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Novel DNA methods and instrumentation for species monitoring in fisheries

Hansen, Brian Klitgaard

Publication date:2019

Document VersionPublisher's PDF, also known as Version of record

Link back to DTU Orbit

Citation (APA):Hansen, B. K. (2019). Novel DNA methods and instrumentation for species monitoring in fisheries. TechnicalUniversity of Denmark.

Novel DNA methods and instrumentation for species monitoring in fisheries

PhD Thesis

Brian Klitgaard Hansen

DTU Aqua National Institute of Aquatic Resources

Novel DNA methods and instrumentation for species monitoring in fisheries

PhD thesis by

Brian Klitgaard Hansen

March 2019

National Institute of Aquatic Resources

Section for Marine Living Resources

Technical University of Denmark

Cover photo: Final visual inspection of the 2G ESP prior to a subsurface deployment off the coast of

San Diego, USA. Photo kindly provided with permission from the Monterey Bay Aquarium Research

Institute.

Main supervisor:

Professor Einar Eg Nielsen

National Institute of Aquatic Resources

Section for Marine Living Resources

Technical University of Denmark

Co-supervisor:

Senior Researcher Dorte Bekkevold

National Institute of Aquatic Resources

Section for Marine Living Resources

Technical University of Denmark

Evaluation panel:

Senior Researcher Naiara Rodríguez-Ezpeleta

Marine Research Division

AZTI, Spain

Lecturer Jens Carlsson

School of Biology and Environmental Science

University College Dublin, Ireland

Senior Researcher Jacob Hemmer-Hansen

National Institute of Aquatic Resources

Technical University of Denmark

Table of Contents

Preface i

Thesis summary iii

Dansk resumé v

Chapter I 1

1. Introduction 2

2. Identification of fish species 5

3. Methodology for analyzing complex samples 13

4. Devices for field applications 26

5. Summary of studies and discussion of the results 32

Chapter II 49 The sceptical optimist: challenges and perspectives for the application of

environmental DNA in marine fisheries

Chapter III 69

Remote, autonomous real-time monitoring of environmental DNA from commercial fish

Chapter IV 91

From DNA to biomass: species quantification of bulk fisheries products

Chapter V 135

Possible Uses of Genetic Methods in Fisheries Under the EU Landing Obligation

Other contributions 157

Preface

This thesis was submitted to the DTU Aqua PhD School at the Technical University of Denmark for

partial fulfillment of the requirements for the Doctor of Philosophy Degree (PhD). The thesis consists

of a general introduction (chapter I) and four original manuscripts (chapter II-V). The PhD was

conducted from October 2015 to March 2019. My PhD education has mainly taken place at DTU

Aqua in Silkeborg, but also included an external research stay at Monterey Bay Aquarium Research

Institute (Moss landing, California, USA), where I received training in the using the 2G ESP from

February to May 2017. Funding for this PhD was provided by DTU Aqua, the Danish agrifish agency

green development and demonstrations programme (GUDP-VIND), European Union’s Horizon 2020

research and innovation programmes, AtlantOS (grant no. 633211) and DiscardLess (grant no.

633680), as well as the Aage V. Jensen Foundation. My external research stay was funded by the

Civil engineer Frans Allings foundation and the Augustinus foundation.

The overarching goal of this thesis was to examine the applicability of modern DNA based methods

and instrumentation for detection and quantification of fish species in complex (mixed) samples

relevant to fisheries monitoring and surveillance. The research conducted had a strong emphasis on

methodological challenges and opportunities for DNA based tools as monitoring and surveillance

tools in relation to marine fisheries management. The completion of this PhD thesis has only been

possible through the invaluable help and collaboration from numerous inspiring people in Denmark

and abroad that I would like to thank.

First, I would like to thank my supervisors Einar Eg Nielsen and Dorte Bekkevold for their help during

my PhD and for giving me this unique opportunity. I thank them for their dedication, insightful

comments and valuable discussions and for always having their door open. They believed in me,

even when I did not.

Life in Silkeborg would not have been so joyful without my fellow students and colleagues with whom

I have been privileged to work beside. Many thanks to Jacob Hemmer-Hansen, Dorte Meldrup, Britta

i

Sønderskov Pedersen, Maj-Britt Jacobsen, Trine Rohde, Camilla From Kristensen, Nikita Susan

Hartvig Nielsen and Karen-Lise Dons Mensberg for insightful suggestions and support in the

laboratory. I am also grateful to have worked with technicians Jes Dolby and Mikkel Skafte, who

provided enthusiastic and specialized expertise when the ESP required a specific solution. I thank

Magnus Wulf Jacobsen, for always staying positive and laugh with me, especially in times when

there definitely was nothing to laugh about (ESP). Further, to all the people working at DTU Aqua

who helped me in all sorts of peculiar ways or just dedicated some time to chat - thank you. I also

thank the numerous supportive and encouraging collaborative project partners in DiscardLess,

AtlantOS, GUDP-VIND and MONIS.

Further, I would like to dedicate a special thanks to Christina Preston, Brent Roman, Roman Marin

III, Scott Jensen, Kevan Yamahara, Doug Pargett, Christopher A. Scholin and James M. Birch at the

Monterey Bay Aquarium Research Institute (MBARI) for their endless patience and dedication to

make the ESP project succeed. I would also like to express my sincere and deepest gratitude for

giving me the opportunity to work in a truly unique and inspiring research environment.

Finally, I thank my ever-encouraging family and friends, who have been extraordinarily patient and

supportive of me during the last three years. Last, but definitely not least, the outcome of this PhD

would not have been possible without the sacrifices and unconditional support from my partner Anne.

You are my anchor when seas are rough and my wind when nothing is moving. Words cannot justify

how deeply grateful I am to have you by my side.

Silkeborg, March 2019

Brian Klitgaard Hansen

ii

Thesis summary

Marine fish play a vital role for global food biosecurity and economy, whose continued productivity

necessitates proper fisheries management. Successful management relies on the ability to

accurately determine and monitor how many fish can be sustainably caught and subsequently

ensure correct reporting of catches through fisheries surveillance. However, current monitoring of

marine fish is expensive and limited to annual surveys, if conducted at all. Moreover, current fisheries

surveillance is reliant on visual assessment of the catch, which make advantageous onboard bulk

storage and processing methods illegal. Thus, tools to improve current monitoring and fisheries

surveillance will benefit both commercial fisheries, management and conservation of marine fish

stocks.

This present thesis examines novel genetic methods and instruments as an alternative approach or

supplement to established fisheries monitoring and surveillance techniques. Utilizing advanced

genetic instrumentation and methodologies, the overarching goals of this PhD were to (1) examine

the current knowledge, challenges and perspectives for using modern DNA monitoring and

surveillance techniques in applications relevant to fisheries, (2) test a fully automated 2. Generation

Environmental Sample Processor (2G ESP) for in-situ environmental DNA (eDNA) analysis, and (3)

assess the ability of DNA based methods to identify and quantify species composition in complex

bulk fisheries products.

The thesis opens with a general introduction briefly describing the history and present state of

genetic applications of relevance to species monitoring and surveillance in fisheries management

(chapter I). The introduction extends to review relevant knowledge, contemporary instrumentation

and methodological issues in order to put the content of the thesis into an overarching state of the

art research context. Hereafter, four manuscripts are provided in chapter II-V. In chapter II we review

the wealth studies which have revealed great potential but also challenges related to the use of

eDNA in marine fish monitoring. In chapter III we demonstrate that a 2G ESP has the ability to

iii

perform autonomous, remote in situ eDNA analysis for real-time monitoring of fish. Further, in

chapter IV we show how DNA based methods can detect and quantify proportions of marine fish

from complex tissue mixtures in bulk fisheries products, such as fish silage and frozen fish blocks.

Finally, chapter V describes and discusses the potential of genetic methods in relation to the

European landing obligation in order to prevent, assess and control the catch of non-targeted

species.

Overall, this thesis presents the immense potential of modern genetics and the rapidly developing

applications in marine fisheries, but also provides critical insights to strengths and weaknesses

related to various methods and applications. Further, the thesis highlights the potential of using novel

methodologies and instrumentation applicable to fisheries monitoring and surveillance. Of particular

note, chapter III provides the first example of a fully autonomous eDNA analysis using the 2G ESP,

a finding which has broad perspectives for future monitoring in remote and inaccessible marine

areas.

iv

Dansk resumé

Marine fiskebestande spiller en vital rolle for den globale fødevaresikkerhed og økonomi, og deres

fortsatte produktivitet nødvendiggør en passende fiskeriforvaltning. Succesfuld forvaltning afhænger

blandt andet af evnen til at bestemme hvor mange fisk der er af en given art, hvor mange det er

bæredygtigt at fange og sikring af at fangster bliver afrapporteret korrekt gennem fiskerimonitering.

Den nuværende monitering af marine fisk er bekostelig og begrænset til årlige undersøgelser, hvis

den overhovedet bliver udført. Moniteringen er betinget af visuel identifikation af de landede fisk,

hvilket gør landing af stærkt forarbejdet fisk, hvor arten ikke længere er kan identificeres, ulovligt.

Dette betyder at visse ”masse” produkter, som ellers kunne være fordelagtige i forhold til håndtering

og opbevaring ikke kan fremstilles. Derfor vil nye redskaber til forbedring af den nuværende

monitering, kontrol og overvågning både gavne fiskerierhvervet, fiskeriforvaltningen og bidrage til

bevarelse af sunde fiskebestande.

Den aktuelle afhandling undersøger nye genetiske metoder og instrumenter som et alternativ eller

supplement til de etablerede fiskerimonitering og overvågnings teknikker. Ved hjælp af avancerede

DNA baserede instrumenter og metoder har de overordnede mål med denne afhandling være at: (1)

gennemgå og give en status for den nuværende viden, herunder udfordringer og perspektiver, for at

anvende moderne DNA moniterings og overvågnings teknikker i formål, der er fiskeri-relevante, (2)

afprøve en fuldt automatiseret 2. Generation Environmental Sample Processor (2G ESP) til analyse

af miljøDNA (mDNA) i havet og (3) vurdere evnen af DNA-baserede metoder til at identificere og

kvantificere artssammensætningen i komplekse fiskeprodukter, såsom fiske-ensilage og frosne

fiskeblokke.

