Predation of Marine Mammals by Grey Seals (Halichoerus Grypus

University of Veterinary Medicine Hannover Institute for Terrestrial and Aquatic Wildlife Research

Predation of marine mammals by grey seals (Halichoerus grypus) – Assessment of the background, the extent and the potential effects on the ecosystem Prädation mariner Säugetiere durch Kegelrobben (Halichoerus grypus) – Untersuchungen zu den Hintergründen, dem Ausmaß und den potentiellen Effekten auf das Ökosystem

INAUGURAL DOCTORAL THESIS in partial fulfilment of the requirements of the degree of Doctor of Natural Sciences - Doctor rerum naturalium - (Dr. rer.nat.)

submitted by Abbo van Neer, MConBio Norden, Ostfriesland

Büsum 2019

Scientific supervision: Prof. Prof. h. c. Dr. Ursula Siebert, Institute for Terrestrial and Aquatic Wildlife Research, University of Veterinary Medicine Hannover, Foundation

Prof. Dr. Gerd Bicker, Institute for Physiology and Cell Biology University of Veterinary Medicine Hannover, Foundation

1st supervisors:

Prof. Prof. h. c. Dr. Ursula Siebert, Institute for Terrestrial and Aquatic Wildlife Research, University of Veterinary Medicine Hannover, Foundation

Prof. Dr. Gerd Bicker, Institute for Physiology and Cell Biology, University of Veterinary Medicine Hannover, Foundation

Prof. Dr. Rune Dietz 2nd supervisor: ……………………………………………

07. Mai 2019 Day of the oral examination: ……………………………………………

I The present dissertation was carried out as part of the projects "Pilotstudie zur Erfassung von Kegelrobbenrissen bei marinen Säugetieren in der deutschen Nordsee" and "Umfassende weiterführende Untersuchungen zur Kegelrobbenprädation auf marine Säugetiere in deutschen Gewässern" (Funded by the Ministry of Energy, Agriculture, the Environment, Nature and Digitalization Schleswig-Holstein, Germany).

Results of the dissertation were published in the following articles: van Neer, A., Jensen, L.F., Siebert, U. (2015). Grey seal (Halichoerus grypus) predation on harbour seals (Phoca vitulina) on the island of Helgoland, Germany. Journal of Sea Research 97, 1–4. https://doi.org/10.1016/j.seares.2014.11.006 This work was primarily conducted by myself. My participation in this study involved the provisioning of the necessary conceptual background with regards to the topic and the collection of data on Helgoland as well as during necropsies. Further, I primarily wrote the manuscript, but in collaboration with the named co-authors and was involved in funding acquisition.

Heers, T., van Neer, A., Becker, A., Grilo, M. L., Siebert, U., & Abdulmawjood, A. (2017). Loop-mediated isothermal amplification (LAMP) assay—A rapid detection tool for identifying red fox (Vulpes vulpes) DNA in the carcasses of harbour porpoises (Phocoena phocoena). PLOS ONE, 12(9), e0184349. https://doi.org/10.1371/journal.pone.0184349 My participation in this study involved the provisioning of the necessary conceptual background with regards to the topic, as well as any samples of marine mammals and foxes used. Further, I participated in writing the manuscript and was involved in funding acquisition.

Heers, T., van Neer, A., Becker, A., Gross, S., Hansen, K. A., Siebert, U., & Abdulmawjood, A. (2018). Loop-mediated isothermal amplification (LAMP) as a confirmatory and rapid DNA detection method for grey seal (Halichoerus grypus) predation on harbour porpoises (Phocoena phocoena). Journal of Sea Research 140, 32–39. https://doi.org/10.1016/j.seares.2018.07.008 My participation in this study involved the provisioning of the necessary conceptual background with regards to the topic, as well as any samples of marine mammals used. Further, I participated in writing the manuscript and was involved in funding acquisition. van Neer, A., Gross, S., Kesselring, T., Wohlsein, P., Leitzen, E., & Siebert, U. (2019). Behavioural and Pathological Insights into a Case of Active Cannibalism by a Grey Seal (Halichoerus grypus) on Helgoland, Germany Journal of Sea Research, 148–149, 12–16. https://doi.org/10.1016/j.seares.2019.03.004 This work was primarily conducted by myself. My participation in this study involved the provisioning of the necessary conceptual background with regards to the topic and the collection of data on Helgoland. Further, I primarily wrote the manuscript, but in collaboration with the named co-authors and was involved in funding acquisition.

Further results of the dissertation are to be published in the following articles: van Neer, A., Gross, S., Kesselring, T., Grilo, M., Ludes-Wehrmeister, E., Roncon, G., & Siebert, U. (in preparation). Putting together the pieces of the puzzle - Part I - Assessing seal carcasses potentially subjected to grey seal (Halichoerus grypus) predation. This work was primarily conducted by myself. My participation in this study involved the provisioning of the necessary conceptual background with regards to the topic and the collection of data on Helgoland

II as well as during necropsies. Further, I primarily wrote the manuscript, but in collaboration with the named co-authors and was involved in funding acquisition. van Neer, A., Gross, S., Kesselring, T., Grilo, M., Ludes-Wehrmeister, E., Roncon, G., & Siebert, U. (in preparation). Putting together the pieces of the puzzle - Part II - Assessing harbour porpoise carcasses potentially subjected to grey seal (Halichoerus grypus) predation. This work was primarily conducted by myself. My participation in this study involved the provisioning of the necessary conceptual background with regards to the topic and the collection of data on Helgoland as well as during necropsies. Further, I primarily wrote the manuscript, but in collaboration with the named co-authors and was involved in funding acquisition.

The following parts of the dissertation were also presented at international conferences or in the form of a letter and a report van Neer, A., Jensen, L. F., Blädel, R., & Siebert, U. (2014). If you can’t beat them, eat them - Behavioural observations of grey seal (Halichoerus grypus) and harbour seal (Phoca vitulina) interactions on the island of Helgoland, Germany. In 28th Conference of the European Cetacean Society, 05.-09.04.2014. Liège, Belgium. (Abstract und Poster; Winner of the prize “Best scientific poster 2014”) This work was primarily conducted by myself. My participation in this study involved the provisioning of the necessary conceptual background with regards to the topic and the collection of data on Helgoland as well as during necropsies. Further, I primarily wrote the respective manuscript, but in collaboration with the named co-authors and was involved in funding acquisition. van Neer, A., Wohlsein, P., & Siebert, U. (2015). Shedding light on the phenomenon of grey seal predation on marine mammals In 29th Conference of the European Cetacean Society, 23.-25.03.2015. St Julian’s, Malta. (Abstract und Poster; Winner of the prize “Best scientific poster 2015”) This work was primarily conducted by myself. My participation in this study involved the provisioning of the necessary conceptual background with regards to the topic and the collection of data on Helgoland as well as during necropsies. Further, I primarily wrote the respective manuscript, but in collaboration with the named co-authors and was involved in funding acquisition.

ICES. (2017). Report of the Workshop on Predator-prey Interactions between Grey Seals and other marine mammals (WKPIGS). Middelfart, Denmark: (N. Hanson, A. van Neer, A. Brownlow, & J. Haelters). Available under http://www.ices.dk/sites/pub/Publication Reports/Expert Group Report/SSGEPD/2017/01 WKPIGS - Report of the Workshop on Predator-prey Interactions between Grey Seals and other marine mammals.pdf This report summarises the findings resulting from the respective workshop which was organised by the named people as part of the 31st Conference of the European Cetacean Society in Middelfart, Denmark. My participation in this workshop involved the initiation, collaborative organisation of the event, chairing of and presenting in sessions, collation and evaluation of the joint European data as well as the acquisition of the funding. Further, I contributed major parts to the manuscript.

Haelters, J., Kerckhof, F., van Neer, A., & Leopold, M. (2015). Letter to the Editor Exposing Grey Seals as Horses and Scientists as Human. Aquatic Mammals, 41(3), 351–353. https://doi.org/10.1578/AM.41.3.2015.351 My participation in this publication involved the collaborative scientific and creative conception and writing of the manuscript.

III

Table of contents

1. Background ...... 1 The phenomenon of grey seal predation on marine mammals ...... 2 2. The first report of grey seal predation on marine mammals in German waters ..... 4 Grey seal (Halichoerus grypus) predation on harbour seals (Phoca vitulina) on the island of Helgoland, Germany ...... 4 3. Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour ...... 14 a. Behavioural and Pathological Insights into a Case of Active Cannibalism by a Grey Seal (Halichoerus grypus) on Helgoland, Germany ...... 14 b. Putting together the pieces of the puzzle – Part I - Assessing seal carcasses potentially subjected to grey seal (Halichoerus grypus) predation ...... 28 c. Putting together the pieces of the puzzle - Part II - Assessing harbour porpoise carcasses potentially subjected to grey seal (Halichoerus grypus) predation ...... 50 4. Using molecular methods as a complementary tool of the macroscopic assessment ...... 69 a. Loop-mediated isothermal amplification (LAMP) assay - A rapid detection tool for identifying red fox (Vulpes vulpes) DNA in the carcasses of harbour porpoises (Phocoena phocoena) ...... 69 b. Loop-mediated isothermal amplification (LAMP) as a confirmatory and rapid DNA detection method for grey seal (Halichoerus grypus) predation on harbour porpoises (Phocoena phocoena) ...... 86 5. Overall Discussion ...... 104 6. Conclusion & Outlook ...... 109 7. Zusammenfassung...... 111 8. Summary ...... 113 9. References ...... 114 10. Supplementary materials ...... 122 a. Development of the local harbour and grey seal stock ...... 122 b. Other publications concerning this topic ...... 123 If you can’t beat them, eat them - Behavioural observations of grey seal (Halichoerus grypus) and harbour seal (Phoca vitulina) interactions on the island of Helgoland, Germany...... 123 Shedding light on the phenomenon of grey seal predation on marine mammals...... 123 Letter to the Editor - Exposing Grey Seals as Horses and Scientists as Human...... 124 11. Permissions to include presented publications ...... 127 12. Statutory declaration ...... 129 13. Acknowledgements ...... 130

List of abbreviations

cf. confer DNA deoxyribonucleic acid e.g. exempli gratia et al. et alii ITAW Institute for Terrestrial and Aquatic Wildlife Research LAMP Loop-mediated isothermal amplification Ministry of Energy, Agriculture, the Environment, Nature and Digitalization, Schleswig MELUND Holstein PCR Polymerase chain reaction PDV Phocine distemper virus

List of figures

Figure caption Page Chapter Fig1. Grey seal catching (A) and feeding (B, C) on a young harbour seal 6 2 (Pictures by courtesy of S. Fuhrmann). Fig. 2. Harbour seal carcasses retrieved in 2013 (B) and 2014 (A, C). 7 Fig. A.1. Grey seal that was observed preying on harbour seals on Helgoland. 13 Fig. A.2. First harbour seal carcass retrieved in 2013. 13 Fig. A.3. Interspecific sexual interaction of a grey and a harbour seal. 13 Chapter Figure 1: Carcass of a juvenile male grey seal, observed being predated by a 3a sub adult grey seal bull. With A repositioned skin of the animal with the helical course of the laceration; B complete carcass as it was retrieved; C fully 17 repositioned skin indicating no tissue loss; D head injury and several bite and scratch wounds behind the right eye. Pictures courtesy of Dominik Nachtsheim. Figure 2: Right side of the head with several bite/scratch wounds marked with 18 arrows. Picture courtesy of Dominik Nachtsheim. Figure 3: Blubber tissue showing a diffuse uneven surface with an irregular 18 blubber depth. Picture courtesy of Dominik Nachtsheim. Figure A.1: 09:50h, the first major amount of blood was visible. Picture courtesy 26 of Katrin Wiese. Figure A.2: Subadult grey seal bull feeding from a hunted juvenile grey seal using the pectoral flippers to push the carcass away while scraping of blubber 27 using the teeth, Helgoland, 26.03.2018 Figure A.3: Sample of the cranial side of the laceration showing intact hair shafts 27 extending over the wound margin. Wound margin indicated by the arrow. Chapter Figure 1: Number of suspected grey seal predation and fox related cases for the 3b years 1995 - 2017; categorised by likelihood of grey seal predation (“likely”, “possibly”, “unlikely”), cases that have only been observed but without recovery of the carcass (“observation only”) and suspected fox interactions (“fox”). Cases related to fox interaction have been included in the category “unlikely” in terms 36 of grey seal predation, but are also shown separately right of the dashed red line. The definite case from 2018 is not included in this figure. Number of grey seal victims are shown in dark grey, number of harbour seal victims in light grey bars.

Figure 2: Percentage of occurrence of the 11 parameters in the different categories for suspected grey seal predation cases (“definite”, “likely”, “possibly”, “unlikely“) and for suspected fox interactions (“fox”). Cases related to 37 fox interaction are only shown in the category “fox”, despite also being “unlikely” with regards to grey seal predation. Parameters framed with a red rectangle are indicative for an interaction with a fox. Figure 3: Subadult grey seal bull with a juvenile harbour seal as prey and shown while inducing the typical helical lacerations by tearing apart the tissue using its 40 pectoral flipper and jaws. Helgoland, 04.10.2017. Picture courtesy of Susanne Hauswaldt. Figure 4: Harbour seal with severe injuries in the area of the throat. Helgoland, 41 Germany, September 2018. Figure A.1: Newly developed protocol to be used for the documentation of suspected grey seal predation cases in its updated version based on the results 45-49 presented here. Chapter Figure 1: Healed lesions on a porpoise fluke potentially induced by grey seal 54 3c teeth. Figure 2: Number of suspected grey seal predation and fox related cases for the years 1990 - 2017; categorised by likelihood of grey seal predation (“likely”, “possibly”, “unlikely”), cases that have only been observed but without recovery of the carcass (“observation only”) and suspected fox interactions (“fox”). Cases 58 related to fox interaction have been included in the category “unlikely” in terms of grey seal predation, but are also shown separately right of the dashed red line. The definite case is not included in this figure. Figure 3: Percentage of occurrence of the 11 parameters in the different categories for suspected grey seal predation cases (“definite”, “likely”, “possibly”, “unlikely“) and for suspected fox interactions (“fox”). Cases related to 59 fox interaction are only shown in the category “fox”, despite also being “unlikely” with regards to grey seal predation. Parameters framed with a red rectangle are indicative for an interaction with a fox. Figure A.1: Protocol used for the documentation of suspected grey seal 65-68 predation cases in its updated version. Chapter Fig1. Alignment of the cytochrome b sequences of red fox (Vulpes vulpes) 4a (accession number AM181037.1) and dog (Canis lupus familiaris (Beagle)) (accession number AY729880.1). The dots represent the same base pair, while 72 the red letters show different base pairs. The positions of the LAMP primers for the red fox are colour-coded. Fig 2. Application of the LAMP assay on a stranded harbour porpoise. (A) Carcass of the stranded harbour porpoise. (B) Sampling locations of the 76 different MSwab™swabs (1–5). Locations were selected at random. Fig 3. Limit of detection of the LAMP assay. (A) Typical amplification curves of ten-fold serially diluted red fox DNA. (B) The standard curve was generated from a dilution series of the DNA of Vulpes vulpes by plotting the amplification time 78 versus the log of the DNA concentration. At concentrations below 1.45E-02 pg/μl the data falls off. The correlation is a linear response. Fig 4. Application of the LAMP assay on a stranded harbour porpoise. Gel electrophoresis of all LAMP products of the stranded harbour porpoise. Directly tested MSwab™samples (without DNA isolation) and DNA isolation of the MSwab™medium using DNeasy® blood and tissue kit. As expected, the 79 detection time of the directly positive 4 and 5 swab samples was longer (swab no. 4 6:00 min longer and swab no. 5 3:30 min longer) than the detection time of the isolated DNA.

Chapter Fig. 1. Alignment of the cytochrome b sequences of grey seal (Halichoerus 4b grypus) (accession number GU167293.1) and harbour porpoise (Phocoena phocoena) (accession number U72039.1). Same base pairs are represented by dots and different base pairs are marked by red letters. The positions of the six 91 species-specific LAMP primers are indicated in colour. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Fig. 2. Spiking experiment. Inoculation of grey seal saliva in the triangular 93 imitated bite mark in the skin preparation of a harbour porpoise. Fig. 3. Application of the LAMP assay on a stranded harbour porpoise (found in September 2017). Carcass of the stranded harbour porpoise as found on the 94 beach on the coastline of Sylt (picture by courtesy of Sönke Lorenzen). Fig. 4. Limit of detection. (A) Typical amplification curves of eight-fold serially diluted grey seal DNA. (B) Mean values (n=6) with standard deviation of the limit 96 of detection of the LAMP assay using serial dilutions of isolated grey seal DNA. Fig. 5. Application of the LAMP assay on a stranded harbour porpoise. Sampling point of four MSwab™ samples. The other swab samples were located on the 99 other side of the harbour porpoise. Fig. 6. Application of the LAMP assay on a stranded harbour porpoise (found in September 2017). Gel electrophoresis of the LAMP amplicons of isolated DNA of the MSwab™ medium using the specific primer set. Lane 1–8 stand for the 100 MSwab™ samples 1–8. Lane “P, GS” display grey seal DNA as positive control. Lane “N, HP” show the negative control harbour porpoise, while lane “N, MM” stand for the mastermix as negative control. In lane “M” the marker is shown. Chapter Figure A. 1: Count data grey seals. 122 10 Figure A. 2: Count data harbour seals. 122

VII

List of tables

Figure caption Page Chapter Table A.1: Caloric content of different species taken from the literature. 26 3a Chapter Table 1: Categories and their respective description used for rating the 31 3b likelihood of grey seal predation and fox interaction. Table 2: Table showing 11 parameters, which have been added to the catalogue and are now routinely used for rating the likelihood of grey seal predation as 33-35 origin of a trauma in seals. Pictures in parameters 1 – 8 courtesy of Dominik Nachtsheim. Chapter Table 1: Categories and their respective description used for rating the 53 3c likelihood of grey seal predation and fox interaction. Table 2: Table showing 11 parameters, which have been added to the catalogue and are now routinely used for rating the likelihood of grey seal predation as 55-58 origin of a trauma in porpoises. Chapter Table 1. Sequences of the six specific primers for detecting red fox DNA. 73 4a Table 2. Spiking experiments with the three serial dilutions of the DNA of Vulpes 78 vulpes. Table 3. LAMP results of the first stranded harbour porpoise investigated for red 79 fox DNA residues. S1 Table. Dog breed samples (n= 19) investigated in the present study as 82 negative controls for the exclusivity test. S2 Table. Limit of detection of the LAMP assay by using serial dilutions of the 82 DNA of Vulpes vulpes. Chapter Table 1 Sequences of the six speciﬁc primers for the detection of grey seal 92 4b DNA. Table 2 Sequences of the six common primers as internal ampliﬁcation control 92 (IAC) for detecting grey seal and harbour porpoise DNA. Table 3 Spiking experiment with four saliva samples of two different grey seals. 96 Chapter Table 1: Summary-table showing parameters suggested for the assessment of 5 suspicious carcasses. As some parameters are specific for either seals or porpoises, respective suitability is indicated. Shown parameters are indicative 106 for grey seal predation unless stated differently (indicators for fox interaction). For details see chapter 3.b & 3.c.

VIII Background

1. Background

In the south eastern part of the North Sea, three marine mammal species are considered to reproduce regularly. Besides the harbour porpoise (Phocoena phocoena) as the only cetacean species (Gilles et al., 2016), two phocid seals occur frequently in these waters (CWSS, 2017). The smaller of the two is the harbour seal (Phoca vitulina) which is currently the most abundant in the Wadden Sea (Galatius et al., 2017). Harbour seals have a broad distribution with subspecies also occurring in the Pacific (Phoca vitulina richardii) and even in freshwater habitat (Phoca vitulina mellonae). They show little sexual dimorphism with female adult individuals from the Atlantic having an average size between 140 and 146 cm and weighing between 67 and 83 kg (Teilmann & Galatius, 2017). Males of this region have an average size between 153 and 156 cm with an average weight between 75 and 104 kg (Teilmann & Galatius, 2017). The harbour seal population in the Wadden Sea has since the beginning of coordinated aerial surveys increased continuously from approximately 3,840 seals during moult in 1975 to 25.036 counted individuals in 2017 (Galatius et al., 2017). Irrespective of the positive net growth since 1975, the population has experienced severe mortality events with two phocine distemper virus (PDV) outbreaks which have caused a sudden population decline of around 57 % in 1988 and 47 % in 2002 respectively and an influenza A (H10N7) outbreak in 2014 which caused a moderate decline of up to 10 % locally (Bodewes et al., 2015; Härkönen et al., 2006). Interestingly, throughout the last years the constant increase of the population has slowed down and seemingly even stopped (development of count data between 2013 and 2017: -1 %; see Figure A. 1). If this pattern persists, indicating a density dependent decrease of growth or if the observed development is within the naturally occurring fluctuation of the population needs to be seen during the coming years.

The second seal species occurring in German waters is the grey seal (Halichoerus grypus). Grey seals occur throughout the Atlantic with three distinct populations, namely the Eastern Atlantic, the western Atlantic and the Baltic Sea population (Hall & Russell, 2017). In contrast to harbour seals, grey seals have been locally extinct due to human exploitation and only started to recolonise the Wadden Sea in the end of the 1960s (Czeck & Paul, 2008). Since their return the population has constantly increased to 5,445 counted seals during moult in 2017 (Brasseur et al., 2017). The two biggest grey seal colonies in German waters are on the Kachelotplate between the islands Borkum and Juist (422 individuals in 2017) and on Helgoland (616 individuals in 2017) (Brasseur et al., 2017). For details on the population development of the two seal species in recent years, also see Figure A. 2 & Figure A. 1 in the Supplementary materials. Opposed to harbour seals, grey seals show a strong sexual dimorphism. Whereas adult female individuals from the North Sea region average in size of up

1 Background to 200 cm weighing between 105 to 186 kg, adult males in our region grow up to 230 cm with a weight of up to 310 kg (Hall & Russell, 2017; Jefferson et al., 2008).

Harbour porpoises occur throughout the continental shelf waters of the Northern Atlantic, the North Pacific, the Black Sea as well as the Baltic Sea and are regarded as an abundant species in most of its range (Hammond et al., 2008). Within a large scale survey which was conducted in 2016 throughout European Atlantic waters, the abundance of porpoises was estimated to be around 466,569 animals (Hammond et al., 2017). Porpoises feed on a variety of prey species including fish and cephalopods but prey preferences are considered variable depending e.g. on region or season (Santos et al., 2004). They too show sexual dimorphism with the females growing on average to 160 cm and weighing around 60 kg whereas the males only grow to around 145 cm weighing on average 50 kg (Bjørge & Tolley, 2009).

Even though no significant decrease in abundance is evident for any of the three marine mammal species frequently occurring in the North Sea, anthropogenic induced threats like habitat degradation, noise and chemical pollution or bycatch persist (IJsseldijk et al., 2018). With the aim to reduce the number and degree of threats, all three species are integrated in different legislative frameworks assigning different levels of protection (Santos & Pierce, 2015). All three species are considered opportunistic top predators in the ecosystem and mainly utilise a diverse range of different fish species (Andreasen et al., 2017; Das et al., 2003; Hammond & Wilson, 2016; Sharples et al., 2012). Besides this piscivorous prey resource, reports of seals preying on water birds as well as infrequent reports of cannibalism by grey seals have been published (Bedard et al., 1993; Lucas & McLaren, 1988; Tallman & Sullivan, 2004).

The phenomenon of grey seal predation on marine mammals In 2011 the first incidences of lesions on a harbour porpoise hypothesised to have been induced by a grey seal, were recorded (Haelters et al., 2012). This study reported of two porpoise carcasses showing severe lesions of unknown aetiology. To elucidate the cause of the detected lesions, the measurements of the wounds taken during the necropsies were compared to skull measurements and inter-teeth distances of grey and harbour seals. Results strongly suggested the grey seal as a potential perpetrator. Despite a first scepticism in the scientific community (Haelters et al., 2015), more publications reporting of similar cases followed (Bouveroux et al., 2014; Jauniaux et al., 2014; Stringell et al., 2015). Investigations which were initiated as a consequence of the first reports, showed the potential extent of the phenomenon with at least 17 % of investigated porpoise carcasses from the Dutch coast being categorised as likely grey seal predation cases (Leopold et al., 2015b). In August 2013 the first case of a harbour seal preyed on by a male grey seal was reported by a local seal ranger from Helgoland, Germany (van Neer et al., 2014, 2015a). Eventually Scottish scientists reported of active cannibalism with a male grey seal preying on juvenile grey seals in a breeding colony

2 Background on the Isle of May (Bishop et al., 2016; Brownlow et al., 2016). Until now, spatially limited studies could show, that the suspected rate of this behaviour in certain areas may well have measurable negative effects on the local part of the population with unknown consequences for the ecosystem (Brownlow et al., 2016; Leopold et al., 2015b). As a result of the first reports of instances of grey seal predation on marine mammals in German waters, two projects funded by the Ministry of Energy, Agriculture, the Environment, Nature and Digitalization, Schleswig Holstein were initiated and conducted in collaboration with different institutes of the University of Veterinary Medicine Hannover, Foundation under the lead of the Institute for Terrestrial and Aquatic Wildlife Research (ITAW) in Büsum.

The aim of these projects was to investigate the phenomenon of grey seal predation on marine mammals in detail with respect to pathological, behavioural and ecological aspects.

This dissertation was conducted within the frame of the above mentioned projects and results will be presented accordingly.

3 The first report of grey seal predation on marine mammals in German waters

2. The first report of grey seal predation on marine mammals in German waters

The following part was published as:

Grey seal (Halichoerus grypus) predation on harbour seals (Phoca vitulina) on the island of Helgoland, Germany

Written by: van Neer, A., Jensen, L. F. & Siebert, U. (2015);

Journal of Sea Research, 97, 1–4. doi:10.1016/j.seares.2014.11.006

Picture by Susanne Hauswaldt

4 The first report of grey seal predation on marine mammals in German waters

Abstract

The prey spectrum of grey seals has to date been described as largely consisting of different ﬁsh, cephalopod and shrimp species. On the German island of Helgoland Düne, where harbour seals (Phoca vitulina) and grey seals (Halichoerus grypus) co-occur, a young male grey seal was observed in 2013 and again in 2014 preying upon young harbour seals. A harbour seal carcass with severe traumatic lesions was retrieved and post-mortem examinations were performed. In the following weeks several carcasses showing similar lesions were found. Sightings of grey seals assumed to be preying on harbour porpoises have increased around the North Sea. Increased competition as well as individualised behaviour could explain the increased number of observations, but former cases of abnormal lesions of marine mammals attributed to for example predation by sharks or mechanical processes should be revisited with regard to the emerging knowledge.

Introduction

The two seal species that reproduce in the southern North Sea, the harbour seal (Phoca vitulina) and the grey seal (Halichoerus grypus) are, amongst others, top predators of the Wadden and North Sea (Hammond et al., 2002). The growing populations of both harbour and grey seals and the overlapping habitat result in an increased rate of interspeciﬁc interactions and presumably an increased intensity of competition (Abt and Engler, 2008; Brasseur et al., 2013a, 2013b, 2012; Svensson, 2012).

Grey seals used to be abundant in the Wadden Sea area until their local extinction around 1500 AD (Lotze, 2005; Reijnders et al., 1995; Wolff, 2005) and recolonised the southern North Sea only around 1970 (Scheibel and Weidel, 1988). Following the decline of harbour seal numbers due to hunting, the population recovered since the protection of the species but the population size varies in the region with recent epizootics causing sudden and substantial declines (Härkönen et al., 2006). In contrast, grey seals are less susceptible to the phocine distemper virus and are thus less affected than the harbour seals (Hall et al., 2006; Härkönen et al., 2006). The harbour seal population in the Wadden Sea was estimated (2014) to be 39,100 seals, and the grey seal population counts (2014) resulted in 4276 seals (Brasseur et al., 2014; Galatius et al., 2014).

Grey seals use the beaches of the island Düne (near Helgoland) all year round but especially in winter, to give birth and mate and all age classes are present (Abt and Engler, 2008; Härkönen et al., 2007). Harbour seals in contrast are most often present during spring and summer months and mainly consist of groups of younger animals (Thomsen, 2009). They pup around June and predominantly on the sandbanks of the nearby Wadden Sea national park, closer to the mainland (Liebsch et al., 2006).

5 The first report of grey seal predation on marine mammals in German waters

Fig1. Grey seal catching (A) and feeding (B, C) on a young harbour seal (Pictures by courtesy of S. Fuhrmann).

Predatory interaction

On the 31st of July 2013 the local seal ranger noticed a young male grey seal (6–7 years old) surrounded by blood in the water, approximately 30 m away from the beach of the island Düne.

After looking around, the grey seal disappeared under water and more blood came to the surface. Later this seal was brieﬂy seen again, this time surfacing and bringing up a dead animal (cf. Fig. 1B). Two hours later, possibly the same young male grey seal was observed, retaining a young harbour seal with a neck-bite and spinning the animal around. At times the harbour seal managed to escape the grip of the grey seal, but it was immediately attacked again. On the following day a carcass of a young harbour seal with severe traumatic lesions was found in the vicinity of the area (Fig. A.2).

6 The first report of grey seal predation on marine mammals in German waters

In the period between July and September 2013 and numerous times throughout 2014, predation on young harbour seals was observed and several carcasses showing similar lesions were found (Fig. 2).

Fig. 2. Harbour seal carcasses retrieved in 2013 (B) and 2014 (A, C).

Post mortem examination of the ﬁrst harbour seal conducted according to Siebert et al. (2007, 2001) showed that the thoracic cavity was opened. Skin, blubber and muscle tissue were missing from an area which extended from the head to the forelimbs. The scapula was partly missing and teeth marks were found in the bone. Besides these lesions, the main pathological ﬁnding was moderate parasitic infection of nematodes in the bronchial tree and pulmonary blood vessels.

Discussion

Video recordings show the grey seal holding the harbour seal with its pectoral ﬂippers and feeding from it (cf. Fig. 1B, C) for a minimum of 24 min. The large quantities of muscle and blubber missing from the retrieved carcasses (Fig. 2 & A.2) indicate that these are incidents of active predatory, rather than aberrant behaviour.

On Helgoland, young male grey seals, engaged in interspeciﬁc sexual aggression with young harbour seals are regularly observed (Fig. A.3). For other pinniped species such behaviour has been reported to lead to the inferior individual being killed or even eaten (Campagna et al.,1988; Harris et al., 2010; Miller et al., 1996; Mortenson and Follis,1997). However, the behaviour reported here – the catching of the animal in the water opposed to the land based

7 The first report of grey seal predation on marine mammals in German waters sexual interaction and especially the extensive feeding behaviour – is not likely to represent an abnormal escalation of sexual behaviour. Rather, the observed behaviour represents feeding behaviour.

The prey spectrum of grey seals largely consists of ﬁsh, cephalopods and shrimps (Bowen and Harrison, 1994; Gilles, 2009; Gilles et al., 2008; Smout et al., 2014), with occasional reports of predation on birds (Lucas and McLaren, 1988) and recently harbour porpoises, Phocoena phocoena (Bouveroux et al., 2014; Haelters et al., 2012; Leopold et al., 2015) as well as one report of cannibalism (Bedard et al., 1993).

Comparing diet composition of harbour seal, grey seal and harbour porpoise, interspecific competition for food resources could take place depending on population sizes, geographical location or change due to seasonal variation in prey abundance and in the long term due to increased overfishing or climate change (Gilles et al., 2008; Gosch et al., 2014; Hauksson and Bogason, 1997; Sharples et al., 2012; Smout et al., 2014; Thomsen, 2009; Tollit and Thompson, 1996). With the populations of grey seals, harbour seals and harbour porpoises growing in the southern North Sea, the intra- as well as interspecific competition is increasing (Das et al., 2003; Don Bowen et al., 2003; Matthiopoulos et al., 2014; Svensson, 2012).

