The relationship between diet and individual size

The comparison between SFI and ML in two years revealed that each SFI was distributed in all ML intervals, but SFI was different concentrating in each interval (Fig.

8). In 2018, individuals in 150-200 mm and 200-250 mm had higher percentage in SFI 3-5. In 2019, individuals in 200-250 mm had higher percentage in SFI 1-3.

The relationship between ML and individual FO% was shown (Fig. 9). The squid with larger ML tended to have higher cephalopod consumption. Individual FO% of fishes and cephalopods in two years increased as ML became larger. In 2018, cephalopod mostly appeared in the stomachs of individual in 300-350 mm. Crustaceans had lower individual FO% from 250-300 mm to 300-350 mm. In 2019, squid in 300-350 mm did not consume fishes. Individual FO% of crustacean was similar in each ML interval. Evidently, there was higher individual FO% of cephalopod in 200-300 mm in 2019.

In 2018, fishes and cephalopods were similarly concentrated in individual FO% 0, indicating most squids did not consume these species (Fig. 10). Individual FO% of crustaceans concentrated in 100 at the left side of ML, indicating most smaller individuals only consume crustaceans. There was similar distribution at individual FO% 50 in the diets of fish and crustacean, therefore the represented diets of fish and crustacean were included in some individuals. Large squid consumed more cephalopod since the FO%

value above 0 in cephalopods located at the right side of ML. Individual FO% of fishes and cephalopods increased along with individual ML. In contrast, the individual FO% of crustaceans decreased while ML increased (Fig. 10). In 2019, other than the similar concentration of individual FO% 0 in fishes and cephalopods as well as 100 in crustaceans, the distribution was more scattered than the one in 2018 (Fig. 11). There was more distribution at the individual FO% 30 in three main diets, which indicated more individuals were consumed as diets including fish, cephalopod and crustacean. In addition, individual FO% 50 observed more in the diets of cephalopod and crustacean.

Regression lines of cephalopod and crustacean were nearly horizontal and showed no trend between FO and ML. The regression line of fish had slightly upward trend, which indicated individual FO slightly rose as ML increased. The relationship between cephalopod and crustacean showed no trend between FO and ML (Fig. 11). While, fish weak positive relationship with ML.

3.3 Artifact detection 3.3.1 Artifact classification

A total of 76 artifacts were found in stomachs of 300 individuals from 2018 and 2019

samples. Three types of artifact were observed in this study, including 69 fibers, 3 films and 4 fragments. Various colors in artifacts were detected, which contained blue in 61 artifacts, red in 9 artifacts, green in 2 artifacts and Black (Grey) in 4 artifacts. The most abundant artifacts among all was blue fiber.

3.3.2 Stomach fullness and diet of artifact consuming individuals

Most individuals within the two years of study had only consumed crustaceans while some individuals had no food remained in their stomachs (Table 3). The proportional distribution of SFI among artifact-consuming individuals was distinctive in two years. In 2018, individuals with SFI 4 and 5 had higher artifact ingestion. Comparing with the percentage in 2018, individuals in 2019 had greater artifact ingestion at SFI 1 and 2.

Artifact-consuming individuals in 2018 had similar SFI with overall individuals in 2018 (Fig. 12). In 2019, the percentage of SFI 0 in artifact-consuming individuals was two times as large as percentage in overall individuals, while percentage of SFI 4 in 2019 was nearly 3 times less.

3.3.3 Artifact composition

A total of 6 artifacts were identified as plastics including nylon 66 and polyethylene terephthalate (PET) (Table 4). Most artifacts were unidentified, only 13.33% in overall FO% in 2018 was plastic. Squid had greater artifact ingestion in 2019 as individual FO%

and individual mean N of artifacts were higher than that in 2018. Nevertheless, individual mean N in two years was less than 0.5 which indicated there were small amount of artifact ingestion in squid in this study. In addition, mean contamination on petri dishes was approximately 0.1 which were non-artifacts and were not be used in this study. Thus, the

airborne contamination was not a serious problem.