Afhandlingen indledes med en generel introduktion, der beskrivelser den historiske udvikling og

nuværende status for anvendelse af genetiske metoder til monitering og overvågning i

fiskeriforvaltning (kapitel I). Introduktionen gennemgår relevant viden, moderne instrumentering og

metodiske problemstillinger, samt sætter afhandlingen i en overordnet forskningskontekst. Herefter

v

foreligger fire manuskripter, kapitel II-IV. I kapitel II gennemgår vi den store mængde af studier, der

har påvist et stort potentiale, men også mange udfordringer i forbindelse med anvendelsen af mDNA

i fiskerimonitering. I kapitel III demonstrerer vi, at en 2G ESP kan udføre fjernstyrede, autonome in

situ mDNA analyser til realtidsmonitering af fisk. Kapitel IV beskriver, hvordan vi via DNA-baserede

metoder kan detektere og kvantificere proportioner af marine fiskearter i komplekse fiskeprodukter,

såsom ensilage og fiskeblokke. Til slut, kapitel V, beskriver og diskuterer vi potentialet for genetiske

metoder i relation til den Europæiske landings forpligtigelse for at forebygge, vurdere og kontrollere

fangster af fisk med lav værdi.

Samlet set, bidrager denne afhandling med et indblik i det enorme potentiale for moderne genetiske

metoder og deres mange og hurtigt voksende anvendelsesmuligheder i forbindelse med marint

fiskeri, men giver også kritisk indsigt i styrker og svagheder relateret til forskellige metoder og

anvendelser. Endvidere fremhæver afhandlingen potentialet for brugen af nye instrumenter, der kan

anvendes til fiskeri monitering og overvågning. Specielt giver kapitel III det første eksempel på en

fuldstændig fjernstyret, autonom mDNA analyse ved hjælp af en 2G ESP, et forskningsresultat der

har brede perspektiver for fremtidig monitering i fjerntliggende og utilgængelige marine områder.

vi

Chapter I

General introduction

1

1. Introduction

‘Counting fish is like counting trees, except that they are invisible and keep moving’

(John Sheperd, University of South Hampton)

Genetics has assisted management of fisheries and conservation efforts for over half a century

(Allendorf, F., Ryman, N., Utter, 1987; Ward, 2000). During this time, fisheries management and

genetics have changed considerably. The number of questions considered by the managers have

increased substantially, so besides traditional single species assessment and management, a more

comprehensive approach is warranted, where managers need to consider additional issues such as

ecosystem effect of fishing as well as monitoring, control and surveillance (MCS) of illegal fishing

practices (Ovenden et al., 2015). Similarly, genetics has undergone a rapid revolution driven forward

by advances in the biomedical sector. This has spiraled into many applications in fisheries

management including assessment of fisheries stock structure, evolutionary responses to fishing,

monitoring of species using environmental DNA (eDNA) and postharvest species identification

(Ovenden et al., 2015). However, despite the diversity of applications, they are presently only utilized

to a low extent in relation to fisheries management. In this light, there is a need to refine and reassess

ways in which genetics can find broader utilization. Thus, developing, assessing and implementing

new genetic tools and instruments in management of fishing practices and marine monitoring may

contribute to achieving improved sustainability and utilization of our marine resources and the

surrounding marine environment (Ward, 2000; Casey et al., 2016).

1.1. Aim

Responsible marine management and policy require a steady flow of information on the ecology of

fish in their environment and harvest from commercial fisheries, but monitoring ecological variation

2

and monitoring catches are difficult and expensive (Biber, 2011). DNA based approaches offer a

promising path forward for providing more flexibility regarding the types of samples which are

applicable for species identification (Dormontt et al., 2018). Hence, the development of DNA based

approaches are moving from traditional barcoding of tissue samples from well-defined individual

specimen to highly complex samples, where many species are potentially contributing, such as

water, gut, feces and processed mixed fish products (Nagase et al., 2010; Pompanon et al., 2012).

The present thesis focuses on the development of novel genetic methodologies and instruments and

how they can be applied to species identification and quantification in the context of marine fisheries

management. Specifically, the thesis centers on two areas within this field: 1) Application of eDNA

in monitoring of commercial fish species and 2) Identification and quantification of species in

processed mixed tissue fish samples for Monitoring, Control and Surveillance (MCS) of fishing

practices under the European Union’s (EU) landing obligation. The two areas of research have many

parallels in their methodology. Still, the processed products (silage, frozen blocks, surimi, etc.)

consist primarily of tissue, with an expected high number of DNA copies, while the eDNA

methodology utilizes environmental sampling (water), where the number of DNA copies generally

are very low. Thus, often the challenges are somewhat different, with a higher attention towards the

ability to detect species in eDNA analysis and more focus on quantification for mixed tissue samples.

However, working on both types of analysis can provide synergistic benefits as controlled

experiments with mixed tissue can serve as a very good evaluation of the reliability of quantification

in eDNA studies, as the tissue input proportions are known.

Overall, the primary objectives of the thesis have been to:

• Review and evaluate the current knowledge and challenges of eDNA methodology and

applications specifically aimed towards marine fisheries with emphasis on the current

monitoring and management paradigm (chapter II).

3

• Modify, test and evaluate the potential use of a novel and fully automated “ecogenomic

sensor”, the 2. Generation Environmental Sample Processor (2G ESP), for conducting in

situ genetic analysis and archiving of eDNA (chapter III).

• Develop and assess qPCR and a MinION analysis of DNA from processed mix-species

products (silage & a frozen fish block) for detection and quantification of fish species content

(chapter IV).

• Suggest and evaluate the potential for the application of genetic methods under the

European landing obligation, to prevent unwanted catches, identify catches and improve

MCS of mixed bulk products from unwanted catches (chapter V).

In section 2 of this thesis, I will briefly present how species identification is currently conducted. It

will discuss the application and advantages of modern genetic approaches, ranging from analysis of

simple single species samples to complex multispecies samples, as well as their applications into

the current management and policy framework. Section 3, first provides an introduction to the

ecology and biology of DNA in the environment and presents the current progress and knowledge

gaps in this field of research. Hereafter, the thesis outlines the methodology and technology related

to analyses of both natural and artificial complex mixed species samples and discusses the

associated progress, challenges and perspectives of different analytical approaches and methods.

In section 4, I present the current state-of-the-art of field-based instrumentation and its present

progress for in situ sample collection, archiving and analysis. Hereafter, section 5 provides a

summarized overview of the results and insights obtained in this thesis and concludes on the likely

implications for future research within the field. Finally, the progress of the research is evaluated in

relation to current and future marine fisheries management perspectives. Combined, these sections

lay out the framework for the research conducted and put the main findings of the four original

manuscripts (chapter II, III, IV and V) into a general context and research perspective.

4

2. Identification of fish species

Many aspects of fisheries management rely on accurate species identification of harvested marine

organisms. Fish caught need to be identified to maintain precise and accurate records of harvest to

inform management. Similarly, established fisheries surveys obtain information on distributions,

abundance, size and fecundity, which feeds into assessment models and even elaborate ecosystem

models. Their data provide vital information to management, which critically depends on the

accuracy and precision of species identification, which holds similar importance for post-harvest

MCS purposes, for correct labeling to determine product substitution fraud and ensure food safety

(Rasmussen et al., 2009; Aranceta-Garza et al., 2011). Hence, accurate and precise species

identification is fundamental for optimal resource management across the whole fisheries sector.

2.1. Morphological species identification

The majority of species identifications conducted today is based on visual inspection of

morphological characteristics from whole specimen. However, currently a significant proportion of

taxonomic identification of fish from surveys or for post-harvest control is conducted by relatively

untrained personnel often with limited taxonomic expertise beyond the most common or

morphological distinct species (Hopkins and Freckleton, 2002; Fischer, 2013). Hence, in surveys it

is common that biological species are simply pooled under a single name, e.g. sand eel (Ammodytes

spp.), or even species from several higher taxonomic groups are placed under a single common

name, e.g. gobies (Gobiidae spp.). However, proper taxonomic identification and validation of

uncommon species are time consuming and there is a general trend that taxonomic expertise is in

decline (Hopkins and Freckleton, 2002). Further, the systematic complexity of the marine organisms

with numerous cryptic and sibling species and the lack of diagnostic characters for especially larval

stages of fish, constitute important impediments to confident visual identification (Daan, 2001;

Bucklin et al., 2016; Djurhuus et al., 2018). Further, visual identification is also reliant on the

5

specimens being fresh, whole and otherwise with relatively intact external features for best precision

and accuracy. Combined, these challenges have narrowed the use of visual taxonomic identification

and caused species to be misidentified or not identified at all significantly biasing valuable databases

(Daan, 2001; Fischer, 2013). However, in the past two decades, new synergies between taxonomists

and geneticists have emerged, most notably illustrated by the advent and implementation of the so-

called DNA barcoding (hereafter barcoding) based on traditional Sanger sequencing (Arnot et al.,

1993; Hebert et al., 2003). The concept, which later leads to barcoding, originated in 1993 from Arnot

and colleagues (1993) who developed a method to identify species using DNA. The method

exploited the natural diversity among DNA sequences originating from different species. 10 years

later the methodology was standardized and advanced further by Herbert and colleagues (2003) to

what today is known as barcoding.

2.2. Single species DNA barcoding

All organisms contain DNA and hence even when morphological characteristics are absent, DNA

can be extracted and analyzed to infer species identity (Galal-Khallaf et al., 2016b). The process of

inferring species identity, DNA barcoding, can be divided into three basic steps: (1) DNA extraction,

(2) PCR, (3) sequencing and analysis. (1) The process of isolating DNA is the process known as

DNA extraction. The most common sample sources used from fish are muscle, fin or gill tissue. DNA

extraction is commonly conducted using standardized protocols, which utilize both physical and

chemical methodologies to purify the DNA. (2) Hereafter, a short standardized DNA fragment of

mitochondrial DNA (mtDNA) from a specific gene region is amplified using PCR. The most commonly

designated DNA barcode for animal species identification is the >650 bp fragment of the cytochrome

c oxidase subunit 1, but any gene region (mtDNA or nuclear DNA) can be used provided it is

diagnostic (Hebert et al., 2003). However, in some cases samples can suffer from DNA degradation,

causing long intact DNA fragments to be absent (Piskata et al., 2017), preventing analysis of full-

length (i.e. ~650bp) barcodes. Accordingly, some studies have been focusing on the development

6

of short barcodes (e.g. 100-200 bp), which increases the likelihood of obtaining PCR amplification

of DNA templates from the study material, but has the downside of often limiting the taxonomic

resolution among closely related species (Shokralla et al., 2015; Thomsen et al., 2016). (3) Once

the target DNA segment has been amplified, the fragments can be sequenced, traditionally using a

Sanger sequencing platform. The obtained DNA sequence is subsequently compared to a database

with reference DNA sequences. If a robust match is made, the identity of the sample specimen can

be determined (Hajibabaei et al., 2011). However, if a match cannot be inferred, the sample likely

belongs to a species which remains undescribed or is simply not represented in the DNA sequence

database (Thomsen and Willerslev, 2014). Species with missing reference DNA sequence

information continues to pose a knowledge gap, at least for the nearest future, impairing the

usefulness of DNA barcoding databases at lower taxonomic levels (species or genus) (see section

3.3) (Kvist, 2013). Although fish species, in general, are well represented in public databases, there

are still many, even very important socio-economical, fish species, such as species within the genus

of sandeels (Ammodytes spp.), where limited barcoding information is available (Pinnegar et al.,

2016). Hence, the precision and accuracy of barcoding, for inferring species identity, rests crucially

on the comprehensiveness and validity of reference DNA databases. FISH-BOL (www.fishbol.org)

and Genbank (www.ncbi.nlm.nih.gov/genbank) are two widely used public DNA sequence reference

databases, also for fish. Genbank is a repository for all available DNA sequences regardless of

taxon, whereas FISH-BOL focuses only on DNA sequences from the world’s fish species. FISH-

BOL is part of the larger International Consortium of the Barcode of Life (www.ibol.org) and provides

some level of quality assurance for the publicly available DNA sequences. Thus, it covers fewer, yet

better validated, fish species than Genbank. Accordingly, for DNA barcoding of unknown

specimen, there can be a trade-off between having a complete reference database or having an

incomplete but well-validated reference DNA database. Reliance on DNA sequence data which

does not originate from a well-curated and vouchered specimen pose risks of incorrect

assignment, such as unvouchered specimen DNA sequences from Genbank (Smith et al., 2016;

Balakirev et al., 2017; Li et al., 2018).