Additionally the observed behaviour could be related to growing population sizes, increasing the likelihood of noticing infrequent behavioural patterns. As grey seal populations are increasing, the chance of noticing behaviour that is only shown by a few single individuals increases too. Thus, the incident on Helgoland could be regarded as a case of individual specialisation with respect to feeding behaviour. The lack of published literature on incidences like this suggests that it is not common behaviour. However, the recent increase in reports from all states bordering the North Sea of harbour porpoises possibly being preyed upon by grey seals, as well as an older report of a grey seal preying on conspeciﬁc pups, together with anecdotic reports from Scotland and Canada on predatory interactions between grey and harbour seals, suggest that the feeding behaviour of grey seals might be more diverse than previously anticipated (Bedard et al., 1993; Bouveroux et al., 2014; Haelters et al., 2012; Leopold et al., 2015).

Given that the ability to learn from conspecifics has been proven to increase the energy gain of individuals, providing there is a competitive advantage to performing a specific behaviour (Whiten et al., 2005), it will be interesting to see if the observed behaviour is subject to social learning within and between the different grey seal populations. And even though our knowledge regarding this behaviour and the resulting risk for humans is yet insufficient, when comparing this case to other predators targeting marine mammals (e.g. the Orca), it seems unlikely that grey seals will pose a serious risk to humans in the future as there is no evidence that would support such an assumption.

8 The first report of grey seal predation on marine mammals in German waters

Considering the pattern of lesions of the retrieved carcasses, other cases certainly need to be reconsidered in the light of the new knowledge. For example the lesions described in cases of presumed shark predation on harbour and grey seals around Sable Island (Lucas and Natanson, 2010; Lucas and Stobo, 2000) as well as some of the lesions presented in Thompson et al. (2013, 2010) and Bexton et al. (2012) partly show a striking similarity to the lesions caused by the observed grey seal predation (cf. Figure 7 and 9 in Thompson et al. (2010) and Figure 3a, c, e in Lucas and Natanson (2010)).

Conclusion

The described interspeciﬁc interactions between a grey seal and harbour seals could be caused by a multitude of factors and may play a crucial role for the population development of the two sympatric seal species and the harbour porpoise. Further research is needed to explain this behaviour, to quantify it and to qualify which ecological effects these interactions might have. Data on diet composition needs to be gathered in more detail to incorporate the spatial, seasonal and inter-annual differences in different grey seal populations as well as more detailed information on the diet composition of likely competitors such as the harbour seal and the harbour porpoise. Additionally, detailed data on the habitat use and foraging grounds of these three species of marine mammals would be helpful in order to assess potential competitive mechanisms. To gain a better understanding and to be able to quantify the extend of such behaviour, scientists working in areas with grey seals should revisit cases of unusual injuries of marine mammals, establish a standard method (e.g. van Bleijswijk et al., 2014) to clarify if a carcass has been subject to predation or scavenging by a larger predator (e.g. the grey seal) and closely monitor the behaviour of grey seals in the future.

Acknowledgements

We would like to thank all observers and especially the seal rangers of Helgoland for their help and reports on the incidences, Sebastian Fuhrmann for providing his recorded footage, as well as the Ministry of Energy, Agriculture, the Environment and Rural Areas Schleswig-Holstein for funding the state wide stranding network.

References

Abt, K., Engler, J., 2008. Rapid increase of the grey seal (Halichoerus grypus) breeding stock at Helgoland. Helgol. Mar. Res. 63, 177–180. http://dx.doi.org/10.1007/s10152-0080143-6. Bedard, C., Kovacs, K., Hammill, M., 1993. Cannibalism by grey seals, Halichoerus grypus, on Amet Island, Nova Scotia. Mar. Mamm. Sci. 9, 421–424. http://dx.doi.org/10. 1111/j.1748- 7692.1993.tb00474.x. Bexton, S., Thompson, D., Brownlow, A., Barley, J., Milne, R., Bidewell, C., 2012. Unusual mortality of pinnipeds in the United Kingdom associated with helical (corkscrew) injuries of

9 The first report of grey seal predation on marine mammals in German waters anthropogenic origin. Aquat. Mamm. 38, 229–240. http://dx.doi.org/10. 1578/AM.38.3.2012.229. Bouveroux, T., Kiszka, J.J., Heithaus, M.R., Jauniaux, T., Pezeril, S., 2014. Direct evidence for gray seal (Halichoerus grypus) predation and scavenging on harbor porpoises (Phocoena phocoena). Mar. Mamm. Sci. 30, 1542–1548. http://dx.doi.org/10.1111/ mms.12111. Bowen, W.D., Harrison, G.D., 1994. Offshore diet of grey seals Halichoerus grypus near Sable Island. Mar. Ecol. Prog. Ser. 112, 1–11. http://dx.doi.org/10.3354/meps112001. Brasseur, S., Borchardt, T., Czeck, R., Jensen, L.F., Galatius, A., Ramdohr, S., Siebert, U., Teilmann, J., 2012. Aerial Surveys of Grey Seals in the Wadden Sea in the Season of 2011– 2012. Wilhelmshaven, Germany. Brasseur, S., Czeck, R., Diederichs, B., Galatius, A., Jensen, L.F., Körber, P., Siebert, U., Teilmann, J., Klöpper, S., 2014. Grey Seal Surveys in the Wadden Sea and Helgoland in 2013– 2014. Wilhelmshaven, Germany. Brasseur, S., Diederichs, B., Czeck, R., Jensen, L.F., Galatius, A., Ramdohr, S., Siebert, U., Teilmann, J., Körber, P., Klöpper, S., 2013a. Aerial Surveys of Grey Seals in the Wadden Sea in the season of 2012–2013. Wilhelmshaven, Germany. Brasseur, S., Diederichs, B., Czeck, R., Jensen, L.F., Galatius, A., Ramdohr, S., Siebert, U., Teilmann, J., Körber, P., Klöpper, S., 2013b. Aerial Surveys of Harbour Seals in the Wadden Sea in 2013. Wilhelmshaven, Germany. Campagna, C., Le Boeuf, B.J., Cappozzo, H.L., 1988. Pup abduction and infanticide in Southern sea lions. Behaviour 107, 44–60. http://dx.doi.org/10.1163/156853988X00188. Das, K., Lepoint, G., Leroy, Y., Bouquegneau, J.M., 2003. Marine mammals from the southern North Sea: feeding ecology data from δ13C and δ15N measurements. Mar. Ecol. Prog. Ser. 263, 287–298. http://dx.doi.org/10.3354/meps263287. Don Bowen, W., Ellis, S.L., Iverson, S.J., Boness, D.J., 2003. Maternal and newborn lifehistory traits during periods of contrasting population trends: implications for explaining the decline of harbour seals (Phoca vitulina), on Sable Island. J. Zool. 261, 155–163. http://dx.doi.org/10.1017/S0952836903004047. Galatius, A., Brasseur, S., Czeck, R., Diederichs, B., Jensen, L.F., Körber, P., Siebert, U., Teilmann, J., Klöpper, S., 2014. Aerial Surveys of Harbour Seals in the Wadden Sea in 2014. Wilhelmshaven, Germany. Gilles, A., 2009. Characterisation of Harbour Porpoise (Phocoena phocoena) Habitat in German Waters. Christian-Albrechts-Universität zu Kiel. Gilles, A., Andreasen, H., Müller, S., Siebert, U., 2008. Nahrungsökologie von marinen Säugetieren und Seevögeln für das Management von NATURA 2000 Gebieten. Teil: Marine Säugetiere (No. FKZ: 805 85 018). Büsum. Gosch, M., Hernandez-Milian, G., Rogan, E., Jessopp, M., Cronin, M., 2014. Grey seal diet analysis in Ireland highlights the importance of using multiple diagnostic features. Aquat. Biol. 20, 155–167. http://dx.doi.org/10.3354/ab00553. Haelters, J., Kerckhof, F., Jauniaux, T., Degraer, S., 2012. The grey seal (Halichoerus grypus) as a predator of harbour porpoises (Phocoena phocoena)? Aquat. Mamm. 38, 343–353. http://dx.doi.org/10.1578/AM.38.4.2012.343. Hall, A.J., Jepson, P.D., Goodman, S.J., Härkönen, T., 2006. Phocine distemper virus in the North and European Seas — data and models, nature and nurture. Biol. Conserv. 131, 221– 229. http://dx.doi.org/10.1016/j.biocon.2006.04.008.

10 The first report of grey seal predation on marine mammals in German waters

Hammond, P.S., Berggren, P., Benke, H., Borchers, D.L., Collet, A., Heide-Jørgensen, M.P., Heimlich, S., Hiby, A.R., Leopold, M.F., Oien, N., 2002. Abundance of harbour porpoise and other cetaceans in the North Sea and adjacent waters. J. Appl. Ecol. 39, 361–376. Härkönen, T., Brasseur, S., Teilmann, J., Vincent, C., Dietz, R., Abt, K., Reijnders, P., 2007. Status of grey seals along mainland Europe from the Southwestern Baltic to France. Sci. Publ. 6, 57–68. Härkönen, T., Dietz, R., Reijnders, P., Teilmann, J., Harding, K., Hall, A., Brasseur, S., Siebert, U., Goodman, S.J., Jepson, P.D., Rasmussen, T.D., Thompson, P., 2006. A review of the 1988 and 2002 phocine distemper virus epidemics in European harbour seals. Dis. Aquat. Org. 68, 115–130. http://dx.doi.org/10.3354/dao068115. Harris, H.S., Oates, S.C., Staedler, M.M., Tinker, M.T., Jessup, D.A., Harvey, J.T., Miller, M.A., 2010. Lesions and behavior associated with forced copulation of juvenile Pacific harbor seals (Phoca vitulina richardsi) by southern sea otters (Enhydra lutris nereis). Aquat. Mamm. 36, 219–229. http://dx.doi.org/10.1578/AM.36.4.2010.219. Hauksson, E., Bogason, V., 1997. Comparative feeding of grey (Halichoerus grypus) and common seals (Phoca vitulina) in Coastal Waters of Iceland, with a note on the diet of hooded (Cystophora cristata) and harp seals (Phoca groenlandica). J. Northwest Atl. Fish. Sci. 22, 125–135. http://dx.doi.org/10.2960/J.v22.a11. Leopold, M.F., Begeman, L., van Bleijswijk, J.D.L., IJsseldijk, L.L., Witte, H.J., Gröne, A., 2015. Exposing the grey seal as a major predator of harbour porpoises. Proc. Biol. Sci. 282, 20142429. http://dx.doi.org/10.1098/rspb.2014.2429. Liebsch, N., Wilson, R., Adelung, D., 2006. Utilisation of time and space by harbour seals (Phoca vitulina vitulina) determined by new remote-sensing methods. In: von Nordheim, H., Boedeker, D., Krause, J.C. (Eds.), Progress in Marine Conservation in Europe — NATURA 2000 Sites in German Offshore Waters. Springer Berlin Heidelberg, Berlin Heidelberg New York, pp. 179–188 http://dx.doi.org/10.1007/3540-33291-X_11. Lotze, H.K., 2005. Radical changes in the Wadden Sea fauna and flora over the last 2,000 years. Helgol. Mar. Res. 59, 71–83. http://dx.doi.org/10.1007/s10152-004-0208-0. Lucas, Z., McLaren, I.A., 1988. Apparent predation by grey seals, Halichoerus grypus, on seabirds around Sable Island. Nova Scotia. Can. Field-Naturalist 102, 675–678. Lucas, Z., Stobo, W.T., 2000. Shark-inflicted mortality on a population of harbour seals (Phoca vitulina) at Sable Island, Nova Scotia. J. Zool. 252, 405–414. http://dx.doi.org/ 10.1017/S0952836900000157. Lucas, Z.N., Natanson, L.J., 2010. Two shark species involved in predation on seals at Sable Island, Nova Scotia. Canada. Proc. Nov. Scotian Inst. Sci. 45, 64–88. Matthiopoulos, J., Cordes, L., Mackey, B., Thompson, D., Duck, C., Smout, S., Caillat, M., Thompson, P., 2014. State-space modelling reveals proximate causes of harbour seal population declines. Oecologia 174, 151–162. http://dx.doi.org/10.1007/ s00442-013-2764-y. Miller, E.H., Ponce de Leon, A., Delong, R.L., 1996. Violent interspecific sexual behavior by male sea lions (Otariidae): evolutionary and phylogenetic implications. Mar. Mamm. Sci. 12, 468–476. http://dx.doi.org/10.1111/j.1748-7692.1996.tb00601.x. Mortenson, J., Follis, M., 1997. Northern elephant seal (Mirounga angustirostris) aggression on harbor seal (Phoca vitulina) pups. Mar. Mamm. Sci. 13, 526–530. http://dx. doi.org/10.1111/j.1748-7692.1997.tb00663.x. Reijnders, P.J.H., van Dijk, J., Kuiper, D., 1995. Recolonization of the Dutch Wadden Sea by the grey seal Halichoerus grypus. Biol. Conserv. 71, 231–235. http://dx.doi.org/10. 1016/0006- 3207(94)00032-L.

11 The first report of grey seal predation on marine mammals in German waters

Scheibel, W., Weidel, H., 1988. Zum Vorkommen von Kegelrobben (Halichoerus grypus Fabricius, 1791, Phocidae, Pinnipedia) in Schleswig–Holstein. Zool. Anz. 220, 65–70. Sharples, R.J., Moss, S.E., Patterson, T.A., Hammond, P.S., 2012. Spatial variation in foraging behaviour of a marine top predator (Phoca vitulina) determined by a large-scale satellite tagging program. PLoS ONE 7, e37216. http://dx.doi.org/10.1371/journal.pone. 0037216. Siebert, U., Wohlsein, P., Lehnert, K., Baumgärtner, W., 2007. Pathological ﬁndings in harbour seals (Phoca vitulina): 1996–2005. J. Comp. Pathol. 137, 47–58. http://dx.doi. org/10.1016/j.jcpa.2007.04.018. Siebert, U., Wünschmann, A., Weiss, R., Frank, H., Benke, H., Frese, K., 2001. Post-mortem ﬁndings in harbour porpoises (Phocoena phocoena) from the German North and Baltic Seas. J. Comp. Pathol. 124, 102–114. http://dx.doi.org/10.1053/jcpa.2000.0436. Smout, S., Rindorf, A., Hammond, P.S., Harwood, J., Matthiopoulos, J., 2014. Modelling prey consumption and switching by UK grey seals. ICES J. Mar. Sci. 71, 81–89. Svensson, C.J., 2012. Seal dynamics on the Swedish west coast: scenarios of competition as Baltic grey seal intrude on harbour seal territory. J. Sea Res. 71, 9–13. http://dx. doi.org/10.1016/j.seares.2012.03.005. Thompson, D., Bexton, S., Brownlow, A., Wood, D., Patterson, T., Pye, K., Lonergan, M., Milne, R., 2010. Report on Recent Seal Mortalities in UK Waters Caused by Extensive Lacerations. St Andrews, Scotland. Thompson, D., Culloch, R., Milne, R., 2013. Current State of Knowledge of the Extent, Causes and Population Effects of Unusual Mortality Events in Scottish Seals. St Andrews, Scotland. Thomsen, K., 2009. Nahrungsökologie der Helgoländer Robben, Seehund (Phoca vitulina) und Kegelrobbe (Halichoerus grypus). Universität Hamburg. Tollit, D.J., Thompson, P.M., 1996. Seasonal and between-year variations in the diet of harbour seals in the Moray Firth. Can. J. Zool. 74, 1110–1121. http://dx.doi.org/10.1139/ z96-123. Van Bleijswijk, J., Begeman, L., Witte, H., IJsseldijk, L., Brasseur, S., Gröne, A., Leopold, M., 2014. Detection of grey seal Halichoerus grypus DNA in attack wounds on stranded harbour porpoises Phocoena phocoena. Mar. Ecol. Prog. Ser. 513, 277–281. http://dx. doi.org/10.3354/meps11004. Whiten, A., Horner, V., de Waal, F.B.M., 2005. Conformity to cultural norms of tool use in chimpanzees. Nature 437, 737–740. http://dx.doi.org/10.1038/nature04047. Wolff, W.J., 2005. The exploitation of living resources in the Dutch Wadden Sea: a historical overview. Helgol. Mar. Res. 59, 31–38. http://dx.doi.org/10.1007/s10152-0040204-4.

12 The first report of grey seal predation on marine mammals in German waters

Supplementary Materials

Fig. A.1. Grey seal that was observed preying on harbour seals on Helgoland.

Fig. A.2. First harbour seal carcass retrieved in 2013.

Fig. A.3. Interspecific sexual interaction of a grey and a harbour seal.

13 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

3. Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

Marine mammal carcasses with lesions induced by a grey seal often show a high degree of mutilation and lesions can be misinterpreted as lesions induced by knife cuts or propeller strikes. Additionally, a differentiation between lesions induced by a grey seal and other causes of trauma such as scavenging by terrestrial predators is essential for an objective interpretation. Therefore, it is important to establish standardised assessment criteria which allow a harmonisation of different data sets and thus a base for the assessment of the ecological relevance of this phenomenon.

For the development of such standardised assessment criteria, a valuable data source are definite cases of grey seal predation (defined as observed cases or cases with genetic proof). Additionally these cases offer a chance to gain insights into the behavioural patterns behind this behaviour.

The following part was published as:

a. Behavioural and Pathological Insights into a Case of Active Cannibalism by a Grey Seal (Halichoerus grypus) on Helgoland, Germany

Written by: van Neer, A., Gross, S., Kesselring, T., Wohlsein, P., Leitzen, E. & Siebert, U. (2019);

Journal of Sea Research, 148–149, 12–16. https://doi.org/10.1016/j.seares.2019.03.004.

Picture by Abbo van Neer 14 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

Abstract

First reports on cases of grey seal predation on other marine mammals from different parts of Europe have been published in recent years, but few cases provide sufficient detail. Here we report a case of active cannibalism by a grey seal, which has been witnessed and recorded in detail on the German island of Helgoland, describing particular behavioural aspects and lesions. In March 2018, a subadult male grey seal was observed catching, killing and feeding extensively from a juvenile grey seal. The carcass showed severe cutaneous lacerations, starting in the head region and following around the trunk in a circular pattern. These results are discussed with regard to the previously reported cases to form a solid knowledge base for retrospective and future assessments of carcasses potentially subjected to grey seal predation. The correct assignment of predated seals is important to determine the potential influence this behaviour may have on seal populations.

Introduction

Grey seals (Halichoerus grypus) have been considered to rely on fish as major prey resource (Hammond and Wilson, 2016) with the exception of cases where seals have been reported to prey on water birds (Lucas and McLaren, 1988; Tallman and Sullivan, 2004) as well as two published reports on cannibalism by a grey seal from Canada (Bedard et al., 1993; Kovacs et al., 1996). A hitherto unknown behaviour of grey seals was reported in 2012 and 2013 for the first time suggesting grey seal predation on harbour porpoises inducing particular lesions in porpoise carcasses (Bouveroux et al., 2014; Haelters et al., 2012). During the following years additional reports documented that grey seals prey on harbour porpoises (Phocoena phocoena) in a potentially ecological relevant extent with the cause of death of 17 % of stranded porpoises in the Netherlands being attributed to grey seal predation (Leopold et al., 2015). Further the predation on harbour seals (van Neer et al., 2015) as well as the utilisation of individuals of their own species as prey through active cannibalism was reported (Bishop et al., 2016).

Cannibalism and infanticide have been reported to occur in various marine mammals (Patterson et al., 1998; Ryazanov et al., 2017; Towers et al., 2018; Wilkinson et al., 2000; Zheng et al., 2016). Different hypotheses on the motivation behind such behaviour have been suggested such as reproductive advantages, acquisition of food or to influence the structure of a social unit (Balme and Hunter, 2013; Derocher and Wiig, 1999; Kawanaka, 1981).

Here, we describe a case of active cannibalism by a grey seal bull on the German island of Helgoland with detailed documentation of the predatory behaviour shown by the bull and the lesions of the retrieved carcass recorded during necropsy.

15 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

Material and Methods

On the 26th of March 2018, a case of grey seal predation on a juvenile male grey seal was observed and documented with photographs and video recordings on the island of Helgoland, Germany. Following the attack, the carcass was retrieved immediately with no scavengers feeding from it in the meantime. A post mortem examination was conducted according to Siebert et al. (2007) around four hours and 40 minutes after the presumed death of the animal.

The age was determined as described in Lockyer et al. (2010). A representative spectrum of organ samples including skin from wound margins was collected during necropsy and immediately fixed in 10% neutral-buffered formalin, embedded routinely in paraffin wax, cut at 3 - 10 µm thickness and tissue sections were subsequently stained with haematoxylin and eosin (HE) for light microscopical examination.

Results

Behavioural observations

On March the 26th 2018 a five year old male grey seal was observed preying on a juvenile male grey seal. The male was identified to have been born on Helgoland in the winter 2012/13 by the number on the flipper tag which is attached to the animal. A first observation was made at approximately 09:40 h by a local ranger on the southern beach of the island Düne close to the shore in about 1.5 m water depth. The grey seal bull was seen holding the body of the juvenile grey seal with both pectoral flippers parallel to its own body while biting into the throat of the juvenile and pushing the body of the smaller seal repeatedly under water. During the first minutes of the observation, the juvenile seal managed to free itself from the grip of the older bull but was immediately captured again and once more held under water. After around one minute into the observation, low amounts of blood were initially visible in the water. After around ten minutes (09:50 h) the surrounding water was largely turning red, presumably due to extensive blood loss from the prey’s throat (Figure A.1), accompanied by cessation of movements only one minute later. Following the presumed death of the prey, the bull changed its behaviour and started feeding from the carcass (Figure A.2). In order to gain access to the energy rich blubber tissue the bull used its pectoral flippers to hold and push away the carcass while biting into the skin of the head region and pulling back its head. This resulted in the tearing of the tissue and exposure of the blubber. From what could be observed, the bull used its lower jaw to scrape pieces of blubber off the skin, swallowing them whole. The skin of the juvenile seal was nearly fully detached from the body and turned inside out exposing the majority of underlying blubber. The bull fed nearly continuously until 11:20 h (ca. 90 min).

16 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

Figure 1: Carcass of a juvenile male grey seal, observed being predated by a sub adult grey seal bull. With A repositioned skin of the animal with the helical course of the laceration; B complete carcass as it was retrieved; C fully repositioned skin indicating no tissue loss; D head injury and several bite and scratch wounds behind the right eye. Pictures courtesy of Dominik Nachtsheim.

Pathomorphological findings

The total length of the carcass was 128 cm, the weight of the remaining body was 25.4 kg, the nutritional status was good and the age less than one year based on the dental development.

Except for the areas around the caudal end of the body, the pectoral flippers, as well as the rostral part of the head, the dermis including the remaining blubber was detached from the body and turned inside out (Figure 1B). The detached skin was repositionable and the fully straight, cut-like wound margin along the torso of the animal could be well approximated (Figure 1A & C). Here no skin was missing. The laceration followed a helical course, starting just ventrally of the right eye, extending to the throat, circling over the dorsum of the animal to the ventral mid-point of the body and ending on the left lateral side, just behind the left pectoral flipper. On the cranial side of the laceration, hairs extended over the wound margin. In nine of nine skin samples examined, the vast majority of hair was macroscopically intact (for example see Figure A.3).

17 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

Around the throat and the lower jaw, pieces of skin including the subcutaneous fatty tissue were missing (Figure 1D). The width of the tissue defect at the start of the helical wound below the right eye was around 5 cm, which is within the published range of inter-canine distances reported for male grey seals (Haelters et al., 2012). The right mandibular Figure 2: Right side of the head with several bite/scratch wounds marked with arrows. Picture courtesy of Dominik Nachtsheim. bone was partially exposed. Bite/scratch marks were present in the area of the right ear and eye with one perforation of the epidermis below the ear (diameter: ~ 1 cm, lesion#1 in Figure 2), as well as another 5.5 cm long straight superficial laceration ventral to the above mentioned perforation (lesion#2 in Figure 2) and a subjacent round laceration with a diameter of around 1.6 cm (lesion#3 in Figure 2). The lateral side of the right eye showed two superficial lesions (lesion#4 & 5 in Figure 2). Ventrally to the eye, a 2 cm long and rather deep laceration was visible (lesion#6 in Figure 2). Macroscopically, scratch and bite marks do not necessarily show the same straight (cut-like) wound margin as can be found in the large lacerations found. The remaining blubber tissue of the animal had a diffuse uneven surface with an irregular blubber depth. The blubber surface of the detached skin showed a pattern with cobblestone-like appearance with numerous roundish structures surrounded by areas of reduced blubber depth, likely caused by the teeth of the bull. Blubber depth along the fringes of the skin flaps appeared to be reduced compared with the middle areas (Figure 3). Multifocal openings of the intercostal spaces with oligofocal lacerations of the surrounding muscle tissue were present. Furthermore, parts of the superficial muscle tissue were missing in the area of the neck, as well as from a smaller area in the transition between detached and attached skin. For the latter the missing tissue was Figure 3: Blubber tissue showing a diffuse uneven surface with an found to be still attached to the irregular blubber depth. Picture courtesy of Dominik Nachtsheim.

18 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour respective area of the blubber. The remaining musculature was intact. The right scapula was partly detached from the underlying tissue. Skeletal trauma was not detected.

The stomach and small intestine were partially prolapsed due to the opening of the peritoneal cavity. Besides yellowish foam no stomach content was present. The liver showed four deep lacerations but no signs of a removal of tissue. Besides a mild hepatic hyperaemia, multifocal, superficial, whitish spots were present. The lungs were severely collapsed and highly oedematous. In the left bronchus up to the bifurcation, viscous yellowish fluid was present. All other organs showed no further relevant gross pathological findings in the context of grey seal predation. Overall, no significant disease or other abnormalities were detected.

Histological examination revealed that the wound margins were slightly irregular with disorganised collagenous fibres. Cellular infiltrates were not present in any of the examined skin samples. Within one of the small circular defects from the head region (lesion#3 in Figure 2), multifocal to coalescing haemorrhages within the dermis and subcutaneous adipose tissue accompanied by marked hyperaemia of dermal vessels were present suggestive of intravital tissue damage. Further, a partial, subtotal atelectasis of the lung accompanied by single proteinaceous coacervates and small numbers of extravascular erythrocytes within deeper airways was detected. In addition a severe, focal, acute, perithyroidal haemorrhage was found. Within the small and large intestine, a mild to moderate lympho-plasmacytic and eosinophilic inflammation was diagnosed. Several larval nematodes were detected in colonic crypts. Comparable inflammatory and reactive changes as well as remnants of parasitic structures were found in the liver. The spleen showed a granulomatous and eosinophilic inflammation as well as a mild extramedullary haematopoiesis. All other investigated organs (bone, brain, heart, aorta, skeletal muscle, tongue, oesophagus, tonsils, lymph nodes, stomach, pancreas, pituitary and adrenal gland, trachea, spinal cord, eye, urinary bladder and thymus) were histologically unremarkable in the context of this case.

Discussion

Behavioural observations

The behavioural observations described here are in general comparable to the observations reported by van Neer et al. (2015) and also in broad terms to the reports by Bishop et al. (2016) and Kovacs et al. (1996). On Helgoland, the observation was made close to the beach with the prey being captured and further handled in shallow water by a subadult bull. This is in contrast to the reports from Scotland, where the prey was caught on land within the breeding colony and dragged (and presumably drowned) in a nearby freshwater pool by an adult male (Bishop et al., 2016). Results of the retrospective assessment of earlier events that were recorded throughout the years on the Isle of May described by Brownlow et al. (2016) suggest

19 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour though, that it is not uncommon that animals in Scotland are also preyed on in shallow water close to shore.

With regard to the behavioural observations reported here, it is likely that the prey died due to either exsanguination (c.f. Figure A.1) or due to asphyxiation. In the Scottish cases, the majority (64 %) has been reported to have died of asphyxiation (Brownlow et al., 2016). The report from Canada also suggests asphyxiation as likely cause of death (Kovacs et al., 1996), as was also reported by Leopold et al. (2015) for porpoises. Even though this is feasible, as the calculated aerobic dive limit of a one-year old grey seal has been reported to be 6.1 ± 0.3 min (Noren et al., 2005) and will most likely be less in a stress situation, the behavioural observations in this case (cessation of movements following the appearance of a larger amount of blood), would rather support exsanguination as cause of death.

There is similarity among previous described cases of grey seal predation on seals with the described case in this paper (Bishop et al., 2016; van Neer et al., 2015), including fairly stereotyped behaviour with the prey being held with the pectoral flippers and using the jaws to open up the carcass near the neck and throat area. After opening, flaps of skin are torn and thus detached from the body by holding the one end of the strip in the jaws and pulling the head back while pushing the carcass away with the flippers (Figure A.2). Blubber is removed by scrapping it off the detached flaps of skin using the teeth of the lower jaw (c.f. Bishop et al., 2016).

The time that was spent feeding from the prey was in the case described in this study around 90 minutes. Thus, here it is obvious that a considerable time is spent utilising the prey. It should be noted though, that an objective quantification of the amount of blubber that has been removed is difficult to realise practically due to the extensive manipulation and naturally varying depth of the blubber throughout the body of seals (Mellish et al., 2007). With regard to the reports from Scotland, it is evident that not all prey items are extensively utilised as in the reported cases the carcasses are left on average 21 min after starting to consume from the prey (Bishop et al., 2016). This is also comparable to the earlier report from Helgoland where the animal was observed to have fed from the prey for at least 24 min (van Neer et al., 2015).

In order to assess the energetic gain of such behaviour, we took as a coarse approximation the average caloric content of seal blubber derived from values published on ringed seals (Phoca hispida) and compared them to e.g. Atlantic cod (Gadus morhua) or Atlantic herring (Clupea harengus). Following this comparison, a grey seal would need to consume around 28 g of seal blubber to take up the same amount of calories as 100 g of herring. Accordingly, 12 g of blubber provides the same amount of calories as 100 g of cod (for details on used values please refer to Table A.1 in the supplementary materials). Thus, despite the risk and energetic

20 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour cost associated with such behaviour, the net energetic gain is potentially large and might therefore be reasonable in light of the prey choice model (Stephens et al., 2007).

It is still unknown, whether the shown behaviour purely resembles energy acquisition or if any other motivation, like aggression, is involved. Nevertheless, for the case described here, any reproductive or socially related reasons seem unlikely due to the timing of the shown behaviour. Grey seals in German waters have their reproductive season between November and January (Brasseur et al., 2018). March resembles the start of the moulting season on Helgoland, which is a period of high-energy demand (Paterson et al., 2012), again indicating a possible energy related motivation. Helgoland is not known to be an area of acute food shortage and the marine communities throughout the German bight have not shown considerable changes in the last years (Neumann et al., 2013). Reproductive success and population development of the local grey seals suggest a healthy subpopulation not suppressed by resource limitation (Brasseur et al., 2018). Thus, even though acute food shortage seems to be unlikely, this behaviour can simply resemble a means of acquiring comparably easy to access high-energy prey in comparison to catching fish offshore.

Pathomorphological findings

The carcass showed a large tissue defect resulting from the tearing and thus detaching of the skin as most prominent finding. Similar cutaneous lesions have been described in the cases from Scotland (Brownlow et al., 2016). Lacerations induced by tearing of the skin showed a single cut-like, smooth and linear wound margin, which follows in a helical (corkscrew like) manner for one or more rotations around the body. In contrast to Brownlow et al. (2016), we did not detect extensive loss of skin but rather well adaptable tissue margins (Figure 1C).

By detaching the skin, the predator exposes the energy rich blubber tissue, which is then subsequently scraped off the skin using the teeth leaving a distinct uneven pattern in the blubber tissue. In earlier cases for which the retrieval of the carcass was not possible directly after the observation (van Neer et al., 2015), the reported extensive loss of muscle tissue seems to be largely the result of post mortem scavenging e.g. by birds. This is supported by the case reported here, as well as by the results presented in Brownlow et al. (2016). The oligofocal alterations of muscle tissue in some areas seem to be the result of detaching the skin using the jaws as well as holding and pushing the carcass using the pectoral flippers. It was not observed, that larger quantities of muscle tissue were actively consumed.