3.3.4 The relationship between artifact ingestion and individual size

Larger squid in two years had greater individual FO%, though the percentage was different in some ML intervals between the two years (Fig. 13). In 2018, the Individuals with 150-200 mm and 200-250 mm ML had similar FO%. Also, the individual FO%

increased as ML increased from 200-250 mm, 250-300 mm to 300-350 mm. In 2019, individual FO% were similar in 200-250 mm and 250-300 mm, though the percentage grew with apparently increasing ML from 250-300 mm to 300-350 mm. FO% of artifacts in two years were concentrated in 0 and represented most individuals had no artifact ingestion (Fig. 14). In addition, the individual FO% and larger ML showed a positive relationship. More evident trend in 2019 was shown due to the steeper slope of regression line.

Chapter 4 Discussion

4.1 Diet analysis

Crystalline lens is necessary for identifying the diet in squid. However, cephalopod and fish have similar appearance of lens. In this study, we differentiated the lens in the stomachs by soft stabbing with tweezers. If the lens could easily separate into two parts, it would be the lens of cephalopod (West et al., 1995). Fish lens is usually spherical and cannot be separated. Previous studies about diet of cephalopod rarely used lens to identify cephalopod. Therefore, adding lens as the key for identifying cephalopod would make the diet of cephalopod more clearly. The statoliths of cephalopod were seldom found in the stomach directly, while they were often found in a translucent tissue. It seems like that the statoliths are more susceptible by gastric acid than otoliths of fish and correspond with the negative effect of ocean acidification in the development of statolith (Kaplan et al., 2013). Conventional estimation would lead to smaller amount of crustacean counting.

Nevertheless, putting all fragments of crustacean into counting would have greater error as the fragments of three main diets in two years were 235 pieces of fish, 65 pieces of fish and 11955 pieces of crustaceans.

In this study, the overall FO% of fish in two years in this study were 10% higher than the percentage in the previous study in 1992 (Ivanovic & Brunetti, 1994). In contrast, the overall FO% of cephalopod in 1992 was 10% greater than the value in 2018 and the percentage of crustacean was 10% higher than the value in 2019. The difference between three main diets would indicate there was more predation of fish by the squid in this area in recent years, though the predation of cephalopod and crustacean was fluctuated. The relationship of ML and diets in 1992 was only apparent in the diet of crustacean. The

overall FO% of amphipod became higher as the ML increased but the percentage of euphausiid decreased. However, the percentage of amphipod decreased along with ML increased in two years. The relationship between overall FO% and ML in 1992 was similar to the trend in 2019, while it was contrary to the trend in 2018. The euphausiid seemed to have fluctuating population in these years, hence amphipod would be less important in the diet of larger squid. Individual FO% of fish and crustacean had lower value in the previous study in 1998 (Mouat et al., 2001), while the percentage of cephalopod was approximately 9 times larger than the value in 2018 in this study. Lower value of fish and crustacean in 1998 might result from lack of food as percentage of cannibalism reached up to 12.24% in 1998. The SFI distribution in the previous study in 2012 was similar to the distribution in 2018, which represented most stomachs of both years were full (Rosas-Luis et al., 2014). The individual FO% in the study in 2012 had 3 to 4 times larger value in fish and about 7 times less value in crustaceans. The possible reason of difference in FO% would be the more southern sampling location in 2012 which might lead to different diet composition of squid.

Due to lower catch in each location, the sampling size in 2019 was 3 times less than the size in 2018. Aside from the sampling size in two years, environmental data could provide possible explanation about variation between diet composition of the two years.

Abundance of cephalopod population was influenced by the SST in their habitat and higher SST led to greater abundance of cephalopod population (Waluda & Pierce, 1998;

Doubleday et al., 2016). There was lower SST in 2019 from the end of February to the middle of March (Fig. 15), which resulted in smaller population of squid. Less competition in smaller population lets squid in 2019 become larger and can consume species in greater size such as fish and cephalopod. Lower chl-a in sampling period of

2019 (Fig. 16) may cause lower primary productivity, which leads to reducing population of planktonic crustacean (Legendre & Rassoulzadegan, 1995). Planktonic crustacean such as amphipod is the necessary diet of Illex argentinus. From lower diet species composition and dispersed SFI, there may have less food abundance in sampling area of 2019. This may also be explained by the related day of environmental data. In 2018, three days before sampling were the most related to ML in 2018 which may indicate squids in 2018 consume more foods as well as the foods remained in their stomach for 2 - 3 days.

