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Thompson FISH495

Thompson FISH495

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R. Thompson FISH495 paper
R. Thompson FISH495 paper

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Development of non-invasive stress biomarkers in octopuses Rachel Thompson June 1, 2009 Abstract Octopuses are an important part

of the Pacific Northwest ecosystem. Recently, local populations have been experiencing declines due to pollution and large-scale climate processes. Non-invasive sampling techniques were developed in an attempt to characterize the physiological condition of adult octopuses in a controlled environment. Potential biomarkers of stress (proteins in epidermal mucus and behavior patterns) were identified, and could be useful in identifying a stressed state in an octopus in the wild or in captivity. Larval developmental patterns were established by analysis of the expression of two important developmental genes, orthodenticle-like protein and hedgehog. This study serves to provide researchers and aquarists with baseline physiological data that could be used to identify a stressed state in an adult octopus, or an altered developmental pattern in larvae, using techniques that are less invasive than hemolymph or tissue sampling. Introduction Octopuses are advanced invertebrates with a strong tie with the Pacific Northwest. Two local species are the Red Octopus (Octopus rubescens) and the Giant Pacific Octopus (Enteroctopus dofleini). Both are benthic species, with ranges along the Eastern Pacific Ocean from Mexico to Alaska. Recently, populations have been declining due to several factors, including pollution and large-scale climate processes affecting the Puget Sound region (Rigby et al. 2005, Villanueva and Norman 2008). Octopus species are important prey items for higher predators in Puget Sound. Their conservation is essential to the maintenance of the region’s trophic structure (Onthank and Cowles 2008).

Octopuses are oviparous organisms that brood and care for their eggs during development (Kaneko et al. 2006). Red Octopus larvae hatch approximately 6 to 8 weeks after they are laid, and enter the water column as planktonic predators (von Boletzky 2003). The timing and success of development is dramatically affected by environmental variables, such as changes in temperature and exposure the chemicals (Clarke et al. 2009). In a laboratory setting, increased temperatures (to 23 degrees Celsius from 18 degrees Celsius) accelerated the development of octopus hatchlings, and shortened their life spans by up to 20% (Forsythe et al. 1988). The effect on adult octopuses has yet to be determined. However, as cephalopods are poikilothermic organisms, their physiology during all life stages is dramatically influenced by water temperature. The challenge lies in identifying and quantifying these influences. Responses to stress in octopuses can be obvious, such as color changing when faced by a predator, but indicators of chemical or temperature stress are not often visual. Stress can impair immune function in many invertebrates, leading to decreased disease resistance (Malham et al. 2003). The cephalopod immune system consists of humoral and cellular mechanisms (Ford 1992). The common stress response of animals is intended to maintain homeostasis when threatened by an environmental change. In cephalopods, this response releases catecholamines into the hemolymph, which shut down body functions related to growth, reproduction and immunity with the intent of allocating energy resources towards more immediate concerns (Malham et al. 2002). Specific effects of stressors, such as exposure to air, include decreased hemocyte counts, and a decrease in phagocytotic activity in those remaining cells. Since immune defenses are downregulated in this manner while the animal is in a stressed condition, it is left more susceptible to disease.

The introduction of temperature and chemical stressors can have significant effects on the physiology of aquatic organisms, including but not limited to, disruption of development, reproduction and growth (Zala and Penn 2004). It has also been found that exposure to chemical pollution, specifically endocrine-disrupting chemicals (EDCs), such as those found in urbanized areas of the Puget Sound, can result in abnormal behavior in vertebrates (Zala and Penn 2004). EDCs can have adverse effects on a range of behaviors that are controlled by hormones, including reproductive and sexual behavior, activity level, aggression, communication and motivation (Zala and Penn 2004). It is therefore likely that analyzing changes in behavior patterns could be used as an indicator, or biomarker, for detecting harmful environmental contaminants (Zala and Penn 2004). It has been suggested that behavior might be a more useful indicator or “biomarker” than standard assays. Behavioral assays are non-invasive, inexpensive and potentially more powerful than other methods, since behavior is the outcome of many complex developmental and physiological processes (Zala and Penn 2004). On the other hand, behavior can be difficult to measure and highly variable. The combination of behavioral and physiological data could provide a comprehensive method for characterizing the octopus stress response. Gene expression patterns are reflective of the timing of particular events in development. Many developmental genes are highly conserved in the animal kingdom (Ingham and McMahon 2001). Two important genes are those that code for orthodenticlelike protein and hedgehog, both of which play critical roles in body patterning and morphogenesis. Analysis of gene expression changes can provide insight into the physiological state of an organism. For example, gene expression “rhythms” in rocky intertidal mollusks allow them to adapt to low-tide heat stress events (Gracey et al. 2008).