Puncture lesions likely induced by teeth and claws can be found especially in the area of the head. According to the behavioural observations, these were potentially inflicted when the animal was held by the jaws of the predator during the process of catching and killing the prey. Presumably due to these different mechanical ways of infliction (tear vs. separation by

21 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour claw/teeth), puncture and scratch lesions did not show the same straight (cut-like) wound margin as the long helical laceration. In contrast to Brownlow et al. (2016), we did not detect any skeletal trauma, neither in the area of the head, nor in other areas of the body. While in the area of the single linear lesion caudal to the head, no skin is missing, the opposite is true for the area around the head and throat. Here, skin as well as the subcutaneous fatty tissue is missing and the width of the detected wound of approximately 5 cm matches with the range of the inter-canine distance of grey seal maxilla reported before (Haelters et al., 2012). In addition, when comparing the two opposing sides of the wound margin, largely intact hair extended over the edge of the wound margin densely. Assessing the condition of the hair allows for a first evaluation if the documented lesion was induced by tearing or cutting of tissue using a sharp knife. In contrast to torn tissue, the extending hair in cut tissue often shows structural damage of the hair shafts (Brownlie and Munro, 2016; Ressel et al., 2016). For seals, this is only expected on the cranial side of the wound margin due to the growth direction of the fur.

Histopathological findings

Histopathological changes indicative for intravital trauma could be observed within one of the skin samples from the right ear (lesion#3 in Figure 2), suggesting that the bull was attacking the head area of the living prey. This can be supported when considering the behavioural observations; the recordings also suggest that the initial attack was focused on the right side of the head. For all other samples taken from the wound margin of the helical laceration this could not be shown and might be the result of the quick transition time between life and death as well as the severe blood loss. Therefore, it is regarded as important that especially lesions in the head area are sampled for further histological examination in order to verify the time of infliction (intra-vitam vs. post-mortem). The extravasation of erythrocytes within the alveoli of the lung as well as the perithyroidal hemorrhage can be indicative of both an inhalation of blood as well as a trauma to the throat. The observed inflammatory changes within the gastrointestinal tract, liver and spleen are most likely caused by the observed parasitic infection but were not considered as significant.

Histopathological investigation revealed no evidence of underlying severe disease that could be indicative for a poor general condition of the prey, making this young seal particularly vulnerable and predisposed as an easy prey.

Assessing marine mammal carcasses in order to clarify the origin of severe trauma is not straightforward. This is due to the possibility of confusing trauma induced by grey seal predation with e.g. anthropogenic induced trauma or trauma as a result of predation and / or post mortem scavenging by terrestrial predators (Brownlow et al., 2016; IJsseldijk and Geelhoed, 2016; Leopold et al., 2015). This differentiation is a common challenge in forensic assessments (de Siqueira et al., 2016) and even though the amount of literature on the

22 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour phenomenon of grey seal predation on marine mammals is steadily growing, reports on full scale pathological assessments of definite grey seal predation cases are mainly lacking (only known report on a definite case Brownlow et al., 2016).

Conclusion

There is an increasing amount of information aiding with the post-mortem examination of potential grey seal predation cases. Nevertheless, the task of objectively differentiating grey seal predation as cause from other sources of trauma remains difficult. The findings described here such as the helical laceration with its smooth wound margin starting in the area of the head, the detached skin and the broadly manipulated blubber show extensive similarities to prior findings. Therefore, they should be seen as an important set of indicators for retrospective as well as future assessments of seal carcasses potentially subjected to grey seal predation. Further behavioural studies should be conducted in order to document the underlying mechanisms of this phenomenon including the processes explaining the origin of specific lesions as well as better understand the motivation and the ecological role of this behaviour.

Acknowledgements

We thank especially Katrin Wiese for her observations as well as the local ranger Michael Janssen and colleagues for their help retrieving the carcass. We thank also the Ministry of Energy, Agriculture, the Environment, Nature and Digitalization Schleswig-Holstein Germany for funding of this work and the necropsy team of the ITAW for their great effort.

References

Balme, G.A., Hunter, L.T.B., 2013. Why leopards commit infanticide. Anim. Behav. 86, 791– 799. https://doi.org/10.1016/j.anbehav.2013.07.019 Bedard, C., Kovacs, K., Hammill, M., 1993. Cannibalism by grey seals, Halichoerus grypus, on Amet Island, Nova Scotia. Mar. Mammal Sci. 9, 421–424. https://doi.org/10.1111/j.1748- 7692.1993.tb00474.x Bishop, A.M., Onoufriou, J., Moss, S., Pomeroy, P.P., Twiss, S.D., 2016. Cannibalism by a male grey seal (Halichoerus grypus) in the North Sea. Aquat. Mamm. 42, 137–143. https://doi.org/10.1578/AM.42.2.2016.137 Bouveroux, T., Kiszka, J.J., Heithaus, M.R., Jauniaux, T., Pezeril, S., 2014. Direct evidence for gray seal (Halichoerus grypus) predation and scavenging on harbor porpoises (Phocoena phocoena). Mar. Mammal Sci. 30, 1542–1548. https://doi.org/10.1111/mms.12111 Brasseur, S., Cremer, J., Czeck, R., Galatius, A., Jeß, A., Körber, P., Pund, R., Siebert, U., Teilmann, J., Klöpper, S., 2018. TSEG grey seal surveys in the Wadden Sea and Helgoland in 2017-2018 - More than ten years of growth in the Wadden Sea area. Wilhelmshaven, Germany. Brouwer, E., 1965. Report of sub-committee on constants and factors, in: Blaxter, K. (Ed.), Energy Metabolism. Academic Press, pp. 441–443.

23 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

Brownlie, H.W.B., Munro, R., 2016. The Veterinary Forensic Necropsy: A Review of Procedures and Protocols. Vet. Pathol. 53, 919–928. https://doi.org/10.1177/0300985816655851 Brownlow, A., Onoufriou, J., Bishop, A., Davison, N., Thompson, D., 2016. Corkscrew Seals: Grey Seal (Halichoerus grypus) Infanticide and Cannibalism May Indicate the Cause of Spiral Lacerations in Seals. PLoS One 11, e0156464. https://doi.org/10.1371/journal.pone.0156464 de Siqueira, A., Cuevas, S.E.C., Salvagni, F.A., Maiorka, P.C., 2016. Forensic Veterinary Pathology: Sharp Injuries in Animals. Vet. Pathol. 53, 979–987. https://doi.org/10.1177/0300985816655850 Derocher, A.E., Wiig, Ø., 1999. Infanticide and Cannibalism of Juvenile Polar Bears (Ursus maritimus) in Svalbard. ARCTIC 52, 307–310. https://doi.org/10.14430/arctic936 Haelters, J., Kerckhof, F., Jauniaux, T., Degraer, S., 2012. The grey seal (Halichoerus grypus) as a predator of harbour porpoises (Phocoena phocoena)? Aquat. Mamm. 38, 343–353. https://doi.org/10.1578/AM.38.4.2012.343 Hammond, P.S., Wilson, L.J., 2016. Grey Seal Diet Composition and Prey Consumption. Scottish Mar. Freshw. Sci. 7, 1–28. https://doi.org/10.7489/1799-1 IJsseldijk, L., Geelhoed, S.C. V, 2016. Fox scavenging mutilations on dead harbour porpoises (Phocoena phocoena). Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University & IMARES - Institute for Marine Resources & Ecosystem Studies. Kawanaka, K., 1981. Infanticide and Cannibalism in Chimpanzees: With Special Reference to the Newly Observed Case in the Mahale Mountains. Afr. Study Monogr. 1, 69–99. https://doi.org/10.14989/67979 Kovacs, K., Lydersen, C., Hammill, M., 1996. Grey Seal Cannibalism. Mar. Mammal Sci. 12, 161. https://doi.org/10.1111/j.1748-7692.1996.tb00316.x Leopold, M.F., Begeman, L., van Bleijswijk, J.D.L., IJsseldijk, L.L., Witte, H.J., Gröne, A., 2015. Exposing the grey seal as a major predator of harbour porpoises. Proc. R. Soc. London B Biol. Sci. 282, 20142429. https://doi.org/10.1098/rspb.2014.2429 Lockyer, C., Mackey, B., Read, F., Härkönen, T., Hasselmeier, I., 2010. Age determination methods in harbour seals (Phoca vitulina) with a review of methods applicable to carnivores. NAMMCO Sci. Publ. 8, 245. https://doi.org/10.7557/3.2688 Lucas, Z., McLaren, I.A., 1988. Apparent Predation by Grey Seals, Halichoerus grypus, on Seabirds around Sable Island, Nova Scotia. Can. Field-Naturalist 102, 675–678. Mellish, J.-A.E., Horning, M., York, A.E., 2007. Seasonal and Spatial Blubber Depth Changes in Captive Harbor Seals (Phoca vitulina) and Steller’s Sea Lions (Eumetopias jubatus). J. Mammal. 88, 408–414. https://doi.org/10.1644/06-MAMM-A-157R2.1 Neumann, H., Reiss, H., Ehrich, S., Sell, A., Panten, K., Kloppmann, M., Wilhelms, I., Kröncke, I., 2013. Benthos and demersal fish habitats in the German Exclusive Economic Zone (EEZ) of the North Sea. Helgol. Mar. Res. 67, 445–459. https://doi.org/10.1007/s10152-012-0334-z Noren, S.R., Iverson, S.J., Boness, D.J., 2005. Development of the Blood and Muscle Oxygen Stores in Gray Seals (Halichoerus grypus): Implications for Juvenile Diving Capacity and the Necessity of a Terrestrial Postweaning Fast. Physiol. Biochem. Zool. 78, 482–490. https://doi.org/10.1086/430228 Paterson, W., Sparling, C.E., Thompson, D., Pomeroy, P.P., Currie, J.I., McCafferty, D.J., 2012. Seals like it hot: Changes in surface temperature of harbour seals (Phoca vitulina) from late pregnancy to moult. J. Therm. Biol. 37, 454–461. https://doi.org/10.1016/j.jtherbio.2012.03.004

24 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

Patterson, I.A.P., Reid, R.J., Wilson, B., Grellier, K., Ross, H.M., Thompson, P.M., 1998. Evidence for infanticide in bottlenose dolphins: an explanation for violent interactions with harbour porpoises? Proc. R. Soc. B Biol. Sci. 265, 1167–1170. https://doi.org/10.1098/rspb.1998.0414 Ressel, L., Hetzel, U., Ricci, E., 2016. Blunt Force Trauma in Veterinary Forensic Pathology. Vet. Pathol. 53, 941–961. https://doi.org/10.1177/0300985816653988 Ryazanov, S.D., Kirillova, A.D., Laskina, N.B., Burkanov, V.N., 2017. Infanticide and cannibalism in Steller sea lions (Eumetopias jubatus). Mar. Mammal Sci. 1–8. https://doi.org/10.1111/mms.12437 Siebert, U., Wohlsein, P., Lehnert, K., Baumgärtner, W., 2007. Pathological Findings in Harbour Seals (Phoca vitulina): 1996–2005. J. Comp. Pathol. 137, 47–58. https://doi.org/10.1016/j.jcpa.2007.04.018 Steimle Jr., F.W., Terranova, R.J., 1985. Energy Equivalents of Marine Organisms from the Continental Shelf of the Temperate Northwest Atlantic. J. Northwest Atl. Fish. Sci. 6, 117–124. https://doi.org/10.2960/J.v6.a11 Stephens, D.W., Brown, J.S., Ydenberg, R.C., 2007. Foraging: Behavior and Ecology. University of Chicago Press, Chicago. Stirling, I., McEwan, E.H., 1975. The caloric value of whole ringed seals (Phoca hispida) in relation to polar bear (Ursus maritimus) ecology and hunting behavior. Can. J. Zool. 53, 1021– 1027. https://doi.org/10.1139/z75-117 Tallman, J., Sullivan, C., 2004. Harbor seal (Phoca vitulina) predation on a male harlequin duck (Histrionicus histrionicus). Northwest. Nat. 85, 31–32. https://doi.org/10.1898/1051- 1733(2004)085<0031:HSPVPO>2.0.CO;2 Towers, J.R., Hallé, M.J., Symonds, H.K., Sutton, G.J., Morton, A.B., Spong, P., Borrowman, J.P., Ford, J.K.B., 2018. Infanticide in a mammal-eating killer whale population. Sci. Rep. 8, 4366. https://doi.org/10.1038/s41598-018-22714-x van Neer, A., Jensen, L.F., Siebert, U., 2015. Grey seal (Halichoerus grypus) predation on harbour seals (Phoca vitulina) on the island of Helgoland, Germany. J. Sea Res. 97, 1–4. https://doi.org/10.1016/j.seares.2014.11.006 Wilkinson, I.S., Childerhouse, S.J., Duignan, P.J., Gulland, F.M.D., 2000. Infanticide and cannibalism in the New Zealand sea lion, Phocarctos hookeri. Mar. Mammal Sci. 16, 494–500. https://doi.org/10.1111/j.1748-7692.2000.tb00942.x Zheng, R., Karczmarski, L., Lin, W., Chan, S.C.Y., Chang, W.-L., Wu, Y., 2016. Infanticide in the Indo-Pacific humpback dolphin (Sousa chinensis). J. Ethol. 34, 299–307. https://doi.org/10.1007/s10164-016-0475-7

25 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

Supplementary Material

Table A.1: Caloric content of different species taken from the literature.

Caloric content [kJ/g wet Species Reference weight] Ringed Seal (pup) 36.4 (Stirling & McEwan, 1975) Ringed Seal (adult) 39.8 (Brouwer, 1965) Atlantic cod (Steimle Jr. & Terranova, 4.6 (Gadus morhua) 1985) Atlantic herring (Steimle Jr. & Terranova, 10.6 (Clupea harengus) 1985)

Figure A.1: 09:50h, the first major amount of blood was visible. Picture courtesy of Katrin Wiese.

26 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

Figure A.2: Subadult grey seal bull feeding from a hunted juvenile grey seal using the pectoral flippers to push the carcass away while scraping of blubber using the teeth, Helgoland, 26.03.2018.

Figure A.3: Sample of the cranial side of the laceration showing intact hair shafts extending over the wound margin. Wound margin indicated by the arrow.

27 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

Wound patterns induced by grey seals naturally show a certain degree of variability. In order to increase the sample size, it is therefore important to also assess cases for which only the carcass was retrieved, but no behavioural observation was possible. Detected wound patterns and related findings should be used in order to account for the respective variability.

The following parts are to be published as:

b. Putting together the pieces of the puzzle – Part I - Assessing seal carcasses potentially subjected to grey seal (Halichoerus grypus) predation

Written by: van Neer, A., Gross, S., Kesselring, T., Ludes-Wehrmeister, E., Roncon, G., Grilo, M. & Siebert, U. (in preparation);

Picture by Ute Pausch

28 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

Abstract

In order to conduct an objective evaluation of potential ecological effects of grey seal predation on other marine mammals, it is essential to establish a broad knowledge base that helps in the thorough identification of such cases during post-mortem examination. With the aim of adding to the existing knowledge, we here report and discuss outcomes resulting from a retrospective evaluation of harbour- and grey seal stranding and necropsy data (n = 3,154) collected on the coast of Schleswig-Holstein, Germany between 1995 and 2017. In addition, the results are compared to a recent case of definite grey seal predation from Germany as well as reports from other countries. Carcasses potentially subjected to grey seal predation show severe lacerations, mainly starting in the head area and following around the trunk in a circular pattern leaving a smooth and cut-like wound margin. Large parts of skin und underlying tissue are detached from the body and loss of blubber tissue is common. Occurrence frequencies of encountered lesions from suspected and definite grey seal predation cases are presented. In addition, a list of parameters to be used for the assessment of similar cases is suggested. Further, the assessed cases are categorised by likelihood of grey seal predation using the absence and presence of the respective parameters. With these parameters and categories, a baseline can be built in order to facilitate the standardised recognition of predation cases during post-mortem examinations of marine mammals

Background

Grey seals (Halichoerus grypus) are the most abundant seal species in the North Sea and in the majority of colonies their numbers are still increasing (ICES, 2018). Especially in areas like the Wadden Sea, where grey seals have been locally extinct mainly due to human exploitation until the late 1980s, numbers are increasing (Reijnders et al., 1995). For example, in the German Wadden Sea the average increase of adult grey seals counted during moult surveys is 13 %, however harbour seals (Phoca vitulina) are still in the majority here (Brasseur et al., 2018; CWSS, 2017; Galatius et al., 2017).

Besides the harbour seal and the harbour porpoise (Phocoena phocoena), grey seals are considered to be the top predator in this ecosystem. Following the conclusions drawn in the literature, it is generally accepted that all three species mainly prey on fish (Andreasen et al., 2017; Das et al., 2003; Hammond & Wilson, 2016; Sharples et al., 2012). When in 2012 and 2013 the first reports of suspected grey seal predation on harbour porpoises were published, it slowly became clear, that the prey spectrum utilised by grey seals is more diverse than previously anticipated (Bouveroux et al., 2014; Haelters et al., 2012). In the beginning scientists throughout the range of grey seals challenged this idea (Haelters et al., 2015). However, during the course of the following years more reports were published showing that grey seals not only

29 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour prey on porpoises, but also on harbour seals and even regularly on individuals of their own species (Bishop et al., 2016; Leopold et al., 2015; van Neer et al., 2015). Recently, scientists from Scotland described cases of juvenile grey seals that were observed to have been preyed on by a grey seal bull. Their gross pathological examination revealed traumatic lesions with detachment of skin and blubber, fractures and canine puncture wounds on the skull. Following a retrospective analysis of the local stranding database, grey seal bulls were assumed to be responsible for a considerable rate of seal mortalities in the area (Brownlow et al., 2016). As a consequence of the fact that predation rates are largely unknown, the ecological significance of this behaviour is still not yet understood and thus recognised entirely.

The big challenge remaining in this context is the diagnostic characterisation of wound patterns induced by grey seals and their differentiation from other wound patterns induced by trauma related to cutting (e.g. propeller strikes) or predation as well as post mortem scavenging by other species particularly foxes and birds. Due to the similarity of the lesions there is the need for standardised diagnostic procedures which utilise the existing knowledge to aid decision making.

Therefore, the aim of this study is to summarise the findings collected during necropsies of harbour and grey seals found dead between 1995 and 2017 on the coasts of Schleswig- Holstein. Results are discussed in comparison to a definite case of predation observed on Helgoland (Germany) as well as to cases reported in the literature and other origins of trauma.

Material and Methods

Carcasses were collected through the local stranding network (Benke et al., 1998; Siebert et al., 2001) and partly necropsied (Siebert et al., 2007). In addition, one well documented case from 2018 (van Neer et al., 2019) was taken for validation of recorded wound patterns. Necropsies included age estimation, determination of sex, weight, length, nutritional state, health status and cause of death. Due to the differences in the decomposition state of the different parts of the body of predation cases, decomposition state of the animal was rated by assessing the remaining intact parts of the body. This gives a more realistic picture as the manipulated parts show an advanced state of decomposition due to the direct exposure to the environment. In most cases, detailed information on the stranding site, condition of the carcass when found, as well as any other useful information was recorded by local volunteers.

Of the 3,154 stranding records available, cases of 353 harbour seals and 64 grey seals with any recorded external wound were rated as suspicious and selected for further investigations with regard to potential grey seal predation. Necropsy results were taken from the stranding database of the ITAW and judged retrospectively.

30 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

Cases, which were considered too decomposed for a thorough evaluation or data deficient, were categorised as “unknown” and excluded from the study.

Since the recognition of the predation phenomenon and the start of a specific research project in 2015, a specific protocol was developed for recording potential cases of grey seal predation as part of the necropsy (see Figure A.1 for the updated version based on the results presented here).

Cases were judged and rated in five prior defined categories (definite, likely, possibly, unlikely and fox) depending on the likelihood of grey seal predation as an origin of documented lesions (Table 1).

Table 1: Categories and their respective description used for rating the likelihood of grey seal predation and fox interaction.

Category Definition Definite The attack has been observed and the carcass was retrieved straight after It is highly likely that the documented trauma is the result of Likely grey seal predation; the majority of parameters 1 - 9 have been found It is possible that the documented trauma is the result of grey Possibly seal predation; some of the parameters 1 - 9 have been found; potentially some indication for a different origin of trauma It is unlikely that the documented trauma is the result of grey Unlikely seal predation; the majority of parameters have not been found; clear indication for a different origin of trauma It is unlikely that the documented trauma is the result of grey Fox seal predation, but indicators for an interaction with a red fox have been found (parameters 10, 11)

Besides any cases for which the carcass has been retrieved and was available for necropsy, anecdotal data consisting of well-documented observational reports from Helgoland for which no carcass was retrieved, have been judged by an experienced researcher and filed and summarised respectively. These cases have been labelled “observation only”.

Results

In total, 417 cases with external wounds were assessed of which 190 were categorised as “unknown” and excluded from further consideration due to the lack of sufficiently detailed information or due to advanced decomposition. Of the remaining 227 cases, 62 were categorised as “observation only” and excluded from any pathological based assessment, leaving 165 cases.

31 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

Gross pathological examination

During necropsies of suspected cases throughout the years 1995 to 2017, different wound patterns have been recorded.

Most characteristic for wounds induced by grey seals is a large laceration with a smooth, cut- like wound margin. The laceration often follows a helical course, starting in the area around the head and extending towards the caudal end of the body. Large parts of the blubber are usually detached from the underlying tissue and manipulation of this tissue is evident. When a carcass is found, the detached tissue can be turned inside out leaving the blubber exposed.

Using these and further findings, a catalogue of common parameters to be used during macroscopic assessment of seal carcasses has been assembled and is updated based on the existing knowledge and experience gathered (Table 2).

32 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

Table 2: Table showing 11 parameters, which have been added to the catalogue and are now routinely used for rating the likelihood of grey seal predation as origin of a trauma in seals. Pictures in parameters 1 – 8 courtesy of Dominik Nachtsheim.

Smooth wound margin: Due to the tearing of the skin, a 1 smooth, cut like wound margin is present throughout large parts of the lesion.

Missing of blubber tissue: Considerable parts of the blubber tissue show signs of manipulation. Often a diffuse uneven blubber surface with an irregular blubber depth (slightly 2 roundish structures surrounded by areas of reduced blubber depth) is evident. Blubber depth along the fringes of the skin flaps is in parts reduced and less than towards the middle areas.

Start of lesion in throat / head area: The origin of the lesion lays on the 3 ventral side of the neck or around the lower jaw / throat area, pieces of skin including the blubber can be missing in this part.

33 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

Undermining / detachment of blubber: Considerable parts of the blubber are detached from the underlying muscular 4 tissue in large parts of the body area. Only in the areas around the caudal end of the body, the pectoral flippers, as well as the rostral part of the head, the skin and underlying tissue is often still attached.

Rake marks in blubber: Rake marks potentially as the result of 5 the incisions by the teeth and/or claws are present in parts of the blubber tissue.

Puncture lesions: 6 Puncture lesions are present in the skin or blubber tissue.

Avulsion of one or both scapulae: An avulsion of one or both scapulae 7 due to the detachment of the skin and blubber (including the pectoral flippers) can be present.

34 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

Helical smooth edged lesion: A smooth edged laceration which 8 follows a helical course, starting in the area of the throat / head and circling backwards once or twice around the body is a common pattern observed.

Skeletal trauma: Fractures of bones with different severity can be present. Fractures of 9 parts of the skull have been observed. Also puncture like fractures in the lower mandible and / or scapula have been observed commonly.

Vast amounts of soft tissue and skin are removed: Lesions which can be situated anywhere on the body with vast 10 amounts of skin, blubber and muscle tissue missing are especially in addition to a ragged wound margin a strong indicator for an interaction with a red fox.

Ragged wound margin (fully or in parts): Considerable parts of the wound 11 margin have a ragged, uneven and not cut-like structure. This is a strong indication for an interaction with a red fox.

35 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

Using the absence and presence of parameters described above, 165 cases of suspected grey seal predation were judged and categorised respectively (Figure 1, Figure 2).

Figure 1: Number of suspected grey seal predation and fox related cases for the years 1995 - 2017; categorised by likelihood of grey seal predation (“likely”, “possibly”, “unlikely”), cases that have only been observed but without recovery of the carcass (“observation only”) and suspected fox interactions (“fox”). Cases related to fox interaction have been included in the category “unlikely” in terms of grey seal predation, but are also shown separately right of the dashed red line. The definite case from 2018 is not included in this figure. Number of grey seal victims are shown in dark grey, number of harbour seal victims in light grey bars.

36 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

Anecdotal data

Additionally, to the cases for which a carcass was retrieved, we filed 62 cases of anecdotal reports of suspected grey seal predation of seals (Figure 1 – category observation only).

Discussion

The phenomenon of grey seal predation of marine mammals has only recently been described in more detail in the scientific literature (Brownlow et al., 2016; Leopold et al., 2015; van Neer et al., 2019, 2015). Yet, the increasing amounts of reports need to be seen as an indicator for the potential ecological relevance of this behaviour. Therefore, in order to gain a better understanding of the scope and thus relevance of this behaviour, it is essential to quantify the number of cases applying standardised assessment criteria.

Post mortem examination

The results presented here summarise the findings of necropsies conducted on 166 cases of suspected grey seal predation from the coasts of Schleswig-Holstein. The respective parameters have been developed during necropsies over the last years and represent the most common lesions recorded.

Most evident in seal carcasses subjected to grey seal predation is a large tissue defect with a long, linear wound margin and partly detached skin resulting from the tearing of the skin

37 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

(parameter 1, 4). Like that, the predator exposes the energy rich blubber tissue which is then subsequently scraped off the skin using the teeth. The scraping of the blubber usually leaves a distinct uneven pattern as well as typical rake marks in the blubber tissue (parameter 2, 5) (van Neer et al., 2019). Lacerations induced by tearing of the skin show a cut-like, smooth and linear wound margin (parameter 1) which often starts in the throat / head area (parameter 3) and follows in a helical manner around the body (parameter 8). Further, puncture wounds potentially induced by the intrusion of the (canine) teeth can be found in the skin/blubber (parameter 6) and bones (parameter 9). The avulsion of one or both scapulae (parameter 7) has been documented commonly and is potentially the result of the force applied when detaching the skin from the body of the prey.

Looking at the frequency of the parameters described (Figure 2), several qualify as good indicators in order to assess the likelihood of grey seal predation as an origin of trauma. Parameters 1 – 7 show very high (> 85 %) and parameter 8 high (> 60 %) occurrence frequencies in “likely” cases and could also be confirmed as “present” in the definite case (van Neer et al., 2019). It needs to be stated though, that some variation is evident as some parameters, despite being found regularly in grey seal predation cases, can also be caused by other means such as scavenging by other predators (e.g. missing blubber (parameter 2)). Parameters 8 – 9 show a lower frequency for the “likely” cases with the parameter “skeletal trauma” occurring least frequent. For the skeletal trauma, this can be explained by using the observational reports. Grey seals seem to mainly target the blubber as a source of high energy content by detaching the skin from the body (van Neer et al., 2019). During this process the skeletal parts of the body are usually not manipulated with exception of the scapulae. If present, skeletal trauma is often found in the area of the skull possibly resulting from the catching and fixation of the prey (Brownlow et al., 2016; van Neer et al., 2019). In addition, skeletal trauma or as well puncture lesions can have many different natural and unnatural causes and thus are not considered as a very good indicator but should be used as additional indicator especially when there is the possibility to measure the inter-canine distance which can be indicative for a specific predator. In contrast, the avulsion of one or both scapulae (parameter 7) and a smooth lesion following a helical course around the body (parameter 1 and 8) can be seen as rather typical even if they are not detected in every single case.

Loss of muscle tissue can most likely in large parts be attributed to scavenging e.g. by birds. This is supported by the definite case (van Neer et al., 2019), as well as an earlier observation made in 2016 on Helgoland, where a potentially predated harbour seal showing the typical lesions was washed ashore and the muscle tissue in the area of the wound which was largely intact, was removed considerably by birds within 90 minutes (Abbo van Neer, personal observation).

38 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

As has been described in the literature before (e.g. van Neer et al., 2019), assessing the condition of the hair bordering the wound margin on the cranial side of the laceration can give first important indications if the tissue defect was induced by cutting or shearing forces and thus, should be considered in any future assessments. As this factor has not been verified throughout all necropsies reported here, it was excluded from the above list of parameters but should be added to the list and used in future assessments (for the updated version of parameters see Figure A.1).

When assessing marine mammal carcasses in the context of grey seal predation which originate from areas with terrestrial predators and/or scavengers present, it should be noted, that lesions induced by terrestrial mammals are often difficult to differentiate from the ones induced by grey seals. On the German island of Sylt, several carcasses have been retrieved also showing extensive defects of the skin and the underlying tissue but have been confirmed by either personal observation through the local seal ranger or by the documentation of tracks and other indicators surrounding the carcass, that either predation (for the observed case) or most likely post mortem scavenging (for the other cases) by the red fox (Vulpes vulpes) was the origin of the lesions. In order to differentiate, again the wound margin should be assessed carefully. As can be seen in Figure 2, while in grey seal predation a linear and fully smooth wound margin (parameter 1, 8) is considered typical, in cases of feeding by foxes a rather ridged wound margin (parameter 11) with considerable areas of soft tissue missing (parameter 10) can be expected. This potentially originates from the separation of the tissue by using their carnassial teeth opposed to tearing as seen in grey seals. Further, the location and course of the wound can be indicative. Wounds produced by grey seals most often start in the area around the head (parameter 3) and follow a rather helical course (parameter 8) often with the possibility of fully repositioning both sides of the wound margin. In contrast, in lesions induced by foxes, most often vast amounts of skin are missing in different areas of the body and wounds resemble areas where most soft tissue has been removed and only the bones are left (parameter 10). In addition to the general macroscopic assessment, wound margins on the cranial side should also be assessed to check for an increased degree of damaged hair shafts which would also be expected in cases of scavenging by terrestrial mammals.

Lesions presented here are consistent with the results reported by Brownlow et al. (2016) and also with an earlier report on grey seal predation of harbour seals from Helgoland (van Neer et al., 2015). Therefore, the resulting parameters should be seen as an important set of

39 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

Figure 3: Subadult grey seal bull with a juvenile harbour seal as prey and shown while inducing the typical helical lacerations by tearing apart the tissue using its pectoral flipper and jaws. Helgoland, 04.10.2017. Picture courtesy of Susanne Hauswaldt. indicators when it comes to evaluating the cause of trauma. Due to the possibility of other factors causing respective alterations, it needs to be emphasised, that no single parameter should be used alone in order to rate the likelihood of grey seal predation. Rather the combination of parameters in conjunction with any other information available (e.g. information on the stranding site and situation or detected predator DNA) allows for a thorough evaluation. Yet, a definite proof that grey seal predation is the origin of a trauma in a seal carcass can to date only be based on an observation and the immediate retrieval of the carcass.

Behavioural observations

Up until today, several observational / anecdotal reports of grey seals preying on marine mammals have been filed in Germany (Figure 1). Such reports originate from different groups ranging from lay people, through semi-professionals to professionals in the field of marine mammal science. Taking into account such information as well as detailed accounts of behavioural observations as recently described in the literature (Bishop et al., 2016; Brownlow et al., 2016; van Neer et al., 2019), the behaviour seems to follow fairly stereotyped steps. Considering the reported observations, most parameters suggested above can be linked to a specific behaviour.

Following the death of the prey (usually either by exsanguination or by asphyxiation), the carcass is opened near the neck and throat area (parameter 3, see also Figure 3) using the pectoral flippers and jaws. The tearing of skin leaves a smooth cut-like laceration (parameter 1 & 8). It also results in the detachment of the blubber from the body (parameter 4) and can

40 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour also cause the avulsion of one or both scapulae (parameter 7). Blubber is scraped of the detached areas of skin using the teeth (parameter 2, 5) and biting can induce puncture lesions throughout the carcass (parameter 6).

