larvae from that of the closely related groups of barnacles within the mixed community of barnacles. Real-time PCR (qPCR) is recognized as an effective device for detection and quantification of different planktonic organisms in a mixed population. In future this tool can be adopted using the B. amphitrite specific primers designed in the present study for quantification of B. amphitrite in the plankton.

ACKNOWLEDGEMENTS

We are grateful to Dr Satish R. Shetye, the Director of the National Institute of Oceanography, Goa for his support and encouragement. We acknowledge the help provided by Dr Rakhee Khandeparker and other colleagues of BBD during this study. This work was carried out as a part of Ballast Water Management Programme, India that was funded by Directorate General of Shipping, Government of India. This is a CSIR-NIO contribution 5661.

References

REFERENCES

Amann, R.I., Krumholz, L. and Stahl, D.A. (1990) Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. Journal of Bacteriology 172, 762–770.Google Scholar
Anil, A.C., Chiba, K., Okamoto, K. and Kurokura, H. (1995) Influence of temperature and salinity on larval development of Balanus amphitrite: implications in fouling ecology. Marine Ecology Progress Series 118, 159–166.CrossRefGoogle Scholar
Baldwin, B.S., Black, M., Sanjur, O., Gustafson, R., Lutz, R.A. and Vrijenhoek, R.C. (1996) A diagnostic molecular marker for zebra mussels (Dreissena polymorpha) and potentially co-occurring bivalves: mitochondrial COI. Marine Molecular Biology and Biotechnology 5, 9–14.Google Scholar
Begum, R.A., Yamaguchi, T. and Watabe, S. (2004) Molecular phylogeny of thoracican barnacles based on the mitochondrial 12S and 16S rRNA genes. Sessile Organisms 21, 47–54.Google Scholar
Bell, J.L. and Grassle, J.P. (1998) A DNA probe for identification of larvae of the commercial surf clam (Spisula solidissima). Marine Molecular Biology and Biotechnology 7, 127–137.Google Scholar
Bertness, M.D., Gaines, S.P., Bermudez, D. and Sandford, E. (1991) Extreme spatial variation in the growth and reproductive output of the acorn barnacle Semibalanus balanoides. Marine Ecology Progress Series 75, 91–100.Google Scholar
Bilodeau, A.L., Lankford, W.S., Kim, T.J., Felder, D.L. and Neigel, J.E. (1999) An ultra sensitive method for detection of single crab larvae (Sesarma reticulatum) by PCR amplification of a highly repetitive DNA sequence. Marine Ecology 8, 683–684.Google Scholar
Bleidorn, C., Vogt, L. and Bartolomaeus, T. (2003) New insights into polychaete phylogeny (Annelida) inferred from 18S rDNA sequences. Molecular Phylogenetics and Evolution 29, 279–288.Google Scholar
Branscomb, E.S. and Vedder, K. (1982) A description of the naupliar stages of the barnacles Balanus glandula Darwin, Balanus cariosus Pallas, and Balanus crenatus Bruguière (Cirripedia, Thoracica). Crustaceana 42, 83–95.Google Scholar
Bucklin, A. (2000) Methods for population genetic analysis of zooplankton. In Harris, R.P., Wiebe, P.H., Lenz, J., Skjoldal, H.R. and Huntley, M. (eds) ICES zooplankton methodology manual. London: Academic Press, pp. 533–570.Google Scholar
Chanley, P. and Andrews, J.D. (1971) Aids for identification of bivalve larvae of Virginia. Malacologia 11, 45–119.Google Scholar
Chen, H.-N., Hoeg, J.T. and Chan, B.K.K. (2013) Morphometric and molecular identification of individual barnacle cyprids from wild plankton: an approach to detecting fouling and invasive barnacle species. Biofouling 29, 133–145.Google Scholar
Clare, A.S., Freet, R.K. and McClary, M. Jr (1994) On the antennular secretions of the cyprid of Balanus amphitrite, and its role as a settlement pheromone. Journal of the Marine Biological Association of the United Kingdom 74, 243–250.CrossRefGoogle Scholar
Coffroth, M.A. and Mulawka, J.M. III (1995) Identification of marine invertebrate larvae by means of PCR-RAPD species specific markers. Limnology and Oceanography 40, 181–189.Google Scholar
Connell, J.H. (1985) The consequences of variation in initial settlement vs. post-settlement mortality in rocky intertidal communities. Journal of Experimental Marine Biology and Ecology 93, 11–45.Google Scholar
Crisp, D.J. (1984) Overview of research on marine invertebrate larvae. In Costlow, J.D. and Tipper, R.C. (eds) Marine biodeterioration: an interdisciplinary study. Annapolis: Naval Institute Press, pp. 103–126.Google Scholar
Deagle, B.E., Bax, N.J., Hewitt, C.L. and Patil, J.G. (2003) Development and evaluation of a PCR-based test for detection of Asterias (Echinodermata: Asteroidea) larvae in Australian plankton samples from ballast water. Marine and Freshwater Research 54, 709–719.Google Scholar