7

A recent study on fish DNA sequences on a widely used barcoding gene (Cytochrome b; Cytb) found

evidence for potential errors in 3.7% of the available DNA sequences from Genbank (Li et al., 2018).

2.3. DNA based identification in complex samples

Sanger sequencing produces a single “consensus” sequence from a sample of DNA. Therefore, the

method only has the ability to infer species identification from well preserved single

specimen/species samples and hence is inadequate for providing species inferences for complex

multispecies samples (Shokralla et al., 2014). However, for a number of fisheries applications

samples consist of tissue originating from many different species where individuals are not easily

distinguishable or morphological identifiable. Therefore, for samples containing species mixtures,

alternative analytical approaches have to be applied in order to identify and potentially quantify the

included species. These include quantitative real-time PCR (qPCR) and metabarcoding (see section

3.3). Studies from both qPCR and metabarcoding have been shown to have numerous applications

of importance to fisheries monitoring and management. It has been demonstrated that these

methods enable cost-efficient, reliable and sensitive assessments for, in theory, an infinite number

of species per sample (Taberlet et al., 2012a; Ji et al., 2013; Miya et al., 2015). Further, combined

with advances in DNA extraction methods allow DNA to be obtained from virtually any state of

degradation (i.e. digested or processed) or substance (i.e., liquid, solid or gas). The only prerequisite

for a successful analysis is that the DNA is relatively intact (Pompanon et al., 2012; Port et al., 2015;

Shokralla et al., 2015). Mixed species samples can be divided into two broad categories consisting

of environmental samples (i.e. water, air or sediment), and mixed tissue samples. In marine research

complex samples of eDNA obtained from water samples have already provided key insights into

marine ecosystem diversity, virtually across the tree of life (e.g. Thomsen et al., 2012; Miya et al.,

2015; Stat et al., 2017). The term eDNA emerged to describe methods where extra organismal

genetic material (i.e., feces, sloughed cells, gametes and other particles) is obtained through

collection of environmental samples and used to infer species presence or absence, rather than by

8

capturing and visually identifying the whole organism. The first use of eDNA methodology was

Ficetola and colleagues (2008), who detected eDNA from the invasive American bullfrog (Rana

catesbeiana) in natural ponds in France (Ficetola et al., 2008). Since then eDNA methodology has

revolutionized the field of modern ecological surveys of aquatic macro-organisms in both fresh (e.g.

Sigsgaard et al., 2014; Tréguier et al., 2014; Lacoursière-Roussel et al., 2016) and marine

environments (e.g. Port et al., 2015; Andruszkiewicz et al., 2017a; Sigsgaard et al., 2017). For

example, Djurhuus and colleagues (2018) used DNA from marine water samples to examine

zooplankton community structure. As zooplankton species composition and availability substantially

influence fish stock productivity, knowledge of zooplankton from DNA provide important biological

indices of importance for stock assessment and fisheries management (e.g. Beaugrand et al., 2003;

Capuzzo et al., 2018; Djurhuus et al., 2018). The general ease and simplicity of obtaining water

samples as a source of eDNA particles has many perspectives related to fisheries monitoring. There

is currently rapid development, in terms of technical optimization to improve and optimize the existing

methodology, through finding better, more sensitive and cost-efficient ways to obtain and process

samples (e.g. Lacoursière-Roussel et al., 2016; Spens et al., 2017; Majaneva et al., 2018). However,

there is also an immense focus on widening the scope of applications and assessing which biological

inferences can be reliably drawn from biological molecules (chapter II; see section 3.4).

Mixed tissue samples of relevance for fisheries applications can be subdivided into two major

categories; diet analysis (i.e., analysis of stomach contents and feces) and artificially mixed tissue

samples (i.e., processed fish products). Diet analysis plays an important role in fisheries assessment

as it provides vital information on predator-prey interactions and food competition. Presently, macro-

or microscopic identification of prey items found in stomachs represent state-of-the-art for revealing

diet composition. However, partial or full digestion of prey items often precludes unambiguous

identification; causing some prey items to be underestimated or not recorded at all (Berry et al.,

2015; Buckland et al., 2017). Recent studies have shown that DNA based approaches can

outperform traditional methods (Berry et al., 2015; Riccioni et al., 2018). For example, Riccioni and

9

colleagues (2018) studied gut contents of European hake, a commercially important fish species,

and showed that metabarcoding provided extended insights into dietary richness and composition

among different hake size classes. Metabarcoding consistently detected a wider spectrum of prey

species as opposed to visual examination, which allowed characterization of feeding strategy for

different size groups at an unprecedented taxonomic resolution.

Illegal, unregulated, unreported fishing and substitution fraud along the fish supply chain is

recognized as a serious impediment to sustainable fisheries (Ogden, 2008). Processed fish products

are particularly susceptible to fraudulent labeling, where highly valuable species may be substituted,

partially or entirely, with cheaper ones (Ogden, 2008; Filonzi et al., 2010). Identification of species

or individuals in processed fish products is most often impossible with visual methods. In contrast,

genetic methods have proven suitable for determining the species identity of a wide array of food

products. These include caviar (Boscari et al., 2014), packaged frozen products (Di Pinto et al.,

2016), fillets (Di Pinto et al., 2015), dried products (Wen et al., 2015), surimi (Galal-Khallaf, Ardura,

Borrell & Garcia-Vazquez et al., 2016b; Giusti et al., 2017) and frozen fish blocks (chapter IV) or

silage (chapter IV). Moreover, the ability to identify species of “industrial” fish used within fishmeal

and other products is particularly important as ~10% of the total fish harvested from the wild ends

up as highly processed products (e.g. fishmeal and fish oil) without distinguishable morphological

characters allowing visual identification (Galal-Khallaf et al., 2016a). For example, Galal-Khallaf and

colleagues (2016a) found that half of the species found in fishmeal samples were considered

overexploited.

2.4. Qualitative and quantitative DNA based tools in fisheries management

Assessment of species composition using eDNA and mixed tissue methodologies are relatively new

applications within fisheries. The two methods have evolved in parallel and are linked through

comparable analytical methodology and instrumentation. The field of eDNA and mixed tissue

10

analysis develops rapidly through expansion of the number of applications thereby improving the

existing scientific foundation for marine resource management (chapter II). Recently, these

approaches have also shown the potential for providing quantitative information on species

composition by using DNA as a proxy for abundance/biomass in either absolute (Fukaya et al., 2018)

or relative quantities (e.g. Kelly et al., 2014) (discussed in chapter II & IV). Quantification of fish

biomass is a key component in many aspects of current fisheries assessment and management.

Hence, the quantitative dimension of DNA analysis of complex samples provides many incentives

for testing eDNA and mixed tissue analysis for implementation in fisheries MCS, which also have

been the motivation for chapter II and IV.

2.4.1. eDNA analysis in fisheries monitoring and stock assessment

In 2012, Thomsen and colleagues demonstrated the first use of particles containing eDNA for

monitoring of fish in a marine setting. Since then, there has been substantial scientific development

within the field, demonstrating advantages of eDNA methodology in comparison to established

methods, the most prominent being cost-efficiency, taxonomic range and sensitivity (e.g. Sigsgaard

et al., 2014; Port et al., 2015; Stat et al., 2018). Within the EU, the Common Fisheries Policy

(European Commission, 2013) and Marine Strategy Framework Directive (MSFD) (European

Commission, 2008), sets the regulatory framework for ensuring sustainable exploitation of marine

living resources, hereunder monitoring and assessment of commercial fish species. While there is a

consensus that eDNA analysis can provide information regarding many issues of relevance to

management and conservation of marine living resources, the focus of the scientific community is

mainly on technological progress rather than on policy and management needs. This has hampered

its effective integration into fisheries management, by widening gaps between science and policy,

governance and management. However, initiatives have emerged under the MSFD, such as utilizing

the eDNA methodology to monitor invasive species (Andersen et al., 2016, 2017), which are more

focused on the end users. For more complex management tasks, such as for stock assessment, the

11

eDNA methodology still has many limitations, but with the present technological status and

management paradigm, it can serve as a complementary tool as discussed in detail in chapter II.

However, in this context studies have started to emerge, which combine eDNA data with

sophisticated computational models to infer both absolute abundance estimates of marine fish

(Fukaya et al., 2018) and ecological quality status through the use of supervised machine learning

(Cordier et al., 2017).

2.4.2. Mixed complex sample analysis in monitoring, surveillance and control

Registration of landings and labeling of seafood throughout the production chain is implemented to

meet regulations regarding food safety and mitigation of fraud. In this context, the EU Commission

Regulation no. 2065/2001 has established detailed rules for labeling of seafood products. However,

tools are required for MCS purposes, which can identify fish species in products. This applies to

products anywhere in the production line from whole fish onboard fishing vessels to highly processed

fish dishes in a restaurant (Ogden, 2008). Especially after the implementation of the landing

obligation under the remit of the Common Fisheries Policy (European Commission, 2013), aiming at

eliminating the discarding of unprofitable fish. MCS is not only important for the prevention of

adulteration, but also for the sustainable management and conservation of overexploited and/or

endangered marine resources (Bunholi et al., 2018). DNA based methodologies have become

increasingly popular and valued for fraud identification providing information on both species and

point-of-origin/population (Nielsen et al., 2012; Helyar et al., 2014). However, also quantitative

estimates, either relative or absolute, are of importance regarding MCS. For example, according to

EU regulation, a prepared fish dish named “vegetables with hake” has to contain at least 8% hake

by weight of the total product (European Commission, 2006). Currently, there are no alternatives to

DNA quantification for assessing species content in products without morphological characteristics.