For porpoises it has been described, that individual animals most likely have escaped attacks by grey seals and apparently survived the inflicted injuries at least for some time (Podt & IJsseldijk, 2017). For seals this is barely documented. One potential case of a harbour seal has been documented on Helgoland. The seal showed severe injuries in the area of the throat which could potentially result from a prior grey seal attack (Figure 4). Nevertheless,

Figure 4: Harbour seal with severe injuries in the area of the despite the similarity of lesions other throat. Helgoland, Germany, September 2018. sources of trauma cannot be excluded.

To date only subadult or young adult male grey seals have been documented showing this behaviour in Germany. They have been observed to prey on harbour seals as well as grey seals, but most often with the prey not older than two years. The oldest animal observed to have been preyed on, was a female grey seal judged by the outer appearance to be three to four years of age. Up until to date, at least four different individuals have been identified to prey on seals, by means of pelage pattern or flipper tag ID. One bull was observed showing this behaviour over consecutive years and as well utilising grey and harbour seals as prey resource on separate occasions (see report of van Neer et al. (2019) & Figure 3, both describe / show the same individual bull). This indicates that such specialisation is not focused on a single prey species but potentially directed towards marine mammals as prey resource in general. It is further unclear, if single individuals utilise both, seals and porpoises, as so far no single individual has been reported preying on seals and porpoises alike. In order to verify this, genetic analysis of e.g. scat samples as well as further observational studies should be conducted.

Conclusion

In order to establish a reliable diagnostic procedure for the differentiation of potential causes of lesions on carcasses of marine mammals with regard to grey seal predation, it is crucial to examine any carcass carefully and in detail using standardised parameters as these suggested here. Additionally, any source of information like reports on indicators on the site of the 41 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour stranding or eye witness reports should be used in the decision making process. Parameters should also be continuously refined based on any new evidence that arises. Further research should be conducted in order to assess the suitability of other methods that could be applied, such as the histological evaluation of wound margins to differentiate between causes of trauma or easy to use methods enabling the detection of grey seal DNA in harbour seal carcasses similar to the ones suggested for porpoise carcasses (Heers et al., 2017, 2018).

Cases of observed grey seal predation should be further used as reliable source of information with regards to the behaviour as well as pathology. Scientists working on this topic should readily join forces to constantly update the list of parameters if necessary and establish an objective method and thus allow for a comprehensive evaluation of the potential ecological effects on the respective populations in the future.

Acknowledgements

We thank especially the local seal rangers throughout Schleswig-Holstein for their invaluable help retrieving carcasses and collecting information, as well as all the people who reported interesting observations to us. We thank also the Ministry of Energy, Agriculture, the Environment, Nature and Digitalization Schleswig-Holstein Germany for funding of this work and the necropsy team of the ITAW for their great effort. Further we would like to thank Dominik Nachtsheim and Susanne Hauswaldt for taking pictures.

References

Andreasen, H., Ross, S. D., Siebert, U., Andersen, N. G., Ronnenberg, K., & Gilles, A. (2017). Diet composition and food consumption rate of harbor porpoises (Phocoena phocoena) in the western Baltic Sea. Marine Mammal Science, 33, 1053–1079. doi:10.1111/mms.12421 Benke, H., Siebert, U., Lick, R., Bandomir, B., & Weiss, R. (1998). The current status of harbour porpoises (Phocoena phocoena) in German waters. Archive Of Fishery And Marine Research, 46(2), 97–123. Bishop, A. M., Onoufriou, J., Moss, S., Pomeroy, P. P., & Twiss, S. D. (2016). Cannibalism by a male grey seal (Halichoerus grypus) in the North Sea. Aquatic Mammals, 42(2), 137–143. doi:10.1578/AM.42.2.2016.137 Bouveroux, T., Kiszka, J. J., Heithaus, M. R., Jauniaux, T., & Pezeril, S. (2014). Direct evidence for gray seal (Halichoerus grypus) predation and scavenging on harbor porpoises (Phocoena phocoena). Marine Mammal Science, 30(4), 1542–1548. doi:10.1111/mms.12111 Brasseur, S., Cremer, J., Czeck, R., Galatius, A., Jeß, A., Körber, P., Pund, R., Siebert, U., Teilmann, J., & Klöpper, S. (2018). TSEG grey seal surveys in the Wadden Sea and Helgoland in 2017-2018 - More than ten years of growth in the Wadden Sea area. Wilhelmshaven, Germany. Retrieved from http://www.waddensea-secretariat.org/sites/default/files/downloads/ TMAP_downloads/Seals/18-07-02_greysealreport2018.pdf Brownlow, A., Onoufriou, J., Bishop, A., Davison, N., & Thompson, D. (2016). Corkscrew Seals: Grey Seal (Halichoerus grypus) Infanticide and Cannibalism May Indicate the Cause of Spiral Lacerations in Seals. PLOS ONE, 11(6), e0156464. doi:10.1371/journal.pone.0156464

42 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

CWSS. (2017). Wadden Sea Quality Status Report. (S. Klöpper, Ed.). Wilhelmshaven, Germany: Common Wadden Sea Secretariat. Retrieved from www.qsr.waddensea- worldheritage.org/reports/ Das, K., Lepoint, G., Leroy, Y., & Bouquegneau, J. M. (2003). Marine mammals from the southern North Sea: feeding ecology data from δ13C and δ15N measurements. Marine Ecology Progress Series, 263, 287–298. doi:10.3354/meps263287 Galatius, A., Brasseur, S., Czeck, R., Jeß, A., Körber, P., Siebert, U., Teilmann, J., & Klöpper, S. (2017). Aerial surveys of Harbour Seals in the Wadden Sea in 2017 - Population counts still in stagnation, but more pups than ever. Wilhelmshaven, Germany. Retrieved from http://www.waddensea-secretariat.org/sites/default/files/downloads/TMAP_downloads/Seals/ 17-11-09_harboursealreport2017.pdf Haelters, J., Kerckhof, F., Jauniaux, T., & Degraer, S. (2012). The grey seal (Halichoerus grypus) as a predator of harbour porpoises (Phocoena phocoena)? Aquatic Mammals, 38(4), 343–353. doi:10.1578/AM.38.4.2012.343 Haelters, J., Kerckhof, F., van Neer, A., & Leopold, M. (2015). Letter to the Editor Exposing Grey Seals as Horses and Scientists as Human. Aquatic Mammals, 41(3), 351–353. doi:10.1578/AM.41.3.2015.351 Hammond, P. S., & Wilson, L. J. (2016). Grey Seal Diet Composition and Prey Consumption. Scottish Marine and Freshwater Science, 7(20), 1–28. doi:10.7489/1799-1 Heers, T., van Neer, A., Becker, A., Grilo, M. L., Siebert, U., & Abdulmawjood, A. (2017). Loop- mediated isothermal amplification (LAMP) assay—A rapid detection tool for identifying red fox (Vulpes vulpes) DNA in the carcasses of harbour porpoises (Phocoena phocoena). PLOS ONE, 12(9), e0184349. doi:10.1371/journal.pone.0184349 Heers, T., van Neer, A., Becker, A., Gross, S., Hansen, K. A., Siebert, U., & Abdulmawjood, A. (2018). Loop-mediated isothermal amplification (LAMP) as a confirmatory and rapid DNA detection method for grey seal (Halichoerus grypus) predation on harbour porpoises (Phocoena phocoena). Journal of Sea Research, 140(July), 32–39. doi:10.1016/j.seares.2018.07.008 ICES. (2018). Report of the working group on marine mammal ecology (WGMME). ICES CM 2018/ACOM:28. La Rochelle, France, 19–22 February 2018: International Council for the Exploration of the Sea. Retrieved from http://www.ices.dk/sites/pub/Publication Reports/Expert Group Report/acom/2018/WGMME/wgmme_2018.pdf Leopold, M. F., Begeman, L., van Bleijswijk, J. D. L., IJsseldijk, L. L., Witte, H. J., & Gröne, A. (2015). Exposing the grey seal as a major predator of harbour porpoises. Proceedings of the Royal Society of London B: Biological Sciences, 282(1798), 20142429. doi:10.1098/rspb.2014.2429 Podt, A. E., & IJsseldijk, L. L. (2017). Grey seal attacks on harbour porpoises in the Eastern Scheldt: cases of survival and mortality. Lutra, 60(2), 105–116. Retrieved from http://rugvin.nl/wp-content/uploads/2018/01/Lutra-602_Podt-IJsseldijk_2017_lr.pdf Reijnders, P. J. H., van Dijk, J., & Kuiper, D. (1995). Recolonization of the Dutch Wadden Sea by the grey seal Halichoerus grypus. Biological Conservation. doi:10.1016/0006- 3207(94)00032-L Sharples, R. J., Moss, S. E., Patterson, T. A., & Hammond, P. S. (2012). Spatial variation in foraging behaviour of a marine top predator (Phoca vitulina) determined by a large-scale satellite tagging program. PloS one, 7(5), e37216. doi:10.1371/journal.pone.0037216 Siebert, U., Wünschmann, A., Weiss, R., Frank, H., Benke, H., & Frese, K. (2001). Post- mortem findings in harbour porpoises (Phocoena phocoena) from the German North and Baltic Seas. Journal of Comparative Pathology, 124(2–3), 102–114. doi:10.1053/jcpa.2000.0436

43 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

Siebert, U., Wohlsein, P., Lehnert, K., & Baumgärtner, W. (2007). Pathological Findings in Harbour Seals (Phoca vitulina): 1996–2005. Journal of Comparative Pathology, 137(1), 47– 58. doi:10.1016/j.jcpa.2007.04.018 van Neer, A., Gross, S., Kesselring, T., Wohlsein, P., Leitzen, E., & Siebert, U., (2019) Behavioural and Pathological Insights into a Case of Active Cannibalism by a Grey Seal (Halichoerus grypus) on Helgoland, Germany. Journal of Sea Research, 148–149, 12–16. https://doi.org/10.1016/j.seares.2019.03.004. van Neer, A., Jensen, L. F., & Siebert, U. (2015). Grey seal (Halichoerus grypus) predation on harbour seals (Phoca vitulina) on the island of Helgoland, Germany. Journal of Sea Research, 97, 1–4. doi:10.1016/j.seares.2014.11.006

44 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

Supplementary Materials

Figure A.1: Newly developed protocol to be used for the documentation of suspected grey seal predation cases in its updated version based on the results presented here.

45 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

46 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

47 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

48 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

49 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

c. Putting together the pieces of the puzzle - Part II - Assessing harbour porpoise carcasses potentially subjected to grey seal (Halichoerus grypus) predation

Written by: van Neer, A., Gross, S., Kesselring, T., Ludes-Wehrmeister, E., Roncon, G., Grilo, M. & Siebert, U. (in preparation);

Picture by Abbo van Neer

50 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

Abstract

As a follow-up on the data presented in van Neer et al. (in preparation -a), we would like to report and discuss outcomes resulting from a retrospective evaluation of harbour porpoise stranding and necropsy data (n = 4,240) collected on the coasts of Schleswig-Holstein, Germany, between 1990 and 2017. As in the prior study, results are compared to a recent case of definite grey seal predation from Germany, which in this case has been confirmed using genetic methods. Further results will also be compared to reports from other countries. Porpoise carcasses potentially subjected to grey seal predation show severe lacerations, mainly starting in the area of the throat. Large parts of skin und underlying tissue are detached from the body and loss of blubber tissue is common. Additionally, puncture lesions are most often present but need to be judged carefully as they can be the result of other none related processes. Based on the occurrence frequencies of encountered lesions from suspected and definite grey seal predation cases, a list of parameters to be used for the assessment of similar cases is suggested. Considered cases are categorised by likelihood of grey seal predation using the absence and presence of the respective parameters. The presented results allow for a standardised assessment of suspected grey seal predation cases with the aim of enabling a joint evaluation of the potential ecological relevance of this behaviour.

Background

Since the year 2012, when the first scientific publication introduced the hypothesis that grey seals (Halichoerus grypus) utilise harbour porpoises (Phocoena phocoena) as prey resource (Haelters et al., 2012), several publications have proven the hypothesis to be true (Bouveroux et al., 2014; Heers et al., 2018; Jauniaux et al., 2014; Leopold et al., 2015a; Podt & IJsseldijk, 2017; Stringell et al., 2015; Van Bleijswijk et al., 2014). Further, it was shown that this behaviour is not limited to specific regions but potentially occurs throughout the North Sea and beyond (ICES, 2017).

Published descriptions of gross pathological examinations of porpoise carcasses include traumatic lesions with large areas of detached skin and blubber, canine puncture wounds throughout the lesions and parallel bite and scratch marks in the skin. Retrospective analysis of the Dutch stranding database could show that at least 17 % of stranded porpoises most likely have died as the result of grey seal predation representing one of the most often detected causes of death (Leopold et al., 2015b). In other areas of the world, predation rates of porpoises are largely unknown and therefore the ecological significance of this behaviour is still not entirely clear.

51 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

An objective differentiation between lesions induced by grey seals in comparison to other sources of trauma is often difficult, thus the situation in porpoises is comparable to the one in seals (van Neer et al., in preparation -a).

To allow for a comparison of predation rates in different areas, standardised assessment criteria should be used. Therefore, the aim of this study is to summarise the findings collected during necropsies of harbour porpoises (Phocoena phocoena) found dead between 1990 and 2017 on the coasts of Schleswig-Holstein. Results are discussed in comparison to a definite case of predation as well as to cases reported in the literature.

Material and Methods

Carcasses were collected through the stranding network (Benke et al., 1998; Siebert et al., 2001) and partly necropsied (Siebert et al., 2007). In contrast to the data available for seals, in German waters no case of a harbour porpoise being preyed on by a grey seal has to date been documented in full comparably to what has been described by van Neer et al. (2019). Therefore, in order to validate the documented wound patterns, a definite case of grey seal predation which has been confirmed using genetic methods was used (for details, see Heers et al., 2018). Necropsies included age estimation, determination of sex, weight, length, nutritional state, health status and cause of death. As was described for seals, decomposition state of the animal was rated by assessing the remaining intact parts of the body (van Neer et al., in preparation -a). In most cases detailed information on the stranding site, condition of the carcass when found, as well as any other useful information was recorded by local volunteers.

Of the 4,240 stranding records available, cases of 1,070 harbour porpoises with any recorded external wounds were rated as suspicious and selected for further investigations. Necropsy results were taken from the stranding database of the Institute for Terrestrial and Aquatic Wildlife Research (Büsum, Germany) and judged retrospectively.

Cases, which were considered too decomposed for a thorough evaluation or data deficient, were categorised as “unknown” and excluded from the study.

Cases were judged and rated in six prior defined categories (definite, likely, possibly, escape, unlikely and fox) depending on the likelihood of grey seal predation as an origin of documented lesions (Table 1).

52 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

Table 1: Categories and their respective description used for rating the likelihood of grey seal predation and fox interaction.

Category Definition The attack has been observed and the carcass was retrieved Definite straight after or the origin of trauma could be verified using genetic methods It is highly likely that the documented trauma is the result of Likely grey seal predation; the majority of parameters have been found It is possible that the documented trauma is the result of grey Possibly seal predation; some of the parameters have been found; potentially some indication for a different origin of trauma

Escape Scar tissue is present indicating prior interactions with a grey seal, but the cause of death is not directly related It is unlikely that the documented trauma is the result of grey Unlikely seal predation; the majority of parameters have not been found; clear indication for a different origin of trauma It is unlikely that the documented trauma is the result of grey Fox seal predation, but indicators for an interaction with a red fox have been found

Besides any cases for which the carcass has been retrieved and was available for necropsy, anecdotal data consisting of well-documented observational reports from Helgoland for which no carcass was retrieved, have been filed and summarised. These cases have been labelled “observation only”.

Results

Of the 4,240, which have been considered, in total 1,070 cases were rated as suspicious due to detected external lesions and assessed accordingly. Of these cases, 835 were categorised as “unknown” and excluded from further consideration due to the lack of sufficiently detailed information or due to advanced decomposition. Of the remaining 235 cases, four were categorised as “observation only” and excluded from any pathological based assessment, leaving 231 cases.

Gross pathological examination

During necropsies of suspected cases throughout the last years, different wound patterns have been recorded. Most characteristic for wounds in harbour porpoises induced by grey seals is a large laceration with a smooth, cut-like wound margin. Large parts of the skin and blubber are usually detached from the underlying tissue and either still partly attached as skin flaps or fully removed. Manipulation of the skin and blubber tissue is evident and often parallel situated puncture and scratch induced lesions can be found. Besides cases for which grey seal

53 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour predation was regarded as the most likely direct cause of death, six cases were found which have died of other causes but showed different amounts of healed lesions evident as scars. Detected scar patterns resembled for example lesions induced by teeth or scratching using the claws (e.g. Figure 1).

Using these and further findings, a catalogue of common parameters to be used during macroscopic assessment of porpoise carcasses has been Figure 1: Healed lesions on a porpoise fluke assembled and is updated based on the existing potentially induced by grey seal teeth. knowledge and experience gathered (Table 2).

54 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

Table 2: Table showing 11 parameters, which have been added to the catalogue and are now routinely used for rating the likelihood of grey seal predation as origin of a trauma in porpoises.

Puncture lesions: 1 Puncture lesions are present in the skin or blubber tissue.

Missing of blubber tissue: Parts of the blubber tissue show signs 2 of manipulation. Blubber depth along the fringes of the skin flaps is often in parts reduced and less than towards the middle areas.

Start of lesion in throat / head area: 3 The origin of the lesion lays on the ventral side of the neck or around the lower jaw / throat area.

55 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

Smooth wound margin: Due to the tearing of the skin, a 4 smooth, cut like wound margin is present throughout large parts of the lesion.

Parallel bite/scratch marks: Parallel running scratches resembling claw scratches or repetitive, parallel 5 puncture lesions resembling bite marks are commonly observed. Bite marks in the area of the tail stock are usually found to be present bilaterally.

Undermining / detachment of blubber: Considerable parts of the blubber are detached from the underlying muscular 6 tissue in large parts of the body area. Only in the areas around the caudal end of the body, the fluke, as well as the rostral part of the head, the skin and underlying tissue is often still attached.

Areas of skin are missing: 7 Larger areas of skin including the underlying blubber tissue can be missing.

56 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

Rake marks in blubber: Rake marks potentially as the result of 8 the incisions by the teeth and/or claws are present in parts of the blubber tissue.

Avulsion of one or both scapulae: An avulsion of one or both scapulae 9 due to the detachment of the skin and blubber (including the pectoral fins) can be present.

Skeletal trauma: Fractures of bones with different severity can be present. Fractures / removal of parts of or whole extremities 10 has been observed commonly in fox related cases. Also puncture-like fractures in the lower mandible and / or scapula have been observed commonly.

57 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

Ragged wound margin (fully or in parts): Considerable parts of the wound 11 margin have a ragged, uneven and not cut-like structure. This is a strong indication for an interaction with a red fox or other scavengers.

Using the absence and presence of parameters described above as well as the observed cases, 234 cases of suspected grey seal predation were judged and categorised respectively (Figure 2). Categorising solely based on the pathological assessment has been conducted for 230 cases as well as the definite case (Figure 3).

Figure 2: Number of suspected grey seal predation and fox related cases for the years 1990 - 2017; categorised by likelihood of grey seal predation (“likely”, “possibly”, “unlikely”), cases that have only been observed but without recovery of the carcass (“observation only”) and suspected fox interactions (“fox”). Cases related to fox interaction have been included in the category “unlikely” in terms of grey seal predation, but are also shown separately right of the dashed red line. The definite case is not included in this figure.

58 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

Figure 3: Percentage of occurrence of the 11 parameters in the different categories for suspected grey seal predation cases (“definite”, “likely”, “possibly”, “unlikely“) and for suspected fox interactions (“fox”). Cases related to fox interaction are only shown in the category “fox”, despite also being “unlikely” with regards to grey seal predation. Parameters framed with a red rectangle are indicative for an interaction with a fox.

Anecdotal data

Additionally, to the cases for which a carcass was retrieved, we filed four cases of anecdotal reports of suspected grey seal predation of harbour porpoises (Figure 2).

Discussion

When assessing the status and the development of populations, one important factor to consider is the mortality rate and its underlying sources (Wade, 1998). If unsatisfactory, this information can then be used to develop and implement specific management measures for example addressing the major sources of unnatural mortality (Loughlin & York, 2000). With regard to the potential ecological relevance of the phenomenon of grey seal predation, it is therefore also important to have reliable estimates of harbour porpoise mortalities resulting from grey seal predation as one natural cause. To allow for a standardised assessment of lesions found in suspected grey seal predation cases, this study aims to summarise the knowledge that has been described and gathered throughout the last six years.

Post mortem examination

The parameters described above resemble the most commonly detected lesions in 231 suspected cases of grey seal predation from the coasts of Schleswig-Holstein including the definite case of grey seal predation. With regard to grey seal predation, parameters 1 – 9

59 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour represent typical lesions, whereas the presence of parameters 10 and 11 is indicative for an interaction with a red fox (Vulpes vulpes) or other scavengers.

In contrast to lesions detected in seals, lesions in porpoises resemble most often puncture lesions in the skin and blubber (parameter 1). Yet, visually most striking is the commonly detected large tissue defect with straight, cut-like wound margins and often flaps of skin and blubber remaining only partly attached to the body (parameter 4, 6, 7). Missing blubber (parameter 2) is also recorded, either as reduced blubber thickness on the flaps of skin or as fully removed parts of blubber and skin. As has been described for seals, the lesion most often originates in the area of the head / throat (parameter 3). A difference that has been detected between the lesions in seals and porpoises is the rate of clear parallel running bite and / or scratch marks in the skin of the animals. Whereas this is rarely detected in seals, most porpoises show respective marks (parameter 5). One potential explanation for this dissimilarity could be the different physical properties of the two types of skin. Seal skin is in contrast to porpoise skin very dense and elastic and tearing and rupturing the skin requires a considerable amount of force (Grear et al., 2018). Porpoise skin, however, is fairly fragile and puncturing or tearing it requires comparably little force (Kipps et al., 2002). These different mechanical properties might also be the explanation why rake marks are found in the blubber (parameter 8) more often in seals than in porpoises. In seals usually little to no skin is missing whereas in porpoises a considerable number of cases have been found where skin is missing (parameter 7). Grey seals have been described to mainly target the energy rich blubber tissue of their prey (van Neer et al., in preparation -a, 2019). For the elastic and robust seal skin this is done by scraping of the blubber using the teeth. For the fragile porpoise skin, we suggest with reference to the existing recordings (e.g. Stringell et al., 2015) and the findings during the necropsies, that the skin including the underlying blubber is more often fully removed by the grey seal and swallowed in whole. If true, this may be related to the net energetic gain which is acquired by the predator. Scraping of blubber tissue from seal skin will likely yield less tissue and cost more energy than being able to tear of whole pieces of blubber (including skin) and thus, may result in a lower energetic gain. However, it is still unclear if the process of catching a porpoise in comparison to younger seals might also cost a considerably larger amount of energy negatively influencing the net gain.

Similar to what has been described for seals, the avulsion of one or both scapulae (parameter 9) can be found and is also likely the result of the force applied when detaching the skin from the body of the prey.

For porpoises, all nine suggested parameters have been found in the definite case of grey seal predation. Parameters 1 – 4 show a very high (≥ 95 %) and parameters 5 - 6 a high rate (90 %) of occurrence in likely cases. Parameters 7 – 9 occur less frequent but still have been

60 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour found in ≥ 50 % of all likely predation cases. These high rates of occurrence throughout all parameters suggest that wound patterns found in porpoises are less variable than the patterns found in seals. To state, if this difference is a result of the different mechanical skin properties or if other factors are responsible, is beyond the scope of this study.

While for seals a skeletal trauma is used as an indicator for grey seal predation, for respective harbour porpoise cases, this is hardly ever (10 %) documented. In contrast, for porpoises a skeletal trauma (parameter 10) is quite frequently (51 %) detected in cases related to scavenging by foxes where for example extremities can be manipulated (Haelters et al., 2016). Like to what has been found in seals, the most often detected parameter in fox related cases is with 94 % the ragged wound margin (parameter 11). Therefore, this can be seen as a good fox indicator in porpoises. This is also confirmed by a definite case of fox scavenging which was confirmed using genetic methods (for details see Heers et al., 2017). It needs to be stated though, that scavenging by birds can result in similar looking lesions increasing the chance of misinterpretation. If rake marks are present, the origin of the lesion can be assessed by measuring the distances of the scratches and comparing them to published values of grey seal/fox teeth distances (e.g. Haelters et al., 2012).

Single puncture lesions in turn, are not considered a very good indicator despite being present in the definite and all of the likely cases. Mainly due to the fragility of the porpoise skin, such lesions can have many different causes (e.g. feeding by birds).

Whether a loss of muscle tissue can be attributed to grey seal predation or is largely caused by scavengers like gulls as has been suggested for seals (van Neer et al., in preparation -a) is still not entirely clear. In German as well as bordering waters, no clear pattern is prevailing. Carcasses with mainly intact as well as fully removed muscle tissue have been documented (c.f. Leopold et al., 2015b). However, the reports by Stringell et al. (2015) suggest that not only the blubber tissue is targeted, but there seems to be some behavioural variation.

Findings and the resulting parameters described here are in line with wound patterns described in earlier publications from other areas (Bouveroux et al., 2014; Haelters et al., 2012; Jauniaux et al., 2014; Leopold et al., 2015b). This shows, that the documented wound patterns make a reliable set of parameters when assessing harbour porpoise carcasses potentially predated by a grey seal and should be used in future assessments.

Besides cases for which the attack of a grey seal directly led to the death of the animal, interestingly, it seems not unusual that porpoises escape this physically superior predator. Several cases have been described in the literature (Jauniaux et al., 2014; Leopold et al., 2015b; Podt & IJsseldijk, 2017) and also in German waters six cases could be documented (Figure 1, Figure 2). Although there has been the odd case of a severely injured seal showing

61 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour comparable lesions to what is associated with grey seal induced lesions (van Neer et al., in preparation -a), such high rates of escape cases as for porpoises have not been reported for seals.

Some of the observed behaviour of grey seals when catching a porpoise can be directly linked to the detected lesions. Stringell et al. (2015) as well as Bouveroux et al. (2014) for example described the grey seal acting as an ambush predator and attacking the porpoise from below using its’ jaw to retain the prey. Lesions starting in the throat area (parameter 3) combined with parallel multifocal puncture lesions (parameter 5) resemble what would be expected as the result of such an attack.

Despite a lower rate of variability in detected wound patterns in porpoise carcasses, care should be applied when assessing lesions as there is always the chance of other factors being involved. Therefore, if possible, a combination of data sources (necropsy results, indicators on the site of the stranding, eye witness reports, etc.) should be used for a systematic evaluation.

With regard to grey seal predation, future research should focus on continuing thorough investigations of stranded marine mammal carcasses in order to further update and refine the suggested set of parameters. Additionally, results of current as well as retrospective analysis of stranding data should be used to support an evaluation of the ecological relevance of this behaviour.

Conclusion

For the pathological assessment of harbour porpoise carcasses, similar to what has been described for seals, challenges prevail when it comes to the differentiation of lesions induced by a grey seal from other sources of trauma. Therefore, it is important to constantly refine sets of parameters deemed useful in this context as soon as new knowledge arises. For a thorough investigation, it is crucial to examine any carcass in detail using standardised parameters as well as consider any source of information like reports on indicators on the site of the stranding or eye witness reports. Additionally, to the pathological assessment, the use of supporting methods such as the molecular verification of specific predator DNA is highly encouraged (Heers et al., 2017, 2018; Van Bleijswijk et al., 2014).

Future research should also focus on elucidating the, to date predominantly unknown behavioural mechanisms behind this phenomenon using observational and bio-logging techniques.

Despite the many questions that remain unanswered, it needs to be emphasised, that up until today a solid knowledge base has been built by researches around the North Sea and beyond. This was done in a joint afford to document and publish new findings with regard to the

62 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour phenomenon of grey seal predation on marine mammals aiming at reliable future assessments of the ecological relevance.

Acknowledgements

References

Benke, H., Siebert, U., Lick, R., Bandomir, B., & Weiss, R. (1998). The current status of harbour porpoises (Phocoena phocoena) in German waters. Archive Of Fishery And Marine Research, 46(2), 97–123. Bouveroux, T., Kiszka, J. J., Heithaus, M. R., Jauniaux, T., & Pezeril, S. (2014). Direct evidence for gray seal (Halichoerus grypus) predation and scavenging on harbor porpoises (Phocoena phocoena). Marine Mammal Science, 30(4), 1542–1548. doi:10.1111/mms.12111 Grear, M. E., Motley, M. R., Crofts, S. B., Witt, A. E., Summers, A. P., & Ditsche, P. (2018). Mechanical properties of harbor seal skin and blubber − a test of anisotropy. Zoology, 126, 137–144. doi:10.1016/j.zool.2017.11.002 Haelters, J., Everaarts, E., Bunskoek, P., Begeman, L., Hinrichs, J. W. J., & IJsseldijk, L. L. (2016). A Suspected Scavenging Event by Red Foxes (Vulpes vulpes) on a Live, Stranded Harbour Porpoise (Phocoena phocoena). Aquatic Mammals, 42(2), 227–232. doi:10.1578/AM.42.2.2016.227 Haelters, J., Kerckhof, F., Jauniaux, T., & Degraer, S. (2012). The grey seal (Halichoerus grypus) as a predator of harbour porpoises (Phocoena phocoena)? Aquatic Mammals, 38(4), 343–353. doi:10.1578/AM.38.4.2012.343 Heers, T., van Neer, A., Becker, A., Grilo, M. L., Siebert, U., & Abdulmawjood, A. (2017). Loop- mediated isothermal amplification (LAMP) assay—A rapid detection tool for identifying red fox (Vulpes vulpes) DNA in the carcasses of harbour porpoises (Phocoena phocoena). PLOS ONE, 12(9), e0184349. doi:10.1371/journal.pone.0184349 Heers, T., van Neer, A., Becker, A., Gross, S., Hansen, K. A., Siebert, U., & Abdulmawjood, A. (2018). Loop-mediated isothermal amplification (LAMP) as a confirmatory and rapid DNA detection method for grey seal (Halichoerus grypus) predation on harbour porpoises (Phocoena phocoena). Journal of Sea Research, 140(July), 32–39. doi:10.1016/j.seares.2018.07.008 ICES. (2017). Report of the Workshop on Predator-prey Interactions between Grey Seals and other marine mammals (WKPIGS). (N. Hanson, A. van Neer, A. Brownlow, & J. Haelters, Eds.). Middelfart, Denmark: ICES. Retrieved from http://www.ices.dk/sites/pub/Publication Reports/Expert Group Report/SSGEPD/2017/01 WKPIGS - Report of the Workshop on Predator-prey Interactions between Grey Seals and other marine mammals.pdf Jauniaux, T., Garigliany, M.-M., Loos, P., Bourgain, J.-L., Bouveroux, T., Coignoul, F., Haelters, J., Karpouzopoulos, J., Pezeril, S., & Desmecht, D. (2014). Bite injuries of grey seals (Halichoerus grypus) on harbour porpoises (Phocoena phocoena). PloS one, 9(12), e108993. doi:10.1371/journal.pone.0108993

63 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

Kipps, E. K., Mclellan, W. A., Rommel, S. A., & Pabst, D. A. (2002). Skin density and its influence on buoyancy in the manatee (Trichechus manatus latirostris), harbor porpoise (Phocoena phocoena), and bottlenose dolphin (Tursiops truncatus). Marine Mammal Science, 18(3), 765–778. doi:10.1111/j.1748-7692.2002.tb01072.x Leopold, M. F., Begeman, L., Heße, E., van der Hiele, J., Hiemstra, S., Keijl, G., Meesters, E. H., Mielke, L., Verheyen, D., & Gröne, A. (2015a). Porpoises: From predators to prey. Journal of Sea Research, 97, 14–23. doi:10.1016/j.seares.2014.12.005 Leopold, M. F., Begeman, L., van Bleijswijk, J. D. L., IJsseldijk, L. L., Witte, H. J., & Gröne, A. (2015b). Exposing the grey seal as a major predator of harbour porpoises. Proceedings of the Royal Society of London B: Biological Sciences, 282(1798), 20142429. doi:10.1098/rspb.2014.2429 Loughlin, T. R., & York, A. E. (2000). An accounting of the sources of Steller sea lion, Eumetopias jubatus, mortality. Marine Fisheries Review, 62(4), 40–45. Retrieved from http://aquaticcommons.org/9764/1/mfr6242.pdf Podt, A. E., & IJsseldijk, L. L. (2017). Grey seal attacks on harbour porpoises in the Eastern Scheldt: cases of survival and mortality. Lutra, 60(2), 105–116. Retrieved from http://rugvin.nl/wp-content/uploads/2018/01/Lutra-602_Podt-IJsseldijk_2017_lr.pdf Siebert, U., Wünschmann, A., Weiss, R., Frank, H., Benke, H., & Frese, K. (2001). Post- mortem findings in harbour porpoises (Phocoena phocoena) from the German North and Baltic Seas. Journal of Comparative Pathology, 124(2–3), 102–114. doi:10.1053/jcpa.2000.0436 Siebert, U., Wohlsein, P., Lehnert, K., & Baumgärtner, W. (2007). Pathological Findings in Harbour Seals (Phoca vitulina): 1996–2005. Journal of Comparative Pathology, 137(1), 47– 58. doi:10.1016/j.jcpa.2007.04.018 Stringell, T., Hill, D., Rees, D., Rees, F., Rees, P., & Morgan, G. (2015). Short Note: Predation of Harbour Porpoises (Phocoena phocoena) by Grey Seals (Halichoerus grypus) in Wales. Aquatic Mammals, 41(2), 188–191. doi:10.1578/AM.41.2.2015.188 Van Bleijswijk, J. D. L., Begeman, L., Witte, H. J., IJsseldijk, L. L., Brasseur, S. M. J. M., Gröne, A., & Leopold, M. F. (2014). Detection of grey seal Halichoerus grypus DNA in attack wounds on stranded harbour porpoises Phocoena phocoena. Marine Ecology Progress Series, 513, 277–281. doi:10.3354/meps11004 van Neer, A., Gross, S., Kesselring, T., Grilo, M., Ludes-Wehrmeister, E., Roncon, G., & Siebert, U. (in preparation -a). Putting together the pieces of the puzzle - Part I - Assessing seal carcasses potentially subjected to grey seal (Halichoerus grypus) predation. in preparation. van Neer, A., Gross, S., Kesselring, T., Wohlsein, P., Leitzen, E., & Siebert, U. (2019). Behavioural and Pathological Insights into a Case of Active Cannibalism by a Grey Seal (Halichoerus grypus) on Helgoland, Germany. Journal of Sea Research, 148–149, 12–16. https://doi.org/10.1016/j.seares.2019.03.004. Wade, P. R. (1998). Calculating limits to the allowable human-caused mortality of cetaceans and pinnipeds. Marine Mammal Science, 14(1), 1–37.