However, the most related day in 2019 was the day of sampling which indicated that the squids in 2019 only consumed less foods on that day, therefore no remains was observed in their stomachs (Table 5 & 6). Lack of food resource resulted in cannibalism in Illex argentinus in 2019. This is for the purpose of maintaining the stability of their population (Ibánez & Keyl, 2010).

4.2 Artifact detection

Squids in two years of this study ingested less artifacts than the opah fish in similar area (Jackson et al., 2000). Comparing with artifact ingestion of penguin in southern region, there were similar numbers of artifact ingestion in the squid in 2018 and penguin in 2009 (Bessa et al., 2019). Artifact ingestion in squid in 2019 were greater than numbers in penguin in the Antarctic area. Since the opah fish and penguin have larger size and more variable diet than squid, we can infer squid ingest less amounts of artifacts in the sampling area.

The first basis for determining artifacts was their unusual colors in stomachs of the squid and their tough texture after the dissolution using KOH. The absorbance spectrum by FTIR confirm the artifact is plastic or non-plastic. When the apparent peak appears in

2800-3000 cm^-1 of the absorbance spectrum, it would firstly classify as plastic with C-H bond and go through database and literature comparison to check up its type of plastic.

The possible reason why there are many unidentified artifacts in this study is most artifacts are not plastic and may be some processed materials in nature such as cotton or rayon. These artifacts from natural material often appear the peak which represents pentose in the spectrum, though most artifacts would combine with some chemical associated procedures which make them unidentifiable. Moreover, filter paper and biofilm on the artifacts may disturb the signal of spectrum.

Larger squid in this study had higher percentage of artifact ingestion, while individuals with artifact intake mostly consumed only crustaceans. The possible reason may be main diet of the squids in this area is crustaceans and there are also some artifacts ingested by crustaceans in nearby area (Jones-Williams et al., 2020). Artifact ingestion in 2019 was more than that in 2018, which may result to larger size of squid or the changes of environment. The effect from environment can be proven by the SFI in 2019. The SFI of individuals with artifact ingestion in 2019 has lower percentage of SFI 4 and higher value of SFI 0 when comparing with SFI of all individuals in 2019. As stated above, we suggest that the artifact ingestion in 2019 has less relationships with the squid diet. This is associated with the artifacts in the environment or remaining artifacts during migration.

4.3 Conclusion

Diet analysis with artifact detection of cephalopods in multiple years is a novel topic.

This study indicates that main diet of Illex argentinus remained the same in these years, which can infer the diet is not influenced by climate change. However, the diet may be effected by short-term change of environment such as primary productivity. The results

of artifact detection indicate that there is less artifact ingestion in Illex argentinus, which suggest that the cephalopods in the southwest Atlantic is safe to eat.

References

Abidli, S., Lahbib, Y., El Menif, N. T. (2019). Microplastics in commercial molluscs from the lagoon of Bizerte (Northern Tunisia). Marine Pollution Bulletin, 142, 243-252.

Acuner, E., Dilek, F. (2004). Treatment of tectilon yellow 2G by Chlorella vulgaris.

Process Biochemistry, 39(5), 623-631.

Atkinson, A., Schmidt, K., Fielding, S., Kawaguchi, S., Geissler, P. (2012). Variable food absorption by Antarctic krill: Relationships between diet, egestion rate and the composition and sinking rates of their fecal pellets. Deep Sea Research Part II:

Topical Studies in Oceanography, 59, 147-158.

Atkinson, A., Hill, S. L., Pakhomov, E. A., Siegel, V., Reiss, C. S., Loeb, V. J., Steinberg, D. K., Schmidt, K., Tarling, G. A., Gerrish, L. (2019). Krill (Euphausia superba) distribution contracts southward during rapid regional warming. Nature Climate Change, 9(2), 142-147.