Since developing larvae are particularly vulnerable to temperature stress, changes in gene expression could be dramatic and interesting. The overall goal of this project is to develop techniques to characterize the stress response of octopus species (O. rubescens and E. dofleini) using epidermal mucus and developmental gene expression patterns. Information is lacking on stress response in octopuses and more importantly, non-lethal, non-invasive means to detect it. Understanding the physiological response of octopuses to stress could be valuable for conservation purposes, predicting effects of environmental stress on local food chains, and simply gaining a better understanding of the lives of these popular, local species. It would be highly useful to isolate biological markers from epidermal surfaces rather than internal body tissues. A method such as this would allow the animal to live, as well as save time and cost of sampling. Potential behavioral and molecular stress biomarkers will be identified in adults using these non-invasive methods, and the gene expression patterns of two important developmental genes will be analyzed in larvae. Ultimately, this study will serve to establish a baseline physiological data set for both adult and larval Red Octopuses Methods Epidermal mucus collection Epidermal mucus was collected from a female Red Octopus (O. rubescens). Three approaches were attempted to determine the most effective sampling procedure. The first approach was to collect mucus using a plastic transfer pipet with the octopus still submerged in the water. The second was to remove the octopus from the water and place it into a sampling container. The third approach was to remove the liquid left in the bottom of the sampling container after the octopus was removed. This liquid theoretically

contained a mixture of seawater and mucus. Protein analysis Samples were combined with SDS, heated to 100 degrees Celsius for 10 minutes, and run on an SDS PAGE gel (Pierce 4-20% Tris-Hepes) for 45 minutes at 150 Volts. Gels were stained with Coomassie Blue Stain for approximately one hour. It was determined that silver staining was more sensitive to the detection of protein bands, and this procedure was used for later staining procedures. Non-invasive techniques were used to identify possible protein biomarkers of stress. Proteins were identified using two approaches. Discovery-based approach: Mass spectroscopy Prominent bands were excised from a gel containing samples collected using each of the three techniques attempted. Bands were chosen based on intensity, pattern and whether or not they were present in all samples. The excised bands were de-stained according to Invitrogen protocol. Trypsin digestion was performed according to the Goodlett lab protocol. The digested proteins were sequenced using mass spectroscopy, and a list of proteins and short sequences found in the bands was received. Stress or immune-related proteins were noted, and investigated to determine possible use as biomarkers of stress.

Lanes: 1

2

3

4

5

6

7

8

Figure 1. Protein gel of epidermal mucus samples collected using various sampling techniques. Lanes 2,3, 7 and 8 contain a 1:1 ratio of mucus collected from the sampling container and 2X SDS. Lane 4 contains mucus collected underwater. Lanes 5 and 6 contain mucus collected with octopus in the sampling container. Targeted approach: Western blotting Heat shock protein 70 (HSP 70) was targeted using the Western blot technique. It was determined that mucus samples contained protein concentrations too low for the HSP 70 antibodies to detect. Leg tissue samples were collected weekly from this point on. Protein was extracted from the leg tissue using 500 uL of Cell Lytic. A Bradford protein assay was performed in order to determine the protein concentration of the extracted samples. A total of seven leg tissue protein samples containing 9.7 ug of protein were run on an SDS PAGE gel for 45 minutes at 150 Volts. A Western blot was done using an Invitrogen Western Breeze (anti-mouse) kit and the anti-HSP 70 antibody. The Western was then developed according to the Western Breeze protocol.

Behavioral assessment A web-cam was installed outside of the octopus aquarium. This provided a 24 hour video feed of its activity. Time lapse videos were available online. The octopus’ behavior at randomly determined time intervals was categorized by certain activities characteristic of varying levels of behavior. These included swimming, crawling, and remaining stationary on the side of the aquarium. Prior to imposing environmental stress, baseline behavior was categorized. An online viewer survey was associated with the live video feed. Viewers were able to check boxes corresponding to these same activity categories, the results of which were imported into an Excel spreadsheet for later analysis.