Some quantitative studies of multispecies fish products have shown good correlations between

12

species-specific DNA and weight content, but this area of research is still developing (Nagase et al.,

2010; Sánchez et al., 2019).

3. Methodology for analyzing complex samples

The potential for using analysis of eDNA and mixed species samples is wide reaching, ranging from

questions related to identification of multiple species (e.g. Miya et al., 2015),

biomass/density/proportion estimates (e.g. Nagase et al., 2010), population genetics (e.g. Sigsgaard

et al., 2016) and migration patterns (e.g. Stoeckle et al., 2017) to name a few. A number of different

methods are currently used in the steps from sample to result, which will be discussed in the following

sections. However, before embarking on the methodology there is a strong need to discuss the

inherent challenges related to the biology and ecology of DNA, which becomes increasingly

important in relation to analysis of complex samples and when trying to provide complex biological

inferences.

3.1. Biology and ecology of DNA

The aim of developing new methodologies for eDNA and mixed tissue samples is that with time the

tools can be implemented to support decision making by authorities. For example, by providing

relevant advice on fisheries quotas or used as evidence in court regarding fish substitution cases.

Therefore, reliability and resolution of the inferences which can be drawn from these complex

samples are dependent on the ability of DNA quantification to provide an accurate representation of

the species within a sample at any point in time. The quantity and persistence of DNA, or knowledge

hereof, within these complex samples is therefore of critical importance in order to assess contents

within a processed fishery product or assessing biodiversity in the ocean. For example, the DNA

based method provides no signal for the species of interest, does this represent at true signal of lack

of presence? Or is it a false negative, which in turn requires the consideration of two possibilities:

13

Firstly, that the species are in fact present but not detected due to technical limitations of the method,

or secondly, that the species are present but are not detected due to severe degradation of DNA.

Knowledge of how much DNA is present and how long DNA is likely to persist in a given surrounding

is therefore of importance to understand both of these scenarios. These factors can be termed, the

biology and ecology of DNA respectively, where the biology of DNA refers to the factors within an

organism which explains variation in quantities of DNA, whereas the ecology of DNA refers to how

the DNA is affected by the abiotic and biotic factors in the surrounding environment (Strickler et al.,

2015). The biology and ecology of eDNA particles is a central theme of chapter II.

3.1.1. Biology of DNA

Complex organisms consist of trillions of cells, which are diversified into numerous specialized cell

types in the organismal tissue. Tissue can, in general, be divided into two major groups; hard tissue

which consist of bone, tooth, enamel, dentin and cementum and soft tissue, which is muscle,

epithelial, nervous and connective tissue. All cells in these tissue types contain both mtDNA and

nuclear DNA (Hartmann et al., 2011). Current research in eDNA and mixed tissue samples rests on

the relatively unexplored assumption that all organisms and individuals contain approximately the

same concentration of DNA (i.e. DNA copies per gram organism tissue) and hence are directly

comparable (Evans et al., 2015; Floren et al., 2015; Thomas et al., 2016; Thomsen et al., 2016).

Therefore, ignoring factors such as species, size, ontogenetic stage, physiologic state, source tissue

type could potentially influence any inferences drawn based on quantitative assessments of DNA.

Variations in template numbers can occur on three levels: (1) cellular, (2) intraspecific and (3)

interspecific. (1) On the cellular level there are typically many mtDNA copies (~1-10) in each

mitochondrion and many mitochondria in each cell, but only one nucleus and one nuclear genome

(Cole, 2016). Hence, mtDNA has generally been preferred for DNA barcoding as it is easier to obtain.

However, the abundance of mitochondrial genomes varies greatly within and among cell types

(Hartmann et al., 2011; Cole, 2016). For example, Hartmann and colleagues (2011) found that the

14

number of mtDNA copies in different tissue types varied almost a 10 fold for the turquoise killifish

(Nothobranchius furzeri) and that mtDNA copies decreased significantly (more than 44%) with age

for brain, fin, liver, muscle and skin tissue. Finally, the density of cells (i.e. cells per weight unit tissue)

across different tissue types varies considerably, adding another level of complexity (Kozłowski et

al., 2010). (2) Differences in the number of DNA copies among individuals of the same species could

also pose a challenge for quantitative DNA analysis as the composition of tissue types may vary for

individuals within species (Cole, 2016). For example distribution of fat and muscle tissue might be

different among individuals simply due to sex, nutrition, ontogenetic stage etc. (Leaf et al., 2018). (3)

The interspecies variation in DNA content relates to general differences in tissue composition across

species. For example, some species of fish have more and larger fins, containing more DNA than

other tissue types, thus obscuring direct comparisons of DNA copies per weight unit. Until now DNA

based quantification has predominantly focused on finding sufficient intact DNA copies of species-

specific mtDNA segments. Hence, the relatively basic insights regarding tissue composition, cells

types and numbers and associated intra- and interspecies variations highlighted here, remains

largely unexplored in terms of their importance regarding quantitative differences of DNA content for

eDNA and mixed tissue samples. On top of these basic biological differences are the environmental

effects, which subsequent to the DNA release from the organism alters the number of copies present

for detection.

3.1.2. Ecology of DNA

When DNA is released into the environment or when an organism dies, the DNA starts to degrade

(e.g. Thomsen et al., 2012; Merkes et al., 2014). Depending on the surrounding environment the

gradual process of DNA degradation can occur at different speeds, from hours to thousands of years

before degradation affects DNA detectability (Pedersen et al., 2015; Strickler et al., 2015). Several

factors may affect the speed at which this breakdown occurs, including abiotic factors (e.g.

temperature, sunlight, pH, salinity) and biotic factors (e.g. extracellular enzymes and

15

microorganisms) (chapter II; Strickler et al., 2015; Andruszkiewicz et al., 2017b). It is important to

emphasize that these factors are equally important for eDNA and mixed tissue studies and hence

may cause variation to both qualitative (false negatives) and quantitative assessments.

DNA based assessment of tissue composition within processed fish products is not uniform across

product type as they undergo different treatments during processing and subsequent storage (high

temperature, high pressure, acidic treatments, addition of certain preservatives and ingredients,

etc.), which to a large extent influence the integrity of DNA. Piskata and colleagues (2017) found

DNA yield from a variety of unprocessed and processed sample types, i.e. raw, canned, boiled,

smoked, spread and pâté, varied 100 fold between processing types. Further, some processed

products had suffered from severe DNA fragmentation hampering detection of mtDNA fragments

≥300 bp. In contrast, shorter 100bp mtDNA fragments were consistently detected for all samples.

Assuming equal treatment of species to the mixture DNA degradation should not affect relative

proportion estimates among species. However, direct DNA to biomass approximations among

samples would require corrections factors accounting for both storage time and product type.

In comparison, eDNA particles are affected by much more complex and dynamic environmental

factors, which is a main theme of chapter II. Recently, much basic eDNA research has focused on

how individual environmental factors affect eDNA particle preservation. These studies have enabled

a better understanding of the number and magnitude of factors influencing marine eDNA particles.

For example, sunlight (Strickler et al., 2015; Andruszkiewicz et al., 2017b) and bacteria (Barnes et

al., 2014; Tsuji et al., 2017) does not seem to significantly change DNA degradation rates in the

ocean, which was hypothesized in the past. In contrast, temperature is currently considered the most

influential factor related to the rate of eDNA particle decay, with lower temperatures preserving the

eDNA particles longer (Tsuji et al., 2017; Collins et al., 2018). Collins and colleagues (2018)

compared eDNA particle half-time between inshore and offshore samples separated by ~40 km and

found that half-life of eDNA in inshore samples was significantly higher (1.6x) than for offshore

samples. The currently available literature have found the half-life of eDNA particles to range

16

between 0.7 – 332.7 hours (Avg.= 47.33 hours SD±60.60, N=68) and between 6.9-71.1 (Avg.= 27.66

hours SD±17.05, N=23) for marine environments (Collins et al., 2018). In addition, environmental

variation (temperature, oxygen, food availability, etc.) may also affect the eDNA shedding rate of the

source organisms, which in turn is also affected by other factors such as diet or ontogenetic stage

(Klymus et al., 2014; Dunn et al., 2017). Moreover, temperature (Lacoursière-Roussel et al., 2016)

and stress (Pilliod et al., 2014) also seem to affect the shedding of eDNA particles. Generally, there

is limited evidence that a given biomass should result in an uniform amount of eDNA particles being

released across individuals and species. However, Sassoubre and colleagues (2016) recently found

that there was no statistical difference in shedding rates among three marine species, Northern

Anchovy (Engraulis mordax), Pacific Sardine (Sardinops sagax) and Pacific Chub Mackerel

(Scomber japonicas), in single species mesocosms. However, it should be noted that they find highly

variable estimates for individual species with standard deviations approximately equal to or higher

than the mean concentrations. Likewise, a number of illustrative studies of fish maintained under

static environmental conditions have shown that shedding rates can change between a few pg. of

DNA to hundreds of thousands of pg (Klymus et al., 2014; Sassoubre et al., 2016). A major concern

regarding studies of eDNA in open marine systems is the origin of the measured DNA. I.e. is the

eDNA produced locally or represent migration from other areas. Fortunately, most evidence

suggests that eDNA particles found in marine environments are fairly contemporary representation

of the local community, even with many potential factors influencing its production, degradation and

transport (Port et al., 2015; O´Donnell et al., 2017; Stoeckle et al., 2017). However, these

confounding factors may become more important for applications beyond merely determining

occurrences, for example when trying to assess distribution boundaries or to estimate biomass (e.g.

Thomsen et al., 2016; Jo et al., 2017). Recently, Fukaya and colleagues (Fukaya et al., 2018) used

a numerical hydrodynamic model which simulated generation, transport and decay of eDNA particles

on the basis of physical conditions within the study area, essentially simulating the ecology of eDNA

particles, and found abundance estimates to be within one order of magnitude of a hydroacoustic

stock assessment.