64 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

Supplementary Materials

Figure A.1: Protocol used for the documentation of suspected grey seal predation cases in its updated version.

65 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

66 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

67 Characterisation of grey seal induced lesions in marine mammal carcasses and associated behaviour

68 Using molecular methods as a complementary tool of the macroscopic assessment

4. Using molecular methods as a complementary tool of the macroscopic assessment

Besides direct observations and the detection of typical wound patterns, an additional source of information by means of molecular diagnostics can be used. This can be especially helpful if a differentiation between lesions induced by a grey seal or a terrestrial scavenger is needed.

The following parts were published as:

a. Loop-mediated isothermal amplification (LAMP) assay - A rapid detection tool for identifying red fox (Vulpes vulpes) DNA in the carcasses of harbour porpoises (Phocoena phocoena)

Written by: Heers, T., van Neer, A., Becker, A., Grilo, M.L., Siebert, U. & Abdulmawjood, A. (2017);

PLoS ONE 12(9): e0184349. https://doi.org/10.1371/journal. pone.0184349

Picture by Thomas Diedrichsen

69 Using molecular methods as a complementary tool of the macroscopic assessment

Abstract

Carcasses of wild animals are often visited by different scavengers. However, determining which scavenger caused certain types of bite marks is particularly difficult and knowledge thereof is lacking. Therefore, a loop-mediated isothermal amplification (LAMP) assay (target sequence cytochrome b) was developed to detect red fox DNA in carcasses of harbour porpoises. The MSwab™ method for direct testing without prior DNA isolation was validated. As a detection device, the portable real-time fluorometer Genie® II was used, which yields rapid results and can be used in field studies without huge laboratory equipment. In addition to in vitro evaluation and validation, a stranded and scavenged harbour porpoise carcass was successfully examined for red fox DNA residues. The developed LAMP method is a valuable diagnostic tool for confirming presumable red fox bite wounds in harbour porpoises without further DNA isolation steps.

Introduction

When it comes to forensic studies it is not only important to confirm the actual “offender”, but also to identify and distinguish between predation and post mortem scavenging. Haelters et al. [1] described a case of a live stranded harbour porpoise (Phocoena phocoena), which had suffered several injuries. However, these wounds did not meet the typically described injuries of grey seal (Halichoerus grypus) attacks, which are known to hunt harbour porpoises [2]. These injuries usually appear as “puncture injuries left by teeth and claws; strips of blubber removed or hanging loose; sometimes muscle partly removed” [1]. Several investigations demonstrated that harbour porpoises are victims of attacks from grey seals [2–6] and in some cases, the DNA of grey seals was isolated from the bite marks of harbour porpoises and confirmed via PCR [2,3]. However, in the case study of Haelters et al. [1], the authors suspected that wounds were caused by a red fox scavenging the animal. Unfortunately, it was not possible to prove this assumption because a PCR yielded negative results. Similar problems occurred on harbour porpoises collected through the stranding network of Schleswig- Holstein, Germany where some wound patterns could not be assigned properly. In this context, IJsseldijk and Geelhoed [7] published video recordings showing a red fox actually fed on the carcass of a harbour porpoise. However, red fox DNA detection within the wounds was not performed. Due to the fact that the injuries inflicted by grey seals and red foxes can be confused (“fat and muscle tissues removed; straight wound margin; bite wound in skin and bones” (A. van Neer, pers. comm., March 2017)), for aquatic wildlife researcher it is of great importance to know if the bite marks are related to a grey seal or a red fox. In these cases molecular identification using LAMP (loop-mediated isothermal amplification) assays can be helpful for verification where the predator species identification is only based on bite marks.

70 Using molecular methods as a complementary tool of the macroscopic assessment

The approach of the present study was therefore to establish a novel DNA detection method that is easy to perform, sound, fast and which can be used during forensic field studies without heavy laboratory equipment. Whereas PCR based methods are time-consuming and special equipment is needed, the LAMP assay is a highly specific method which initially used four different primers [8]. Nagamine et al. [9] optimised the LAMP method to include six different primer pairs with two additional loop primers, which ultimately led to an acceleration of the reaction and markedly increased the sensitivity [9]. With the six designed primers a number of eight distinct sequences can be recognised. Moreover, the LAMP method can be performed at a constant temperature and will be complete after 30 minutes. A portable real-time isothermal fluorometer Genie® II (Optigene, United Kingdom) can be used as a detection device during field studies to facilitate and accelerate reliable results.

The LAMP method is already validated for various topics in many research areas. In the field of food safety, bacterial pathogens [10] and fungal contaminants [11] were detected and in case of urgent human health issues, evidence of emerging pathogens like tuberculosis [12] or zika virus disease [13] were possible. Furthermore, identifying animal species in meat products, such as ostrich or pork, was conducted with the LAMP technology [14,15].

To save even more time and make the system more practical, a swab method, described by Abdulmawjood et al. [14], was evaluated to determine the DNA without a previous DNA isolation step.

In the present study, a target sequence of the cytochrome b gene of red fox (Vulpes vulpes) was selected, primers were designed and the LAMP method was validated in vivo and successfully tested on a harbour porpoise which had been stranded on the coastline of Northern Germany and showed possible red fox bite marks. These are typically deep, focal, the blubber being penetrated with bleeding occurring in the underlying tissue [1]. Applying this method it was possible to identify the red fox as a scavenger by directly testing swab samples without previous DNA isolation.

71 Using molecular methods as a complementary tool of the macroscopic assessment

Materials and methods

Design of LAMP primer set

Six specific LAMP primers (forward outer primer VV-CY-F3, backward outer primer VV-CYB3, forward inner primer VV-CY-FIP, backward inner primer VV-CY-BIP, forward loop primer VV- CY-LoopF and backward loop primer VV-CY-LoopB) were designed using Primer-Explorer V4 (Eiken Chemical Co., Japan). A comparison of the two cytochrome b sequences of red fox and dog was performed, using the Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information (NCBI) (Maryland, USA). For the breed beagle (Canis

Fig1. Alignment of the cytochrome b sequences of red fox (Vulpes vulpes) (accession number AM181037.1) and dog (Canis lupus familiaris (Beagle)) (accession number AY729880.1). The dots represent the same base pair, while the red letters show different base pairs. The positions of the LAMP primers for the red fox are colour-coded.

72 Using molecular methods as a complementary tool of the macroscopic assessment lupus familiaris), which was considered as a model species, the accession number AY729880.1 and for the red fox (Vulpes vulpes) the accession number AM181037.1 were used. The alignment of the cytochrome b sequences of beagle and red fox resulted in the gene sequences of both animals being 84% identical (Fig 1). The homology for the fox amplicon against the canine amplicon was 85%. The differences in the cytochrome b nucleic acid sequence of red foxes and dogs were considered in the selection of a species-specific primer set. All primers were synthesised by Eurofins Genomics (Ebersberg, Germany). A detailed list of the primer sequences is given in Table 1.

Positive and negative control material

The DNA was isolated from muscle samples of Vulpes vulpes (n = 5) which were provided by the Institute for Terrestrial and Aquatic Wildlife Research, University of Veterinary Medicine Hannover, Foundation. The muscle samples of Canis lupus familiaris were obtained from the Institute of Pathology, University of Veterinary Medicine Hannover, Foundation. The red fox samples came from the North Sea Coast off Germany (region Eiderstedt). As there are numerous dog breeds in Germany, which can be considered as scavengers, different dog breeds and crossbreeds (n = 19) as representative examples for the variety of breeds in Germany, were investigated (S1 Table). All samples were thoroughly dissected by the staff members of the institutes named above.

Table 1. Sequences of the six specific primers for detecting red fox DNA.

MSwab™method

The MSwab™ system (Copan Italia S.p.A., Brescia, Italy) was used to extract DNA without previous DNA purification techniques. In this quick and simple method a flocked swab was stroked across the muscle tissue and subsequently submerged in 1 ml of MSwab™ medium, containing Tris HCl, EDTA, TRIS Base, Dimethyl Sulfoxide (DMSO), Bovine Serum Albumin and distilled water. Finally, the reaction tube was shaken by hand and 8 μl was used directly in the LAMP reaction.

DNA isolation

73 Using molecular methods as a complementary tool of the macroscopic assessment

The DNA extraction of the muscle samples was performed with the commercial DNeasy® blood and tissue kit (Qiagen, Darmstadt, Germany), in accordance with the manufacturer’s instructions, using the protocol “Purification of Total DNA from Animal Tissues (Spin-Column Protocol)”. In brief, 25 mg of the red fox or dog muscle was mixed with 200 μl lysis buffer and 20 μl proteinase K and incubated. After lysis, the DNA was processed according to the manufacturer’s instructions. All positive and negative DNA controls used during the validation and application of the LAMP assay were isolated from muscle samples.

To increase the sensitivity of the MSwab™ method mentioned above, the DNA from the MSwab™ medium was also extracted using the protocol “Purification of Total DNA from Animal Blood or Cells (Spin-Column Protocol)”. Subsequently, the DNA concentration was measured with the spectrophotometer NanoDrop2000c (Thermo Fisher Scientific, Massachusetts, USA).

LAMP assay

The loop-mediated isothermal amplification assay was carried out with a real-time fluorometer (Genie® II, Optigene, United Kingdom). The Genie® II is a battery-powered and portable device with two heating blocks. In each heating block eight samples can be investigated simultaneously so that a total number of sixteen samples can be amplified at any one time. The device provides the opportunity to observe the amplification process in real-time on the screen and the analysis will be completed after 30 min.

In accordance with the manufacturer’s instructions, for each sample, 15 μl Isothermal Master Mix ISO-001 (Optigene, United Kingdom) was used with 0.4 μM of the F3-Primer and the B3- Primer. Furthermore, 0.8 μM of the LoopF-Primer and the LoopB-Primer and 1.6 μM of the FIP-Primer and BIP-Primer were admixed and 8 μl of sample DNA were added so that the total reaction volume was 30 μl.

Genie® II works under an isothermal amplification temperature and can be programed for different temperatures. For the specific primer set the optimum temperature was determined as 65˚C with detection duration of approximately twenty minutes. The subsequent melting process (in Genie® II called annealing process) starts with 98˚C and ends with a temperature of 80˚C with a ramp rate of 0.05˚C/sec.

Limit of detection

To determine the limit of detection of the LAMP reaction, a serial dilution with DNA concentrations from 1.45E+04 pg/μl to 1.45E-05 pg/μl was prepared using red fox DNA, isolated from muscle tissue, and AE buffer (10 mM Tris-Cl, 0.5 mM EDTA; pH 9.0). The limit of detection was calculated based on a six-fold verification of the DNA serial dilution.

74 Using molecular methods as a complementary tool of the macroscopic assessment

Sensitivity and specificity

The sensitivity is the proportion of true positive red fox samples that are correctly identified by the LAMP assay. The specificity is the proportion of true negative dog samples that are correctly identified by the LAMP assay [16]. To verify whether the primer set amplifies red fox DNA correctly as positive, muscle samples of five different animals were tested using the MSwab™ protocol. Three swab samples were taken from each thigh muscle sample. The specificity was determined to rule out that the specific primers amplify canine DNA. In each case, three swab samples of 19 muscle samples from different dogs were also tested with the MSwab™ method. Each swab was tested directly, without further DNA isolation, with the LAMP assay.

Spiking experiments

To verify whether the LAMP reaction was working properly under simulated in vivo conditions, a skin preparation (thickness 2 cm) of a harbour porpoise was manually incised with a scalpel (approx. 0.5 cm wide and 1 cm deep), imitating red fox bite marks. Subsequently, 10 μl of the isolated red fox DNA from one fold (mean DNA concentration 1.45E+04 pg/μl) to eight fold dilution (mean DNA concentration 1.45E-03 pg/μl) was pipetted thoroughly in the imitated bite mark lesions. After 10 minutes, the tissue was swabbed and the samples were amplified directly using LAMP without prior DNA isolation. The spiking procedure was repeated in triplicate with three independent serial dilutions.

Necropsy and application of the LAMP assay on a stranded harbour porpoise

One harbour porpoise carcass (Fig 2A), which was presumably scavenged by a red fox (significant red fox related tracks were observed around the carcass) was collected from the coastline of Northern Germany (Sylt) in October 2016 and transported to the Institute for Terrestrial and Aquatic Wildlife Research in Büsum. The carcass was stored frozen (-20˚C) until the necropsy in December 2016. Before sampling, the carcass was thawed at room temperature for three days. During the necropsy, several sterile MSwab™ samples were taken from one large wound lesion. Prior to sampling, the dissecting table was treated with the DNA decontamination solution DNA-ExitusPlus™ (AppliChem GmbH, Darmstadt, Germany) to eliminate potential previous, undesirable DNA residues. To rule out contamination, the table surface was also tested for red fox DNA after this procedure. After sampling (Fig 2B), the necropsy was performed according to Siebert et al. [17].

75 Using molecular methods as a complementary tool of the macroscopic assessment

Fig 2. Application of the LAMP assay on a stranded harbour porpoise. (A) Carcass of the stranded harbour porpoise. (B) Sampling locations of the different MSwab™swabs (1–5). Locations were selected at random.

The MSwab™ samples were analysed by LAMP after the necropsy without prior DNA extraction. Additionally, the DNA within the MSwab™ medium was purified and another LAMP reaction was performed to increase the sensitivity of the assay. Concerning the similarity of injuries of the grey seal and the red fox, in both runs the isolated DNA of a grey seal muscle was used as a negative control. Additionally isolated DNA of a dog muscle and isolated DNA of a harbour porpoise muscle were applied as negative control as well.

Finally, the LAMP products of the non-extracted and the extracted DNA were confirmed with gel electrophoresis. Additionally, four other stranded carcasses, including two harbour

76 Using molecular methods as a complementary tool of the macroscopic assessment porpoises, one harbour seal and one grey seal were investigated for red fox DNA, using the developed LAMP assay.

Gel electrophoresis

To detect the LAMP amplicons, a gel electrophoresis was conducted with 8 μl of the LAMP products, using 2% agarose gel (peqGOLD Universal agarose gel, PeQlab, Erlangen, Germany) and Tris-Borat-EDTA-buffer (pH 8.4). A DNA marker for 50 bp-2 kb (Biozym, Hessisch Oldendorf, Germany) was used.

Results

Limit of detection, sensitivity and specificity

Typical amplification curves of the diluted red fox DNA, using the real-time fluorometer Genie® II, are shown in Fig 3A. The detection probability was 100% up to a DNA concentration of 1.45E-02 pg/μl with a detection time of 09:02 min (± 00:20 min) (Fig 3). The detection probability dropped to 83% at a DNA concentration of 1.45E-03 pg/μl (mean detection time 13:12 minutes) (S2 Table). The mean of the annealing temperature was 83.1˚C (± 0.1˚C).

For the sensitivity, five muscle samples of different red foxes were tested directly (without DNA isolation) with the MSwab™ method. With the specific primer set each of the samples gave a correctly positive amplification. The mean of the detection time was 13:09 min (± 03:58 min) and the mean temperature of the annealing curve was 82.3˚C (± 0.2˚C). For the specificity a number of 57 MSwab™ samples of 19 different dog muscles were tested without further DNA isolation step. All samples were negative as tested.

Spiking experiments

The detection of the DNA of the first trial was possible up to the concentration 1.45E+02 pg/μl DNA/bite mark. The mean detection time in that case was 18:07 min (±01:07 min) (Table 2). The minimum DNA concentration of second trial which could be detected was 1.29E+03 pg/ μl DNA/bite mark and the mean detection time was 18:22 min (± 00:37 min). The third trial showed a detection time of 18:37 min (± 00:37 min) with a minimum concentration of 1.6E +03 pg/μl. None of the swab sample was extracted with a further DNA isolation step.

Necropsy

The female adult harbour porpoise, with a length of 159 cm, presented a large tissue defect in the right dorso-lateral side of the cranial part of the body. The defect extended from the caudal

77 Using molecular methods as a complementary tool of the macroscopic assessment

Fig 3. Limit of detection of the LAMP assay. (A) Typical amplification curves of ten-fold serially diluted red fox DNA. (B) The standard curve was generated from a dilution series of the DNA of Vulpes vulpes by plotting the amplification time versus the log of the DNA concentration. At concentrations below 1.45E-02 pg/μl the data falls off. The correlation is a linear response. side of the blowhole to the area dorsal to the right front flipper, extending from the dorsum of the animal to the level of the right ramus of the mandible. The majority of skin, blubber and muscular tissue in the wound area was missing. The wound’s margins in its left and cranial right side were regular, whereas the other edges were irregular. In several sites of the wound margin it was possible to detect triangular-shaped edges where the margin had a regular surface. Table 2. Spiking experiments with the three serial dilutions of the DNA of Vulpes vulpes.

Application of the LAMP assay on a stranded harbour porpoise

Initially, a total number of eight swabs were tested without DNA isolation (Table 3). The three control swab samples of the dissecting table were all negative. Swabs 1, 2 and 3 did not show an amplification signal with the specific primer. However, the swab samples 4 and 5 were positive after 16:30 min and 15:45 min, respectively. The positive control was already detected after 5:30 min. All negative controls were not amplified with the selected primer set.

78 Using molecular methods as a complementary tool of the macroscopic assessment

Table 3. LAMP results of the first stranded harbour porpoise investigated for red fox DNA residues.

Besides the direct sample testing, DNA was isolated from the Mswab™ samples and subsequently tested with the LAMP assay (Table 3). With this additional purification step, the DNA detection was possible in all bite mark samples. The results of both direct and isolated samples were confirmed by gel electrophoresis (Fig 4).

As expected, the detection time of the directly positive 4 and 5 swab samples was longer (swab no. 4 6:00 min longer and swab no. 5 3:30 min longer) than the detection time of the isolated DNA. The other two investigated harbour porpoises, the harbour seal and the grey seal were negative for red fox DNA residues.

Fig 4. Application of the LAMP assay on a stranded harbour porpoise. Gel electrophoresis of all LAMP products of the stranded harbour porpoise. Directly tested MSwab™samples (without DNA isolation) and DNA isolation of the MSwab™medium using DNeasy® blood and tissue kit. As expected, the detection time of the directly positive 4 and 5 swab samples was longer (swab no. 4 6:00 min longer and swab no. 5 3:30 min longer) than the detection time of the isolated DNA.

79 Using molecular methods as a complementary tool of the macroscopic assessment

Discussion

The results of this project show that it is possible to detect red fox DNA residues in the wounds of a harbour porpoise, using the LAMP technology. In this case it was presumed to find red fox DNA in the bite marks, as significant tracks of a red fox were viewed around the stranded carcass. In the other four cases, of negatively tested marine mammals it was not known if a red fox visited the carcasses. These results indicate that no red fox was feeding on those carcasses or the DNA of the red fox in the bite marks was degraded.

On a Dutch coastline the red fox was identified as a scavenger with video-recording proof [7]. However, molecular biological identification of a predator can confirm an observation or identity of the predator without visual proof [18]. Besides that, the predation itself cannot be observed regularly anyhow, so a test based on DNA analysis is needed.

Confirmation of DNA residues in wounds is not an easy task and brings challenges for the sampling and analysis process. Although previous studies succeeded in isolating predator DNA of grey seals from wounds of harbour porpoises via PCR [3,5], this was not successful for detecting red fox DNA [1]. This shows that PCR analyses might not always be the method of choice when the objective is to detect scavenger DNA residues. A good alternative to PCR is the loop-mediated isothermal amplification assay. This relatively modern method is reported to be rapid and easy to apply [8]. LAMP is a highly specific method with particular high target selectivity, due to the fact that six primers bind to eight specific target sequences [9], this being the main reason for using the LAMP technology in the present study. Additionally, the use of Genie® II as a portable detection device offers the possibility of performing analysis during field studies in areas where there is no easy access to laboratories. As DNA within wounds is very sensitive, this also has the advantage of avoiding degradation or contamination during the transportation process [19]. Another benefit of the LAMP assay is its greater sensitivity compared to the PCR [20,21]. This plays an important role when working with saliva samples, as they contain relatively low quantities of DNA, but offer a good opportunity for solving forensic questions in wildlife areas [22]. Usually, the DNA must be isolated from a sample before initiating a PCR. With the LAMP assay in combination with a swab method, no further DNA extraction step is necessary and thus the swab samples can be tested directly and user-friendly [23].

Alongside red foxes, dogs are known to be potential scavengers of harbour porpoises and in some cases even the most abundant ones in Australia [1,4,5,24]. As there is a close relation between the mitochondrial DNA of red foxes and dogs, the cytochrome b sequence of dogs was considered and successfully ruled out during the primer design and amplification process.

80 Using molecular methods as a complementary tool of the macroscopic assessment

The results of our study confirm that taking DNA samples is an effective method to clarify forensic questions in the field of wildlife research, since we succeeded in detecting the DNA of Vulpes vulpes in the bite lesion of a stranded harbour porpoise. In various wildlife projects, researchers were able to show that saliva samples can be used to identify different predators. For example, DNA of coyotes [19,25] or wolves [26] was detected in sheep carcasses. A substantial problem is that DNA originating from saliva is subject to different types of degradation, like enzymes, bacteria or weather conditions [27,28]. Furthermore, Pääbo et al. [28] pointed out that low temperatures and dry conditions lead to a slower DNA degradation. Moreover, due to sea water there may be a dilution of the DNA.

Besides using the LAMP method, this may explain the unsuccessful evidence of red fox DNA via PCR in the project of Haelters et al. [1] and the negative results of the other four tested stranded marine mammals in this study in comparison to our positive results.

In this context, it has to be pointed out that in our study only two out of the five directly tested swab samples of the harbour porpoise carcass showed a positive amplification. This may be the result of a low DNA quantity due to degradation, as after DNA isolation of the swab samples and the associated higher yield or accessibility of DNA all samples were detected as positive.

We recommend to swab over the whole lesion or in the middle of the lesion as these were the locations of the directly positive swabs in our case. It can also be beneficial to take several samples within these locations or increase the amplification time from 20 to 30 minutes. In the performed application of the LAMP assay each sample of the dissecting table yielded negative results for red fox DNA residues. Thus, it is further recommended to take swab samples of the dissecting table as quality assurance and to avoid false positive results.

Although the MSwab™ method offers the advantage of direct DNA testing, it was astonishing that the detection limit in the spiking experiment was rather poor (1.45E+02 pg/μl) in comparison to 1.45E-03 pg/μl during the limit of detection testing. This shows that there is a significant DNA quantity lost during sampling because of a dilution in 1 ml MSwab™ medium and a huge matrix effect due to inhibitor factors like fat and blood within the wound lesion, making it even more difficult to catch particularly low concentrations of DNA residues within deep wounds. Nevertheless, with the stranded harbour porpoise we were able to demonstrate that the DNA quantity is still sufficient to detect red fox DNA with the MSwab™ under simulated in vivo conditions.

Fast and specific DNA detection methods will always be a major topic for forensic studies and with the developed LAMP assay it is possible to detect the red fox as the scavenger of marine mammals. The field-use of the Genie® II fluorometer is simple as it is possible to blend the master mix in advance so there is only the need for the MSwab™, a pipette and the portable

81 Using molecular methods as a complementary tool of the macroscopic assessment and battery-powered Genie® II device on-site. The samples can then be transferred directly into the LAMP assay to identify the scavenger or predator immediately but can also be purified to increase sensitivity.

Conclusion

The developed LAMP method is a valuable diagnostic tool for wildlife researchers to identify red fox DNA in carcasses of marine mammals. In this study we could successfully confirm that wound lesions, presumably caused by red fox bites, were actually red fox-related. The presented method can be performed on-site, fast and without the need for large laboratory equipment.

Supporting information

S1 Table. Dog breed samples (n= 19) investigated in the present study as negative controls for the exclusivity test.

S2 Table. Limit of detection of the LAMP assay by using serial dilutions of the DNA of Vulpes vulpes.

82 Using molecular methods as a complementary tool of the macroscopic assessment

Acknowledgments

We are grateful to the staff members of the Institute for Terrestrial and Aquatic Wildlife Research in Büsum, University of Veterinary Medicine Hannover, Foundation, for sample preparation and help during sampling of the stranded and scavenged harbour porpoise. Furthermore, we would like to thank staff members of the Institute of Pathology, University of Veterinary Medicine Hannover, Foundation, for providing the dog muscle samples.

References

1. Haelters J, Everaarts E, Bunskeok P, Begeman L, Hinrichs JWJ, Ijsseldijk LL (2016) A Suspected Scavenging Event by Red Foxes (Vulpes vulpes) on a Live, Stranded Harbour Porpoise (Phocoena phocoena). Aquatic Mammals 42: 227–232. 2. Leopold MF, Begeman L, van Bleijswijk JDL, Ijsseldijk LL, Witte HJ, Gröne A (2014) Exposing the grey seal as a major predator of harbour porpoises. Proceedings of the Royal Society B: Biological Sciences 282. 3. van Bleijswijk JDL, Begeman L, Witte HJ, Ijsseldijk LL, Brasseur S, Gröne A, et al. (2014) Detection of grey seal Halichoerus grypus DNA in attack wounds on stranded harbour porpoises Phocoena phocoena. Marine Ecology Progress Series 513: 277–281. 4. Haelters J, Kerkchof F, Jauniaux T, Degraer S (2012) The Grey Seal (Halichoerus grypus) as a Predator of Harbour Porpoises (Phocoena phocoena)? Aquatic Mammals: 343– 353. 5. Jauniaux T, Garigliany MM, Loos P, Bourgain JL, Bouveroux T, Coignoul F, et al. (2014) Bite injuries of grey seals (Halichoerus grypus) on harbour porpoises (Phocoena phocoena). PLoS One 9: e108993. https://doi.org/10.1371/journal.pone.0108993 PMID: 25461599 6. Stringell T, Hill D, Rees D, Rees F, Rees P, Morgan G (2015) Short Note: Predation of Harbour Porpoises (Phocoena phocoena) by Grey Seals (Halichoerus grypus) in Wales. Aquatic Mammals 41: 188–191. 7. IJsseldijk L, Geelhoed S (2016) Fox scavenging mutilations on dead harbour porpoises (Phocoena phocoena). IMARES Report [LXXX/JJ]. 8. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28: E63. PMID: 10871386 9. Nagamine K, Hase T, Notomi T (2002) Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes 16: 223–229. 10. Sheet OH, Grabowski NT, Klein G, Abdulmawjood A (2016) Development and validation of a loop mediated isothermal amplification (LAMP) assay for the detection of Staphylococcus aureus in bovine mastitis milk samples. Mol Cell Probes 30: 320–325. https://doi.org/10.1016/j.mcp.2016.08.001 PMID: 27495132 11. Abd-Elsalam K, Bahkali A, Moslem M, Amin OE, Niessen L (2011) An optimized protocol for DNA extraction from wheat seeds and Loop-Mediated Isothermal Amplification (LAMP) to detect Fusarium graminearum contamination of wheat grain. Int J Mol Sci 12: 3459– 3472. https://doi.org/10.3390/ ijms12063459 PMID: 21747688 12. Organization WH (2016) The Use of Loop-Mediated Isothermal Amplification (TB- LAMP) for the Diagnosis of Pulmonary Tuberculosis: Policy Guidance. WHO Verlag, World Health Organization, Genf, Schweiz.

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13. Lee D, Shin Y, Chung S, Hwang KS, Yoon DS, Lee JH (2016) Simple and Highly Sensitive Molecular Diagnosis of Zika Virus by Lateral Flow Assays. Anal Chem 88: 12272– 12278. https://doi.org/10.1021/ acs.analchem.6b03460 PMID: 28193014 14. Abdulmawjood A, Grabowski N, Fohler S, Kittler S, Nagengast H, Klein G (2014) Development of loop mediated isothermal amplification (LAMP) assay for rapid and sensitive identification of ostrich meat. PLoS One 9: e100717. https://doi.org/10.1371/journal.pone.0100717 PMID: 24963709 15. Lee S-Y, Kim M-J, Hong Y, Kim H-Y (2016) Development of a rapid on-site detection method for pork in processed meat products using real-time loop-mediated isothermal amplification. Food Control 66: 53–61. 16. Altman DG, Bland JM (1994) Diagnostic tests 1: sensitivity and specificity. BMJ 308: 1552. PMID: 8019315 17. Siebert U, Wunschmann A, Weiss R, Frank H, Benke H, Frese K (2001) Post-mortem findings in harbour porpoises (Phocoena phocoena) from the German North and Baltic Seas. Journal of Comparative Pathology 124: 102–114. https://doi.org/10.1053/jcpa.2000.0436 PMID: 11222006 18. Mumma MA, Soulliere CE, Mahoney SP, Waits LP (2013) Enhanced understanding of predator-prey relationships using molecular methods to identify predator species, individual and sex. Mol Ecol Resour 14: 100–108. https://doi.org/10.1111/1755-0998.12153 PMID: 23957886 19. Williams CL, Blejwas K, Johnston JJ, Jaeger MM (2003) A Coyote in Sheep’s Clothing: Predator Identification from Saliva. Wildlife Society Bulletin Vol. 31: 926–932. 20. Parida M, Posadas G, Inoue S, Hasebe F, Morita K (2004) Real-Time Reverse Transcription Loop-Mediated Isothermal Amplification for Rapid Detection of West Nile Virus. Journal of Clinical Microbiology 42: 257–263. https://doi.org/10.1128/JCM.42.1.257-263.2004 PMID: 14715762 21. Parida M, Horioke K, Ishida H, Dash PK, Saxena P, Jana AM, et al. (2005) Rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription- loop-mediated isothermal amplification assay. J Clin Microbiol 43: 2895–2903. 22. Harms V, Nowak C, Carl S, Muñoz-Fuentes V (2015) Experimental evaluation of genetic predator identification from saliva traces on wildlife kills. Journal of Mammalogy 96: 138–143. 23. Kaneko H, Kawana T, Fukushima E, Suzutani T (2007) Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances. J Biochem Biophys Methods 70: 499–501. https://doi.org/10.1016/j.jbbm.2006.08.008 PMID: 17011631 24. Schlacher TA, Weston MA, Lynn D, Schoeman DS, Huijbers CM, Olds AD, et al. (2014) Conservation gone to the dogs: when canids rule the beach in small coastal reserves. Biodiversity and Conservation 24: 493–509. 25. Blejwas K, Williams CL, Shin GT, McCullough DR, Jaeger MM (2006) Salivary DNA Evidence Convicts Breeding Male Coyotes of Killing Sheep. Journal of Wildlife management 70: 1087–1093. 26. Caniglia R, Fabbri E, Mastrogiuseppe L, Randi E (2013) Who is who? Identification of livestock predators using forensic genetic approaches. Forensic Sci Int Genet 7: 397–404. https://doi.org/10.1016/j. fsigen.2012.11.001 PMID: 23200859 27. Williams CL, Johnston JJ (2004) Using Genetic Analyses to Identify Predators. Sheep & Goat Research Journal 19: 85–88.