Larval gene expression Egg samples were collected weekly. RNA was extracted using the Tri-Reagent isolation protocol. A Bradford nucleic acid assay was performed in order to determine the RNA concentration of the samples. cDNA was made by reverse transcription. Primers for two developmental genes were designed, orthodenticle-like protein (OTX, Octopus bimaculoides) and hedgehog (HH, Octopus bimaculoides). Real-Time PCR was done using an Opticon system. Data was analyzed according to Miner. Results Success varied in relation to the sampling technique used. The underwater sampling method resulted in mucus samples that were too dilute with seawater to produce bands after gel electrophoresis (Figure 2). Samples collected from the sampling container had sufficient protein concentrations to produce easily visible bands. Mucus pipetted

directly from the epidermal surface produced bands similar in intensity and pattern to the samples collected from the sampling container. Lanes: 1 2 3 4 5 6 7

Figure 2. Mucus samples collected using three sampling techniques were visualized using gel electrophoresis. Lanes 2,3, 6 and 7 contain mucus collected from the sampling container. Lane 4 contains mucus collected underwater. Lane 5 contains mucus collected with the octopus removed from the water.

Identification of protein biomarkers

Discovery-based approach Proteins present in the excised bands were identified using mass spectroscopy.

Table 1. Representative mass spectroscopy results after trypsin digestion. See appendix for complete list of mass spectroscopy results.
Protein Total Description number of peptides 20 (P02662) Alpha-S1casein precursor 3 (P02754) Betalactoglobulin precursor (Q00215) Actin, cytoplasmic (P02666) Betacasein precursor (P02808) Statherin precursor (P08670) Vimentin (P79020) pHresponse regulator protein (Q7VQL4) Chaperone protein dnaK (Heat shock protein 70) Peptide sequence

CAS1_BOVIN

YLGYLEQ

LACB_BOVIN

VLVLDTDYK

ACT_HYDAT,ACTC_STYPL

2

GYSFTTTAER

CASB_BOVIN STAT_HUMAN VIME_HUMAN,VIME_PANTR PALA_EMENI

25 14 111 2

DM[147]PIQAF FGYGYGPYQPVPEQPLYPQPYQPQY QQYTF ILLAELEQLK DDISSALVR

DNAK_BLOFL

1

HSQVFSTAEDNQSAVTIHVLQGER

These lists of proteins were sorted through and condensed into a short list of proteins possibly related to stress and immune function. They were thus assumed to be

potentially useful as biomarkers of stress (Lahov and Regelson 1996, Barak et al. 2005).

Table 2. Selected proteins identified from epidermal mucus samples by mass spectroscopy with functions related to stress or immune function. These proteins have potential use as biomarkers. Protein Casein Heat Shock Protein 70 (Chaperone protein dnaK) Dermcidin Function Antioxidant peptide, radical scavenging activity, inhibits growth of E. coli Chaperone and monitor for other proteins, maintains conformations, disposal of degraded proteins Antimicrobial, limits skin infection by potential pathogens

Targeted protein analysis The antibody for Heat Shock Protein identified the presence of the protein in the

extracted leg tissue samples (Figure 3). Lanes: 1 2 3 4 5 6 7 8 9 10 11 12

Figure 3. Nitrocellulose membrane following Western blotting. The dark marker indicates the presence of HSP 70 in the leg tissue samples. Lanes 2 through 8 contain 9.7 ug of leg tissue protein, loaded into wells in chronological order. The lane 2 sample was collected on 10/24/2008, the lane 8 sample was collected on 4/1/2009. Lanes 9 through 12 contain 20 ul of mucus collected from the skin with the octopus removed from the water. The lane 9 sample was collected on 1/28/2009 and the lane 12 sample was collected on 4/8/2009. Heat shock protein is known to be produced in increased amounts during stressful conditions. Therefore, HSP 70 could be a useful biomarker for identifying stress in octopus species. Identification of behavior biomarkers

Crawling Swimming On Side of Aquarium

Figure 4. Baseline activity patterns were established after time lapse video analysis. Prior to experimentation, behavior patterns were categorized. The octopus was quite active. At the three randomly determined time intervals, it was often crawling and swimming in the aquarium. 40% of time was spent crawling on the substrate or side of aquarium, 40% of time was spent swimming, and a minimal amount of time (20%) was spent stationary on the side of the aquarium (Figure 3). These patterns will serve as a baseline level of activity (control) to be used for comparison after stressful conditions are imposed.

