Innate immune responses of lumpfish (Cyclopterus lumpus) : Transcriptome analysis and characterization of pro-inflammatory cytokines

In recent years, lumpfish and different species of wrasse are used as cleaner fishes for removal of sea lice from farmed Atlantic salmon in Europe and Canada. The production is successful, but there are challenges with high mortality due to bacterial infections. In-depth knowledge of the immune system in lumpfish, particularly immune responses upon bacterial infections and identification of immune genes, will make an important basis for development of immune-prophylactic measures. Prior to this study, there were no available sequences of immune genes in lumpfish in public databases. To obtain sequence information from lumpfish, RNA sequencing of head kidney leukocytes (HKLs) exposed to Vibrio anguillarum O1, followed by de novo transcriptome assembly, was performed. The transcriptome encompassed 221659 trinity genes, 9033 differentially expressed genes (DEG) at 6 hours post exposure (hpe) and 15225 DEG at 24 hpe. The DEG analysis of the lumpfish transcriptome clearly showed that the alternative pathway of the complement cascade is one of the chief mechanisms in the Vibrio anguillarum response of lumpfish HKLs. Furthermore, the DEG analysis also clearly showed that the TLR signaling through the canonical NFκB pathway was the major pathway in the adaptation of the innate immune response. Further, a wider MAPK pathway regulation was observed, involving all of its sub pathways, however, at much less potent regulation levels compared with NFκB. Globally, the DEG analysis displayed a picture of a pro-inflammatory reaction, initiated by toll-like receptor TLR5S, and the alternative complement pathway, and resulting in high up-regulation of the cytokines interleukin (IL)-1β, IL-6, CXCL8 (also known as IL-8) and tumor necrosis factor (TNF)-α. Further characterization of the lumpfish IL-1 family, both ligands and receptors (IL-1R), were performed. Downstream signaling pathways were also characterized. Full-length sequences of the ligands IL-1β, IL-18, and the fish-specific IL-1 family members nIL-1F and IL-1Fm2, the receptors IL-1R1, IL-1R2, IL18-R1, suppression of tumorgenicity (ST2), and partial sequences of double immunoglobulin IL-1R related molecule (DIGIRR) and IL- receptor accessory protein (IL-Ra) were identified. In vitro stimulation of lumpfish leukocytes with a selection of PAMPs, showed that lumpfish IL-1β and nIL-1F were upregulated, most potently by flagellin. The phylogenetic analysis of the IL-1 family ligands showed that IL-1β, nIL-1F1 and IL1Fm2 are more similar to each other than to IL-18. Furthermore, it placed sequences from lobe-finned fish and shark within the nIL-1F clade, suggesting that nIL-1F, together with IL-1β are ancestral genes. This is the first report describing the occurrence of nIL-1F in non-teleost species. Characterization of lumpfish TNF-α revealed that it contained the hallmark properties of TNF-family at nucleotide and peptide levels. Characterization of lumpfish IL-6 revealed a novel gene arrangement, with one less exon, than other described IL-6 teleost sequences. Furthermore, the predicted peptide sequence of lumpfish IL-6 contained a signal peptide first revealed after proteolytic cleavage. Caspase 1 was predicted to cleave in this region of lumpfish IL-6. It is therefore hypothesized that lumpfish IL-6 may be dependent on caspase 1 processing. In the normal tissues and unstimulated leukocytes, the levels of TNF-α transcripts were higher than IL-6, except for eye and brain where the transcript levels of IL-6 were unexpectedly high. Both genes displayed a similar induction pattern to PAMP stimulation in vitro and both were most potently stimulated by flagellin. IL-6 was more potently stimulated that TNF-α. In conclusion, the lumpfish innate immune responses are potent and consist of most of the molecules for a modern teleost. The alternative complement pathway and TLR signaling pathway are fundamental to the in vitro response to Vibrio anguillarum O1 by lumpfish leukocytes. The identification and characterization of the major pro-inflammatory cytokines and design of qPCR assays in lumpfish provides a valuable tool to measure innate immune responses in lumpfish e.g. upon immune modulation, such as vaccination, microbial disease or physiological trials

Behind the Veil of Choice: Testing the Effect of Decision Information and Decision Structure Interventions in a Self-Service Food Setting

