Prey Remains From Stomachs of Killer Whales ( <scp> <i>Orcinus orca</i> </scp> ) Stranded in Alaska, 2005–2021

Three genetically and behaviorally distinct killer whale (Orcinus orca) ecotypes are found in Alaskan waters: resident, transient, and offshore. These ecotypes are known to occupy different habitats and differ in their morphology, ecology, social structure, foraging behavior, and vocalizations (de Bruyn et al. 2013; Ford 2019; Matkin and Saulitis 1994; Morin et al. 2024). They have different prey preferences and are provisionally recognized as distinct subspecies (The Society for Marine Mammalogy 2024). In Alaska, resident killer whales range from Southeast Alaska to the Bering Sea and are more predictable in their movements than transient and offshore ecotypes. Two stocks of residents frequent Alaska waters: Eastern North Pacific Alaska Residents (AR) found from Southeast Alaska to the Aleutian Islands and Bering Sea, and Eastern North Pacific Northern Residents (NR) found in Southeast Alaska (Figure 1). Previous studies indicate that residents eat fish (including salmon, Oncorhynchus spp.) and cephalopods (mostly squid), but are not known to eat marine mammals (Ford et al. 1998; Saulitis et al. 2000; Van Cise et al. 2024). Transient, killer whales (also known as Bigg's; Morin et al. 2024) are found in all waters surrounding Alaska, with some whales making extensive movements into the Chukchi Sea (Madrigal et al. 2021; Willoughby, Ferguson, et al. 2022; Willoughby, Stimmelmayr, et al. 2022). There are at least three stocks of transient killer whales in Alaskan waters (Figure 1): (1) Gulf of Alaska, Aleutian Islands, and Bering Sea Transients (GAT), (2) AT1 Transients occurring in Prince William Sound and Kenai Fjords (overlapping but not known to associate with GAT), and (3) West Coast Transients (WCT; formerly Eastern North Pacific Transients, ENP) occurring in Southeast Alaska (Muto et al. 2021). Transient killer whales prey on marine mammals, including pinnipeds (Ford et al. 1998; Matkin et al. 2007; Vos et al. 2006), small cetaceans (Dahlheim and White 2010; Ford et al. 1998; Frost et al. 1992; Matkin et al. 2007; Saulitis et al. 2000), large cetaceans (Ford et al. 2005; Matkin et al. 2007; Willoughby, Ferguson, et al. 2022; Willoughby, Stimmelmayr, et al. 2022), and sea otters (Enhydra lutris). Other aquatic species such as river otters (Lontra canadensis) and seabirds have also been documented as prey (Ford et al. 1998; Vos et al. 2006), as have some terrestrial species (e.g., moose, Alces alces and Sitka black-tailed deer, Odocoileus hemionus sitkensis; Ford and Ellis 1999; Matkin and Saulitis 1994). Transients are not known to eat fish; however, squid have been found in their stomachs (Ford et al. 1998; Vos et al. 2006). Offshore killer whales are the least known of the three ecotypes. The Eastern North Pacific Offshore stock is thought to be a single stock with individuals that summer in Alaska (Figure 1), with a distribution that includes Southeast Alaska and the Aleutian Islands (Dahlheim et al. 2008; Zerbini et al. 2007). Little is known about their diet, but it is thought that they feed on sharks and other fishes, including Pacific halibut (Hippoglossus stenolepis), salmon, and sculpin (Cottidae family; Dahlheim et al. 2008; Ford et al. 2011; Heise et al. 2003; Jones 2006). Most killer whale diet information comes from observations of feeding, fecal prey analysis, and stomach contents recovered from dead whales (Ford et al. 2016; Heise et al. 2003; Saulitis et al. 2000). Here, we identified prey items recovered from the stomachs of killer whale carcasses opportunistically collected in Alaska between 2005 and 2021. Killer whale stomachs were either collected whole and then frozen, or the contents were removed and then frozen by Alaska Stranding Network responders before being shipped to the Alaska Department of Fish and Game (ADF&G) laboratory in Fairbanks. In the laboratory, stomach contents were weighed to the nearest 0.1 g and then rinsed with fresh water over stacked 1.0 and 0.5 mm sieves. For whole stomachs, all parts of the stomach were opened and rinsed over the sieves to ensure all hard parts such as otoliths, fish bones, and cephalopod beaks that tend to adhere to the stomach lining were collected. Prey were identified based on diagnostic morphological characteristics by taxonomic experts or using molecular genetic approaches. Fish otoliths were identified when possible (e.g., Morrow 1979) and fish were tabulated by adding the maximum count of left or right otoliths or by using half of the count of otoliths that could not be identified as left or right. All recovered otoliths were too broken or degraded to be useful for estimating fish size. In some cases, fish bones were the only remaining parts. Cephalopod beaks were identified (e.g., Clarke 1986) and the maximum count of upper or lower beaks was used to determine the minimum number of cephalopods consumed. Other invertebrates were identified using reference collections and taxonomic keys (e.g., Foster 1991; Kozloff 1999). Mammal parts were primarily identified by comparing them