C rawling Brooding On Side of Aquarium No Data

Figure 5. The activities of the octopus were categorized after eggs were laid. After the eggs were laid, behavior was again categorized to determine the impact of post-reproductive senescence (a biological rather than environmental stressor) on behavior. The general behavioral pattern showed a shift from active crawling and swimming to stationary activities. Immediately after the eggs were laid, the female octopus began a constant routine of brooding. Brooding entailed positioning itself on top of the eggs and blowing water through the siphon to oxygenate the egg strands. Approximately 90% of time was spent brooding the eggs on the side of the aquarium, while 2% of time was spent crawling. For 8% of the time, data was not available due to camera malfunction (Figure 4).

Larval gene expression

Expression (Fold over Minimum)

1000000 100000 10000 1000 100 10 1
1/ /0 28 9 2/ 4/ 09 / 11 2/ 09 / 18 2/ 09 / 25 2/ 09 3/ 4/ 09 / 11 3/ 09 / 18 3/ 09 / 25 3/ 09 09 09 1/ 8/ 4/ 4/

Date

Figure 6. Orthodenticle-like protein expression (log of fold over minimum) in octopus eggs collected weekly from 1/28/09 to 4/12/09. There was a 100,000-fold increase in expression of the gene for orthodenticle-like protein (OTX) over the course of the 10 week sampling period. There was zero gene expression on 1/28/09, which was the day the eggs were laid. A minimum expression (Plotted as zero in Figure 5) was quantified on 2/4/09. From 2/4/09 to 4/12/09, the expression of OTX increased fairly steadily up to its maximum value of 100,000 fold over minimum.

Expression (Fold over Minimum)

1000

100

10

1
28 1/ /0 9 2/ 4/ 09 1 2/ 0 1/ 9 1 2/ 0 8/ 9 2 2/ 0 5/ 9 09 09 09 09 4/ 1/ 8/ 5/ 3/ /2 /1 /1 3 3 3 4/ 1/ 09 8/ 4/ 09

Date

Figure 7. Hedgehog expression (log of fold over minimum) in octopus eggs collected weekly from 1/28/09 to 4/12/09.

The hedgehog gene was expressed relatively late in development. A minimum expression was quantified on 3/25/09, with zero expression up until that date. Maximum expression was reached on 4/8/09, which represents an approximate 300-fold increase over minimum over the course of several weeks. Expression declined after 4/8/09, and reached a final value of less than a 100-fold increase over minimum at the end of the sampling period.

Discussion The development of non-invasive sampling techniques was fairly successful. 3 methods were tested, and results varied. It was determined that collecting epidermal mucus from the sampling container, and directly from the epidermal surface with the octopus removed from the water were the two most useful methods. In this sense, usefulness was measured by the intensity and number of protein bands present after protein gel electrophoresis. Mucus samples collected underwater were not useful, as no bands were visible. Therefore, it is likely that collecting mucus from the skin and from the sampling container could be useful techniques as a first step to identifying protein biomarkers. Both of these methods were minimally invasive. Stress was imposed on the octopus during both procedures, as the animal was removed from the water and was exposed to air for approximately 5 minutes. This air exposure could also introduce thermal stress, as the temperature of the air was approximately 10 degrees higher than the temperature of the aquarium water. Therefore, it is possible that stress-related proteins could be present in the control samples. Differences in the expression of many proteins will have to be measured, rather than simply noting the presence of one or two stressrelated proteins in the experimental samples. The non-invasive methods developed were used prior to identification of proteins. The discovery-based approach provided an extensive list of proteins after mass spectroscopy sequencing. Several interesting stress and immune related proteins were present, and further investigation into their functions identified them as possible biomarkers. Alpha, beta, and kappa caseins composed a large percentage of the proteins identified. Caseins are milk proteins that function as antioxidants. Short casein peptides participate in various biological activities. Some function as immunomodulators