Endo, N., Sato, K., Matsumura, K., Yoshimura, E., Odaka, Y. and Nogata, Y. (2010) Species-specific detection and quantification of common barnacle larvae from the Japanese coast using quantitative real time PCR. Biofouling 26, 901–911.Google Scholar
Fernando, S.A. (2006) Monograph on Indian barnacles. Kochi: OSTC Marine Benthos-02, Ocean Science and Technology Cell, CUSAT, 219 pp.Google Scholar
Flowerdew, M.W. (1985) Indices of genetic identity and distance in three taxa within the Balanus amphitrite Darwin complex (Cirripedia, Thoracica). Crustaceana 49, 7–15.Google Scholar
Frischer, M.E., Danforth, J.M., Tyner, L.C., Leverone, J.R., Marelli, D.C., Arnold, W.S. and Blake, N.J. (2000) Development of an Argopecten-specific 18S rRNA targeted genetic probe. Marine Biotechnology 2, 11–20.Google Scholar
Goffredi, S.K., Jones, W.J., Scholin, C.A., Marin, R. and Vrijenhoek, R.C. (2006) Molecular detection of marine invertebrate larvae. Marine Biotechnology 8, 149–160.Google Scholar
Hare, M.P., Palumbi, S.R. and Butman, C.A. (2000) Single-step species identification of bivalve larvae using multiplex polymerase chain reaction. Marine Biology 137, 953–961.CrossRefGoogle Scholar
Hebert, P.D.N. (2002) Life in polar waters. Integrative and Comparative Biology 42, 1242.Google Scholar
Hebert, P.D.N., Cywinska, A., Ball, S.L. and deWaard, J.R. (2003a) Biological identifications through DNA barcodes. Proceedings of the Royal Society of London B 270, 313–321.Google Scholar
Hebert, P.D.N., Ratnasingham, S. and deWaard, J.R. (2003b) Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings of the Royal Society of London B 270, 596–599.Google Scholar
Henry, D.P. and McLaughlin, P.A. (1975) The barnacles of the Balanus amphitrite complex (Cirripedia, Thoracica). Zoologische Verhandelingen Leiden 141, 1–254.Google Scholar
Hillis, D.M., Mable, B.K. and Moritz, C. (1996) Applications of molecular systematics: the state of the field and a look to the future. In Hillis, D.M., Moritz, C. and Mable, B.K. (eds) Molecular systematics. Sunderland, MA: Sinauer Associates, pp. 515–543.Google Scholar
Holm, E.R. (1990) Attachment behavior in the barnacle Balanus amphitrite amphitrite (Darwin): genetic and environmental effects. Journal of Experimental Marine Biology and Ecology 135, 85–98.Google Scholar
Jones, W.J., Preston, C., Marin, R., Scholin, C.A. and Vrijenhoek, R.C. (2008) A robotic molecular method for in situ detection of marine invertebrate larvae. Molecular Ecology Resources 8, 540–550.Google Scholar
Karande, A.A. (1967) On cirripede crustaceans (barnacles), an important fouling group in Bombay waters. Proceedings of the Symposium on Crustaceans. Marine Biological Association of India 2, 1942–1953.Google Scholar
Khandeparker, L. and Anil, A.C. (2011) Role of conspecific cues and sugars in the settlement of cyprids of the barnacle, Balanus amphitrite. Journal of Zoology 284, 206–214.Google Scholar
Khandeparker, L., Anil, A.C. and Raghukumar, S. (2003) Barnacle larval destination: piloting possibilities by bacteria and lectin interaction. Journal of Experimental Marine Biology and Ecology 289, 1–13.CrossRefGoogle Scholar
Khandeparker, L., Anil, A.C. and Raghukumar, S. (2006) Relevance of biofilm bacteria in modulating the larval metamorphosis of Balanus amphitrite Darwin. FEMS Microbiology Ecology 58, 425–438.Google Scholar
Klinbunga, S., Ampayup, P., Tassanakajon, A., Jarayabhand, P. and Yoosukh, W. (2000) Development of species-specific markers of the tropical oyster (Crassostrea belcheri) in Thailand. Marine Biotechnology 2, 476–484.Google Scholar
Larsen, J.B., Frischer, M.E., Rasmussen, L.J. and Hansen, B.W. (2005) Single-step nested multiplex PCR to differentiate between various bivalve larvae. Marine Biology 146, 1119–1129.Google Scholar
Levin, L.A. (1990) A review of methods for labeling and tracking marine invertebrate larvae. Ophelia 32, 115–144.Google Scholar
Maki, J.S., Rittschof, D., Costlow, J.D. and Mitchell, R. (1988) Inhibition of attachment of larval barnacles, Balanus amphitrite, by bacterial films. Marine Biology 97, 199–206.Google Scholar
MaKinster, J.G., Felder, D.L., Chlan, C., Boudreaux, M. and Neigel, J.E. (1999) PCR amplification of a middle repetitive element detects larval stone crabs (Crustacea: Decapoda: Menippidae) in estuarine plankton samples. Marine Ecology Progress Series 181, 161–168.Google Scholar
Morgan, T.S. and Rogers, A.D. (2001) Specificity and sensitivity of microsatellite markers for the identification of larvae. Marine Biology 139, 967–973.Google Scholar
Nichols, S.J. and Black, M.G. (1994) Identification of larvae: the zebra mussel (Dreissena polymorpha), quagga mussel (Dreissena rosteriformis bugensis), and Asian clam (Corbicula fluminea). Canadian Journal of Zoology 72, 406–417.Google Scholar