Avio, C. G., Gorbi, S., Milan, M., Benedetti, M., Fattorini, D., d'Errico, G., Pauletto, M., Bargelloni, L., Regoli, F. (2015). Pollutants bioavailability and toxicological risk from microplastics to marine mussels. Environmental Pollution, 198, 211-222.

Baalkhuyur, F. M., Dohaish, E.-J. A. B., Elhalwagy, M. E., Alikunhi, N. M., AlSuwailem, A. M., Røstad, A., Coker, D. J., Berumen, M. L., Duarte, C. M. (2018). Microplastic in the gastrointestinal tract of fishes along the Saudi Arabian Red Sea coast. Marine Pollution Bulletin, 131, 407-415.

Bessa, F., Ratcliffe, N., Otero, V., Sobral, P., Marques, J. C., Waluda, C. M., Trathan, P.

N., Xavier, J. C. (2019). Microplastics in gentoo penguins from the Antarctic region.

Scientific Reports, 9(1), 1-7.

Bidder, A. M. (1950). The digestive mechanism of the European squids Loligo vulgaris, Loligo forbesii, Alloteuthis media and Alloteutihis subulata. Journal of Cell Science, 3(13), 1-43.

Bidder, A. M. (1966). Feeding and digestion in cephalopods. The Mollusca: Physiology, Part 2, 5, 149.

Boschi, E. E., Fischbach, C. E., Iorio, M. I. (1992). Catálogo ilustrado de los crustáceos estomatópodos y decápodos marinos de Argentina.

Breiby, A. (1985). Predatory role of the flying squid (Todarodes sagittatus) in north Norwegian waters. NAFO Sci. Coun. Studies, 9, 125-132.

Caddy, J., Rodhouse, P. (1998). Cephalopod and groundfish landings: evidence for ecological change in global fisheries? Reviews in Fish Biology and Fisheries, 8(4), 431-444.

Chapman, J. W. (2007). Amphipoda: chapter 39 of the light and smith manual: intertidal invertebrates from Central California to Oregon. Completely Revised and Expanded.

Clarke, M. R. (1986). A handbook for the identification of cephalopod beaks. Clarendon Press, Oxford.

Cole, M., Lindeque, P., Fileman, E., Halsband, C., Galloway, T. S. (2015). The impact of polystyrene microplastics on feeding, function and fecundity in the marine copepod Calanus helgolandicus. Environmental Science Technology, 49(2), 1130-1137.

Collins, M. A., Rodhouse, P. G. (2006). Southern ocean cephalopods. Advances in Marine Biology, 50, 191-265.

Cury, P., Bakun, A., Crawford, R. J., Jarre, A., Quinones, R. A., Shannon, L. J., Verheye, H. M. (2000). Small pelagics in upwelling systems: patterns of interaction and structural changes in “wasp-waist” ecosystems. ICES Journal of Marine Science,

57(3), 603-618.

Dehaut, A., Cassone, A.-L., Frère, L., Hermabessiere, L., Himber, C., Rinnert, E., Rivière, G., Lambert, C., Soudant, P., Huvet, A. (2016). Microplastics in seafood:

Benchmark protocol for their extraction and characterization. Environmental Pollution, 215, 223-233.

Doubleday, Z. A., Prowse, T. A., Arkhipkin, A., Pierce, G. J., Semmens, J., Steer, M., Leporati, S. C., Lourenço, S., Quetglas, A., Sauer, W. (2016). Global proliferation of cephalopods. Current Biology, 26(10), R406-R407.

FAO. (2019). FAO yearbook. Fishery and Aquaculture Statistics 2017/FAO annuaire.

Statistiques des pêches et de l’aquaculture 2017/ FAO anuario. Estadísticas de pesca y acuicultura 2017. Rome/Roma.

Foekema, E. M., De Gruijter, C., Mergia, M. T., van Franeker, J. A., Murk, A. J., Koelmans, A. A. (2013). Plastic in north sea fish. Environmental Science Technology, 47(15), 8818-8824.

Frederiksen, M., Edwards, M., Richardson, A. J., Halliday, N. C., Wanless, S. (2006).