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28. Pääbo S, Poinar H, SerreD, Jaenicke-Despres V, Hebler J, Rohland N, et al. (2004) Genetic analyses from ancient DNA. Annu Rev Genet 38: 645–679. https://doi.org/10.1146/annurev.genet.37.110801.143214 PMID: 15568989

85 Using molecular methods as a complementary tool of the macroscopic assessment

b. Loop-mediated isothermal amplification (LAMP) as a confirmatory and rapid DNA detection method for grey seal (Halichoerus grypus) predation on harbour porpoises (Phocoena phocoena)

Written by: Heers, T., van Neer, A., Becker, A., Gross, S., Hansen, K. A., Siebert, U. & Abdulmawjood, A. (2018);

Journal of Sea Research, 140, 32–39. doi:10.1016/j.seares.2018.07.008

Picture by Katharina Tilly

86 Using molecular methods as a complementary tool of the macroscopic assessment

Abstract

An increasing number of harbour porpoises along the coastlines of northern Europe are victims of attacks by preying grey seals. To confirm grey seal attacks at a molecular level, a fast loop- mediated isothermal amplification (LAMP) method was developed, based on the amplification of the cytochrome b target gene. With this method, saliva residues within bite marks, presumably originating from grey seal attacks, can be detected onsite, using a swab-based method in combination with a portable real-time fluorometer (Genie® II). The developed LAMP assay included an internal amplification control and was validated for the sensitivity, specificity, limit of detection and a spiking experiment was performed with saliva samples from two grey seals. Finally, three stranded harbour porpoises, with injury patterns presumably originating from grey seal attacks, were analysed. Grey seal DNA was successfully determined in a bite mark of one harbour porpoise with the LAMP method. For direct on-site molecular biological investigations without the opportunity to perform more time-consuming and equipment intensive PCR investigations, the developed rapid LAMP assay offers the possibility to confirm that injuries, probably caused by a grey seal, indeed resulted from a grey seal attack.

1. Introduction

During the last few years, an increasing number of stranded harbour porpoises (Phocoena phocoena) with massive loss of tissue [‘strips of blubber removed or hanging loose; sometimes muscle partly removed’ (Haelters et al., 2016)] were found along the coastlines of northern Europe. Initially, it was assumed that those animals were victims of fishery bycatch or a ship's propeller (Camphuysen & Siemensma, 2011; IAMMWG et al., 2015). However, Bouveroux et al. (2014) observed and photographed grey seal (Halichoerus grypus) attacks on harbour porpoises on the coast of northern France. Stringell et al. (2015) also provided evidence of the phenomenon of a grey seal predation on a harbour porpoise in Wales with the aid of photo and video material. Thus, there was the presumption that these serious injuries, which led to death, might be attributed to grey seal attacks (Haelters et al., 2012; Jauniaux et al., 2014; Leopold et al., 2014; van Bleijswijk et al., 2014; Stringell et al., 2015). To date, assessing carcasses mainly relies on macroscopic inspection and the resulting judgment of the researcher, posing the risk of respective occasional misjudgement (Hanson et al., 2017). In some areas, grey seal predation was held responsible for the death of at least 17% of stranded deceased porpoises, representing one of the major causes of death in the southeastern North Sea (Leopold et al., 2014). The reasons why grey seals attack harbour porpoises are not fully clarified. Stringell et al. (2015) assumed that harbour porpoises function as food because blubber and skin were consumed by an attacking grey seal and did not preclude a competition for food between both species. On the other hand, Jauniaux et al. (2014) and Haelters et al. (2012) cannot rule out aggressive behaviour of grey seals. To confirm the assumption of grey

87 Using molecular methods as a complementary tool of the macroscopic assessment seal attacks, researchers analysed bite marks of harbour porpoises for residues of grey seal DNA via PCR and successfully determined DNA of grey seals in harbour porpoise carcasses (Jauniaux et al., 2014; van Bleijswijk et al., 2014). The issue of frequently occurring severely injured or dead harbour porpoises on the coastline of northern Germany raised the question whether these wounds also originated from grey seal attacks. Therefore, a rapid detection technique, which can be used during forensic field studies, was needed. The highly sensitive and fast loop-mediated isothermal amplification (LAMP) method is a relatively new and rapid molecular biological method requiring six different primers, binding on eight target regions, which results in high sensitivity and specificity (Notomi et al., 2000; Nagamine et al., 2002). The easy-to-use method can be performed with a real-time fluorometer (Genie® II, Optigene, Horsham, United Kingdom), which allows the parallel testing of 16 samples. Furthermore, the reaction process can be observed in real-time and after 30 min the entire run is finished. The LAMP technology has al-ready been used in numerous research projects, for example, for detecting human blood-borne infectious disease like HIV (Vanjari et al., 2014) or hepatitis B (Moslemi et al., 2009). Moreover, it has been used to provide evidence of foodborne pathogens like Staphylococcus aureus or Listeria monocytogenes (Wang et al., 2010; Sheet et al., 2016) as well as animal species identification of ostrich, pork or horse in meat (Abdulmawjood et al., 2014; Cho et al., 2014; Yang et al., 2014). Furthermore, the detection of red fox (Vulpes vulpes) DNA in bite marks of a harbour porpoise carcass was possible with the LAMP technology (Heers et al., 2017). The latter case is related to the issue of grey seal predation on harbour porpoises as the bite lesions of red foxes displayed a similarity to bite marks of grey seals and both can hardly be differentiated when relying solely on macroscopic assessment (Heers et al., 2017). Heers et al. (2017) showed that by using a certain swab technique, a DNA purification step is not needed prior to LAMP, which accelerates and simplifies the procedure considerably. This swap technique was also used in the present study. The aim of the study was the development of a rapid and sensitive method to detect grey seal DNA in stranded harbour porpoises directly on the beach. Based on the cytochrome b sequence, six specific LAMP primers were created and for the internal amplification control, six common LAMP primers were generated. The method was validated and subsequently tested on three stranded harbour porpoises, showing typical injury patterns of grey seal predations. Using this assay, it was feasible to detect predator DNA residues of a grey seal in a bite lesion of one harbour porpoise. The LAMP assay is therefore of enormous benefit for wildlife researchers. Not only is it time-saving and field testing can be carried out with little equipment, but it also enables researchers to detect DNA traces of grey seals in bite marks of harbour porpoises and clarify forensic issues.

88 Using molecular methods as a complementary tool of the macroscopic assessment

2. Material and methods

2.1. Design of LAMP primer sets

By using the software PrimerExplorer V4 (Eiken Chemical Co., Japan), six specific LAMP primers (forward outer primer HG-F3, backward outer primer HG-B3, forward inner primer HG- FIP, backward inner primer HG-BIP, forward loop primer HG-LoopF and backward loop primer HG-LoopB) were designed. The cytochrome b sequences of grey seal and harbour porpoise were compared with the Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information (NCBI) (Maryland, USA). For grey seals (Halichoerus grypus) the accession number GU167293.1 and for harbour porpoises (Phocoena phocoena) the accession number U72039.1 were used (Fig. 1). To select a species-specific primer set, the differences in the cytochrome b sequences between grey seals and harbour porpoises were considered. To rule out false negative results, an internal amplification control (IAC) with six common primers was designed (forward outer Primer F3-IAC, backward outer primer B3-IAC, forward inner primer FIP-IAC, backward inner primer BIP-IAC, forward loop primer LoopF-IAC and backward loop primer LoopB-IAC), to amplify the DNA of grey seals as well as the DNA of harbour porpoises. This was conducted with the 16S sequences of harbour porpoise (accession number AB481401.1) and grey seal (accession number ×72004.1).

All primers were produced by Euroﬁns Genomics (Ebersberg, Germany). The detailed primer sequences of the speciﬁc primers are listed in Table 1 and the sequences of the common primers (IAC) are displayed in Table 2.

2.2. Positive and negative control material

The DNA of the positive and negative control material was isolated from muscle samples. The tissue samples were taken from stranded marine mammal carcasses during routine necropsy by the Institute for Terrestrial and Aquatic Wildlife Research, University of Veterinary Medicine Hannover, Foundation. The samples were collected in the frame of the states' stranding network funded and permitted by the responsible Ministry of Energy Transition, Agriculture, Environment, Nature and Digitalisation of Schleswig-Holstein (MELUND), Germany. As positive control, material muscle samples of grey seals (n = 8) and as negative control material muscle samples of harbour porpoises (n = 24) were extracted by employees of the aforementioned institute.

89 Using molecular methods as a complementary tool of the macroscopic assessment

2.3. Swab method

The swab system was used to forego a previous DNA puriﬁcation step and utilise the DNA directly in the LAMP reaction. To select the best swab type with the highest sensitivity, a preliminary test with three commercially available swab types (MSwab™, Copan, Italy; eSwab™, Copan, Italy; hyplex® LPTV, Amplex BioSystems GmbH, Gießen) was performed (data not shown). In the swab validation experiment, the MSwab™ swab (Copan Italia S.p.A., Brescia, Italy) yielded the fastest ampliﬁcation times, therefore it was used for further research work. The swab was stroked across the muscle tissue and subsequently sub-merged in the associated medium (1 ml MSwab™ medium contains Tris HCl, EDTA, TRIS Base, Dimethyl Sulfoxide (DMSO), Bovine Serum Albumin and distilled water). Finally, the reaction tube was shaken by hand and 8 μl of the MSwab™ medium was used directly in the LAMP reaction.

2.4. DNA isolation

The DNA of the muscle samples was extracted with the commercial DNeasy® blood and tissue kit (Qiagen, Darmstadt, Germany), in accordance with the manufacturer's instructions set forth in the protocol “Puriﬁcation of Total DNA from Animal Tissues (Spin-Column Protocol)”. In brief, 25 mg of muscle tissue, 200 μl lysis buﬀer and 20 μl proteinase K were mixed and incubated subsequently. The following steps were processed in accordance with the manufacturer's instructions.

To increase the sensitivity of the swab method (mentioned above), it is also possible to extract the DNA from the MSwab™ medium. Therefore, 100 μl of the MSwab™ medium was used and isolated in accordance with the protocol “Puriﬁcation of Total DNA from Animal Blood or Cells (Spin-Column Protocol)”.

Subsequently, after DNA isolation, the DNA concentration was determined, using the spectrophotometer NanoDrop2000c (Thermo Fisher Scientiﬁc, Dreieich, Germany).

2.5. LAMP assay

The loop-mediated isothermal amplification assay was performed with a real-time fluorometer Genie® II, which is a battery-operated, portable device with two heating blocks, allowing a total number of 16 samples to be amplified simultaneously. The amplification process can be monitored in real-time on screen.

90 Using molecular methods as a complementary tool of the macroscopic assessment

Fig. 1. Alignment of the cytochrome b sequences of grey seal (Halichoerus grypus) (accession number GU167293.1) and harbour porpoise (Phocoena phocoena) (accession number U72039.1). Same base pairs are represented by dots and different base pairs are marked by red letters. The positions of the six species-specific LAMP primers are indicated in colour. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

91 Using molecular methods as a complementary tool of the macroscopic assessment

Table 1 Sequences of the six speciﬁc primers for the detection of grey seal DNA.

In accordance with the manufacturer's instructions, 15 μl Isothermal Master Mix ISO-001 (Optigene, Horsham, United Kingdom) was used with 0.4 μM of the F3-Primer and the B3- Primer for each sample. Furthermore, 0.8 μM of the LoopF-Primer and the LoopB-Primer as well as 1.6 μM of the FIP-Primer and BIP-Primer were added. Finally, a volume of 8 μl template DNA was admixed. The LAMP assay worked with an isothermal ampliﬁcation temperature of 65 °C and a detection duration of roughly twenty minutes. The following annealing process with a ramp rate of 0.05 °C/s started with a temperature of 98 °C and ﬁnished with 80 °C.

2.6. Sensitivity and speciﬁcity

The sensitivity was determined to verify the amplification of the DNA from different grey seal individuals with the selected specific primer set. With the MSwab™ technique three swab samples were taken from each muscle sample of different grey seals (n = 8). To rule out the amplification of harbour porpoise DNA with the specific primer set, the specificity was determined. Three swab samples Table 2 Sequences of the six common primers as internal amplification control (IAC) for detecting grey seal and harbour porpoise DNA. of each muscle sample from different harbour porpoises (n = 24) were also tested using the MSwab™ method. To eliminate potentially available and undesired residues of grey seal DNA, the surface of the harbour porpoise muscle samples was treated with the DNA decontamination product DNA-ExitusPlus™ (AppliChem GmbH, Darmstadt, Germany). Subsequently, the muscles were sliced with sterile scalpels and the MSwab™ samples were taken from the inside of the muscles. To rule out false negative results and ensure that DNA-ExitusPlus™ did not destroy the harbour porpoise DNA in the MSwab™ medium, each swab sample was tested with the common primer set (IAC). Each swab sample was tested directly with the LAMP assay without further DNA isolation steps.

92 Using molecular methods as a complementary tool of the macroscopic assessment

2.7. Limit of detection

The limit of detection of the LAMP assay was determined with isolated grey seal DNA originating from a muscle sample. A serial dilution with concentrations ranging from 1.00E + 03 pg/μl to 1.00E¬04 pg/μl were prepared using AE buﬀer (10 mM Tris-Cl, 0.5 mM EDTA; pH 9.0). The dilution series was tested six-fold with the LAMP method.

Fig. 2. Spiking experiment. Inoculation of grey seal saliva in the triangular imitated bite mark in the skin preparation of a harbour porpoise.

2.8. Spiking experiment with grey seal saliva

A spiking experiment in accordance with Heers et al. (2017) was modified and performed with four saliva samples of two different grey seals from the Marine Biological Research Centre, University of Southern Denmark in Kerteminde. The animals were housed with permission from the Danish Food and Drug Administration. All applicable international, national and institutional guidelines for the care and use of animals were followed. The saliva samples were collected during medical training sessions and taken from the oral cavity of the grey seals with MSwab™ swabs. To ensure there was actually grey seal DNA in the four swab samples, the MSwab™ medium of the native saliva samples had been previously tested with the specific primer set. Subsequently, lesions caused by grey seal teeth, were imitated by incising approximately 0.5 cm wide and 0.5 cm deep triangular injuries with a sterile scalpel in a 2 cm thick skin preparation of a harbour porpoise. 50 μl of the MSwab™ medium of each saliva sample and 50 μl water (as negative control) were carefully pipetted in the imitated bite lesions (Fig. 2). After an impact time of 10 min, the saliva sample and the water were swabbed with an MSwab™ and the samples were tested without need for a further DNA extraction step. The time for the amplification process was increased to 30 min and every saliva sample was tested sixfold. Subsequently, the DNA of the native saliva samples was extracted and the DNA yield was measured with the spectrophotometer Nano-Drop2000c.

93 Using molecular methods as a complementary tool of the macroscopic assessment

2.9. Necropsy and application of the LAMP assay on stranded harbour porpoises

In September 2017, a harbour porpoise carcass, potentially predated by a grey seal, was found on the coastline of northern Germany (Sylt) (Fig. 3) and transported to the Institute for Terrestrial and Aquatic Wildlife Research in Büsum. For the molecular biological investigation with the developed LAMP assay, eight MSwab™ samples were taken from eight different bite marks of the stranded harbour porpoise. In a following step, the necropsy was performed in accordance with Siebert et al. (2001). First, the swab samples were tested without further DNA purification and with an amplification time of 30 min. Furthermore, a DNA extraction of the MSwab™ samples as described above was performed Fig. 3. Application of the LAMP assay on a stranded harbour porpoise and the isolated DNA was also (found in September 2017). Carcass of the stranded harbour porpoise as found on the beach on the coastline of Sylt (picture by Sönke tested with a 30-min amplification Lorenzen). time. Isolated DNA of a grey seal muscle was applied as positive control, while an MSwab™ sample of a harbour porpoise muscle was used as negative control. To rule out false negative results due to inhibitors, the native as well as the isolated swab samples were investigated with the common primer set (IAC).

To conﬁrm the results of the LAMP experiment with the speciﬁc primer set, a gel electrophoresis with the LAMP amplicons of the isolated DNA was subsequently carried out. For this, 2% agarose gel (peqGOLD Universal agarose gel, PeQlab, Erlangen, Germany), TrisBorat-EDTA (pH 8.4) and a DNA marker ranging in size from 50 bp to 2 kb (Biozym, Hessisch Oldendorf, Germany) were utilised.

94 Using molecular methods as a complementary tool of the macroscopic assessment

In addition to the above mentioned stranded harbour porpoise, two further harbour porpoises (found in August 2016 and July 2017 on the coastline of Sylt), presumably having been attacked by grey seals, were investigated with the LAMP method for residues of grey seal DNA.

According to Heers et al. (2017) there can be great similarity between the bite marks of the scavenger red fox (Vulpes vulpes) and the predator grey seal. For this reason, the native as well as the isolated DNA of all three harbour porpoises were investigated for red fox DNA with the LAMP assay in accordance with Heers et al. (2017).

3. Results

3.1. Alignment

The alignment of the cytochrome b sequences of grey seal and harbour porpoise resulted in identities of 78%, while the grey seal amplicon compared with the harbour porpoise amplicon resulted in a homology of 19%.

3.2. Sensitivity, speciﬁcity and limit of detection

Eight muscle samples of different grey seals were tested directly in triplicate with the MSwab™ method to determine the sensitivity. All samples were detected as positive with a mean detection time of 10:49 min (± 01:33 min). The mean annealing temperature of the grey seal amplification was 83.3 °C (± 0.13 °C). Furthermore, 24 different harbour porpoise muscle samples were tested for the specificity. Each sample was tested in triplicate as well. All MSwab™ samples were tested correctly as negative with the specific primer set and correctly as positive with the common primer set (IAC).

The limit of detection was tested six-fold with a dilution series of isolated grey seal DNA and with the specific primer set. An example of the amplification curves using the real-time fluorometer Genie® II, is shown in Fig. 4A. The results of the mean detection times of each dilution stage are shown in Fig. 4B. The higher the dilution step, the longer the amplification time (Fig. 4B). A DNA concentration of 1.00E + 03 pg/μl was detected after 05:55 min (± 00:07 min). Up to a DNA concentration of 1.00E-01 pg/μl, the DNA was determined with a 100% detection probability with a mean detection time of 09:37 min (± 00:30 min). The detection probability declined to 50% at a DNA concentration of 1.00E-02 pg/μl (mean detection time 12:20 min), finally reaching 0% after the subsequent dilution step (1.00E-03 pg/μl).

3.3. Spiking experiment with grey seal saliva

The detection of grey seal DNA from saliva samples was possible in all imitated bite marks. All results are shown in Table 3. The negative control water was not ampliﬁed. The detection times

95 Using molecular methods as a complementary tool of the macroscopic assessment

Table 3 Spiking experiment with four saliva samples of two different grey seals.

of the spiked saliva samples were longer than the ampliﬁcation times of the native tested saliva samples.

3.4. Necropsy of the stranded harbour porpoise (found in September 2017)

The male adult harbour porpoise, with a length of 131.5 cm and moderate body condition, presented large tissue defects over the entire body. Only the snout, the dorsal part of the head, both front flippers, partially the ventral abdomen, the base of the fluke and the fluke itself were covered by skin. The rest of the skin was attached to the ventral abdomen in large flaps, with some parts were missing. Especially the skin at the head showed multiple parallel punctual and elongated incisions. The wound margin of the skin flaps was regular with some triangular- shaped tears. The majority of the blubber on the skin flaps was intact, with some punctual lesions of up to 5 mm in diameter in the fringes of the flaps. In contrast, most of the exposed muscle tissue was missing. The thoracic and abdominal cavities were opened and some abdominal organs were missing partly or completely.

3.5. Application of the LAMP assay on stranded harbour porpoises

The bite lesions of the stranded harbour porpoise (found in September 2017) were investigated for grey seal DNA. With the speciﬁc primer set the molecular biological investigation of the eight MSwab™ samples without further DNA extraction step did not lead to a positive

Fig. 4. Limit of detection. (A) Typical amplification curves of eight-fold serially diluted grey seal DNA. (B) Mean values (n=6) with standard deviation of the limit of detection of the LAMP assay using serial dilutions of isolated grey seal DNA.

96 Using molecular methods as a complementary tool of the macroscopic assessment amplification of residues of grey seal DNA in the different bite lesions of the harbour porpoise. After DNA isolation of the eight MSwab™ mediums, the swab number 5 yielded a positive amplification after 12:45 min and consequently the molecular biological identification of predator DNA in the bite mark. The positive control of the grey seal DNA was determined after 7:00 min, while the negative control was not amplified. The sampling point of swab 5 was located in the blubber of the neck area. In the skin of the area around the neck and the head, many bite marks were found (Fig. 5). With the gel electrophoresis the positive amplification of the swab number 5 (lane 5) could be confirmed (Fig. 6).

To rule out false negative results, all native swab samples as well as the isolated DNA samples were tested with the common primer set (IAC). All samples were detected correctly as positive.

In addition, two further harbour porpoises were investigated with the LAMP method for residues of grey seal DNA. Grey seal DNA was not detected in any of the swab samples.

Due to the risk of confusion between the bite marks originated by grey seals or red foxes, all three stranded harbour porpoises were tested for red fox DNA with the LAMP assay according to Heers et al. (2017). In no case residues of scavenger DNA of red fox were detected in any of the bite marks.

4. Discussion

For providing evidence of grey seal DNA in bite lesions of harbour porpoises, a rapid and easy- to-perform DNA detection method with the possibility of an on-site investigation was developed and successfully tested on a predated harbour porpoise. The system can be useful for wildlife researchers to detect predator DNA of grey seals in harbour porpoises without the need for cumbersome laboratory equipment in the ﬁeld. With this LAMP assay we successfully conﬁrmed the assumption of a grey seal predation on a harbour porpoise at a molecular level. In addition to predation, this new method can also be used to clarify the rarer phenomenon of the post-mortem scavenging activities of grey seals on harbour porpoises (Leopold et al., 2014).

Applying the method of Heers et al. (2017) we could additionally rule out the presence of scavenger DNA of the red fox in all investigated bite marks. Moreover, a spiking experiment was conducted with three aims in mind: First, to imitate the attack of a grey seal as realistically as possible; second, to ensure that the developed primer set is specific, despite the high quantity of background DNA of the closely related species; third, to assess whether good results in difficult matrices like blood and fat can be obtained. We succeeded in simulating a bite lesion with grey seal saliva samples during the spiking experiment and determined grey seal DNA in all imitated bite marks. Therefore, we could confirm the specificity of the LAMP assay despite the large amount of background DNA of the harbour porpoise. The measured

97 Using molecular methods as a complementary tool of the macroscopic assessment

DNA yield could not be fully related to the amplification times as the saliva samples with the highest DNA concentrations resulted in the longest amplification times. It can be assumed there was not only grey seal but also foreign DNA in the saliva samples as the saliva samples were taken while feeding the grey seals with fish (Kamodyová et al., 2013). This resulted in the DNA of grey seal and fish being isolated simultaneously during the DNA isolation step of the saliva samples and measured simultaneously with the NanoDrop2000c. During the spiking experiment it was notable that the detection time in the imitated bite marks was significantly higher than the amplification time of the native tested saliva samples. This increase is possibly attributable to certain matrix effects and inhibiting factors like fat and blood (Schrader et al., 2012). A further aspect in this context was the high dilution effect due to the 1 ml MSwab™ medium. Furthermore, the spiking experiment clearly showed that even with a highly similar saliva inoculum and a simple incision lesion, there was a considerable variation between the detection times within the six runs. This indicates that the swab sampling process was difficult to perform consistently, in particular, when it came to deep lesions and small DNA quantities. We therefore recommend taking at least three swab samples per wound lesion. As there were some samples in the spiking experiment which were detected only after the regular amplification time of 20 min, it is advisable to increase the time of the amplification process to 30 min in the field.

Within the spiking experiment it was possible to identify grey seal DNA in the imitated bite marks without need for a further DNA isolation step. Also in the study of Heers et al. (2017) the DNA of a red fox was detected in a scavenged harbour porpoise without extracting the DNA. Comparing these results with the stranded harbour porpoise (found in September 2017) in the present study, it is apparent that the direct tested MSwab™ samples did not manage to achieve a positive amplification. However, after isolating the DNA samples to obtain a higher DNA yield, the residues of grey seal DNA were amplified as positive. Regarding the other two investigated harbour porpoises, no grey seal DNA was identified. Negative results could indicate that no grey seal attack had taken place or the DNA of the grey seals was degraded after all. Overall, we can confirm the difficulty of grey seal DNA detection in carcasses of harbour porpoises as reported by Jauniaux et al. (2014), showing that in only two out of the five tested harbour porpoises, DNA residues of grey seals were determined. Various reasons

98 Using molecular methods as a complementary tool of the macroscopic assessment

Fig. 5. Application of the LAMP assay on a stranded harbour porpoise. Sampling point of four MSwab™ samples. The other swab samples were located on the other side of the harbour porpoise. may be considered for the challenging evidence of DNA in real bite marks compared to the imitated bite marks during the spiking experiment. First, the seawater is an added aggravating factor as the grey seal attack usually happens in the sea and the seawater can wash out the DNA following predation by a grey seal (Bouveroux et al., 2014; Jauniaux et al., 2014; van Bleijswijk et al., 2014; Stringell et al., 2015). Furthermore, an inflammatory reaction and the bleeding lesion can lead to the loss of any DNA residues (Jauniaux et al., 2014; Haelters et al., 2016). Thus, Pääbo et al. (2004) and Wayne et al. (1999) described various causes of DNA degradation. Whereas enzymes, bacteria, insects or UV radiation increase the degradation, low temperature and dry conditions lead to a reduction in DNA degradation. While the carcasses of the harbour porpoises were lying on the beach, rain can also lead to a loss of DNA residues (Brinkman et al., 2010). Another reason for a negative evidence of grey seal DNA residues might be a long lapse of time between an attack and sampling. Leopold et al. (2014) assumed an ever-increasing challenge of DNA evidence in marine mammals the longer the time between an attack and necropsy. Regarding terrestrial predation animals, Harms et al. (2015) indicated that saliva samples from bite marks should be taken as soon as possible and recommended a sample collection within 24–48 h after predation. Against the background of higher DNA degradation due to a long period of time between predation and taking swabs (Haelters et al., 2016), it is of great importance to lose as little time as possible between predation and sampling. Furthermore, we recommend testing the carcass directly on-site with the LAMP assay. The portable LAMP method, with the advantage of being field-usable and requiring little equipment, offers an excellent opportunity to save precious time until necropsy. A further advantage of the cost-effective LAMP method is the detection of small quantities in the femtogram range (Dhama et al., 2014). With the developed LAMP method we even

99 Using molecular methods as a complementary tool of the macroscopic assessment

managed to detect 1.00E-01 pg/μl grey seal DNA with a detection probability of 100%. The results of previous LAMP publications, dealing with animal species identiﬁcation in meat, obtained an analytical sensitivity of 1 pg/μl(Yang et al., 2014; Lee et al., 2016) or 0.35 pg/μl (Abdulmawjood et al., 2014). Compared to these studies, the LAMP method in the present study achieved a better limit of detection. This is particularly important for detecting DNA in saliva samples, where low DNA quantities are

Fig. 6. Application of the LAMP assay on a stranded harbour available (Harms et al., 2015). Due to porpoise (found in September 2017). Gel electrophoresis of the the rising number of grey seal attacks LAMP amplicons of isolated DNA of the MSwab™ medium using the specific primer set. Lane 1–8 stand for the MSwab™ samples 1–8. on harbour porpoises the developed Lane “P, GS” display grey seal DNA as positive control. Lane “N, HP” show the negative control harbour porpoise, while lane “N, MM” fast and sensitive LAMP method stand for the mastermix as negative control. In lane “M” the marker constitutes an optimal possibility to is shown. test stranded harbour porpoises for DNA residues of grey seals directly on-site and at places where only little equipment can be taken. For forensic ﬁeld studies, researchers can verify within a very short timeframe if encountered lesions on a harbour porpoise carcass are the result of grey seal predation by applying this method or using the method of Heers et al. (2017) in combination if lesions result from red fox scavenging.

5. Conclusion

The newly developed LAMP method enables small concentrations (1.00E-01 pg/μl) of grey seal DNA to be detected, which is particularly important when investigating DNA in saliva samples or bite lesions. We successfully determined grey seal DNA in an attacked harbour porpoise and could consequently confirm an incident of grey seal predation with the LAMP assay. With the advantage of being field-usable, the LAMP method is feasible for marine wildlife researchers to confirm rapid grey seal predation on a harbour porpoise via wound swapping at a molecular level and clarify forensic issues.

100 Using molecular methods as a complementary tool of the macroscopic assessment

Acknowledgments

We wish to thank the staﬀ of the Institute for Terrestrial and Aquatic Wildlife Research in Büsum, University of Veterinary Medicine Hannover, Foundation, for preparing samples and sampling the stranded harbour porpoises. Furthermore, we are grateful to the seal ranger for taking pictures and retrieving the stranded harbour porpoise. The investigations were funded by the Ministry of Energy, Agriculture, Environment and Rural Areas of Schleswig-Holstein, Germany (grant number 60079084).