Patil, J.G., Gunasekera, R.M., Deagle, B.E. and Bax, N.J. (2005a) Specific detection of Pacific oyster (Crassostrea gigas) larvae in plankton samples using nested polymerase chain reaction. Marine Biotechnology 7, 11–20.Google Scholar
Patil, J.G., Gunasekera, R.M., Deagle, B.E., Bax, N.J. and Blackburn, S.I. (2005b) Development and evaluation of a PCR based assay for detection of the toxic dinoflagellate, Gymnodinium catenatum (Graham) in ballast water and environmental samples. Biological Invasions 7, 983–994.Google Scholar
Peplies, J., Glockner, F.O., Amann, R. and Ludwig, W. (2004) Comparative sequence analysis and oligonucleotide probe design based on 23S rRNA genes of Alphaproteobacteria from North Sea bacterioplankton. Systematic and Applied Microbiology 27, 573–580.Google Scholar
Pitombo, F.B. (2004) Phylogenetic analysis of the Balanidae (Cirripedia, Balanomorpha). Zoologica Scripta 33, 261–276.Google Scholar
Pradillon, F., Schmidt, A., Peplies, J. and Dubilier, N. (2007) Species identification of marine invertebrate early stages by whole larvae in situ hybridisation of 18S ribosomal RNA. Marine Ecology Progress Series 333, 103–116.Google Scholar
Raimondi, P.T. (1991) Settlement behavior of Chthamalus anisopoma larvae largely determines the adult distribution. Oecologia 85, 349–360.Google Scholar
Rittschof, D., Branscomb, E.S. and Costlow, J.D. (1984) Settlement and behavior in relation to flow and surface in larval barnacles, Balanus amphitrite Darwin. Journal of Experimental Marine Biology and Ecology 82, 131–146.Google Scholar
Rogers, A. (2001) Molecular ecology and identification of marine invertebrate larvae. In Atkinson, D. and Thorndyke, M. (eds) Environment and animal development: genes, life histories and plasticity. Oxford: BIOS Scientific Publishers, pp. 29–69.Google Scholar
Shanks, A.L. (1986) Tidal periodicity in the daily settlement of intertidal barnacle larvae and an hypothesized mechanism for the cross-shelf transport of cyprids. Biological Bulletin 170, 429–440.Google Scholar
Simon-Blecher, N., Huchon, D. and Achituv, Y. (2007) Phylogeny of coral inhabiting barnacles (Cirripedia; Thoracica; Pyrgomatidae) based on 12S, 16S and 18S rDNA analysis. Molecular Phylogenetics and Evolution 44, 1333–1341.Google Scholar
Stach, T. and Tubeville, J.M. (2002) Phylogeny of Tunicata inferred from molecular and morphological characters. Molecular Phylogenetics and Evolution 25, 408–428.Google Scholar
Strathmann, R.R., Branscomb, E.S. and Vedder, K. (1981) Fatal errors in set as a cost of dispersal and the influence of intertidal flora on set of barnacles. Oecologia 48, 13–18.Google Scholar
Sutherland, J.P. (1990) Recruitment regulates demographic variation in a tropical intertidal barnacle. Ecology 71, 955–972.Google Scholar
Thiyagarajan, V., Venugopalan, V.P., Nair, K.V.K. and Subramoniam, T. (1997a) Larval description of Balanus reticulatus Utinomi (Cirripedia, Balanidae), reared in the laboratory. Journal of Experimental Marine Biology and Ecology 209, 215–231.Google Scholar
Thiyagarajan, V., Venugopalan, V.P., Subramoniam, T. and Nair, K.V.K. (1997b) Description of the naupliar stages of Megabalanus tintinnabulum (Cirrepedia: Balanidae). Journal of Crustacean Biology 17, 332–342.Google Scholar
Thompson, J.D., Gibson, T.J., Plewnaik, F., Jeanmougin, F. and Higgins, D.G. (1997) The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25, 4876–4882.Google Scholar
Vadopalas, B., Bouma, J.V., Jackels, C.R. and Friedman, C.S. (2006) Application of real-time PCR for simultaneous identification and quantification of larval abalone. Journal of Experimental Marine Biology and Ecology 334, 219–228.CrossRefGoogle Scholar
Wagh, A.B. and Bal, D.V. (1970) Observations on systematic of sessile barnacles from the west coast of India-1. Journal of the Bombay Natural History Society 71, 109–123.Google Scholar
Webb, K.E., Barnes, D.K.A., Clark, M.S. and Bowden, D.A. (2006) DNA barcoding: a molecular tool to identify Antarctic marine larvae. Deep-Sea Research II 53, 1053–1060.CrossRefGoogle Scholar
Winnepenninckx, B., Backeljau, T. and de Wachter, R. (1995) Phylogeny of protostome worms derived from 18S rRNA sequences. Molecular Biology and Evolution 12, 641–649.Google Scholar
Wood, A., Krogran, N.J., Dover, J., Schneider, J., Heidt, J., Boateng, M.A., Dean, K., Golshani, A., Zhang, Y., Greenblatt, J.F., Johnston, M. and Shilatifard, A. (2003) Bre1, an E3 ubiquitin ligase required for recruitment and substrate selection of Rad6 at a promoter. Molecular Cell 11, 267–274.Google Scholar
Figure 0