(immunostimulators in cows and humans) (Fiat and Jolles 1988). Others have radical scavenging activity (Clausen et al. 2009) and have been found to inhibit the growth of E.coli, staphylococcus, and streptococcus bacteria (Lahov and Regelson 1996). It is possible that caseins with similar functions could be found in octopus species. Exposure to bacterial pathogens would certainly result in casein production. It is possible that thermal or chemical stress could induce the production of casein proteins as part of a stress response. Another interesting protein with similar function is dermcidin. Dermcidins are potent antimicrobial proteins that defend against pathogenic microorganisms including S. aureus and E. coli (Barak et al. 2005). It is likely that dermcidin production would increase when faced with a pathogenic stressor, and possible that thermal or chemical stress could induce a response. Heat shock protein 70 was detected in epidermal mucus samples. Heat shock proteins are the most conserved proteins in both prokaryotes and eukaryotes (Schmitt et al. 2007). They accumulate in cells exposed to thermal or other stressful stimuli. They function as molecular chaperones and help cells to survive potentially lethal conditions, as well as regulate apoptosis (Didelot et al. 2006). Heat shock proteins assist other proteins in maintaining correct conformations during environmental stress, and transport degraded proteins to proteasomes for disposal (Schmitt et al. 2007). Because heat shock protein 70 is highly conserved and directly related to stress response, it was suitable for a targeted-protein analysis using the Western blot technique. HSP 70 was detected by its antibody in protein extracted from octopus leg tissue under the control sampling conditions. It is potentially the most useful protein biomarker identified, as its function is well-known and applicable to an experiment in which thermal stress is imposed.

However, the sampling technique required to perform a Western blot was more invasive than the collection of epidermal mucus for mass spectroscopy. A Western blot was attempted using epidermal mucus, but it was determined that the protein concentrations of the samples were too low to get a clear result. It is also possible that HSP 70 is not found in mucus. Small leg tissue samples were collected, which seemed to have little effect on the well-being of the octopus. Its activity level remained high after the procedure. Octopus leg tissue regenerates after damage, and after several days the leg began to re-grow. As this procedure did not require anesthesia and did not cause longterm damage to the octopus, it is still considered to be minimally invasive. The techniques developed to analyze and categorize octopus behavior provided a general, baseline pattern of daily activity. The difference between the activity patterns before and after egg-laying was dramatic. The most obvious change was a shift from active swimming and crawling to near constant brooding. The shift is a clear indicator of the level of parental care provided by female octopuses to their young. The technique of categorizing behavior based on time-lapse videos at randomly determined intervals could be useful in evaluating the effect of environmental stress on octopus physiology. Behavior is a direct result of physiological condition (Zala and Penn 2004). Behavioral analysis is practical for many reasons. It is inexpensive, comprehensive and most importantly for this study, non-invasive. Future work would incorporate a controlled experiment to test whether the production of casein, dermcidin, and heat shock protein 70 differs between control (nonstressed) and experimental (stressed) conditions. The baseline physiological data could be used for comparison purposes. This experiment would also validate the effectiveness of the non-invasive sampling techniques developed.

The analysis of gene expression in octopus paralarval development revealed interesting patterns. The expression patterns of the two genes investigated, orthodenticle-like protein (OTX) and hedgehog (hh), varied greatly over time. Orthodenticle-like protein is a DNA binding protein with the essential function of regulating head and central nervous system development (Klein and Li 2002). OTX proteins are widespread throughout the animal kingdom, and have evolved specialized roles in some taxonomic groups. OTX appears to be expressed early in O. rubescens development. This could indicate that morphogenesis of the anterior neural structures of octopuses occurs close to the onset of development. The hedgehog gene family has been found in vertebrates as well as several invertebrate taxa, including mollusks. Hedgehog is expressed in both embryonic and adult tissues, where it mediates development, growth, patterning and morphogenesis (Grimaldi et al. 2007). Several studies have shown that hedgehog genes are highly conserved in invertebrates (Ingham and McMahon 2001). Grimaldi et al. (2007) showed that the hedgehog pathway is involved in the differentiation of striated muscle fibers in the cuttlefish (Sepia officinalis) mantle. It is possible that hedgehog could also be involved in muscle differentiation of octopus, since cuttlefish and octopuses are in the same phylum. Hedgehog is expressed later in development (relative to OTX), which could indicate that it is involved with patterning and morphogenesis at later stages of development. This could be the time frame in which muscle fiber development occurs. However, it is possible that hedgehog was expressed at low levels throughout development, but the concentration was too low to be detected by the Real-Time PCR equipment.