References

Abdulmawjood, A., Grabowski, N., Fohler, S., Kittler, S., Nagengast, H., Klein, G., 2014. Development of loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive identification of ostrich meat. PLoS One 9 (6), e100717. https://doi.org/10. 1371/journal.pone.0100717. PubMed PMID. 24963709. PubMed Central PMCID: PMCPMC4071038. Bouveroux, T., Kiszka, J.J., Heithaus, M.R., Jauniaux, T., Pezeril, S., 2014. Direct evidence for gray seal (Halichoerus grypus) predation and scavenging on harbor porpoises (Phocoena phocoena). Mar. Mamm. Sci. 30 (4), 1542–1548. Brinkman, T.J., Schwartz, M.K., Person, D.K., Pilgrim, K.L., Hundertmark, K.J., 2010. Effects of time and rainfall on PCR success using DNA extracted from deer fecal pellets. Conserv. Genet. 11 (4), 1547–1552. https://doi.org/10.1007/s10592-009¬9928-7. Camphuysen, C.J., Siemensma, M.L., 2011. Conservation plan for the Harbour Porpoise Phocoena phocoena in The Netherlands: towards a favourable conservation status. In: NIOZ Report 2011-07. Royal Netherlands Institute for Sea Research, Texel, pp. 1–186. Cho, A.R., Dong, H.J., Cho, S., 2014. Meat species identification using loop-mediated isothermal amplification assay targeting species-specific mitochondrial DNA. Kor. J. Food Sci. Anim. Resour. 34 (6), 799–807. https://doi.org/10.5851/kosfa.2014.34.6. 799. PubMed PMID. 26761677. PubMed Central PMCID: PMC4662195. Dhama, K., Karthik, K., Chakraborty, S., Tiwari, R., Kapoor, S., Kumar, A., et al., 2014. Loop- mediated isothermal amplification of DNA (LAMP): a new diagnostic tool lights the world of diagnosis of animal and human pathogens: a review. Pak. J. Biol. Sci. PJBS 17 (2), 151–166. Epub 2014/05/03. PubMed PMID. 24783797. Haelters, J., Kerkchof, F., Jauniaux, T., Degraer, S., 2012. The Grey seal (Halichoerus grypus) as a predator of harbour porpioses (Phocoena phocoena)? Aquat. Mamm. 343–353. Haelters, J., Everaarts, E., Bunskeok, P., Begeman, L., Hinrichs, J.W.J., Ijsseldijk, L.L., 2016. A suspected scavenging event by red foxes (Vulpes vulpes) on a live, stranded harbour porpoise (Phocoena phocoena). Aquat. Mamm. 42 (2), 227–232. https://doi. org/10.1578/AM.42.2.2016.227. Hanson N, van Neer A, Brownlow A, Haelters J. Report of Workshop on Predator-prey Interactions between Grey Seals and other marine mammals (WKPIGS). Middelfart, Denmark: ICES. Retrieved from http://www.ices.dk/sites/pub/Publication Reports/ Expert Group Report/SSGEPD/2017/01 WKPIGS -Report of Workshop on Predator prey Interactions between Grey Seals and other marine mammals.pdf.2017. Harms, V., Nowak, C., Carl, S., Muñoz-Fuentes, V., 2015. Experimental evaluation of genetic predator identification from saliva traces on wildlife kills. J. Mammal. 96 (1), 138–143. https://doi.org/10.1093/jmammal/gyu014. 101 Using molecular methods as a complementary tool of the macroscopic assessment

Heers, T., van Neer, A., Becker, A., Grilo, M.L., Siebert, U., Abdulmawjood, A., 2017. Loop- mediated isothermal amplification (LAMP) assay—a rapid detection tool for identifying red fox (Vulpes vulpes) DNA in the carcasses of harbour porpoises (Phocoena phocoena). PLoS One 12 (9), e0184349. https://doi.org/10.1371/journal. pone.0184349. IAMMWG, Camphuysen CJ, Siemensma ML. A Conservation Literature Review for the Harbour Porpoise (Phocoena phocoena). JNCC Report. 2015;No. 566, Peterborough: 96 pp. Jauniaux, T., Garigliany, M.M., Loos, P., Bourgain, J.L., Bouveroux, T., Coignoul, F., et al., 2014. Bite injuries of grey seals (Halichoerus grypus) on harbour porpoises (Phocoena phocoena). PLoS One 9 (12), e108993. https://doi.org/10.1371/journal.pone.0108993. PubMed PMID. 25461599. Kamodyová, N., Durdiaková, J., Celec, P., Sedláčková, T., Repiská, G., Sviežená, B., et al., 2013. Prevalence and persistence of male DNA identified in mixed saliva samples after intense kissing. Forens. Sci. Int.: Genet. 7 (1), 124–128. https://doi.org/10. 1016/j.fsigen.2012.07.007. Lee, S.-Y., Kim, M.-J., Hong, Y., Kim, H.-Y., 2016. Development of a rapid on-site detection method for pork in processed meat products using real-time loop-mediated isothermal amplification. Food Control 66, 53–61. https://doi.org/10.1016/j. foodcont.2016.01.041. Leopold, M.F., Begeman, L., van Bleijswijk, J.D.L., Ijsseldijk, L.L., Witte, H.J., Gröne, A., 2014. Exposing the grey seal as a major predator of harbour porpoises. Proc. R. Soc. B Biol. Sci. 282 (1798). Moslemi, E., Shahhosseiny, M.H., Javadi, G., Praivar, K., Sattari, T.N., Amini, H.K., 2009. Loop mediated isothermal amplification (LAMP) for rapid detection of HBV in Iran. Afr. J. Microbiol. Res. 3 (8), 439–445 August, 2009. 3(8):439–45. Nagamine, K., Hase, T., Notomi, T., 2002. Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol. Cell. Probes 16 (3), 223–229. Epub 2002/07/30. PubMed PMID. 12144774. Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., et al., 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28 (12), E63. Epub 2000/06/28. PubMed PMID. 10871386. PubMed Central PMCID: PMCPMC102748. Pääbo, S., Poinar, H., Serre, D., Jaenicke-Despres, V., Hebler, J., Rohland, N., et al., 2004. Genetic analyses from ancient DNA. Annu. Rev. Genet. 38, 645–679. https://doi.org/ 10.1146/annurev.genet.37.110801.143214. PubMed PMID. 15568989. Schrader, C., Schielke, A., Ellerbroek, L., Johne, R., 2012. PCR inhibitors -occurrence, properties and removal. J. Appl. Microbiol. 113 (5), 1014–1026. https://doi.org/10. 1111/j.1365- 2672.2012.05384.x. PubMed PMID. 22747964. Sheet, O.H., Grabowski, N.T., Klein, G., Abdulmawjood, A., 2016. Development and validation of a loop mediated isothermal amplification (LAMP) assay for the detection of Staphylococcus aureus in bovine mastitis milk samples. Mol. Cell. Probes 30 (5), 320–325. https://doi.org/10.1016/j.mcp.2016.08.001. PubMed PMID. 27495132. Siebert, U., Wunschmann, A., Weiss, R., Frank, H., Benke, H., Frese, K., 2001. Post mortem findings in harbour porpoises (Phocoena phocoena) from the German north and Baltic seas. J. Comp. Pathol. 124 (2–3), 102–114. https://doi.org/10.1053/jcpa. 2000.0436. PubMed PMID. 11222006. Stringell, T., Hill, D., Rees, D., Rees, F., Rees, P., Morgan, G., 2015. Short note: predation of harbour porpoises (Phocoena phocoena) by Grey seals (Halichoerus grypus) in Wales. Aquat. Mamm. 41 (2), 188–191. https://doi.org/10.1578/am.41.2.2015.188. van Bleijswijk, J.D.L., Begeman, L., Witte, H.J., Ijsseldijk, L.L., Brasseur, S., Gröne, A., et al., 2014. Detection of grey seal Halichoerus grypus DNA in attack wounds on stranded harbour

102 Using molecular methods as a complementary tool of the macroscopic assessment porpoises Phocoena phocoena. Mar. Ecol. Prog. Ser. 513, 277–281. https://doi.org/10.3354/meps11004. Vanjari, L., Vemu, L., Dandona, L., Schneider, J.A., 2014. Application of real time loop mediated isothermal amplification (RT LAMP) as a point of care assay using dried blood spots for rapid detection of HIV infection. BMC Infect. Dis. 14 (Suppl 3), O11. https://doi.org/10.1186/1471-2334-14-s3-o11. Wang, D., Huo, G., Ren, D., Li, Y., 2010. Development and evaluation of a loop-mediated isothermal amplification (Lamp) method for detecting Listeria monocytogenes in raw milk. J. Food Saf. 30 (2), 251–262. https://doi.org/10.1111/j.1745-4565.2009. 00196.x. Wayne, R.K., Leonard, J.A., Cooper, A., 1999. Full of sound and fury: history of ancient DNA. Annu. Rev. Ecol. Syst. 30, 457–477. Yang, L., Fu, S., Peng, X., Li, L., Song, T., Li, L., 2014. Identification of pork in meat products using real-time loop-mediated isothermal amplification. Biotechnol. Equip. 28 (5), 882–888. https://doi.org/10.1080/13102818.2014. 963789. PubMed PMID. 26019573. PubMed Central PMCID: PMC4433938.

103 Overall Discussion

5. Overall Discussion

For marine mammals, with all three species occurring in German waters being under the umbrella of some sort of legislative protection framework, there is the need to verify causes of death particularly if of unnatural nature (Bernaldo de Quirós et al., 2018). This can only be achieved by implementing a comprehensive stranding network and performing specific necropsies of stranded animals by specialists in this field (Haelters et al., 2018; Siebert et al., 2001, 2007; ten Doeschate et al., 2017). This is especially relevant if e.g. the source of a detected lesion is deemed to be directly related to human actions (Bernaldo de Quirós et al., 2018). In this regard, for example mortality as a consequence of marine mammal – fisheries interaction has been identified as one of the major causes of death in different areas of the globe (Carretta et al., 2018; Hamilton & Baker, 2019; Northridge, 2018; Reeves et al., 2013). Due to the delicate nature of this topic with regards to the social and political consequences, a rigorous scientific assessment of stranded marine mammals is crucial in order to identify sources of e.g. fatal lesions. Reliable results originating from these assessments can than in turn support the development and implementation of sound management measures. Within this context the phenomenon of grey seal predation on marine mammals has played a prominent role in the area surrounding the North Sea.

This phenomenon can still be regarded as a fairly new discovery. In 2012 the first publication introduced this hypothesis (Haelters et al., 2012) and despite first scepticism within the scientific community (Haelters et al., 2015), several publications followed from different areas confirming that grey seals prey on harbour seals, harbour porpoises as well as conspecifics (Bishop et al., 2016; Leopold et al., 2015a; van Neer et al., 2019, 2015a).

As a consequence of this emerging knowledge, studies conducting retrospective analysis of necropsy data found, that in some cases, a considerable amount of marine mammals that have been ascribed a cause of death directly related to anthropogenic actions such as fisheries bycatch or propeller strike by ships, now had to be reconsidered as natural cause of death induced by grey seal predation (Brownlow et al., 2016; Leopold et al., 2015b). In this regard, the emerging knowledge has already considerably improved the situation as standardised parameters that have been confirmed to be good indicators for grey seal predation are now available to be used for future assessments.

Likewise, through the common use of standardised methods, a future cross-national comparison of the recorded data should be made possible. This is particularly relevant in the context of international agreements and conservation efforts, such as the Marine Strategy Framework Directive or the Flora-Fauna-Habitat Directive, since assessments of the state of

104 Overall Discussion the ecosystem as well as the distinction between anthropogenic induced changes and natural ones must be made (Santos & Pierce, 2015).

Besides the relevance in terms of the management of human-wildlife conflicts, reliable estimates of the rate of grey seal predation on the respective marine mammal species are also crucial for any ecological studies of competition, related food-web-effects and their respective population consequences. Despite the growing knowledge base, it is still unknown whether predation rates are high enough to have effects on a population scale for grey seals as well as their prey (Brownlow et al., 2016; ICES, 2017; Leopold et al., 2015a). Within this context the prey composition of grey seals needs to be re-assessed utilising the emerging knowledge and included when applying model approaches such as the one recently used for harbour seals (Aarts et al., 2018).

Therefore, the aim of this thesis was to acquire and provide the necessary background with regard to grey seal predation on marine mammals and to develop and present different tools for the standardised assessment of carcasses during necropsies as well as during retrospective analyses of stranding data.

To gain a better understanding of the situation in German waters with regard to grey seal predation, we summarised the necropsy findings of suspicious cases by using the available data of harbour (n = 128) and grey seals (n = 36) as well as harbour porpoises (n = 230) that were collected at the coasts of Schleswig-Holstein between 1990 and 2017. Based on the detected wound patterns and any other available information (e.g. stranding reports), the cases were categorised by likelihood of grey seal predation as origin of lesions (“definite”, “likely”, “possibly”, “unlikely” and “fox”). Findings were than used to develop and present parameters related to specific lesions (see Table 1 below), which should be utilised in future necropsies to rate the likelihood of grey seal predation in a standardised approach. As a way of verifying the validity of the suggested parameters, they were compared to definite cases of grey seal predation which were either directly observed (van Neer et al., 2019) or confirmed using molecular methods (Heers et al., 2018). Besides the predation of grey seals, further parameters and methods are suggested which can be used to differentiate between grey seal predation and the predation or post-mortem scavenging by terrestrial predators such as the red fox (Heers et al., 2017; van Neer et al., in preparation -a, in preparation -b).

Carcasses potentially subjected to grey seal predation show severe lacerations, mainly starting in the cervical area and having a smooth and cut-like wound margin. For seals this laceration often follows around the trunk in a helical pattern whereas for porpoises, often considerable areas of skin and blubber tissue are missing entirely. Further large parts of skin und underlying tissue are detached from the body and the loss of blubber tissue is common. The patterns of lesions that have been recorded in German waters are consistent with the findings from other

105 Overall Discussion regions indicating that hunting and feeding techniques used by the different individuals are likely to be very similar throughout the different regions (Brownlow et al., 2016; Haelters et al., 2012; Jauniaux et al., 2014; Leopold et al., 2015b).

Table 1: Summary-table showing parameters suggested for the assessment of suspicious carcasses. As some parameters are specific for either seals or porpoises, respective suitability is indicated. Shown parameters are indicative for grey seal predation unless stated differently (indicators for fox interaction). For details see chapter 3.b & 3.c.

suitable for suitable for parameter seals porpoises

A smooth wound margin X X

Missing blubber tissue X X

The lesion originates in the cervical area X X

Large parts of the blubber and skin are detached from X X the underlying muscular tissue

Linear and often parallel running rake marks are X X present in the blubber tissue

Puncture lesions can be detected X X

Parallel bite and/or scratch marks are present X

Regularly one or both scapulae are found to be X X detached

Often the lesion follows a helical path around the body X

Bone defects X X (indicative for fox)

Large parts of skin and soft tissue are missing X X (indicative for fox)

The wound margin shows a ragged pattern rather X X than smooth (indicative for fox) (indicative for fox)

Although the development of standardised parameters for the macroscopic assessment of suspicious marine mammal carcasses can already be regarded as significant advantage (van Neer et al., in preparation -a, in preparation -b), it needs to be noted that this tool is still based on a potentially observer biased method in need of an experienced specialist. Therefore, future studies should further focus on quantifiable and easily reproducible methods as tools of choice. Besides the presented molecular tools available (for details see chapter 4), histopathological methods may pose considerable potential. Here, a reliable differentiation between sharp force trauma and respective lacerations induced by grey seals would already allow the exclusion of

106 Overall Discussion several anthropogenic induced causes of mortality. Further it needs to be evaluated, if histopathology can also be used to differentiate between lesions induced by grey seals and lesions resulting from the predation or scavenging by terrestrial predators as has been done by molecular means (Heers et al., 2017).

In German waters, to date only male grey seals have shown this behaviour. This is especially interesting in the light of sex-dimorphic feeding strategies. For grey seals it was already shown, that prey selection and area use can show sex-specific patterns when utilising piscivorous prey (Beck et al., 2007; Breed et al., 2009; Russell et al., 2015). With regards to the predation of marine mammals, it also seems that this behaviour is only shown by males, but in order to clarify this, large scale diet studies should be conducted. Due to the expected lack of marine mammal related hard parts in stomach content samples, especially scat based molecular methods such as metabarcoding are deemed useful (Berry et al., 2017; Schwarz et al., 2018). Despite the advances of this technology, this method has not yet been used for this purpose and limitations will still persist as the rate of cannibalism by grey seals would only be detectable exerting an unreasonably high effort with the need to differentiate between the individual based genetic code of predator and prey. Yet, using such molecular methods would additionally enable a better understanding of the general prey preferences of individuals utilising marine mammals as prey. Even though we could show that a single individual uses both seal species as prey (van Neer et al., in preparation -a), it still remains unclear whether single individuals utilise all three marine mammal species and which role marine mammals play in their general prey composition (e.g. the ratio between piscivorous and mammalian prey).

In order to gain a better understanding of temporal persistence of this behaviour, additionally stable isotope analysis as a measurement of long term feeding strategies could be taken (Tucker et al., 2007). By using this method, it should be possible to evaluate the importance of marine mammals as prey for a greater timescale in comparison to the short timescale achieved by scat analysis. Collecting a sufficiently large enough sample size might prove difficult in practice though.

Further discussion and potentially data collection is also needed in order to evaluate this behaviour in light of ecological foraging theories. In prey choice models or more general optimal foraging theories, predators are expected to prefer prey species with the highest net energetic gain and a lowest possible rate of risk involved (Stephens et al., 2007). When comparing average caloric contents of different fish species with a proxy for seal prey, it becomes evident, that the energetic gain related to this behaviour might be large in comparison to the energetic cost and risk-taking (van Neer et al., 2019). Even though such a coarse comparison might have enough validity to be used as a first assessment, future work should focus on a more detailed evaluation. This should include the determination of specific caloric

107 Overall Discussion contents of the different prey species as well as approximations of energy expenditure associated with predating marine mammals by means of bio-logging studies.

It is also unclear what effect the utilisation of marine mammals as prey has with regard to the exposure and accumulation of pollutants. For other marine predators relying on marine mammals as prey as well as for humans consuming marine mammal meat it was shown, that the accumulation of pollutants can have significant negative effects on the general fitness of the individuals (Aubail et al., 2012; Booth & Zeller, 2005; Jepson et al., 2016; McKinney et al., 2013; Sonne et al., 2006).

From what is known to date, it is expected that only a few specialised male grey seals follow this foraging strategy. On Helgoland four different males showing this behaviour have been identified (van Neer et al., in preparation -a). Based on re-sightings, for at least one of them it was also shown, that this behaviour was evident over consecutive years. Further it is noticeable that on Helgoland, carcasses showing the typical lesions occur in bursts with many such carcasses stranding in a short period of time, often followed by weeks and sometimes even month of no cases. During one of such periods of increased numbers of stranded carcasses, one of the identified males was observed during daily counts and by the time the presence of this individual could not be confirmed any more, the stranding of such carcasses ceased. This also supports the hypothesis, that only a few specialists show this behaviour. It further indicates that the respective individuals don’t necessarily remain in one colony but potentially move between colonies. This was also shown by colleagues from Scotland for a case recorded on the Isle of May (Bishop et al., 2016). Here the identified male was equipped with a GPS tag and therefore it was possible to follow the movement. The male left the colony after preying on conspecific pups for some days and eventually moved to a known grey seal haul-out in German waters in a way, which can certainly be interpreted as directed movement (Thompson et al., 2015). First carcasses showing the associated lesions where recorded the day the male arrived in that area. Earlier studies confirmed that male as well as female grey seals show consistent individual variation in behaviour across time and/or contexts, termed animal personality (Bubac et al., 2018; Twiss & Franklin, 2010). For other species, personalities were identified to be reflected in several behavioural traits e.g. in feeding specialisation (Patrick & Weimerskirch, 2014). Whether this type of feeding specialisation is also related to respective personalities in grey seals is possible but entirely unclear.

Except for one case on Helgoland where divers recorded a known grey seal feeding from a harbour seal under water, observations of this behaviour have only been possible from land or boat-based. In order to further elucidate the behavioural mechanisms behind this phenomenon, future studies should take advantage of the recent technical advances. For example have cameras coupled with movement and GPS sensors attached to marine

108 Conclusion & Outlook mammals been used to assess the feeding behaviour as well as prey preferences (Heaslip et al., 2014; Mikkelsen et al., 2019; Volpov et al., 2015). Such method could also be used with regard to this behaviour, in order to gather detailed records of the processes of catching, killing and feeding and to approximate the associated energy expenditure. Further it would be possible to assess the individuals’ prey preference and composition and also relate this to spatio-temporal information recorded. This is highly needed to get a better understanding of the ratio between actually predated animals and the number of individuals found on the beach, which likely resembles only a fraction of the total number predated. Additionally, the spatial data of the stranding records should be further considered with regard to potential hot spots in relation to the abundance estimates of prey species like harbour seals and porpoises.

6. Conclusion & Outlook

Within this thesis we were able to compile a wealth of information with regard to the predation of marine mammals by grey seals. The observations confirm that male grey seals prey on harbour seals and also show cannibalistic behaviour in German waters. Retrospective as well as genetic analysis of strandings additionally confirmed the presence of grey seal predation of harbour porpoises.

Further, different methods which allow for a standardised assessment of suspicious marine mammal carcasses were developed in the course of the study. Besides using molecular methods as a complementary tool when assessing porpoise carcasses, the macroscopic assessment using the suggested parameters is until today regarded as the method of choice for any of the three species. It needs to be acknowledged though, that a prerequisite to allow for a thorough evaluation of cases, is the installation and maintenance of a well-organised local stranding network enabling systematic necropsies of marine mammal carcasses. Additionally, any source of information like reports from the site of the stranding or eye witness reports should be used in the decision-making process. Cases of observed grey seal predation should be further used as reliable source of information with regards to the behaviour as well as pathology.

Despite the advantages of recent years, more research is needed to further optimise and harmonise assessment criteria with the aim of developing a reproducible and objective method for the differentiation of detected lesions and in turn being able to reliably estimate predation rates and prey composition of grey seals and thus, the ecological relevance of this behaviour. Furthermore, research should be conducted in order to assess the suitability of other methods that could be applied, such as the histological evaluation of wound margins to differentiate between causes of trauma. Also, easy to use methods enabling the detection of grey seal DNA

109 Conclusion & Outlook in harbour seal carcasses similar to the ones suggested for porpoise carcasses should be further developed (Heers et al., 2017, 2018).

Regardless of the many questions that remain unanswered, it needs to be emphasised, that up until today a solid knowledge base has been built by researches around the North Sea and beyond. This was done in a joint afford to document and publish new findings with regard to the phenomenon of grey seal predation of marine mammals. Scientists working on this topic should continue to join forces to constantly update the list of parameters if necessary and establish an objective method and thus allow for a comprehensive evaluation of the potential ecological effects on the respective populations in the future. Therefore, also effort to record and assess strandings should be implemented and standardised throughout the regions to allow for a spatially inclusive and comprehensive monitoring.

Grey seals have always been considered as one of the top predators in the North Sea and surrounding areas. Within the last years convincing evidence could be gathered, which supports the hypothesis, that grey seals need to be not only seen as one of the top predators, but rather as the unknown apex-predator in this ecosystem and should be considered as such in future assessments of related ecosystem processes.

110 Zusammenfassung

7. Zusammenfassung van Neer, Abbo: Prädation mariner Säugetiere durch Kegelrobben (Halichoerus grypus) – Untersuchungen zu den Hintergründen, dem Ausmaß und den potentiellen Effekten auf das Ökosystem

Untersuchungen der letzten Jahre haben gezeigt, dass Kegelrobben andere marine Säugetiere wie Seehunde, Schweinswale und andere Kegelrobben im Bereich der Nordsee als Nahrungsressource nutzen. Der erste Bericht, der die Hypothese, dass Kegelrobben andere marine Säuger prädieren wurde 2012 durch belgische Wissenschaftler veröffentlicht. Im Jahr 2013 wurde erstmals die Prädation von Seehunden generell und spezifisch aus deutschen Gewässern beobachtet.

Die vorliegende Arbeit enthält einen umfassenden Überblick über den aktuellen Wissensstand zur Prädation von Meeressäugern durch Kegelrobben in deutschen Gewässern. Mit Hilfe von zwei Fallberichten und einer detaillierten Bewertung der vorhandenen Strandungsdatenbank die eine Zeitreihe von 1990 bis 2017 umfasst, werden aufgetretene Wundmuster beschrieben und in Form verschiedener Bewertungskriterien zusammengefasst. Die vorgeschlagenen Parameter spiegeln die am häufigsten nachgewiesenen Läsionen wider. Sie umfassen unter anderem einen glatten, schnittartigen Wundrand der durch das Zerreißen des Gewebes entstanden ist, große Teile Haut inklusive des darunter liegenden Gewebes, welche vom Körper abgelöst wurden sowie den Verlust von Fettgewebe. Ebenfalls werden häufig punktuelle Wunden im Fettgewebe und bei Schweinswalen auch in der Haut gefunden. Dazu werden auch immer wieder punktförmige Frakturen zum Beispiel an der Skapula oder dem Unterkiefer festgestellt. Neben den durch Kegelrobben verursachten Läsionen gilt insbesondere ein unregelmäßiger Wundrand als relevant in Bezug zu einer Interaktion mit einem Fuchs. Hier. Dargestellte Ergebnisse der makroskopischen Bewertung werden in Bezug zu früheren Publikationen interpretiert und diskutiert. Zusätzlich und ergänzend zu den makroskopischen Auswertungsmethoden werden molekulare Methoden vorgestellt, die eine weitere Charakterisierung des Räubers ermöglichen.

Das dokumentierte Verhalten über die Zeit und zwischen Individuen als weitgehend konsistent betrachtet werden. Ob dies auf ein allgemeines, spezielles Jagdverhalten oder sogar auf die Persönlichkeit eines Individuums zurückzuführen ist, bleibt unklar. Seit dem ersten Erkennen dieses Verhaltens wird auf nationaler und internationaler Ebene an einem besseren Verständnis der Größenordnung und der ökologischen Relevanz gearbeitet. In Anbetracht der dargestellten Ergebnisse ist davon auszugehen, dass dieses Verhalten räumlich zumindest teilweise verbreitet ist. Unser Verständnis reicht jedoch noch nicht aus, um eine umfassende Bewertung der ökologischen Relevanz vorzunehmen und sollte daher einen Schwerpunkt der

111 Zusammenfassung zukünftigen Forschung bilden. Ebenso sollten durch standardisierte Methoden künftige länderübergreifende Vergleiche der erfassten Daten unterstützt werden.

112 Summary

8. Summary van Neer, Abbo: Predation of marine mammals by grey seals (Halichoerus grypus) – Assessment of the background, the extent and the potential effects on the ecosystem

Studies in recent years have shown that grey seals use other marine mammals such as harbour seals, harbour porpoises and other grey seals in the North Sea as food resource. The first report introducing the hypothesis of grey seals predating marine mammals was published by Belgian scientists in 2012. In 2013 predation of harbour seals was observed for the first time and reported from German waters.

The present thesis contains a comprehensive overview of the current state of knowledge on the predation of marine mammals by grey seals in German waters. With the help of two case reports and a detailed evaluation of the existing stranding database encompassing a time series from 1990 until 2017, occurring wound patterns are described and summarised in the form of several assessment criteria. Suggested parameters reflect the most common lesions detected and include besides others, lacerations, a smooth, cut-like wound margin, large parts of skin and underlying tissue that are detached from the body, as well as the loss of blubber tissue. Further, punctate wounds are often found in the blubber tissue and for porpoises also in the skin. Moreover punctuate fractures are repeatedly found, for example on the scapula or the lower jaw. Besides the lesions induced by grey seals, an overview on assessment criteria for an interaction with a fox is also given. Here especially a ragged wound margin is seen as relevant. Presented findings resulting from the macroscopic assessment are interpreted and discussed in light of former publications. In addition and complementary to the macroscopic evaluation methods, molecular methods are presented which allow a further characterisation of the predator.

The documented behaviour can be regarded as largely consistent over time and between individuals. Whether this is related to a general specialised feeding behaviour or is even related to an individuals’ personality remains unclear. Since the first recognition of this behaviour, work has been under way at national and international level to gain a better understanding of its scale and ecological relevance. In view of the presented findings, it must be assumed that this behaviour is at least partially widespread. However, our understanding is not yet sufficient to make a comprehensive assessment of the ecological relevance and should therefore be a major focus of future research. Likewise, through the use of standardised methods, future cross-national comparisons of the recorded data should be supported.

113 References

9. References

Aarts, G., Brasseur, S., Poos, J. J., Schop, J., Kirkwood, R., van Kooten, T., Mul, E., Reijnders, P., Rijnsdorp, A. D., & Tulp, I. (2018). Harbour seals are regaining top-down control in a coastal ecosystem. bioRxiv. doi:10.1101/267567

Aubail, A., Dietz, R., Rigét, F., Sonne, C., & Wiig Øand Caurant, F. (2012). Temporal trend of mercury in polar bears (Ursus maritimus) from Svalbard using teeth as a biomonitoring tissue. Journal of Environmental Monitoring, 14(1), 56–63. doi:10.1039/c1em10681c

Beck, C. A., Iverson, S. J., Bowen, W. D., & Blanchard, W. (2007). Sex differences in grey seal diet reflect seasonal variation in foraging behaviour and reproductive expenditure: evidence from quantitative fatty acid signature analysis. The Journal of Animal Ecology, 76(3), 490–502. doi:10.1111/j.1365-2656.2007.01215.x

Bedard, C., Kovacs, K., & Hammill, M. (1993). Cannibalism by grey seals, Halichoerus grypus, on Amet Island, Nova Scotia. Marine Mammal Science, 9(4), 421–424. doi:10.1111/j.1748-7692.1993.tb00474.x

Bernaldo de Quirós, Y., Hartwick, M., Rotstein, D., Garner, M., Bogomolni, A., Greer, W., Niemeyer, M., Early, G., Wenzel, F., & Moore, M. (2018). Discrimination between bycatch and other causes of cetacean and pinniped stranding. Diseases of Aquatic Organisms, 127(2), 83–95. doi:10.3354/dao03189

Berry, T. E., Osterrieder, S. K., Murray, D. C., Coghlan, M. L., Richardson, A. J., Grealy, A. K., Stat, M., Bejder, L., & Bunce, M. (2017). DNA metabarcoding for diet analysis and biodiversity: A case study using the endangered Australian sea lion (Neophoca cinerea). Ecology and Evolution, 7(14), 5435–5453. doi:10.1002/ece3.3123

Bishop, A. M., Onoufriou, J., Moss, S., Pomeroy, P. P., & Twiss, S. D. (2016). Cannibalism by a male grey seal (Halichoerus grypus) in the North Sea. Aquatic Mammals, 42(2), 137– 143. doi:10.1578/AM.42.2.2016.137

Bjørge, A., & Tolley, K. A. (2009). Harbor porpoise (Phocoena phocoena). In W. F. Perrin, B. Würsig, & J. G. M. Thewissen (Eds.), Encyclopedia of Marine Mammals (2nd ed., pp. 530–533). Amsterdam: Academic Press.