Through a series of three high powered studies, this thesis explores and measures responses to two different nudging interventions related to price display and serving utensils in a self-service food environment. Study 1 (N = 400) reveals that people intuitively think displaying price in kilograms as opposed to hectograms will lead to a reduction in the amount of food purchased, and that using tongs instead of spoons has no effect on purchasing behavior. In contrast to these findings, a pre-registered field study conducted in study 2 (N = 1965) measuring actual purchasing behavior revealed that price display did not affect purchasing behavior, but that swapping spoons for tongs resulted in a statistically significant reduction of food purchased. In exploring the underlying mechanisms of these findings, study 3 (N = 2447) suggests that the relationship between serving utensils and amount of food served is mediated by both satisfaction and serving effort. Altogether, this paper highlights that different serving utensils can alter purchasing behavior, and that people’s intuition regarding behavioral responses does not always correspond to actual behavior. Furthermore, study 3 extends the current literature by suggesting that serving effort alone cannot explain the change in amount of food served caused by swapping serving utensils as thought in previous research, and proposes satisfaction as an additional underlying mechanism. Keywords: Nudge, choice architecture, pre-registration, field study, decision information, decision structure, effort, satisfactio

Behind the Veil of Choice: Testing the Effect of Decision Information and Decision Structure Interventions in a Self-Service Food Setting

Through a series of three high powered studies, this thesis explores and measures responses to two different nudging interventions related to price display and serving utensils in a self-service food environment. Study 1 (N = 400) reveals that people intuitively think displaying price in kilograms as opposed to hectograms will lead to a reduction in the amount of food purchased, and that using tongs instead of spoons has no effect on purchasing behavior. In contrast to these findings, a pre-registered field study conducted in study 2 (N = 1965) measuring actual purchasing behavior revealed that price display did not affect purchasing behavior, but that swapping spoons for tongs resulted in a statistically significant reduction of food purchased. In exploring the underlying mechanisms of these findings, study 3 (N = 2447) suggests that the relationship between serving utensils and amount of food served is mediated by both satisfaction and serving effort. Altogether, this paper highlights that different serving utensils can alter purchasing behavior, and that people’s intuition regarding behavioral responses does not always correspond to actual behavior. Furthermore, study 3 extends the current literature by suggesting that serving effort alone cannot explain the change in amount of food served caused by swapping serving utensils as thought in previous research, and proposes satisfaction as an additional underlying mechanism. Keywords: Nudge, choice architecture, pre-registration, field study, decision information, decision structure, effort, satisfactio

Effect of wind on Svalbard reindeer fur insulation

The heat transfer through Svalbard reindeer (Rangifer tarandus platyrhynchus) fur samples was studied with respect to wind velocity, season and animal age. A total of 33 dorsal fur sections were investigated using a wind tunnel. Insulation varied with season (calving, summer, autumn and winter). At zero wind velocity, fur insulation was significantly different between seasons for both calf and adult fur samples. At the same time, there was no significant difference between calf and adult insulation for the summer, autumn and winter seasons. Calf fur insulated as well as adult fur. Winter insulation of Svalbard reindeer was approximately 3 times that of summer. Increasing wind veloci¬ty increased heat loss, however, the increase was not dramatic. When wind coefficients (slope) of the heat transfer regression lines were compared, between season and between calf and adult, no significant differences were reported. All fur samples showed similar increases in heat transfer for wind velocities between 0 and 10 m.s-1. The conductance of winter fur of Svalbard reindeer was almost half that of caribou fur. Also, conductance was not as greatly influenced by wind as caribou fu

Do seasonal changes in Svalbard reindeer fur have relevance for heat transfer?

Physical characteristics of Svalbard reindeer (Rangifer tarandus platyrhynchus) fur samples were examined with respect to season. A total of 33 dorsal fur sections including adults and calves were investigated. A direct relationship between hair density and heat transfer was not observed, and optimal hair density may depend on an interaction with other fur characteristics. Seasonal changes in fur length and depth were inversely proportional to, and appear to be the main determinants of, seasonal changes in calm air conductance. Fur length and depth, however did not explain the exceptional wind resistance of Svalbard reindeer fur. Since wind has little effect on heat transfer through Svalbard reindeer fur regardless of season or animal age, fur characteristics, which change are not likely critical. Instead, constant physical characteristics, which trap still-air within the fur and resist wind compaction and penetration, are probably responsible. These could include hair stiffness, the crimped wave, hollow hairs, intertwining distal tips and the fine wool fuzz about the hair roots. The extent and means are not well understood at present

Seasonal variations and responses to normal activity of the deep body temperature in the Svalbard reindeer (Rangifer tarandus platyrhynchus)

Deep body temperature was recorded in two female Svalbard reindeer during summer and winter. The reindeer were subjected to naturally occurring weather, photoperiod and stimuli in outdoor pens on Svalbard. A telemetry system was employed using transmitters ingested into the rumen. Mean deep body temperature was 0.3°C higher in winter and while the animals were lying down. This suggests a different strategy for thermoregulation than that employed by other reindeer subspecies