From plankton to top predators: bottom‐up control of a marine food web across four trophic levels. Journal of Animal Ecology, 75(6), 1259-1268.

Golikov, A. V., Sabirov, R. M., Lubin, P. A., Jørgensen, L. L., Beck, I.-M. (2014). The northernmost record of Sepietta oweniana (Cephalopoda: Sepiolidae) and comments on boreo-subtropical cephalopod species occurrence in the Arctic. Marine Biodiversity Records, 7.

Green, S. J., Côté, I. M. (2014). Trait‐based diet selection: prey behaviour and morphology predict vulnerability to predation in reef fish communities. Journal of Animal Ecology, 83(6), 1451-1460.

Haimovici, M., Brunetti, N. E., Rodhouse, P. G., Csirke, J., Leta, R. H. (1998). Illex argentinus. FAO Fisheries Technical Paper, 27-58.

Hays, G. C., Richardson, A. J., Robinson, C. (2005). Climate change and marine plankton.

Trends in Ecology Evolution, 20(6), 337-344.

Hazen, E. L., Jorgensen, S., Rykaczewski, R. R., Bograd, S. J., Foley, D. G., Jonsen, I. D., Shaffer, S. A., Dunne, J. P., Costa, D. P., Crowder, L. B. (2013). Predicted habitat shifts of Pacific top predators in a changing climate. Nature Climate Change, 3(3), 234-238.

Hazen, E. L., Abrahms, B., Brodie, S., Carroll, G., Jacox, M. G., Savoca, M. S., Scales, K. L., Sydeman, W. J., Bograd, S. J. (2019). Marine top predators as climate and ecosystem sentinels. Frontiers in Ecology the Environment, 17(10), 565-574.

Hoving, H. J. T., Gilly, W. F., Markaida, U., Benoit‐Bird, K. J., ‐Brown, Z. W., Daniel, P., Field, J. C., Parassenti, L., Liu, B., Campos, B. (2013). Extreme plasticity in life‐

history strategy allows a migratory predator (jumbo squid) to cope with a changing climate. Global Change Biology, 19(7), 2089-2103.

Ibánez, C. M., Keyl, F. (2010). Cannibalism in cephalopods. Reviews in Fish Biology and Fisheries, 20(1), 123-136.

Ivanovic, M. L., Brunetti, N. E. (1994). Food and feeding of Illex argentinus. Antarctic Science, 6(2), 185-193.

Jackson, G. D., Buxton, N. G., George, M. J. (2000). Diet of the southern opah Lampris immaculatus on the Patagonian Shelf; the significance of the squid Moroteuthis ingens and anthropogenic plastic. Marine Ecology Progress Series, 206, 261-271.

Jamieson, A., Brooks, L., Reid, W., Piertney, S., Narayanaswamy, B., Linley, T. (2019).

Microplastics and synthetic particles ingested by deep-sea amphipods in six of the

deepest marine ecosystems on Earth. Royal Society Open Science, 6(2), 180667.

Jansen, E., Overpeck, J., Briffa, K., Duplessy, J., Joos, F., Masson-Delmotte, V., Olago, D., Otto-Bliesner, B., Peltier, W., Rahmstorf, S. (2007). Paleoclimate. Climate change : the physical science basis contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change.

Jones-Williams, K., Galloway, T., Cole, M., Stowasser, G., Waluda, C., Manno, C. (2020).

Close encounters-microplastic availability to pelagic amphipods in sub-antarctic and antarctic surface waters. Environment International, 140, 105792.

Kaplan, M. B., Mooney, T. A., McCorkle, D. C., Cohen, A. L. (2013). Adverse effects of ocean acidification on early development of squid (Doryteuthis pealeii). PLoS One, 8(5).

Khan, S., Malik, A. (2014). Environmental and health effects of textile industry wastewater. In Environmental deterioration and human health (pp. 55-71): Springer.

Klages, N. T. (1996). Cephalopods as prey. II. Seals. Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences, 351(1343), 1045-1052.

Klein, E. S., Hill, S. L., Hinke, J. T., Phillips, T., Watters, G. M. (2018). Impacts of rising sea temperature on krill increase risks for predators in the Scotia Sea. PLoS One, 13(1), e0191011.