17

Ultimately, both DNA biology and ecology play important roles on what and how we assess DNA in

complex samples and can have wide ranging consequences for both qualitative, but especially on

quantitative applications. Combining modeling with corrections for different types of products or

environmental samples, can improve the ability to accurately transfer DNA copy estimates into

abundance and biomass estimates from complex samples. However, besides the biology and

ecology of DNA, there are also more well studied technical challenges influencing the amount of

DNA detected (discussed in section 3.3) (Thomas et al., 2016; Ushio et al., 2018). Previous attempts

to control for biasing factors, i.e. biological, ecological and technical, have primarily focused on using

a single correction factor for each species, accounting for multiple sources of bias simultaneously

that cause certain species to be overrepresented, underrepresented or absent (Thomas et al., 2014,

2016).

3.2. From sample to DNA

A major advantage of eDNA and mixed species samples methodologies is that they can be applied

for all types of samples, provided that the DNA remains detectable. For the mixed tissue samples,

the methods potentially apply to all steps in the supply chain, from whole fish to highly processed

fish products. For eDNA, the methods apply to all aquatic environments (fresh, brackish and marine),

and often enable sampling at sites otherwise restricted or impossible to access with conventional

methods. Last, for applications, the sampling is non-destructive and easy to collect. A small mixed

tissue sample (~30 mg) or small water sample for eDNA (~2 l) will be sufficient for most applications

(Rees et al., 2014). Hence, even non-specialists can collect samples with limited instructions, making

it an obvious tool for citizen science (e.g. Biggs et al., 2014). The methods can be described in three

steps: (1) Sampling, (2) DNA extraction and (3) analysis. Presently there is little consensus or

standardization regarding how these steps should be performed, thus they are often optimized

specifically for each study or type of sample (Rees et al., 2014; Goldberg et al., 2016).

18

For eDNA analysis, water samples are most commonly filtered to remove the water and hence

concentrating the particulate matter (Rees et al., 2014). Presently, several filter types are used

including polycarbonate, glass fiber, cellulose and nylon filters, with a variety of pore sizes from 0.02

– 180µm (Rees et al., 2014; Turner et al., 2014a). Sample collection is done either by direct filtration

or by collecting the sample in a container followed by on-site or laboratory based filtration (e.g. Pilliod

et al., 2013; Sigsgaard et al., 2016; Thomsen et al., 2016). Sample sizes vary substantially from 15

ml to 45 L, but with the most typical sampling sizes around 1-2 l (Foote et al., 2012; Bergman et al.,

2016). Once the sample is collected and filtered, eDNA can be extracted directly from the filter.

Numerous methods have been described, using both commercial and non-commercial kits (e.g.

Turner et al., 2014b; Deiner et al., 2015; Djurhuus et al., 2017). Generally, the eDNA concentration

for target species is low in water samples collected in the field. Thus, the use of optimized protocols,

combining the most optimal filtration type, sample preservation and DNA extraction, can enhance

eDNA recovery. Accordingly, protocol optimization has been subject to significant development in

recent years with significant improvement of detection probability and quantification (e.g.

Lacoursière-Roussel et al., 2016; Spens et al., 2017; Majaneva et al., 2018).

For mixed tissue samples, the potential starting material is diverse, as it can be in both solid (e.g.

minced meat, surimi etc.) and liquid (silage and fish oil) state. In comparison to eDNA analysis, mixed

tissue samples generally do not suffer from insufficient DNA content for basic species detection,

although the yield varies largely within and between sample types (e.g. Piskata et al., 2017). Hence,

mixed tissue sample analysis have not required similar optimization of sampling protocols as eDNA,

and have generally just been analyzed using a generic commercial kits such as QIAamp® DNA Mini

Kit (Qiagen, Germany) (Galal-Khallaf et al., 2016b) or NucleoSpin® Tissue kit (Macherey-Nagel)

(Bunholi et al., 2018). However, since the scope of mixed tissue analysis is expanding to more

complex samples, the adoption of optimized protocols from eDNA literature for extracting DNA from

products consisting of liquids, e.g. as seen in chapter IV, will likely improve yield (Lacoursière-

Roussel et al., 2016; Majaneva et al., 2018).

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3.3. Genetic tools for species identification and quantification

Since the arrival of the Sanger sequencing method, which has been the workhorse of DNA

barcoding, a lot of development and innovation has taken place regarding genetic/genomic

technology (Goodwin et al., 2016; Besser et al., 2018). Most notably, massive parallel sequencing

technologies became commercially available in 2005, which caused all genomic sequencing centers

to shift sequencing method (Shokralla et al., 2012; Goodwin et al., 2016). Along with the

development of sequencing, the applicability of PCR tools, hereunder qPCR, also emerged as an

alternative to sequencing (Evans et al., 2015). Traditional barcoding was optimized for identification

of species in samples with relatively intact DNA. Thus, new more complex barcoding applications,

such as eDNA mixed tissue samples, initially had to develop and modify methodologies to fit

applications involving degraded and low quantity DNA (Taberlet et al., 2012b; Shokralla et al., 2015).

While this initial challenge has largely been overcome, much research is now focusing on the next

frontier of understanding and overcoming the technical challenges of quantitative applications.

3.3.1. PCR, qPCR & ddPCR

Non-sequencing based PCR approaches has played an important role in the implementation of

genetically based species identification in fisheries and have generally been a more accessible

starting point compared to sequencing. PCR based approaches rely on species-specific assays

targeting diagnostic sequence variation in order to exclusively amplify DNA fragments of the species

of interest. The assays are carefully validated in a three step procedure: in silico, in vitro and finally

in situ. First assays are in silico designed using dedicated software, e.g. Geneious (Biomatters Ltd,

New Zealand) or primer-BLAST (www.ncbi.nlm.nih.gov/tools/primer-blast/). Assays are optimized

to contain as many mismatches as possible in the binding sites between target and closely related

non-target species in order to prevent unspecific amplification. Hereafter, assays are in silico

validated by matching assays against all known DNA sequences, to rule out any non-target species,

potentially reacting with the assays. Then the assays are tested in vitro, with DNA extracted from

20

target and non-target sympatric species, for signs of cross-species amplification. Finally, the assays

are tested in situ, preferably with positive as well as negative samples in order to assess their

specificity (Goldberg et al., 2016).

Three different PCR methods have been used for species identification in the past including endpoint

PCR (e.g. Jerde et al., 2011), qPCR (e.g. Bojolly et al., 2017) and digital droplet PCR (ddPCR) (e.g.

Doi et al., 2015). Endpoint PCR amplification utilizes a set of species-specific primers and after

amplification, positive amplification can be visualized using gel electrophoresis. Thus, this method

only provides presence/absence indices (Ficetola et al., 2008; Jerde et al., 2011). qPCR and ddPCR

methods are generally superior to endpoint PCR because they can add more specificity (with the

addition of a probe), sensitivity and can be used for quantification. qPCR use a relative measurement

of fluorescence for quantification of the number of DNA copies in an unknown sample, which are

compared to a standard curve (e.g. Goldberg et al., 2016). In contrast, ddPCR is a direct method

that circumvents the use of standard curves to estimate DNA target abundance. A sample is

randomly partitioned into thousands of discrete droplets of identical size via microfluidics. Droplets

are then thermally cycled and individually screened for fluorescence which is indicative of target DNA

(Doi et al., 2015). Subsequently, absolute quantification can be achieved from the ratio of positive

and negative droplets using Poisson statistics. qPCR and ddPCR have superior quantitative

precision compared to HTS (Ushio et al., 2018). However, there are multiple other benefits of qPCR

and ddPCR in comparison to sequencing approaches. First, both qPCR and ddPCR are generally

very rapid approaches to get from sample to result (<1 hour for some applications) and requires little

computing power or bioinformatics to generate accurate results (Sepulveda et al., 2018). Secondly,

if complex samples only contain few species or if only a few species are targeted, qPCR and ddPCR

will generally be a more cost-efficient, quick and sensitive approach. However, if many and

potentially unknown species are investigated, such as in metabarcoding, then HTS will be a superior

method. Likewise, even for less complex samples, HTS allow for “tagging” and pooling of many

21

samples in a single sequencing run, leading to fast and efficient analysis of many samples

simultaneously.

3.3.2. High-throughput sequencing

HTS is currently changing the way we characterize assemblages of organisms from virtually all

sample types across the tree of life (Zepeda Mendoza et al., 2015; Stat et al., 2017). State-of-the-

art analysis using a mtDNA metabarcoding approach goes through five analytical steps from

extracted DNA to taxonomic assignment of HTS sequences; (1) PCR using “universal”

(metabarcoding) primers, (2) library preparation, (3) sequencing, (4) data pre-processing and (5)

taxonomic assignment (e.g. Evans et al., 2015; Miya et al., 2015; Thomas et al., 2016).

DNA based assessments of a broad selection of species (metabarcoding) have traditionally been

accomplished by the use of so-called universal primers, which allow amplification of particular DNA

segments for all species within a targeted taxonomic grouping (e.g. vertebrates or invertebrates)

(Miya et al., 2015). In principle, the primers aim to fulfill three basic requirements for successful

application to complex samples, which are: (1) ability to amplify a short DNA fragment allowing

detection from degraded DNA, (2) primer binding site should be well conserved across the targeted

group of organisms while flanking diagnostic regions and (3) primers should be specific to the

targeted group. However, these criteria are often problematic to fulfill leading to insufficient

diagnostic differences between closely related species, hence a lower taxonomic resolution than

species level is achieved (e.g. Kelly et al., 2014; Thomsen et al., 2016). This was illustrated in a

study by Kelly and colleagues (2014) who used universal primers targeting vertebrates and found

successful detection in an aquarium tank was dependent on the taxonomic group (e.g. no detection

for all elasmobranchs, green sea turtle(Chelonia mydas) and ocean sunfish (Mola mola)). Further,

some DNA sequences did not include diagnostic variability. Hence, a lower taxonomic ranking

(genus or family) had to be applied. To circumvent this challenge, recent studies have utilized

22

multiple primers to simultaneously improve detection probability and taxonomic resolution (Miya et

al., 2015; Stat et al., 2017). In addition to detection, DNA sequence read abundance is used as

quantitative estimates as a proxy for relative biomass or abundance. However, several biological

(discussed in section 3.1; chapter II; chapter IV) and technical limitations hamper the use of

metabarcoding sequence abundance for quantitative applications (Evans et al., 2015; Thomsen et

al., 2016). Technically, there are challenges related to sequencing (e.g. sequencing errors, data

processing etc.), but especially the use of the initial mtDNA PCR amplification bias quantification.

Although universal primers are designed to bind to conserved regions of mtDNA, sequence variation

of these binding sites affect amplification efficiency among species thereby skewing qualitative

estimates and even leading to failure to detect some taxa (Kelly et al., 2014; Evans et al., 2015).

