University of Plymouth PEARL https://pearl.plymouth.ac.uk 04 University of Plymouth Research Theses 01 Research Theses Main Collection 2000 A MOLECULAR APPROACH TO CALANUS (COPEPODA:CALANOIDA) DEVELOPMENT AND SYSTEMATICS Lindeque, Penelope Kate http://hdl.handle.net/10026.1/2641 University of Plymouth All content in PEARL is protected by copyright law. Author manuscripts are made available in accordance with publisher policies. Please cite only the published version using the details provided on the item record or document. In the absence of an open licence (e.g. Creative Commons), permissions for further reuse of content should be sought from the publisher or author. A MOLECULAR APPROACH TO CALANUS (COPEPODA:CALANOIDA)DEVELOPMENT AND SYSTEMATICS by Penelope Kate Lindeque A thesis submitted to the University of Plymouth in partial fulfilment for the degree of Doctor gf Philosophy Department of Biological Sciences Faculty of Science In collaboration with Plymouth Marine Laboratory January 2000 UNIVERSITY 01~ PLYMOUTH Item No. C\oO 432708S Date 1 4 SEP 2000S Class No. T .S15 -34 Li"N Contl. No. '{.. '1 0 I+\").. 356 '1 LIBRARY SERVICES '---· U4,RO.~~· I REfERENCE ONLY I LIBRARY STORE ' "'l! .. .·. .. ; { ..; .1 - ~ .I ~. • . --(-I •,- .- o; ' ·- •c Aflcl 1 11 believe.Iove survives death._ .and your1passing,!ilthough;slld an9runl!CC{!ptable; tll!S beeh a reminder 1toms a:n to ·makerthe: most,ot:every ·moment You are forever present:.·' ii .. ·;: \' ..... -. .'r- 'r ~... .. •: I I I. I ,) '· 0 livetGol(lsmi th. t: o', ·' A MOLECULAR APPROACH TO CALANUS (COPEPODA: CALANOIDA) DEVELOPMENT AND SYSTEMATICS Penelope Kate Lindeque ABSTRACT Production and recruitment measurements in manne copepods of the genus Calanus have been addressed via the study of genes involved in early embryogenesis. The first sequence from a Calanus helgolandicus (C. helgolandicus) developmental gene (Cal­ Antp) has been cloned by screening a C. helgolandicus genomic library with a homologous Calanus homeobox probe. Sequencing of an isolated and sub-cloned fragment of this gene, plus further analysis by Inverse Polymerase Chain Reaction (IVPCR), has shown it to be homologous with other Antennapedia homeobox genes. The temporal expression of Cal-Antp was analysed through its messenger RNA (mRNA) complement by Reverse Transcription Polymerase Chain Reaction (RT-PCR). The gene was expressed in tissue taken from eggs over 18 hours old, and in nauplii and copepodite stages, but no expression was detected in eggs less than 18 hours old or adult tissue. Three further homeobox­ containing genes have been identified and analysed through their expression in C. helgolandicus eggs. Two of these are caudal homologues, and the third is homologous to the Antennapedia class of genes. The C. helgolandicus developmental gene sequence data provides a means of developing probes to monitor the temporal expression of such genes and their responses to environmental influence. The applicability of such probes to the investigation of key production and recruitment processes, including egg viability measurement, is discussed. A relatively simple and cost effective method has been developed to identify the four Calanus species common to the North Atlantic. This system involves the PCR amplification of a region of the mitochondrial rRNA gene without prior purification of the DNA, followed by Restriction Fragment Length Polymorphism (RFLP) analysis of the amplified product. The versatility of the method is demonstrated by the unambiguous identification to species of any life stage, from egg to adult, and of any individual body parts. The molecular identification technique has for the first time shown the unexpected presence of three different Calanus species in Lurefjorden, Norway and has proved to be consistently accurate for all individuals tested including geographically distinct conspecific populations. iv LIST OF CONTENTS Page Title page Abstract IV List of Contents V List of Figures ix List of Tables xi Abbreviations XII Acknowledgements xiv Author's declaration XV CHAPTER! Introduction 1 1.1 Copepods and the zooplankton community 3 1.1.1 Zooplankton 3 1.1.2 The contribution of copepods to secondary production 3 1.1.3 Methods of measuring secondary production 5 1.1.4 Calanus egg viability and recruitment 8 1.2 New techniques and technologies 9 1.2.1 Current techniques available 9 1.2.2 Problems associated with using molecular techniques in marine biology 10 1.3 Calanus development 11 1.3.1 Overview 11 1.3.2 Calanus developmental genes 12 1.3.3 Potential use of Calanus developmental genes 19 1.4 Calanus systematics 20 1.4.1 Current molecular applications 20 1.4.2 Potential molecular applications 21 1.5 Aims and objectives 22 CHAPTER2 Materials and Methods 24 2.1 Materials 