to reference materials maintained at ADF&G, the University of Alaska Museum, and the California Academy of Sciences, or by using molecular genetic approaches. Some prey remains (i.e., a rib, two vertebral columns, and skin) could not be positively identified using reference collections, so tissue was removed and submitted for molecular genetic analysis. Genomic DNA was extracted using the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Germantown, MD). A 359 bp region of the cytochrome b gene (cyb) of the mitochondrial genome was amplified to identify potential pinniped species (Kocher et al. 1989; Carr and Marshall 1991; Parson et al. 2000). A longer fragment (655 bp) of the mtDNA cytochrome oxidase subunit I (coxI) gene was targeted to identify any potential fish species (Hyde et al. 2014). Polymerase chain reaction (PCR) products were cleaned using Optima strips (EdgeBio, 8WELL-1) and sequenced on the Applied Biosystems 3500 Series Genetic Analyzer (Thermofisher, Waltham, MA) using BigDye chemistry (Thermofisher, Waltham, MA). Sequence chromatograms were inspected by eye and assembled using Geneious Prime 2023.2.1. To verify species identity, sequences generated from prey parts were compared to publicly available sequences in the NCBI BLAST database (Altschul et al. 1990). To assign carcasses to general age classes (i.e., calf, juvenile, or adult), total length (straight line snout to fluke notch) was used for transient and resident whales. Length of calves ranged from 201 to 360 cm, juveniles 361–500 cm, and adults were greater than 501 cm (Raverty et al. 2020). The offshore killer whale was aged from growth layer groups in tooth cross-sections (Tang et al. 2019). Killer whale carcasses were provisionally assigned to North Pacific killer whale management stocks based upon multiple lines of evidence, including mtDNA sequence data, individual photo-identification (where available; Wells 2018), and stranding location. Ecotype was determined based on molecular genetic sequence analysis of skin samples or photo-identification. Genomic DNA was extracted using the Macherey-Nagel NucleoMag tissue extraction kit (Macherey-Nagel, Pennsylvania, USA). A 965 bp region of the 5′-end of the mtDNA hypervariable control region was amplified as two overlapping fragments using two sets of primer pairs: TRO (Martien et al. 2014) and D (Rosel et al. 1994), and primer pair H497 and L16218 (Martien et al. 2014). Sequencing of the PCR product in both directions was performed with the same primers as above using the ABI 3130XL Automated Sequencer (Applied Biosystems Inc., Foster City, CA). All sequences were aligned using Sequencer v4.1 software (Gene Codes Corp., Ann Arbor, MI) and compared to a compiled worldwide killer whale reference sequence library (SWFSC), in addition to publicly available killer whale sequences in GenBank (Altschul et al. 1990) to determine ecotype. Ecotype was assigned based on 100% identity to the following known, vouchered reference sequences: transient GAT (DQ399081.01; Zerbini et al. 2007), transient WCT, formerly ENP (GU187163.1; Morin et al. 2010); resident NR (DQ399078.01; Zerbini et al. 2007). Caveats to consider when using stomach contents from stranded whales to understand diet include the difficulty of accessing remote coastal locations to find and recover whale carcasses before decomposition occurs. Not all stomachs are collected in their entirety or in good condition, which can further complicate identifying prey items. Stranded whales are often sick or injured and may not be eating normally prior to death. In addition, stomach contents are inherently biased because hard parts (e.g., otoliths and cephalopod beaks) remain in the stomach longer than soft parts and can accumulate through several feedings (Jobling and Breiby 1986; Pitcher 1980), which can over-represent some prey taxa. In contrast, soft-bodied prey digest quickly and can lead to underrepresentation. This is particularly relevant for transient killer whales known to eat the tongue and flesh of the lower jaws of large cetaceans (Willoughby, Ferguson, et al. 2022; Willoughby, Stimmelmayr, et al. 2022), leaving no hard parts to identify. Small invertebrates and fish, unless in large numbers, are likely consumed secondarily and not targeted killer whale prey. Despite these limitations, stomach contents can provide information regarding prey species and feeding behavior (e.g., swallowing prey whole). We analyzed stomach contents recovered from 13 Alaskan killer whales, including six residents, six transients, and one offshore. The six residents included three females (two calves and one adult), two males (one calf and one adult), and one adult of unknown sex (Table 1, Figure 1). 