Bodewes, R., Bestebroer, T. M., van der Vries, E., Verhagen, J. H., Herfst, S., Koopmans, M. P., Fouchier, R. A. M., Pfankuche, V. M., Wohlsein, P., Siebert, U., Baumgärtner, W., & Osterhaus, A. D. M. E. (2015). Avian Influenza A(H10N7) Virus–Associated Mass Deaths among Harbor Seals. Emerging Infectious Diseases, 21(4), 720–722. doi:10.3201/eid2104.141675

Booth, S., & Zeller, D. (2005). Mercury, food webs, and marine mammals: Implications of diet and climate change for human health. Environmental Health Perspectives, 113(5), 521– 526. doi:10.1289/ehp.7603

114 References

Bouveroux, T., Kiszka, J. J., Heithaus, M. R., Jauniaux, T., & Pezeril, S. (2014). Direct evidence for gray seal (Halichoerus grypus) predation and scavenging on harbor porpoises (Phocoena phocoena). Marine Mammal Science, 30(4), 1542–1548. doi:10.1111/mms.12111

Brasseur, S., Borchardt, T., Czeck, R., Jensen, L. F., Galatius, A., Ramdohr, S., Siebert, U., & Teilmann, J. (2011a). Aerial surveys of grey seals in the Wadden Sea in the season of 2010-2011. Retrieved from http://www.waddensea- secretariat.org/sites/default/files/downloads/ final_report_aerial_surveys_of_grey_seals_in_the_wadden_sea_in_2011.pdf

Brasseur, S., Borchardt, T., Czeck, R., Jensen, L. F., Galatius, A., Ramdohr, S., Siebert, U., & Teilmann, J. (2012a). Aerial surveys of Grey Seals in the Wadden Sea in the season of 2011-2012. Wilhelmshaven, Germany. Retrieved from http://www.waddensea- secretariat.org/sites/default/files/downloads/final_report_aerial_surveys_of_grey_seals_i n_the_wadden_sea_in_2012.pdf

Brasseur, S., Borchardt, T., Czeck, R., Jensen, L. F., Galatius, A., Ramdohr, S., Siebert, U., & Teilmann, J. (2012b). Aerial surveys of Harbour Seals in the Wadden Sea in 2012. Wilhelmshaven, Germany. Retrieved from http://www.waddensea-secretariat.org/ sites/default/files/downloads/trilateral_seal_counts_report_2012.pdf

Brasseur, S., Borchardt, T., Czeck, R., Jensen, L. F., Jørgensen, A. G., Ramdohr, S., Siebert, U., & Teilmann, J. (2011b). Aerial surveys of Harbour Seals in the Wadden Sea in 2011 - Solid increases in total number as well as pups. Wilhelmshaven, Germany. Retrieved from http://www.waddensea-secretariat.org/sites/default/files/downloads/ trilateral_seal_counts_report_2011.pdf

Brasseur, S., Cremer, J., Czeck, R., Galatius, A., Jeß, A., Körber, P., Pund, R., Siebert, U., Teilmann, J., & Klöpper, S. (2018). TSEG grey seal surveys in the Wadden Sea and Helgoland in 2017-2018 - More than ten years of growth in the Wadden Sea area. Wilhelmshaven, Germany. Retrieved from http://www.waddensea- secretariat.org/sites/default/files/downloads/TMAP_downloads/Seals/18-07- 02_greysealreport2018.pdf

Brasseur, S., Czeck, R., Diederichs, B., Galatius, A., Jensen, L. F., Körber, P., Siebert, U., Teilmann, J., & Klöpper, S. (2014). Grey Seal surveys in the Wadden Sea and Helgoland in 2013-2014. Wilhelmshaven, Germany. Retrieved from http://www.waddensea- secretariat.org/sites/default/files/CWSS_Internal/TMAP/Marine_Mammals/grey_seal_re port_2014.pdf

Brasseur, S., Czeck, R., Galatius, A., Jensen, L. F., Jeß, A., Körber, P., Pund, R., Siebert, U., Teilmann, J., & Klöpper, S. (2016). TSEG Grey Seal surveys in the Wadden Sea and Helgoland in 2015-2016. Wilhelmshaven, Germany. Retrieved from http://www.waddensea-secretariat.org/sites/default/files/downloads/tmap/ MarineMammals/GreySeals/grey_seal_report_2016.pdf

Brasseur, S., Czeck, R., Galatius, A., Jensen, L. F., Jeß, A., Körber, P., Pund, R., Siebert, U., Teilmann, J., & Klöpper, S. (2017). TSEG Grey Seal surveys in the Wadden Sea and Helgoland in 2016-2017. Wilhelmshaven, Germany. Retrieved from http://www.waddensea-secretariat.org/sites/default/files/CWSS_Internal/TMAP/ Marine_Mammals/17-06-07_greysealreport2017.pdf

Brasseur, S., Czeck, R., Galatius, A., Jensen, L. F., Jeß, A., Körber, P., Siebert, U., Teilmann, J., & Klöpper, S. (2015). Grey Seal surveys in the Wadden Sea and Helgoland in 2014- 2015. Wilhelmshaven, Germany. Retrieved from http://www.waddensea- secretariat.org/sites/default/files/downloads/tmap/ MarineMammals/GreySeals/grey_seal_report_2015.pdf

115 References

Brasseur, S., Diederichs, B., Czeck, R., Jensen, L. F., Galatius, A., Ramdohr, S., Siebert, U., Teilmann, J., Körber, P., & Klöpper, S. (2013a). Aerial surveys of Grey Seals in the Wadden Sea in the season of 2012-2013. Wilhelmshaven, Germany. Retrieved from http://www.waddensea-secretariat.org/sites/default/files/downloads/TMAP_downloads/ Seals/aerial_surveys_of_grey_seals_in_the_wadden_sea_in_the_season_of_2012- 13.pdf

Brasseur, S., Diederichs, B., Czeck, R., Jensen, L. F., Galatius, A., Ramdohr, S., Siebert, U., Teilmann, J., Körber, P., & Klöpper, S. (2013b). Aerial surveys of Harbour Seals in the Wadden Sea in 2013. Wilhelmshaven, Germany. Retrieved from http://www.waddensea- secretariat.org/sites/default/files/downloads/TMAP_downloads/Seals/aerial_surveys_of_ harbour_seals_in_the_wadden_sea_in_2013.pdf

Brasseur, S. M. J. M., Reijnders, P. J. H., Borchardt, T., Siebert, U., Ramdor, S., Czeck, R., Körber, P., Jensen, L. F., & Teilmann, J. (2010a). Aerial surveys of Harbour Seals in the Wadden Sea in 2010 - Strong increase in pups, slight increase in total number. Wilhelmshaven, Germany. Retrieved from http://www.waddensea- secretariat.org/sites/default/files/downloads/ trilateral_seal_counts_report_2010.pdf

Brasseur, S. M. J. M., Reijnders, P. J. H., Borchardt, T., Siebert, U., Stede, M., Ramdor, S., Jensen, L. F., & Teilmann, J. (2009a). Aerial surveys of Harbour Seals in the Wadden Sea in 2009 - Growth of the harbour seal population slowing down? Wilhelmshaven, Germany. Retrieved from http://www.waddensea- secretariat.org/sites/default/files/downloads/ trilateral_seal_counts_report_2009.pdf

Brasseur, S. M. J. M., Reijnders, P. J. H., Borchardt, T., Siebert, U., Stede, M., Ramdor, S., Jensen, L. F., Teilmann, J., & Tougaard, J. (2008). Aerial Surveys of Harbour Seals in the Wadden Sea in 2008 : Back to Pre-epizootic Level , and Still Growing: Wadden Sea Harbour Seal Population in 2008. Wilhelmshaven, Germany. Retrieved from http://www.waddensea- secretariat.org/sites/default/files/downloads/trilateral_seal_counts_report_2008.pdf

Brasseur, S., Reijnders, P., Borchardt, T., Siebert, U., Ramdohr, S., Czeck, R., Körber, P., Jensen, L. F., & Teilmann, J. (2010b). Aerial surveys of grey seals in the Wadden Sea in the season of 2009-2010. Wilhelmshaven, Germany. Retrieved from http://www.waddensea- secretariat.org/sites/default/files/downloads/final_report_aerial_surveys_of_grey_seals_i n_the_wadden_sea_in_2010.pdf

Brasseur, S., Reijnders, P., Borchardt, T., Siebert, U., Ramdohr, S., Stede, M., Jensen, L. F., & Teilmann, J. (2009b). Aerial surveys of grey seals in the Wadden Sea in the seasons of 2007- 2008 and 2008-2009. Wilhelmshaven, Germany. Retrieved from http://www.waddensea- secretariat.org/sites/default/files/downloads/final_report_aerial_surveys_of_grey_seals_i n_the_wadden_sea_in_2009.pdf

Breed, G. A., Jonsen, I. D., Myers, R. A., Bowen, W. D., & Leonard, M. L. (2009). Sex-specific, seasonal foraging tactics of adult grey seals (Halichoerus grypus) revealed by state– space analysis. Ecology, 90(11), 3209–3221. doi:10.1890/07-1483.1

Brouwer, E. (1965). Report of sub-committee on constants and factors. In K. Blaxter (Ed.), Energy metabolism (pp. 441–443). Academic Press.

Brownlow, A., Onoufriou, J., Bishop, A., Davison, N., & Thompson, D. (2016). Corkscrew Seals: Grey Seal (Halichoerus grypus) Infanticide and Cannibalism May Indicate the Cause of Spiral Lacerations in Seals. PLOS ONE, 11(6), e0156464. doi:10.1371/journal.pone.0156464

116 References

Bubac, C. M., Coltman, D. W., Don Bowen, W., Lidgard, D. C., Lang, S. L. C., & den Heyer, C. E. (2018). Repeatability and reproductive consequences of boldness in female gray seals. Behavioral Ecology and Sociobiology, 72(6), 100. doi:10.1007/s00265-018-2515- 5

Carretta, J. V, Helker, V., Muto, M. M., Greenman, J., Wilkinson, K., Lawson, D., Viezbicke, J., & Jannot, J. (2018). Sources of human-related injury and mortality for U.S. Pacific west coast marine mammal stock assessments, 2012-2016. NOAA technical memorandum NMFS; NOAA-TM-NMFS-SWFSC; 601. U.S. Department of Commerce. doi:10.7289/V5/TM-SWFSC-601

CWSS. (2017). Wadden Sea Quality Status Report. (S. Klöpper, Ed.). Wilhelmshaven, Germany: Common Wadden Sea Secretariat. Retrieved from www.qsr.waddensea- worldheritage.org/reports/

Czeck, R., & Paul, M. (2008). Grey seals — a homecoming species in the Wadden Sea. Senckenbergiana maritima, 38(2), 143–146. doi:10.1007/BF03055290

Das, K., Lepoint, G., Leroy, Y., & Bouquegneau, J. M. (2003). Marine mammals from the southern North Sea: feeding ecology data from δ13C and δ15N measurements. Marine Ecology Progress Series, 263, 287–298. doi:10.3354/meps263287

Galatius, A., Brasseur, S., Czeck, R., Diederichs, B., Jensen, L. F., Körber, P., Siebert, U., Teilmann, J., & Klöpper, S. (2014). Aerial surveys of Harbour Seals in the Wadden Sea in 2014. Wilhelmshaven, Germany. Retrieved from http://www.waddensea- secretariat.org/sites/default/files/downloads/TMAP_downloads/Seals/harbour_seal_repo rt_2014_b.pdf

Galatius, A., Brasseur, S., Czeck, R., Jensen, L. F., Jess, A., Körber, P., Pund, R., Siebert, U., Teilmann, J., & Klöpper, S. (2016). Aerial surveys of Harbour Seals in the Wadden Sea in 2016. Wilhelmshaven, Germany. Retrieved from http://www.waddensea- secretariat.org/sites/default/files/downloads/TMAP_downloads/Seals/aerial_surveys_of_ harbour_seals_in_the_wadden_sea_in_2016.pdf

Galatius, A., Brasseur, S., Czeck, R., Jensen, L. F., Jeß, A., Körber, P., Pund, R., Siebert, U., Teilmann, J., & Klöpper, S. (2015). Aerial surveys of Harbour Seals in the Wadden Sea in 2015. Wilhelmshaven, Germany. Retrieved from http://www.waddensea- secretariat.org/sites/default/files/downloads/tmap/MarineMammals/harbour_seal_report _2015.pdf

Galatius, A., Brasseur, S., Czeck, R., Jeß, A., Körber, P., Siebert, U., Teilmann, J., & Klöpper, S. (2017). Aerial surveys of Harbour Seals in the Wadden Sea in 2017 - Population counts still in stagnation, but more pups than ever. Wilhelmshaven, Germany. Retrieved from http://www.waddensea- secretariat.org/sites/default/files/downloads/TMAP_downloads/Seals/ 17-11- 09_harboursealreport2017.pdf

Gilles, A., Viquerat, S., Becker, E. A., Forney, K. A., Geelhoed, S. C. V, Haelters, J., Nabe- Nielsen, J., Scheidat, M., Siebert, U., Sveegaard, S., van Beest, F. M., van Bemmelen, R., & Aarts, G. (2016). Seasonal habitat-based density models for a marine top predator, the harbor porpoise, in a dynamic environment. Ecosphere, 7(6), e01367. doi:10.1002/ecs2.1367

Haelters, J., Kerckhof, F., & Jauniaux, T. (2018). Strandings of cetaceans in Belgium from 1995 to 2017. Lutra, 61(1), 107–126.

117 References

Haelters, J., Kerckhof, F., Jauniaux, T., & Degraer, S. (2012). The grey seal (Halichoerus grypus) as a predator of harbour porpoises (Phocoena phocoena)? Aquatic Mammals, 38(4), 343–353. doi:10.1578/AM.38.4.2012.343

Haelters, J., Kerckhof, F., van Neer, A., & Leopold, M. (2015). Letter to the Editor Exposing Grey Seals as Horses and Scientists as Human. Aquatic Mammals, 41(3), 351–353. doi:10.1578/AM.41.3.2015.351

Hall, A., & Russell, D. J. F. (2017). Gray Seal (Halichoerus grypus). In B. Würsig, J. G. M. Thewissen, & K. Kovacs (Eds.), Encyclopedia of Marine Mammals (3rd ed., pp. 420–422). Academic Press.

Hamilton, S., & Baker, G. B. (2019). Technical mitigation to reduce marine mammal bycatch and entanglement in commercial fishing gear: lessons learnt and future directions. Reviews in Fish Biology and Fisheries, 6, 1–25. doi:10.1007/s11160-019-09550-6

Hammond, P. S., Bearzi, G., Bjørge, A., Forney, K., Karczmarski, L., Kasuya, T., Perrin, W. F., Scott, M. D., Wang, J. Y., Wells, R. S., & Wilson, B. (2008). Phocoena phocoena. The IUCN Red List of Threatened Species 2008, e.T17027A6. Retrieved from https://www.iucnredlist.org/species/17027/6734992

Hammond, P. S., Lacey, C., Gilles, A., Viquerat, S., Börjesson, P., Herr, H., Macleod, K., Ridoux, V., Santos, M. B., Scheidat, M., Teilmann, J., Vingada, J., & Øien, N. (2017). Estimates of cetacean abundance in European Atlantic waters in summer 2016 from the SCANS-III aerial and shipboard surveys. Retrieved from https://synergy.st- andrews.ac.uk/scans3/files/2017/04/SCANS-III-design-based-estimates-2017-04-28- final.pdf

Hammond, P. S., & Wilson, L. J. (2016). Grey Seal Diet Composition and Prey Consumption. Scottish Marine and Freshwater Science, 7(20), 1–28. doi:10.7489/1799-1

Härkönen, T., Dietz, R., Reijnders, P., Teilmann, J., Harding, K., Hall, A., Brasseur, S., Siebert, U., Goodman, S. J., Jepson, P. D., Rasmussen, T. D., & Thompson, P. (2006). A review of the 1988 and 2002 phocine distemper virus epidemics in European harbour seals. Diseases of Aquatic Organisms, 68, 115–130. doi:10.3354/dao068115

Heaslip, S. G., Bowen, W. D., & Iverson, S. J. (2014). Testing predictions of optimal diving theory using animal-borne video from harbour seals (Phoca vitulina concolor). Canadian Journal of Zoology, 318, 309–318. doi:10.1139/cjz-2013-0137

ICES. (2017). Report of the Workshop on Predator-prey Interactions between Grey Seals and other marine mammals (WKPIGS). (N. Hanson, A. van Neer, A. Brownlow, & J. Haelters, Eds.). Middelfart, Denmark: ICES. Retrieved from http://www.ices.dk/sites/pub/Publication Reports/Expert Group Report/SSGEPD/2017/01 WKPIGS - Report of the Workshop on Predator-prey Interactions between Grey Seals and other marine mammals.pdf

118 References

IJsseldijk, L. L., Doeschate, M. T. I. ten, Davison, N. J., Gröne, A., & Brownlow, A. C. (2018). Crossing boundaries for cetacean conservation: Setting research priorities to guide management of harbour porpoises. Marine Policy, 95(July), 77–84. doi:10.1016/j.marpol.2018.07.006

Jauniaux, T., Garigliany, M.-M., Loos, P., Bourgain, J.-L., Bouveroux, T., Coignoul, F., Haelters, J., Karpouzopoulos, J., Pezeril, S., & Desmecht, D. (2014). Bite injuries of grey seals (Halichoerus grypus) on harbour porpoises (Phocoena phocoena). PloS one, 9(12), e108993. doi:10.1371/journal.pone.0108993

Jefferson, T. A., Webber, M. A., & Pitman, R. L. (2008). Marine Mammals of the World - A Comprehensive Guide to their Identification. FAO Species Identification Guide. Elsevier. doi:10.1016/B978-0-12-383853-7.X5001-X

Jepson, P. D., Deaville, R., Barber, J. L., Aguilar, À., Borrell, A., Murphy, S., Barry, J., Brownlow, A., Barnett, J., Berrow, S., Cunningham, A. a, Davison, N. J., ten Doeschate, M., Esteban, R., Ferreira, M., Foote, A. D., Genov, T., Giménez, J., Loveridge, J., Llavona, Á., Martin, V., Maxwell, D. L., Papachlimitzou, A., Penrose, R., Perkins, M. W., Smith, B., de Stephanis, R., Tregenza, N., Verborgh, P., Fernandez, A., & Law, R. J. (2016). PCB pollution continues to impact populations of orcas and other dolphins in European waters. Scientific Reports, 6(1), 18573. doi:10.1038/srep18573

Leopold, M. F., Begeman, L., Heße, E., van der Hiele, J., Hiemstra, S., Keijl, G., Meesters, E. H., Mielke, L., Verheyen, D., & Gröne, A. (2015a). Porpoises: From predators to prey. Journal of Sea Research, 97, 14–23. doi:10.1016/j.seares.2014.12.005

Leopold, M. F., Begeman, L., van Bleijswijk, J. D. L., IJsseldijk, L. L., Witte, H. J., & Gröne, A. (2015b). Exposing the grey seal as a major predator of harbour porpoises. Proceedings of the Royal Society of London B: Biological Sciences, 282(1798), 20142429. doi:10.1098/rspb.2014.2429

Lucas, Z., & McLaren, I. A. (1988). Apparent Predation by Grey Seals, Halichoerus grypus, on Seabirds around Sable Island, Nova Scotia. The Canadian Field-Naturalist, 102, 675– 678.

McKinney, M. A., Iverson, S. J., Fisk, A. T., Sonne, C., Rigét, F. F., Letcher, R. J., Arts, M. T., Born, E. W., Rosing-Asvid, A., & Dietz, R. (2013). Global change effects on the long-term feeding ecology and contaminant exposures of East Greenland polar bears. Global change biology, 19(8), 2360–2372. doi:10.1111/gcb.12241

Mikkelsen, L., Johnson, M., Wisniewska, D. M., van Neer, A., Siebert, U., Madsen, P. T., & Teilmann, J. (2019). Long‐term sound and movement recording tags to study natural behavior and reaction to ship noise of seals. Ecology and Evolution, 9(5), 2588–2601. doi:10.1002/ece3.4923

Northridge, S. (2018). Fisheries interactions. In B. Würsig, J. G. M. Thewissen, & K. M. Kovacs (Eds.), Encylopedia of Marine Mammals (Third Edition) (pp. 375–383). Academic Press. doi:10.1016/B978-0-12-804327-1.00021-2

Patrick, S. C., & Weimerskirch, H. (2014). Personality, Foraging and Fitness Consequences in a Long Lived Seabird. PLoS ONE, 9(2), e87269. doi:10.1371/journal.pone.0087269

Reeves, R. R., McClellan, K., & Werner, T. B. (2013). Marine mammal bycatch in gillnet and other entangling net fisheries, 1990 to 2011. Endangered Species Research, 20(1), 71– 97. doi:10.3354/esr00481

Reijnders, P. J. H., Brasseur, S. M. J. M., Borchardt, T., Siebert, U., Stede, M., & Tougaard, S. (2008). Aerial surveys of Harbour seals in the entire Wadden Sea in 2007: Population

119 References

age-composition returning to a stable age-structure? Wilhelmshaven, Germany. Retrieved from http://www.waddensea-secretariat.org/sites/default/files/downloads/ trilateral_seal_counts_report_2007.pdf

Russell, D. J. F., Mcclintock, B. T., Matthiopoulos, J., Thompson, P. M., Thompson, D., Hammond, P. S., Jones, E. L., Mackenzie, M. L., Moss, S., & Mcconnell, B. J. (2015). Intrinsic and extrinsic drivers of activity budgets in sympatric grey and harbour seals. Oikos, 124(11), 1462–1472. doi:10.1111/oik.01810

Santos, M. B., & Pierce, G. J. (2015). Marine mammals and good environmental status: science, policy and society; challenges and opportunities. Hydrobiologia, 750(1), 13–41. doi:10.1007/s10750-014-2164-2

Santos, M. B., Pierce, G. J., Learmonth, J. A., Reid, R. J., Ross, H. M., Patterson, I. A. P., Reid, D. G., & Beare, D. (2004). Variability in the diet of harbor porpoises (Phocoena phocoena) in Scottish waters 1992–2003. Marine Mammal Science, 20(1), 1–27. doi:10.1111/j.1748-7692.2004.tb01138.x

Schwarz, D., Spitzer, S. M., Thomas, A. C., Kohnert, C. M., Keates, T. R., & Acevedo- Gutiérrez, A. (2018). Large-scale molecular diet analysis in a generalist marine mammal reveals male preference for prey of conservation concern. Ecology and Evolution, (April), 1–17. doi:10.1002/ece3.4474

Sharples, R. J., Moss, S. E., Patterson, T. A., & Hammond, P. S. (2012). Spatial variation in foraging behaviour of a marine top predator (Phoca vitulina) determined by a large-scale satellite tagging program. PloS one, 7(5), e37216. doi:10.1371/journal.pone.0037216

Siebert, U., Wünschmann, A., Weiss, R., Frank, H., Benke, H., & Frese, K. (2001). Post- mortem findings in harbour porpoises (Phocoena phocoena) from the German North and Baltic Seas. Journal of Comparative Pathology, 124(2–3), 102–114. doi:10.1053/jcpa.2000.0436

Sonne, C., Dietz, R., Larsen, H. J. S., Loft, K. E., Kirkegaard, M., Letcher, R. J., Shahmiri, S., & Móller, P. (2006). Impairment of cellular immunity in west Greenland sledge dogs (Canis familiaris) dietary exposed to polluted minke whale (Balaenoptera acutorostrata) blubber. Environmental Science and Technology, 40(6), 2056–2062. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/16570636

Steimle Jr., F. W., & Terranova, R. J. (1985). Energy Equivalents of Marine Organisms from the Continental Shelf of the Temperate Northwest Atlantic. Journal of Northwest Atlantic Fishery Science, 6, 117–124. doi:10.2960/J.v6.a11

Stephens, D. W., Brown, J. S., & Ydenberg, R. C. (2007). Foraging: Behavior and Ecology. Chicago: University of Chicago Press.

Stirling, I., & McEwan, E. H. (1975). The caloric value of whole ringed seals (Phoca hispida) in relation to polar bear (Ursus maritimus) ecology and hunting behavior. Canadian Journal of Zoology, 53(8), 1021–1027. doi:10.1139/z75-117

Stringell, T., Hill, D., Rees, D., Rees, F., Rees, P., & Morgan, G. (2015). Short Note: Predation of Harbour Porpoises (Phocoena phocoena) by Grey Seals (Halichoerus grypus) in Wales. Aquatic Mammals, 41(2), 188–191. doi:10.1578/AM.41.2.2015.188

Tallman, J., & Sullivan, C. (2004). Harbor seal (Phoca vitulina) predation on a male harlequin duck (Histrionicus histrionicus). Northwestern Naturalist, 85(1), 31–32. doi:10.1898/1051-

120 References

1733(2004)085<0031:HSPVPO>2.0.CO;2

Teilmann, J., & Galatius, A. (2017). Harbor Seal (Phoca vitulina). In B. Würsig, J. G. M. Thewissen, & K. Kovacs (Eds.), Encyclopedia of Marine Mammals (3rd ed., pp. 451–455). Academic Press. ten Doeschate, M. T. I., Brownlow, A. C., Davison, N. J., & Thompson, P. M. (2017). Dead useful; methods for quantifying baseline variability in stranding rates to improve the ecological value of the strandings record as a monitoring tool. Journal of the Marine Biological Association of the United Kingdom, 1–5. doi:10.1017/S0025315417000698

Thompson, D., Onoufriou, J., Brownlow, A., & Bishop, A. (2015). Preliminary report on predation by adult grey seals on grey seal pups as a possible explanation for corkscrew injury patterns seen in the unexplained seal deaths. St. Andrews, Scotland. Retrieved from http://www.smru.st-and.ac.uk/documents/2162.pdf

Tucker, S., Bowen, W., & Iverson, S. (2007). Dimensions of diet segregation in grey seals Halichoerus grypus revealed through stable isotopes of carbon (δ13C) and nitrogen (δ15N). Marine Ecology Progress Series, 339, 271–282. doi:10.3354/meps339271

Twiss, S. D., & Franklin, J. (2010). Individually Consistent Behavioural Patterns in Wild, Breeding Male Grey Seals (Halichoerus grypus). Aquatic Mammals, 36(3), 234–238. doi:10.1578/AM.36.3.2010.234 van Neer, A., Gross, S., Kesselring, T., Grilo, M., Ludes-Wehrmeister, E., Roncon, G., & Siebert, U. (in preparation -a). Putting together the pieces of the puzzle - Part I - Assessing seal carcasses potentially subjected to grey seal (Halichoerus grypus) predation. in preparation. van Neer, A., Gross, S., Kesselring, T., Grilo, M., Ludes-Wehrmeister, E., Roncon, G., & Siebert, U. (in preparation -b). Putting together the pieces of the puzzle - Part II - Assessing harbour porpoise carcasses potentially subjected to grey seal (Halichoerus grypus) predation. in preparation. van Neer, A., Gross, S., Kesselring, T., Wohlsein, P., Leitzen, E., & Siebert, U. (2019). Behavioural and Pathological Insights into a Case of Active Cannibalism by a Grey Seal (Halichoerus grypus) on Helgoland, Germany. Journal of Sea Research, 148–149, 12– 16. https://doi.org/10.1016/j.seares.2019.03.004. van Neer, A., Jensen, L. F., Blädel, R., & Siebert, U. (2014). If you can’t beat them, eat them - Behavioural observations of grey seal (Halichoerus grypus) and harbour seal (Phoca vitulina) interactions on the island of Helgoland, Germany. In 28th Conference of the European Cetacean Society, 05.-09.04.2014. Liège, Belgium. van Neer, A., Jensen, L. F., & Siebert, U. (2015a). Grey seal (Halichoerus grypus) predation on harbour seals (Phoca vitulina) on the island of Helgoland, Germany. Journal of Sea Research, 97, 1–4. doi:10.1016/j.seares.2014.11.006 van Neer, A., Wohlsein, P., & Siebert, U. (2015b). Shedding light on the phenomenon of grey seal predation on marine mammals. In 29th Conference of the European Cetacean Society, 23.-25.03.2015. St Julian’s, Malta.

Volpov, B. L., Hoskins, A. J., Battaile, B. C., Viviant, M., Wheatley, K. E., Marshall, G., Abernathy, K., & Arnould, J. P. Y. (2015). Identification of Prey Captures in Australian Fur Seals (Arctocephalus pusillus doriferus) Using Head-Mounted Accelerometers: Field Validation with Animal-Borne Video Cameras. PLOS ONE, 10(6), e0128789. doi:10.1371/journal.pone.0128789

121 Supplementary materials

10. Supplementary materials

a. Development of the local harbour and grey seal stock

Figure A. 2: Count data grey seals.

Figure A. 1: Count data harbour seals.

Figure A.1 & A.2 based on data from trilateral coordinated aerial surveys, with Grey seals: (Brasseur et al., 2009b, 2010b, 2011a, 2012a, 2013a, 2014, 2015, 2016, 2017, 2018). Harbour seals: (Brasseur et al., 2013b, 2011b, 2012b, 2008, 2009a, 2010a, Galatius et al., 2014, 2015, 2016, 2017; Reijnders et al., 2008).

122 Supplementary materials

b. Other publications concerning this topic If you can’t beat them, eat them - Behavioural observations of grey seal (Halichoerus grypus) and harbour seal (Phoca vitulina) interactions on the island of Helgoland, Germany. van Neer, A., Jensen, L. F., Blädel, R. & Siebert, U. (2014) 28th Conference of the European Cetacean Society, 05.-09.04.2014. Liège, Belgium. Winner of the prize “Best scientific poster”

123 Supplementary materials

Shedding light on the phenomenon of grey seal predation on marine mammals. van Neer, A., Wohlsein, P. & Siebert, U. (2015b) 29th Conference of the European Cetacean Society, 23.-25.03.2015. St Julian’s, Malta. Winner of the prize “Best scientific poster”

124 Supplementary materials

Letter to the Editor - Exposing Grey Seals as Horses and Scientists as Human. Haelters, J., Kerckhof, F., van Neer, A. & Leopold, M. (2015) Aquatic Mammals, 41(3), 351–353. doi:10.1578/AM.41.3.2015.351

125 Supplementary materials

126 Permissions to include presented publications

11. Permissions to include presented publications

Permission letter for: Haelters, J., Kerckhof, F., van Neer, A., & Leopold, M. (2015). Letter to the Editor - Exposing Grey Seals as Horses and Scientists as Human. Aquatic Mammals, 41(3), 351–353. https://doi.org/10.1578/AM.41.3.2015.351

127 Permissions to include presented publications

Regarding the articles: van Neer, A., Gross, S., Kesselring, T., Wohlsein, P., Leitzen, E., & Siebert, U. (2019). Behavioural and Pathological Insights into a Case of Active Cannibalism by a Grey Seal (Halichoerus grypus) on Helgoland, Germany Journal of Sea Research, 148–149, 12–16. https://doi.org/10.1016/j.seares.2019.03.004

Heers, T., van Neer, A., Becker, A., Gross, S., Hansen, K.A., Siebert, U., Abdulmawjood, A. (2018). Loop-mediated isothermal amplification (LAMP) as a confirmatory and rapid DNA detection method for grey seal (Halichoerus grypus) predation on harbour porpoises (Phocoena phocoena). Journal of Sea Research 140, 32–39. https://doi.org/10.1016/j.seares.2018.07.008 van Neer, A., Jensen, L.F., Siebert, U. (2015). Grey seal (Halichoerus grypus) predation on harbour seals (Phoca vitulina) on the island of Helgoland, Germany. Journal of Sea Research 97, 1–4. https://doi.org/10.1016/j.seares.2014.11.006

The Journal of Sea Research has agreed to the reproduction of the papers published within this journal under the prerequisite of including the full reference of the respective publications. This request was followed.

Regarding the article:

Heers, T., van Neer, A., Becker, A., Grilo, M.L., Siebert, U., Abdulmawjood, A. (2017). Loop-mediated isothermal amplification (LAMP) assay—A rapid detection tool for identifying red fox (Vulpes vulpes) DNA in the carcasses of harbour porpoises (Phocoena phocoena). PLoS One 12, e0184349. https://doi.org/10.1371/journal.pone.0184349

The paper published in PLoS ONE is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. This request was followed.

I would like to thank all journals for allowing me to reproduce the published material within this thesis!

128 Statutory declaration

12. Statutory declaration

Attachment 3 (in accordance with § 7 section 2 sentence 7 of the Dr. rer. nat. - Doctoral Regulations)

Declaration

Hereby I declare that I wrote the dissertation (Predation of marine mammals by grey seals (Halichoerus grypus) – Assessment of the background, the extent and the potential effects on the ecosystem) on my own. The following support of a third party was sought when completing the doctoral thesis: none ………………………………………………………………………………………………….

I have taken no help against payment from consultancy services (doctoral adviser or other persons). None has received services against payment indirectly or directly for work which is in any way connected with the contents of the submitted dissertation.

I completed the dissertation at the following institutions:

………………………………………………………………………………………………….Institute for Terrestrial and Aquatic Wildlife Research, Büsum ………………………………………………………………………………………………….

The doctoral thesis was not submitted previously for an examination or doctorate or for a similar purpose for assessment. (If the doctoral thesis was completed at an external institution, a declaration of the head of the corresponding institution also has to be enclosed, stating that he/she agrees to the work being submitted in form of a dissertation to the University of Veterinary Medicine Hannover.) I declare to the best of my knowledge that all details are complete and correspond to the truth.

……………………………………………………………………………… Date, own signature

129 Acknowledgements

13. Acknowledgements

I would like to thank my supervisors, the external reviewer and especially Ursula Siebert for their support and trust.

In addition, I would like to thank my girlfriend and my family for the unrestricted support on the various levels! Without your understanding of the many long working days and the associated neglect of social duties, I would have been unable to accomplish this task.

Further I would like to thank all the people who have spent hours in the field observing and recording interesting behaviour as well as collecting carcasses for necropsies and providing dozens of useful pictures. This joint effort deserves a high recognition!!!

Last but not least, I would like to thank the many colleagues in the ITAW and the cooperation partners (especially Teresa Heers) for their fantastic support and good cooperation. Without the many hours in the dissection room and the constructive discussions, I would never have come this far.

THANK YOU!

130