Kühn, S., Van Werven, B., Van Oyen, A., Meijboom, A., Rebolledo, E. L. B., Van Franeker, J. A. (2017). The use of potassium hydroxide (KOH) solution as a suitable approach to isolate plastics ingested by marine organisms. Marine Pollution Bulletin, 115(1-2), 86-90.

Legendre, L., Rassoulzadegan, F. (1995). Plankton and nutrient dynamics in marine waters. Ophelia, 41(1), 153-172.

Lipiński, M., Underhill, L. (1995). Sexual maturation in squid: quantum or continuum?

South African Journal of Marine Science, 15(1), 207-223.

Lombarte, A., Chic, Ò., Parisi-Baradad, V., Olivella, R., Piera, J., García-Ladona, E.

(2006). A web-based environment for shape analysis of fish otoliths. The AFORO database. Scientia Marina, 70(1), 147-152.

Lusher, A., Welden, N., Sobral, P., Cole, M. (2017). Sampling, isolating and identifying microplastics ingested by fish and invertebrates. Analytical Methods, 9(9), 1346-1360.

Markaida, U., Gilly, W. F., Salinas-Zavala, C. A., Rosas-Luis, R., Booth, J. A. T. (2008).

Food and feeding of jumbo squid Dosidicus gigas in the central Gulf of California during 2005-2007. CalCOFI Rep, 49, 90-103.

Markaida, U., Sosa-Nishizaki, O. (2003). Food and feeding habits of jumbo squid Dosidicus gigas (Cephalopoda: Ommastrephidae) from the Gulf of California, Mexico. Journal of the Marine Biological Association of the United Kingdom, 83(3), 507-522.

Mouat, B., Collins, M. A., Pompert, J. (2001). Patterns in the diet of Illex argentinus (Cephalopoda: Ommastrephidae) from the Falkland Islands jigging fishery.

Fisheries Research, 52(1-2), 41-49.

Murphy, E., Watkins, J., Trathan, P., Reid, K., Meredith, M., Thorpe, S., Johnston, N., Clarke, A., Tarling, G., Collins, M. (2007). Spatial and temporal operation of the Scotia Sea ecosystem: a review of large-scale links in a krill centred food web.

Philosophical Transactions of the Royal Society B: Biological Sciences, 362(1477), 113-148.

Piatkowski, U., Pierce, G. J., da Cunha, M. M. (2001). Impact of cephalopods in the food

chain and their interaction with the environment. Fisheries Research, 52(1-2), 1-142.

PlasticsEurope. (2018). Annual review 2017–2018. In: PlasticsEurope AISBL Brussels.

Poloczanska, E. S., Brown, C. J., Sydeman, W. J., Kiessling, W., Schoeman, D. S., Moore, P. J., Brander, K., Bruno, J. F., Buckley, L. B., Burrows, M. T. (2013). Global imprint of climate change on marine life. Nature Climate Change, 3(10), 919-925.

Portner, E. J., Markaida, U., Robinson, C. J., Gilly, W. F. (2019). Trophic ecology of Humboldt squid, Dosidicus gigas, in conjunction with body size and climatic variability in the Gulf of California, Mexico. Limnology Oceanography.

Rodhouse, P. G., Hatfield, E. M. C. (1990). Dynamics of growth and maturation in the cephalopod Illex argentinus de Castellanos, 1960 (Teuthoidea: Ommastrephidae).

Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences, 329(1254), 229-241.

Rodhouse, P. G., Nigmatullin, C. M. (1996). Role as consumers. Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences, 351(1343), 1003-1022.

Rodhouse, P. G. (2013). Role of squid in the Southern Ocean pelagic ecosystem and the possible consequences of climate change. Deep Sea Research Part II: Topical

Studies in Oceanography, 95, 129-138.

doi:https://doi.org/10.1016/j.dsr2.2012.07.001

Rosas-Luis, R., Sánchez, P., Portela, J. M., Del Rio, J. L. (2014). Feeding habits and

Rosas-Luis, R., Sánchez, P., Portela, J. M., Del Rio, J. L. (2014). Feeding habits and