2005054 (26-Aug-05, GBNP-00) 2011020 (UAMMamm: 113864) Raverty et al. (2020) (20170612) 2011008 Raverty et al. (2020) (20110313) Raverty et al. (2020) (20140718) Raverty et al. (2020) (20160410) 2020192 (1228023, UAMMamm: 144574) 2015300 (2015-300) Raverty et al. (2020) (O059); Tang et al. (2019) (O059) Three resident killer whale stomachs were empty, and three contained fish parts and cephalopod beaks. AR 2017053 contained beaks from 42 magister armhook squid, Berryteuthis magister (Table 2). AR 2021017 contained beaks from three squid and fish otoliths, which were broken such that the species of origin could not be determined. The only NR whale (2013001) contained otoliths from two adult Chinook salmon (Oncorhynchus tshawytscha) and an upper and lower beak from one giant Pacific octopus (Enteroctopus dofleini). Magister armhook squid (Berryteuthis magister) beaks from 42 squida Unidentified fish vertebrae from 2 small fishb Crab (Oregoniidae family)b Unidentified fish otoliths from 2 fish. Squid (1 clawed armhook, Gonatus onyx and 2 Gonatus sp.). Chinook salmon (Oncorhynchus tshawytscha) otoliths from 2 fish Giant Pacific octopus (Enteroctopus dofleini) beaks from 1 octopusd Polychaetes (2 Polynoidae family and 10 Nereis sp.)b Amphipod (Gammaridea suborder)b Bivalve (Mytilus sp.)b Sea lion (Steller Eumetopias jubatus or California Zalophus californianus) and harbor seal (Phoca vitulina) claws and whiskers Unidentified fish rib bonesb Unidentified bivalves (10 very small, < 50 mm)b Unidentified invertebrate (possibly a chiton)b Sea lion and harbor seal claws and whiskers Partial amphipodb Harbor seal parts: including claws from 9 individuals, 3 forelimbs, whiskers, and large chunks of skin and blubber Feather shafts from an unidentified bird Steller sea lion forelimb bones (including 2 right humeri indicating 2 individuals), a sternum and ribsf, hindlimb bones, skinf; and whiskers Unidentified sea lion claws and whiskers Elephant seal (Mirounga angustirostris) claws and whiskers. Harbor seal claws and whiskers Harbor porpoise (Phocoena phocoena) caudal vertebrae and vertebral disksf,g, f,g Cephalopod beaks were present in all three resident killer whale stomachs (Table 2) suggesting cephalopods may be important prey for resident killer whales in this study. Magister armhook squid are also prey for Stejneger's beaked whales (Mesoplodon stejnegeri) in Alaska (Savage et al. 2021), identifying prey overlap between these two cetaceans. However, squid consumed by this killer whale were larger, averaging 286.6 mm in length and 714.3 g in weight, compared to those consumed by the Stejneger's beaked whales, which averaged 179.4 mm in length and 211.4 g in weight (Savage et al. 2021). Although empty of prey, AR 2005054 contained two fishing hooks, one J hook approximately 6 cm long and a circle hook approximately 7 cm long. The circle hook was attached to longline fishing gear including the line and snap for a total length of approximately 40 cm. There was a small hole in the stomach lining (0.5 cm) of this whale, but the hooks did not perforate the stomach wall. Another heavy gauge line and a snap trailing from the whale's mouth was attached to an additional fishing hook (treble), which had perforated its throat (Raverty et al. 2020), indicating multiple interactions with fishing gear prior to its death. This whale was emaciated and died of sepsis from these wounds (Raverty et al. 2020). Killer whale depredation on fish during active fishing has previously been reported in Alaska (Matkin and Saulitis 1994; Peterson and Hanselman 2017), and this is not the first resident killer whale carcass found with fishing hooks in its stomach (Heise et al. 2003). All six transient whales contained prey remains (Table 2), although the stomach of a GAT calf (2016025) contained only a single Pacific sand lance (Ammodytes hexapterus) otolith. Non-calf GAT whales were consistent in their diet of harbor seals (Phoca vitulina) and sea lions (Table 2). Harbor seals were found in all five of the non-calf killer whale stomachs (1–9 harbor seals per stomach based on claws). Sea lions, identified by whiskers, were found in three of the stomachs. Based on morphology, these whiskers were most likely from Steller sea lions (Eumetopias jubatus), although some could be from California sea lions (Zalophus californianus). In contrast, the two WCT whales had a more varied diet of pinnipeds, a mustelid, and a small cetacean. WCT 2020246 contained the highest prey species diversity, including at least three pinniped species and a harbor porpoise (Phocoena phocoena; Table 2). The stomach contained claws from at least six individual pinnipeds and whiskers from harbor seals, elephant seals (Mirounga angustirostris), and sea lions. Several Steller sea lion bones were identified visually and genetically. A section of caudal vertebrae found in this stomach was genetically identified as harbor porpoise (99% match). A bullet fragment, jacket, and ballistic tip as well as small pieces of plastic were also found. The bullet fragments likely came from prey because there was no evidence of a bullet wound in the stomach. The other WCT 2020192 contained sea otter and harbor seal bones. All marine mammal prey identified in this study are previously known transient killer whale prey (Fomin et al. 2023; Matkin and Saulitis 1994; Vos et al. 2006). Harbor seals were the predominant prey identified here and are known to be an important component of transient killer whale diet (Heise et al. 2003; Saulitis et al. 2000; Vos