Other factors, which induce PCR bias, is template secondary structure, suboptimal GC content and

number of PCR cycles (Aird et al., 2011). To mitigate PCR bias, Ushio and colleagues (2018)

implemented an internal DNA standard (i.e. known amounts of non-natural occurring short DNA

fragments) and by using a correction equation they could standardize DNA sequence reads allowing

better comparative estimates. Presently there is no consensus regarding laboratory and data

analysis pipelines for metabarcoding, in order to infer species abundances from complex samples.

While accurate quantitative assessment can vastly improve the practical value of metabarcoding,

PCR-bias seems unavoidable on results of taxonomic recovery, leading to obscure potential

correlations to biomass of target species. Accordingly, new research focus on PCR free approaches

known as direct sequencing.

In contrast to metabarcoding, direct sequencing is not restricted to mtDNA amplicons but directly

sequences all DNA fragments within a sample regardless of the source gene. Hence, direct

sequencing is a more unbiased methodology for obtaining qualitative and quantitative inferences

from complex samples. However, the vast majority of DNA sequences will be uninformative and

predominantly from undesired organisms, such microbes in environmental samples (Stat et al.,

2017). Another factor hampering the application of direct sequencing is the lack of proper reference

23

databases for other than mtDNA based barcoding genes. The inefficiency of the approach was

highlighted by Stat and colleagues (2017) who performed direct sequencing on seawater samples

and found that only ~14% of the 22.3 million reads obtained could be assigned to a taxonomic unit

and only 2.4% assigned to eukaryotes. There are several emerging supplementary methods, such

as hybridization based sequence capture (Zhou et al., 2013; Wilcox et al., 2018) or blocking

oligonucleotides (Vestheim and Jarman, 2008; Boessenkool et al., 2012), which can be implemented

into a direct sequencing pipeline and serve to enrich targets of interest.

In conclusion, technological development of HTS coupled with the large amounts of high-resolution

data, likely makes it the future method for investigating DNA from complex samples, in particular

regarding large scale species assessments. As target species groups do not have to be selected a

priori, as for PCR based methods, HTS allow for direct measurement of total biodiversity (Thomsen

et al., 2012; Miya et al., 2015). Finally, as the cost of sequencing continues to decrease it makes

HTS more competitive regarding cost-effectiveness than other genetic analysis methods as well as

established visual survey methods.

3.4. Novel applications

Biological molecules from complex samples contain much more information than mere species

identification. Emerging applications include population and functional genetics as well as insights

from RNA as an alternative to DNA. Analysis of eDNA and mixed species samples relies almost

exclusively on short (<200bp) segments of mtDNA genes. These genetic markers lack the ability to

distinguish hybrids and have low power for identification of fish populations (stocks) and individuals

(e.g. Kelly et al., 2014; Galal-Khallaf et al., 2016b). Yet, two studies have recently illustrated that

single species haplotype diversity is attainable from water samples (Sigsgaard et al., 2016; Baker et

al., 2018; Parsons et al., 2018). Still, as population genetics is frequency based, there is currently

no way of assessing whether a sample is unbiased or contains unequal numbers of sequences from

24

different individuals. Indeed if sequences from some (close by) individuals occur at a high rate in the

sample, then estimates of haplotype frequencies are likely to be significantly biased. Thus,

population differences could likely be inferred from the occurrence of rare or unique haplotypes

rather than minute frequency differences as used in individual based analysis. To obtain higher

resolution through identification of unique haplotypes long DNA sequences are warranted. A recent

study by Deiner and colleagues (2017) found that whole mitochondrial DNA molecules (16kb) could

be extracted from water samples, contradictory to previous beliefs, illustrating the potential to

improve population specific inferences from eDNA. Long DNA sequences are generally rare in the

marine environment, due to their rapid degradation and dilution (chapter II). Similarly, in mixed tissue

analysis, point-of-origin assignment of multispecies fish products would prove highly valuable to

assess fraud regarding catch area. For fisheries products, longer DNA sequences may generally be

more common due to the higher DNA concentration, but successful analysis will largely depend on

the specific processing and storage (Piskata et al., 2017). For most marine fish, low population

differentiation is an inherent feature of the species (See Ward et al., 1994), but can also be caused

by the choice and resolution of genetic marker. Thus, longer DNA sequences will provide more

population-specific information given that the sequences entail an informative genomic region.

It has been shown that eDNA particles can persist for extended periods (half-life ≤71 hours) of time

under specific environmental conditions in marine environments (Strickler et al., 2015; Collins et al.,

2018). Thus in these environments extracted eDNA may not be representative of the contemporary

biodiversity. Moreover, activities such as bottom trawling, stirring up sediments rich in eDNA, and

fish discards may introduce exogenous DNA of species which are not present in the area (chapter

II; Merkes et al., 2014). As DNA is fragmented (degrade) rapidly in the environment, long DNA

fragments (>800bp) represent “fresh” DNA and thus provide the best spatial and temporal snapshot

of contemporary biodiversity (Jo et al., 2017). An alternative approach is to utilize environmental

RNA (eRNA) which is a much less stable molecule degrading rapidly after cell death, thereby

providing a more accurate spatiotemporal representation of the living organisms within an area

25

(Pochon et al., 2017). Further, the use of eRNA could also reveal functional genomics properties by

providing information on transcribed genes in the wild, similar to studies conducted for prokaryotes

(Gilbert and Dupont, 2011). Thus, eRNA could reveal important adaptive information related to e.g.

health, ontogenetic stage or environment stress (Nielsen and Pavey, 2010). However, eRNA studies

of eukaryotes are hampered by the lack of sufficient reference RNA sequence information with the

ability to distinguish species. Eukaryotes have much larger and more complex genomes, compared

to prokaryotes. Hence, sequencing a sufficient number of eukaryotic transcriptomes has previously

been a cost-prohibitive barrier for studying eukaryotic nuclear DNA/RNA (Gilbert and Dupont, 2011).

Additionally, many applications using the eDNA methodology are struggling with low detection

probability in environmental samples even for DNA and hence using RNA which is less stable will

be challenging (Moyer et al., 2014; Minamoto et al., 2015).

In mixed tissue samples it is not unrealistic that RNA (or DNA) potentially could reveal product quality

or help assess when and where an organism was harvested (died), similar to what forensic scientists

have been using to estimate time since deposition of biological material (Bremmer et al., 2012). A

study conducted by Zhao and colleagues (2018) investigated the changes in DNA yield, purity and

integrity in frozen meat over time. They found a significant positive correlation between beef quality

and DNA yield, illustrating that DNA can function as a predictor of freshness. Combining DNA or

RNA based tools as indicators of freshness, genetics could serve as tools for MCS in fisheries,

potentially revealing where and when the fish was caught (Nielsen et al., 2012). While RNA genetics

potentially would work well for fresh samples, it is unlikely that even slightly processed products

would contain any RNA from macro-organisms due to the unstable nature of the molecule.

4. Devices for field applications

Genetics has provided the means for moving biodiversity surveys from the field into the molecular

laboratory. However, there is a need to take genetics back to the field in order to reduce the time

26

from sampling to results. Presently, the turnaround time can be anything from days to months

depending on the type of analysis. This has sparked a general research drive towards miniaturized

portable instruments and protocols, providing the ability to extract and analyze DNA in the field

(Quick et al., 2016; Faria et al., 2017). For both the mixed tissue samples and eDNA analysis,

instruments which enable field usage would be highly valuable for many applications. For example,

fisheries inspectors carry a huge economic responsibility if they decide to put landings or products

in quarantine because of suspected fraud, which can have severe consequences for the product

value. Performing in situ genetic tests could provide immediate indices of catch or product species

content that could facilitate the decision on how to proceed (chapter IV). Similarly, detailed real-time

detection of invasive species in aquatic environments is important, as their eradication is typically

only feasible before populations become established and while they still have a geographically

limited distribution. The eDNA methodology has already shown great prospects as an early detection

tool, e.g. by detection of invasive species in water samples from ports (Brown et al., 2016; Borrell et

al., 2017). Hence reducing the turnaround time to near real-time will likely result in improved

mitigation success.

For more than a decade, qPCR has been used in various field applications since the method is

relatively well suited for in situ applications due to the relatively limited sample preparation

requirements and stability of assays reagents. Presently, qPCR has been used in applications such

as detection of indicator bacteria for fecal contamination, harmful algal blooms (Yamahara et al.,

2015) and detecting an oak pathogen from foliage and stems (Tomlinson et al., 2005). There are

several fully portable qPCR instruments on the market, including Two3 and three9 (Biomeme Inc.,

USA), Mini8 Plus (Coyote Bioscience USA Inc., USA) and Freedom4 (Ubiquitome, New Zealand),

with similar analytical properties as benchtop instruments. Although qPCR seems to be a much more

suitable technology for in situ assessments at the moment, there are currently large research efforts

related the newly introduced miniaturized 3rd generation sequencing instruments MinION and

SmigdION from Oxford Nanopore Technologies (UK) (Milicchio and Prosperi, 2017). These two

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instruments have literally brought sequencing to the palm of the hand. The MinION, the only

commercially available of the two instruments, measures 10 × 3.2 × 2cm, weighs less than 90g and

provides easy data and power transfer through a USB port (Pomerantz et al., 2018). This device has

already been tested at remote sites to e.g. monitor outbreaks of Ebola in Tanzania (Quick et al.,

2016), Zika virus in Brazil (Faria et al., 2017) as well as in situ barcoding of frogs (Menegon et al.,

2017) and snakes (Pomerantz et al., 2018) in the rainforest. Although we are just seeing the first

glimpse of how in situ sequencing can be utilized, the future possibilities for applying this powerful

technology are immense. For eDNA and mixed tissue samples, especially real-time selective

sequencing seems like a promising path forward (Loose et al., 2016). Real-time selective sequencing

is a tool for controlling which sequences are processed based on a real-time comparison between a

database of targeted sequences and the DNA strand being sequenced. The downside of many of

these in situ methodologies is that they can be difficult to adjust to challenging conditions while in

the field, e.g. related to inhibition, whereas procedures and hardware in conventional laboratories

are more easily adjustable and optimizable (e.g. Jane et al., 2014).