Further investigation is necessary to determine the exact roles of orthodenticlelike protein and hedgehog in octopus larval development. General expression patterns were identified, including timing during development and relative increase in expression over time. These increases in expression are likely due to a specific developmental event, such as brain morphogenesis in the case of orthodenticle-like protein, or muscle fiber differentiation in the case of hedgehog. These data could be used for comparison purposes, possibly to identify changes in gene expression due to particular stressors, such as temperature. Future work would involve mapping gene expression in specific body regions and at specific times to determine the correlation between expression and the development of organs. The overall goal of this project was to determine if the stress-response of adult octopuses can be characterized using non-invasive methods. This limited the scope of possible techniques. A genetic analysis would have required tissue or hemolymph samples which are more difficult to obtain, and involve procedures that are often harmful to the animal. For this reason, institutions such as aquariums may value non-invasive methods over hemolymph or tissue collection. Collecting epidermal mucus was fast, inexpensive, and relatively harmless. The techniques used provided valuable information about the protein composition of mucus, and sequencing identified several proteins that may be related to stress-response. Identifying a stressed physiological state in a particular animal could allow aquariums to ensure that the conditions of their exhibits do not negatively affect the animals. Since senescence is a time period characterized by high levels of stress and physiological change, these non-invasive methods could allow researchers and aquarists to predict the onset of senescence, and therefore reproduction

and subsequent death.

References

Acampora, D., Postiglione, MP, Avantaggiato, V., Di Bonito, M., Simeone, A. The role of Otx and Otp genes in brain development. The International Journal of Developmental Biology. 44:669-677. Anderson, RC, Wood, JB., Byrne, RA. 2002. Octopus senescence: The beginning of the end. Journal of Applied Animal Welfare Science. 5(4): 275-283. Barak, O., Treat, JR, James, WD and Gross, PR. 2006. Antimicrobial peptides: effectors of innate immunity in the skin. Advances in Dermatology. 21: 357-374. Burglin, TR. 2008. Evolution of hedgehog and hedgehog-related genes, their origin from Hog proteins in ancestral eukaryotes and discovery of a novel Hint motif. BMC Genomics. 9: 127. Clarke, N, Routledge, EJ, Garner, A, Casey, D, Benstead, R, Walker, D, Watermann, B, Gnass, K, Thomsen, A and Jobling, S. 2009. Exposure to treated sewage effluent disrupts reproduction and development in the seasonally breeding Ramshorn snail (subclass: Pulmonata, Planorbarius corneus). Environmental Science Technology. 43(6): 2092-2098. Clausen, MR, Skibsted, LH and Stagsted, J. 2009. Characterization of major radical scavenger species in bovine milk through size exclusion chromatography and functional assays. Journal of Agricultural and Food Chemistry. 57(7): 2912-2919. Didelot, C., Schmitt, E., Brunet, M., Maingret, L., Parcellier, A. and Garrido, C. 2006. Heat shock proteins: endogenous modulators of apoptotic cell death. Handbook of Experimental Pharmacology. 172: 171-198.

Fiat, A. and Jolles, P. 1988. Caseins of various origins and biologically active casein

peptides and oligosaccharides: Structural and physiological aspects. Molecular and Cellular Biochemistry. 87(1): 5-30. Forsythe, JW and Hanlon, RT. 1988. Effect of temperature on laboratory growth, reproduction, and life span of Octopus bimaculoides. Journal of Experimental Marine Biology and Ecology. 98: 369-379. Gracey, AY, Chaney, ML, Boomhower, JP, Tyburczy, WR, Connor, K and Somero, GN. 2008. Rhythms of gene expression in a fluctuating intertidal environment. Current Biology. 18(19): 1501-1507. Grimaldi, A., Tettamanti, G., Acquati, F., Bossi, E., Guidali, ML, Banfi, S., Valvassori, R., de Eguileor, M. 2007. A hedgehog homolog is involved in muscle formation and organization of Sepia officinalis (mollusca) mantle. Developmental Dynamics. 237(3): 659-671. Ingham, PW, McMahon, AP. 2001. Hedgehog signaling in animal development: paradigms and principles. Genes and Development. 15: 3059-3087. Kaneko, N, Oshima, Y, Ikeda, Y. 2006. Egg brooding behavior and embryonic development of Octopus laqueus (Cephalopoda: Octopodidae). Molluscan Research. 26(3): 113-117. Lahov, E. and Regelson, W. 1996. Antibacterial and immunostimulating casein-derived substances from milk: casecidin, isracidin peptides. Food and Chemical Toxicology. 34(1): 131-145.

Malham, SK, Lacoste, A, Gelebart, F, Cueff, A and Poulet, SA. 2002. A first insight into stress-induced neuroendocrine and immune changes in the octopus Eledone cirrhosa. Aquatic Living Resources. 15: 187-192.