27 2.2 Microbiological methods 28 2.2.1 Media for microbiology 28 2.2.2 Handling and culturing E. coli 29 2.2.3 Preparation of electrocompetent cells 29 2.2.4 Preparation of chemically competent cells 30 2.2.5 Transformation of electrocompetent E. coli cells 31 2.2.6 Transformation of chemically competent E. coli cells 32 2.2.7 Preparation of ampicillin stock solution 32 2.2.8 Lac selection of plasmids 33 2.2.9 Storage of transformed E. coli cells 33 2.3 Nucleic acid methods 33 2.3.1 Buffers and solutions for molecular biology 33 2.3.2 Phenol extraction of DNA 34 V 2.3.3 PhenoUchloroform extraction of DNA 35 2.3.4 Chloroform extraction of DNA 35 2.3.5 Ethanol precipitation of DNA 35 2.3.6 Electrophoresis of DNA 35 2.3.7 Recovery of DNA from agarose gels 36 2.3.8 Preparation of the vector pBiuescript SK for molecular cloning 37 2.3.9 Treatment of DNA to create blunt termini 37 2.3.10 Ligation of blunt-ended PCR product to digested pBluescript SK 38 2.3.11 Identification of recombinant clones by colony PCR 38 2.3.12 Recovery ofrecombinant plasmid DNA from E. coli 40 2.3.13 Preparation of single stranded DNA for sequencing using Dynabeads 40 2.3.14 Sequencing of single stranded DNA using the chain termination method 42 2.3.15 Preparation of acrylamide gel for sequencing 43 2.3.16 Sequencing gel electrophoresis 44 2.3.17 Autoradiography 45 2.3.18 Southern transfer of DNA to Hybondrn-N membranes 45 CHAPTER3 Construction of a Genomic Library from Calanus helgolandicus 47 3.1 Introduction 50 3.1.1 Strategies for gene isolation 50 3.1.2 What type of library? 53 3.1.3 Which type of vector? 54 3.2 Methods 56 3.2.1 Overview of genomic library construction 56 3.2.2 DNA extraction from Calanus helgolandicus 61 3.2.3 Partial restriction endonuclease digestion of genomic DNA 63 3.2.4 Assembly and titration of intact recombinant A. DASH IT bacteriophage 65 3.2.5 Dephosphorylation and size fractionation of partially digested DNA 68 3.2.6 Library amplification 71 3.3 Results 73 3.3.1 Overview of genomic library construction 73 3.3.2 DNA extraction from Calanus helgolandicus 74 3.3.3 Partial restriction endonuclease digestion of genomic DNA 76 3.3.4 Assembly and titration of intact recombinant bacteriophage 78 3.3.5 Increase in titre with size fractionation and dephosphorylation of DNA inserts 79 3.3.6 Library amplification 83 3.5 Discussion 83 vi CHAPTER4 Identification and analysis of specific developmental genes from the genomic library 86 4.1 Introduction 90 4.1.1 Probe selection 90 4.2 Methods 94 4.2.1 Obtaining probes to screen the library 94 4.2.2 Screening the library 96 4.2.3 Further analysis of the positive lambda clones 104 4.2.4 Analysis of expression of C. helgolandicus developmental gene obtained from library 107 4.2.5 Obtaining sequence data outside the 207L sub-clone by Inverse PCR 116 4.3 Results 118 4.3.1 Probes prepared to screen the library 118 4.3.2 Library Screening 119 4.3.3 Further analysis of the positive tailless and 207L A. clones 123 4.3.4 Analysis of expression of the C. helgolandicus developmental gene obtained from the library 133 4.3.5 Obtaining sequence data outside the 207L sub-clone by inverse PCR 135 4.4 Discussion 136 CHAPTERS Identification and analysis of genes expressed during a defined developmental period 142 5.1 Introduction 145 5.1.1 Identification of developmental genes expressed in C. helgolandicus eggs, via reverse transcription PCR analysis 146 5.1.2 Inverse PCR as a method of acquiring homeobox flanking sequences 146 5.2 Methods 149 5.2.1 Identification of developmental genes expressed in C. helgolandicus eggs via reverse transcription PCR analysis 149 5.2.2 Inverse PCR analysis of homeobox flanking sequences 151 5.3 Results 157 5.3.1 Reverse transcription-PeR analysis of developmental genes expressed in C. helgolandicus eggs 157 5.3.2 Inverse PCR analysis of homeobox flanking sequences 161 5.4 Discussion 173 CHAPTER6 Development of a molecular technique to distinguish the identity of Calanus species at any developmental stage 182 6.1 Introduction 185 6.1.1 Why distinguish between the North Atlantic Calanus species? 185 6.1.2 Why use genetic characters for taxonomic discrimination? 186 vii 6.1.3 Genetic strategies for species identification 187 6.2 Methods 191 6.2.1 Sample collection and preservation 191 6.2.2 Development of a Restriction Fragment Length Polymorphism (RFLP) technique to distinguish between Calanus species 191 6.2.3 Extension of the technique for use on individual body parts and at any developmental stage 193 6.3 Results 194 6.3.1 Sample preservation 194 6.3.2 Development of a Restriction Fragment Length Polymorphism (RFLP) technique to distinguish between Calanus species 194 6.3.3 Extension of the technique for use on individual body parts and at any developmental