4.1. Deployable instrumentation

Marine fish have wide spatial distributions ranging from the easily accessible nearshore areas to the

remote deep-sea. Monitoring efforts mainly target commercial species and are limited to a frequency

of annual or semiannual surveys, if surveys are conducted at all (ICES, 2012, 2013). Although the

eDNA methodology has proved to be a cost-competitive alternative or supplement for many

monitoring applications, off-shore sample collection remains an inherent challenge due to the

associated costs of ship-time. Currently, water sampling is based on manual sampling from shore or

boat (e.g. Sigsgaard et al., 2017; Yamamoto et al., 2017). The manual sampling prevents the

acquisition of regular eDNA time series in offshore regions, potentially overlooking important

temporal and spatial changes in occurrence and abundance. Hence, for short-term monitoring (days-

months), time series of eDNA data can be of high value (see chapter III). Currently, there are two

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ways of obtaining time series from water samples in marine ecosystems, either by visiting each

sampling station at intervals or remaining at the site for consecutive sampling. Dependent on the

time covered, sampling intervals and economy, one approach might be more advantageous than the

other, but both are economically very costly, in particular for offshore sites. Hence, instruments that

automatically collect in situ samples, can drastically decrease the costs of sampling and potentially

widen temporal insights compared to the brief snapshots which currently characterize marine

monitoring programs.

4.1.1. Automated samples

Advances in remotely operated instruments for offshore monitoring have made high frequency, long-

term data collection possible for a variety of physical (e.g. temperature and currents), chemical (e.g.

salinity, oxygen), optical (e.g. turbidity and chlorophyll) and biological (e.g. plankton composition)

parameters. These oceanographic instruments can be divided into two large groups; “samplers” and

“ecogenomic sensors”. Most of the devices available for biological analysis are samplers, meaning

that they only collect and filter water samples and store the filter onboard with or without a chemical

preservative. Non-preservative type samplers like the Remote Access Sampler (RAS),

PhytoPlankton Sampler (PPS) and Water and Microplankton Sampler (WaMS) do not utilize

preservatives and hence have limited relevance for eDNA research with their current configuration

due to the rapid degradation of eDNA (Strickler et al., 2015; Ottesen, 2016; McQuillan and Robidart,

2017). Preservative samplers protect genetic material from in situ degradation and have previously

been used to study key functional groups of organisms using transcript and protein quantification.

The preservation of DNA, RNA and protein greatly enhances the analytical possibilities after

deployment especially for analyzing eDNA particles. Preservation-type samplers include Biological

OsmoSampling System (BOSS), the Suspended Particulate Rosette (SPR), the Microbial Sampler-

in situ Incubation Device (MS-SID) (McQuillan and Robidart, 2017). Samplers are usually moored to

a fixed position, but they can also be fitted into autonomous surface vehicles (ASV) and autonomous

29

underwater vehicles (AUV) (Bird et al., 2007; Busquets et al., 2015; Pargett et al., 2015). An

alternative approach for collection of water samples for eDNA analysis is by the use of unmanned

aerial vehicles (UAV; drone) (Ore et al., 2015; Doi et al., 2017). Presently, the operation of UAVs are

manual and confined to freshwater and coastal operations. However, UAVs automation will increase

over time improving the applicability to offshore sampling. Currently, there is limited usage of any of

the above-mentioned instruments in eDNA research although they highlight many new perspectives

for expansion of eDNA sampling. The predominant challenges with these instruments are that they

are generally complex to operate, expensive and not particularly designed for decontamination

standards of the eDNA methodology.

4.1.2. Ecogenomic sensors

In addition to samplers are the ‘ecogenomic sensors’. The term covers instruments which

autonomously collect and perform molecular analysis in order to detect and/or quantify specific

organisms (Ottesen, 2016). These instruments remove the traditional time lag from field sampling to

conventional laboratory analysis and mitigate the traditional challenges related to transporting

precious samples (e.g. degradation and contamination). Ecogenomic sensors include instruments

such as the Autonomous Microbial Genosensor (AMG) and the 2G ESP which has been used for

eDNA analysis in relation to this thesis (chapter III). Both the 2G ESP and the AMG are submersible

and provide a suite of core functions such as communication, water sampling, DNA/RNA purification

and different molecular analysis options. The AMG utilizes Nucleic Acid based Sequence

Amplifications enabling RNA based amplification. By utilizing RNA rather than DNA only

transcriptionally active living cells, and not dead cells, will be detected and enumerated. The AMG

is only designed for shorter subsurface deployments, <3 days, where it can collect 12 samples of

30-50 ml of water (Fries et al., 2007; McQuillan and Robidart, 2017). The 2G ESP (fig. 1) has a more

broad suite of in situ molecular analysis options such as rRNA sandwich hybridization (Harvey et al.,

2012), protein ELISA (Doucette et al., 2009) and qPCR (e.g. Preston et al., 2011).

30

Figure 1: Ecogenomic sensors. Left: Schematic illustration of the 2G ESP in a pelagic mooring setup. Top right:

2G ESP (Photo kindly provided with permission from the Monterey Bay Aquarium Research Institute). Bottom

right: Tethys-class long-range AUVs. The AUV to the right carries a 3G ESP.

The qPCR analysis is implemented through the addition of a separate analytical module, a so-called

Micro Fluidic Block (MFB). The MFB utilizes microfluidics and sequential injection to perform solid

phase extraction and qPCR on the processed samples from the 2G ESP (Preston et al., 2011). The

2G ESP can also store filtered samples on board using a chemical preservative for post-deployment

laboratory based analysis. In the past, both meta-transcriptomics (e.g. Ottesen et al., 2014),

proteomics (Saito et al., 2011) and qPCR (chapter II; Preston et al., 2011; Yamahara et al., 2015)

analysis have been conducted on ESP stored filter samples post-deployment. One of the advantages

of the 2G ESP is its versatility. Due to its multiple options for analysis, it can be modified to address

various hypotheses targeting everything from viruses to whales within a single deployment (chapter

31

III; Preston et al., 2011; Yamahara et al., 2015). The 2G ESP can be deployed for up to six months

and has been tested to a depth of 4000 meters (McQuillan and Robidart, 2017). Figure 1 illustrates

the 2G ESP pelagic mooring setup. Further, in contrast to most of the samplers, the 2G ESP uses

bleach after each sampling and analysis (bleach followed by rinsing with DNase free water) on all

surfaces in contact with the samples, thereby conforming to standards for eDNA methodology.

Further, a third generation of the ESP (3G ESP) is currently under development (Pargett et al., 2015).

The 3G ESP is much smaller than its 2nd generation predecessor enabling it to fit into a Tethys-

class long-range AUV, thus essentially making the ESP mobile (figure 1). The AUV has a range of

1800 km and can move freely in the upper water column to a depth of 300 meters. Both the 2G and

3G ESP can be coupled with environmental sensors enabling trigger sampling when a specific

environmental condition is present. Moreover, the 3G ESP is able to conduct “seek, find, and sample”

missions based on user-defined environmental conditions (Pargett et al., 2015).

5. Summary of studies and discussion of the results

The following sections summarize the content and major findings of each of the four manuscripts in

this thesis (chapter II-V) and discuss how the combined conclusions contribute to an advanced

understanding and development in the respective fields of eDNA and mixed tissue sample analysis.

The thesis opens with a review of the challenges and perspectives in relation to the present state of

the art regarding eDNA methodology for monitoring marine fish (chapter II). This study set the stage

for chapter III, which examine the use of a 2G ESP to perform autonomous in situ eDNA analysis for

real-time monitoring of marine fish. Hereafter, chapter IV assess the ability to infer qualitative and

quantitative content of different fish species from DNA analysis of mixed tissue samples. The thesis

ends with chapter V, which describes the potential use of genetics in relation to the EU landing

obligation to prevent and monitor the catch of non-targeted species.

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Chapter II: The sceptical optimist: challenges and perspectives for the application of

environmental DNA in marine fisheries

This review addresses the challenges and perspectives for the use of the eDNA methodology within

fisheries monitoring and management. Firstly, we describe several inherent challenges associated

with biological and environmental factors related to eDNA, as well as their interactions, and how

these factors influence production, transport and degradation of eDNA. Secondly, we identify five

major areas within marine fisheries and highlight the challenges and perspectives for implementation

of eDNA analysis and point towards immediate needs for knowledge building to improve the

approach. Last, we illustrate several emerging applications and technologies of relevance to the

eDNA application in fisheries, which are expected to improve and expand the use of eDNA in

fisheries. Based on this review, we emphasize that the methodological challenges and present

knowledge gaps need careful consideration for proper use in monitoring, but also argue that with

added insights the eDNA methodology can become a key tool in fisheries assessment and

management.

Chapter III: Remote, autonomous real-time monitoring of environmental DNA from

commercial fish

This study explores the ability of the 2G ESP to completely automate sampling and analysis of eDNA

from 4 marine species in a 4.5 million liter mesocosms at the North Sea Oceanarium, Hirtshals,

Denmark. During the deployment, the 2G ESP conducted 30 sampling events. Each sampling event

was composed of a sample which was analyzed in situ, followed by either one or two samples which

were chemically preserved and stored onboard the 2G ESP for post-deployment laboratory based

analysis. The results demonstrate that the 2G ESP was able to consistently detect and quantify

target molecules from the most abundant species (Atlantic mackerel) in the tank with analytical

results becoming internet accessible 3-4 hours after initiation of sample collection. In contrast,

33

detection of rare species was challenged by both biological and technical aspects coupled to the

nature of eDNA and the 2G ESP instrumentation. In conclusion, the ESP holds much promise for

remote, autonomous in situ analysis of eDNA and for generation of eDNA based monitoring time

series.

Chapter IV: From DNA to biomass: species quantification of bulk fisheries products

This study examines, as a proof of concept, the ability of DNA based analysis to provide qualitative

and quantitative assessments of species composition in bulk multi-species products (silage and fish

blocks) relevant to MCS applications in fisheries. Using qPCR and metabarcoding (MinION), we

demonstrate the ability to provide robust qualitative and quantitative estimates of fish species from

frozen fish block and silage samples. qPCR analysis of silage samples collected over 21 days,

detected all species in very stable quantities and species of gadoids matched input tissue proportions

with high accuracy (≤8% inaccuracy). qPCR and nanopore sequencing analysis of run-off water and

exterior swipes from frozen fish blocks also provided consistent detection of species. Further, the

developed sampling tools allowed quick, easy-to-standardize and non-destructive sampling, which

combined with the analytic performance, demonstrate high potential for MCS applications.

Chapter V: Possible Uses of Genetic Methods in Fisheries Under the EU Landing Obligation

Finally, we assessed DNA based methods under the EU landing obligation to help alleviate capture

of non-target species and to assess catch composition and mixed species products made from low-

value species. We identified three general themes where DNA based applications could provide

relevant information; (1) species identification of by-catch, (2) inferring species from mixed species

products and (3) the potential of using eDNA for assessment of species occurrences prior to fishing

in order to reduce bycatch. We provide specific examples of their potential use in relevant case

34

studies and present the most significant limitations for their implementation in fisheries and fisheries

management. Finally, we present potential future perspectives with an emphasis on portable and

automatic devices.