Malham, SK, Lacoste, A, Gelebart, F, Cueff, A and Poulet, SA. 2003. Evidence for a direct link between stress and immunity in the mollusc Haliotis tuberculata. Journal of Experimental Zoology Part A-Comparative Experimential Biology. 259A(11): 136-144. Nederbragt, AJ, Welscher, P., van den Driesche, S., van Loon, AE, Dictus, WJAG. 2002. Development Genes and Evolution. 212: 330-337. Onthank, KL and Cowles, DL. 2008. Metabolic measurements of energy flow through Octopus rubescens. 2008 Ocean Sciences Meeting: From the Watershed to the Global Ocean. Rigby, PR, Sakurai, Y. 2005. Multidimensional tracking of giant pacific octopuses in Northern Japan reveals unexpected foraging behavior. Marine Technology Society Journal. 39(1): 64-67. Rosa, R., Dierssen, HM, Gonzalez, L., Seibel, BA. 2008. Large-scale diversity patterns of cephalopods in the Atlantic open ocean and deep sea. Ecology. 89(12): 34493461. Villanueva, R. and Norman, MD. 2008. Biology of the planktonic stages of benthic octopuses. Advances in Marine Biology. 46: 105-202. Von Boletzky, S. 2003. Biology of early life stages in cephalopod mollusks. Advances in Marine Biology. 44: 143-203.

Schmitt, E., Gehrmann, M., Brunet, M., Multhoff, G. and Garrido, C. 2007. Intracellular and extracellular functions of heat shock proteins: repercussions in cancer therapy. Journal of Leukocyte Biology. 81(1): 15-27. Klein, WH and Li, X. 2002. Function and evolution of Otx proteins. Biochemical and

Biophysical Research Communications. 258(2): 229-233.

Appendix

Group Probabili ty 1

Protein

Protein Probabili ty

Percent Covera ge 26.2

CAS1_BOV 1 IN CAS1_BOV 1 IN CAS1_BOV 1 IN CAS1_BOV 1 IN CAS1_BOV 1 IN CAS1_BOV 1 IN CAS1_BOV 1 IN CAS1_BOV 1 IN CAS1_BOV 1 IN CAS1_BOV 1 IN Protein Protein Probabili

Numbe r of Unique Peptid es 12

Total Numbe r of Peptid es 20

Percent Share of Spectru m ID’s 3.53

Description

Peptide Sequence

Precurs or Ion Charge 1

1

26.2

12

20

3.53

1

26.2

12

20

3.53

1

26.2

12

20

3.53

1

26.2

12

20

3.53

1

26.2

12

20

3.53

1

26.2

12

20

3.53

1

26.2

12

20

3.53

1

26.2

12

20

3.53

1

26.2

12

20

3.53

Group Probabili

Percent Covera

Numbe r of

Total Numbe

Percent Share

(P02662) Alpha-S1casein precursor (P02662) Alpha-S1casein precursor (P02662) Alpha-S1casein precursor (P02662) Alpha-S1casein precursor (P02662) Alpha-S1casein precursor (P02662) Alpha-S1casein precursor (P02662) Alpha-S1casein precursor (P02662) Alpha-S1casein precursor (P02662) Alpha-S1casein precursor (P02662) Alpha-S1casein precursor Description

YLGYLEQ

EPMIGVNQELAYFY PELFR EPM[147]IGVNQE LAYFYPELFR FFVAPFPEVFGK

2

2

2

HQGLPQEVLNENL LR IGVNQELAYFYPEL FR YLGYLEQLLR

2

2

2

EPMIGVNQELAYFY PELFR EPM[147]IGVNQE LAYFYPELFR HQGLPQEVLNENL LR Peptide Sequence

3

3

3

Precurs or Ion

Group Probabili ty 1

Protein

Protein Probabili ty

Percent Covera ge 23.4

CAS2_BOV 1 IN CASB_BO VIN 1

Numbe r of Unique Peptid es 5

Total Numbe r of Peptid es 6

Percent Share of Spectru m ID’s 1.06

Description

Peptide Sequence

Precurs or Ion Charge 1

1

29

12

19

3.29

(P02663) Alpha-S2casein precursor (P02666) Beta-casein precursor

PITPTLNR

YPVEPFTESQ

1

1 1

CASB_BO VIN CASB_BO VIN

1 1

29 29

12 12

19 19

3.29 3.29

(P02666) Beta-casein precursor (P02666) Beta-casein precursor DM[147] (P02666) Beta-casein precursor (P02666) Beta-casein precursor (P02666) Beta-casein precursor (P02666) Beta-casein precursor DM[147] (P02666) Beta-casein precursor Description