Table 1. Balanus amphitrite specific primers aligned with the corresponding sequence from other available barnacles.

Figure 1

Table 2. Sequences of oligonucleotide primers used for species-specific detection of Balanus amphitrite.

Figure 2

Fig. 1. Agarose gel electrophoresis of PCR amplified 1600 bp product of the 18S rRNA gene from Balanus amphitrite using specific primers. Left lane represents standard size markers (1 Kb DNA ladder, Chromous). Lane 1 is control using Artemia sp., Lane 2 is the PCR product of 18S rRNA gene region using B. amphitrite genomic DNA as template. Lanes 3–6 are genomic DNA from other known available barnacles as template: Megabalanus tintinnabulum (3); Chthamalus malayensis (4); Lepas sp. (5); Ibla sp. (6).

Figure 3

Fig. 2. PCR detection of Balanus amphitrite larvae in plankton sample. Left lane contains standard size markers (1 Kb DNA ladder, Chromous). Lane 1 is control. Lane 2 is zooplankton without cirrepede larvae. Lane 3 is PCR product of zooplankton. Lane 4 is zooplankton (without cirrepede larvae) spiked with 10 B. amphitrite larvae. Lane 5 is PCR product of zooplankton (without cirrepede larvae) spiked with 100 B. amphitrite larvae.