et al. 2006). To our knowledge, this is the first report of an elephant seal as prey for transient killer whales in Alaska. However, they are known prey of transient killer whales in British Columbia, Canada (Baird and Dill 1995; Ford et al. 1998), and elephant seal range includes the Gulf of Alaska (Le Boeuf et al. 2000). A previous study monitoring killer whale foraging behavior in Southeast Alaska noted that elephant seals were available but not targeted by killer whales (Dahlheim and White 2010). Northern fur seals (Callorhinus ursinus) are also a known prey of Pacific transient killer whales (Matkin et al. 2007), although we did not find any evidence of them (note: northern fur seal whiskers are morphologically different and were not identified). Harbor porpoises have been previously identified as transient killer whale prey, from both visual observations and stomach contents in Southeast Alaska and British Columbia, Canada (Dahlheim and White 2010; Ford et al. 1998; McInnes et al. 2024; Saulitis et al. 2000). Harbor porpoise was the only cetacean identified here, but many other cetaceans are known prey of Pacific transient killer whales (Dahlheim and White 2010; Ford et al. 2005; Frost et al. 1992; Matkin et al. 2007; Saulitis et al. 2000; Willoughby, Ferguson, et al. 2022; Willoughby, Stimmelmayr, et al. 2022). Although killer whales have been observed attacking sea otters (Hatfield et al. 1998), our study is only the third report of sea otter remains in killer whale stomachs in the Pacific (Fomin et al. 2023; Vos et al. 2006). The bird feathers found in our study (Table 2) were too digested for species identification. Previous studies have identified cormorants (Phalacrocorax sp.) and sea ducks (Family Anatidae) as transient killer whale prey (Matkin and Saulitis 1994; Vos et al. 2006). No cephalopods were found in transient killer whale stomachs in this study; although, cephalopods have been identified in stomachs from transient killer whales from the North Pacific (Hanson and Walker 2014; Vos et al. 2006). The genetic identification of a salmon shark (Lamna ditropis, 98% match; Table 2) as prey for Offshore 2015-300 is consistent with previous observations (Ford 2019). Unlike resident and transient killer whales, the teeth of adult offshore killer whales are often worn down to the gum line so that pulp cavities are exposed, which has been attributed to the abrasive dermal denticles embedded in shark skin (Ford et al. 2011). Offshore Pacific killer whales grasp sharks with their teeth and shake them to dislodge the large, lipid-rich liver (Ford et al. 2011; Pyle et al. 1999). The liver is soft tissue that would digest quickly and is not likely to be identified in the stomachs of recovered killer whale carcasses. The teeth of this whale showed severe wear with pulp cavity exposure and evidence of endodontic and periodontal disease in 60% of the teeth (Tang et al. 2019). The whale was at least 30–35 years old based on tooth age, and in addition to chronic tooth lesions, showed evidence of cardiovascular disease and was emaciated (Tang et al. 2019). Finding a shark vertebral column in the stomach of this whale indicates that some sharks may be consumed whole or in large pieces, although this practice may be unique to killer whales with worn teeth or other health complications. Regardless of its biases, results from stomach contents of stranded killer whales provided important information about diet, diet overlap with other stocks and species, feeding behavior, and health issues. For example, a shark vertebral column evidence that at least some offshore killer whales more than shark two different of fishing hooks in the stomach of one resident killer whale, known to have died from sepsis by a third and different of a of interactions for this whale and likely for its Killer whale prey identified here is consistent with prior studies of killer whale diet in Alaska. These results (1) in addition to fish, cephalopods are important prey for resident killer whales from both Southeast Alaska and the Aleutian Islands, (2) sea otters may be targeted prey and the of prey consumed by transient killer whales, and (3) salmon sharks are prey for offshore killer whales. Although our results are consistent with previous we that of stomachs from stranded killer whales is to further prey for these ecotypes and diet as in the prey and analysis, and and and analysis, and analysis, and analysis, and and We would to the Alaska Stranding Network for these stomachs, particularly and We the taxonomic of the William Walker identified the cephalopod beaks and many of the we and to for also identifying otoliths and The University of Alaska of the North of and with University of Alaska laboratory, with the University of California and with for regarding some prey and from the California Academy of for of marine mammal bones to as Prey remains from these killer whales are at The Alaska Department of Fish and Game in Alaska. The stomach analysis was by Alaska and and was were collected and to the Marine Mammal and Stranding The no of

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