DMPIQAFLLYQEPV LGPVR PIQAFLLYQEPVLGP VR

2 2

1 1 1 1

CASB_BO VIN CASB_BO VIN CASB_BO VIN CASB_BO VIN CASB_BO VIN Protein

1 1 1 1

29 29 29 29

12 12 12 12

19 19 19 19

3.29 3.29 3.29 3.29

FQSEEQQQTEDEL QDK LLYQEPVLGPVR DMPIQAFLLYQEPV LGPVR PIQAFLLYQEPVLGP VR IHPFAQTQSLVYPFP GPIPN Peptide Sequence

2 2 3 3

1

1

29

12

19

3.29

2

Group Probabili ty 1

Protein Probabili ty 1

Percent Covera ge 29

CASB_BO VIN

Numbe r of Unique Peptid es 12

Total Numbe r of Peptid es 19

Percent Share of Spectru m ID’s 3.29

Precurs or Ion Charge 2

(P02666) Beta-casein

PIQAFLLYQEPVLGP VR

Group Probabili ty 1

Protein

Protein Probabili ty 1

Percent Covera ge 16.3

LACB_BOV IN,LACB_B UBBU

Numbe r of Unique Peptid es 3

Total Numbe r of Peptid es 4

Percent Share of Spectru m ID’s 0.68

Description

Peptide Sequence

Precurs or Ion Charge 1

1 1

STAT_HUM AN STAT_HUM AN STAT_HUM AN TRY1_RAT

1 1

54.8 54.8

5 5

14 14

2.42 2.42

(P02754) Betalactoglobulin precursor (Beta-LG) (Allergen Bos d 5),(P02755) Betalactoglobulin precursor (Beta-LG) (P02808) Statherin precursor (P02808) Statherin precursor (P02808) Statherin precursor (P00762) Anionic trypsin-1 precursor (EC 3.4.21.4) (Anionic trypsin I) (Pretrypsinoge n I) (P00762) Anionic trypsin-1 precursor (EC 3.4.21.4) (Anionic trypsin I) (Pretrypsinoge n I) Description

VLDTDYK

FGYGYGPYQPVPE QPLYPQPYQPQYQ QYTF IGRFGYGYGPYQPV PEQPLYPQPYQPQY QQYTF RIGRFGYGYGPYQP VPEQPLYPQPYQPQ YQQYTF LGEHNINVLEGDE QFINAAK

3 3

1 1

1 1

54.8 8.1

5 2

14 3

2.42 0.51

3 2

1

TRY1_RAT

1

8.1

2

3

0.51

LGEHNINVLEGDE QFINAAK

3

Group Probabili ty

Protein

Protein Probabili ty

Percent Covera ge

Numbe r of Unique

Total Numbe r of

Percent Share of

Peptide Sequence

Precurs or Ion Charge

Group Probabili ty 1 1 1 1

Protein

Protein Probabili ty 1 1 1 1

Percent Covera ge 17.8 17.8 17.8 17.8

VIME_HU MAN,VIME _PANTR VIME_HU MAN,VIME _PANTR VIME_HU MAN,VIME _PANTR VIME_HU MAN,VIME _PANTR VIME_HU MAN,VIME _PANTR

Numbe r of Unique Peptid es 8 8 8 8

Total Numbe r of Peptid es 111 111 111 111

Percent Share of Spectru m ID’s 18.91 18.91 18.91 18.91

Description

Peptide Sequence

Precurs or Ion Charge 2 2 2 2

1

1

17.8

8

111

18.91

(P08670) Vimentin,(Q5R 1W8) Vimentin (P08670) Vimentin,(Q5R 1W8) Vimentin (P08670) Vimentin,(Q5R 1W8) Vimentin (P08670) Vimentin,(Q5R 1W8) Vimentin M[147] (P08670) Vimentin,(Q5R 1W8) Vimentin

ILLAELEQLK LLQDSVDFSLADAI NTE LLQDSVDFSLADAI NTEFK ALDIEIATYR

SSVPGVRLLQDSV DFSLADAINTE

2

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