5.1. Concluding remarks and implications

Overall, the present thesis provides a number of new insights into the application of DNA based tools

of relevance for monitoring and managing marine living resources. A central component of this thesis

has been to convey knowledge of novel genetic tools for fisheries, regarding their perspectives and

associated challenges (chapter II and V). This is especially important now, as many managers and

institutions responsible for fisheries monitoring and advice are faced with deciding on whether to

implement eDNA or mixed tissue DNA analysis in MCS or not. Despite the general attractiveness

and apparent simplicity of these new genetic tools, the level of knowledge of their strength and

limitations is generally highly restricted beyond experienced practitioners within the research field.

However, while we as scientist depend on scientific progress and report on achievements and

perspectives it is crucial, and of equal importance, that we as specialist also inform on the challenges

and uncertainties from which we infer our biological conclusions. Of particular note, chapter II

highlights concrete examples of the known unknowns in the eDNA methodology, which potentially

challenge our biological inferences. With the rapid pace of this field, a wealth of new literature is

quickly emerging which tries to address key issues of uncertainty. However, due to the complexity

of the eDNA methodology and application, the research still display highly variable and contradicting

results and conclusions (e.g. Andruszkiewicz, Sassoubre & Boehm, 2017; Collins et al., 2018; Pilliod

et al., 2014). Nonetheless, this research still improves our knowledge and the results provide clues

to how challenges related to eDNA analysis can be addressed.

This thesis also demonstrates how technological innovations continue to produce entirely new tools

and instruments of relevance to DNA based species identification for fisheries monitoring. The 2G

35

ESP, presented and tested in a mesocosm for eDNA analysis in chapter III, showed robust long-

term performance for autonomous detection and monitoring of abundant fish species. In contrast,

detection of rare species was challenging under the current setup. It should be noted that the 2G

ESP is not optimized to rare targets (<50 DNA copies/ml). Previously, the 2G ESP was used to asses

abundant microorganisms such as phytoplankton and bacteria (Preston et al., 2011; Yamahara et

al., 2015). Hence, optimizing the 2G ESP sensitivity to trace rare eDNA templates for future

deployments is a priority. For this reason, the 2G ESP is at the same time incredibly exciting and

challenging to work on. Further, the margin of error is very small and even minor issues can have

catastrophic consequences for a deployment, which was the main reason for conducting the first

deployment at the mesocosm. However, in October 2018 we successfully conducted our first

subsurface 2G ESP deployment, in Skagerrak on 15 meters depth 5 kilometers from land for a

month. While the current expertise and operation costs associated to the 2G ESP still restrict broad

use and implementation, this first proof of concept to completely automated water collection and

eDNA analysis has very large perspectives for autonomous eDNA monitoring applications.

Chapter IV demonstrated that DNA based tools can be a robust and efficient way of providing

inferences of contents within complex bulk fisheries products. This has broad perspectives for MCS

applications of bulk products where visual identification and quantification is difficult or impossible.

Further, the study provided important insights for understanding the link between fish biomass and

DNA content in complex tissue samples of fish. While the methods did demonstrate robust and

stabile proportion estimates for especially the gadoids in silage samples, the methods will benefit

from further refinement and calibration to ensure sufficient reliability and improve accuracy across

species in practical applications. Chapter IV also demonstrated the first quantitative use of a

metabarcoding approach using a MinION, with promising results comparable to the qPCR analysis.

Accordingly, application of the MinION and similar devices have great perspectives for future MCS

applications due to its portability potentially allowing for on-site analysis. Further, bulk fisheries

samples with high DNA content are particularly suitable for direct sequencing applications as the

36

vast majority of the DNA present comes from the target organisms and not from other organisms

such as bacteria or algae, which currently hampers direct sequencing of environmental samples

(Stat et al., 2017).

Throughout this thesis, it is evident that the past decade has provided major advances in genetic

applications and tools for species detection and quantification in fisheries, and yet we are only at the

very beginning of this technological revolution. Consequently, we now have unprecedented

possibilities for testing new applications (chapter IV) and instruments (chapter III & IV). The

combined surge of new methods and instruments will not only broaden the use of genetics as a

methodology in fisheries management but also improve knowledge and push the boundaries for the

biological inferences obtainable from this novel and excitingly complex field of research. Yet,

identifying and recognizing the limitations and challenges is a prerequisite for successful future

implementation of novel genetic methods in fisheries monitoring and management (chapter II, IV &

V).

5.2. A look into the future

Despite the many genetic tools and applications which can provide information of relevance for

fisheries management, there seems to be a prevailing lack of implementation of even the most

fundamental genetic applications (Casey et al., 2016). I foresee that the application of genetics,

particularly the eDNA and mixed tissue DNA methodologies will increase significantly in the coming

decades. Particularly as the methods become more well proven, cost-effective and as the knowledge

base of fundamental challenges for implementation is improved (chapter II and V). Specifically,

regarding the EU landing obligation, it is apparent that the current legislation, stating that all fish

should be landed as sorted whole fish, is a significant obstacle for developing profitable business

models for unwanted catches in a large number of fisheries. Thus, acceptance of DNA based tools

for MCS will allow bulk products to be landed, resulting in less handling (less crew) for the fishermen.

37

Hence, establishing these links provides incentives for both commercial fishermen and fisheries

managers to take up the technology, which potentially could ease its implementation in the future.

The present state of DNA based methods also coincides with the emergence of portable, easy-to-

use and cheap field instruments, which will increase the ease of implementation. Based on our

findings, it is realistic to imagine that in the not so distant future a network of hundreds or even

thousands of deployable instruments will enable remote real-time DNA based monitoring in the

oceans, essentially providing managers with a livestream to the contemporary status of target

species or ecosystem biodiversity. Such tools would be of high value, as management can be

adjusted according to the current status, instead of relying on annual snapshots, which characterize

current monitoring. Although such a network seems far away, the research conducted here has

provided some of the initial steps of making this vision possible. Further, the 2G ESP can to some

extent be compared to LEGO, in the sense that these systems are customizable. Modifying and

equipping the instruments with additional tools and sensors can also aid biological inferences. For

example, adding modules that measure and integrates environmental factors or in situ HTS.

Although modifying and equipping the instruments is not easy and often require diverse

multidisciplinary expertise (electronics, mechanical, software, molecular, etc.) the possibilities are in

the hands of the users. Although the path to validate and apply new technologies is neither easy nor

smooth, the small steps forward will eventually get us closer to the goal of improving methodologies

and management thereby conserving of our common resources.

38

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enables high-fidelity recovery of biodiversity for bulk arthropod samples without PCR amplification. GigaScience, 2: 4. doi:10.1186/2047-217X-2-4

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Chapter II

The sceptical optimist: challenges and perspectives for the

application of environmental DNA in marine fisheries

Hansen, B.K., Bekkevold, D., Clausen, L.W. & Nielsen, E.E.

Published in Fish and Fisheries

2018

49

Due to copyright, the paper are not included in this version of the thesis.

68

Chapter III

Remote, autonomous real-time monitoring of environmental

DNA from commercial fish

Hansen, B.K., Jacobsen, L.M.W., Middelboe, A.L., Bekkevold, D., Knudsen, S.W., & Nielsen, E.E.

In preparation

Intended for submission to Nature Methods

69

Due to copyright, the paper are not included in this version of the thesis.

90

Chapter IV

From DNA to biomass: species quantification of bulk

fisheries products

Hansen, B.K., Farrant, G., Ogden, R., Humble, E., Ólafsdóttir, G., Bekkevold, D., Knudsen, S.W.,

& Nielsen, E.E.

In preparation

Intended for submission to ICES Journal of Marine science

91

Due to copyright, the paper are not included in this version of the thesis.

134

Chapter V

Possible Uses of Genetic Methods in Fisheries Under the

EU Landing Obligation

Jacobsen, M.W., Hansen, B.K., & Nielsen, E.E.

Published in the book The European Landing Obligation: Reducing Discards in Complex, Multi-

Species and Multi-Jurisdictional Fisheries

2018

135

Due to copyright, the paper are not included in this version of the thesis.

Other contributions Below are other contributions resulting from additional work I have been involved in during my PhD.

Andersen, J. H., Kallenbach, E., Hesselsøe, M., Knudsen, S. W., Møller, P. R., Bekkevold, D.,

Hansen, B. K., et al. 2016. Steps toward nation-wide monitoring of non-indigenous species in Danish

marine waters under the Marine Strategy Framework Directive. 1-122 pp.

doi:10.13140/RG.2.1.3003.6889

Andersen, J. H., Kallenbach, E., Thaulow, J., Hesselsøe, M., Bekkevold, D., Hansen, B. K.,

Jacobsen, L. M. W., et al. 2017. Development of species-specific eDNA-based test systems for

monitoring of non-indigenous species in Danish marine waters. 1-77 pp.

Knudsen, S. W., Ebert, R. B., Hesselsøe, M. , Kuntke, F., Hassingboe, J., Mortensen, P. B.,

Thomsen, P. F., et al. 2018. Species-specific detection and quantification of environmental DNA

from marine fishes in the Baltic Sea. Journal of Experimental Marine Biology and Ecology, 510: 31–

45. doi:10.1016/J.JEMBE.2018.09.004

157

Technical University of Denmark

DTU AquaKemitorvetDK-2800 Kgs. Lyngby

www.aqua.dtu.dk

Marine fish are under increasing pressure from a variety of anthropogenic stressors neces-sitating well-informed fisheries management. Successful management relies on the ability to accurately determine how many fish are there, how many can be sustainably caught and that exploitation rates are reported correctly through fisheries monitoring, control and surveillance. Thus, developing molecular tools that aid this process is of broad interest.

This PhD thesis examines and evaluates the use of novel DNA based methods and instru-mentation for applications of relevance to fisheries monitoring. Specifically, it demon-strates the first use of a submersible and completely autonomous “lab-in-a-can”, an Envi-ronmental Sample Processor (ESP), for remote real-time monitoring of marine fish using environmental DNA (i.e. DNA released in the environment). We show that the instrument can conduct all analysis steps from water collection to analysis of eDNA and can transmit the results in real-time to scientists located more than 200 km away. We also demonstrate the promising use of DNA based methods for identification and quantification of species composition in complex multi-species bulk fisheries products, such as fish silage and fro-zen fish blocks. Overarching, the research demonstrates that DNA based tools are a robust and efficient way to identify and quantify content of fish products where visual assess-ment is impossible. This suggests, that these methods can be a large asset for fisheries monitoring, control and surveillance applications, when properly developed and imple- mented while recognising challenges and shortcomings.