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INTERACTION OF TIME AND ENVIRONMENT ON SHEEPSHEAD M INNOW BY MORGAN MOLINA-MARIN Thesis Submitted to the Division of Natural Sciences New College of Florida In partial fulfillment of the requirements for the degree Bachelor of Arts Under the sponsorship of Alfred Beulig Sarasota, Florida May, 2012
ii Acknowledgments I would like to take a moment to thank those who h ave helped me write this paper. The first word of appreciation must go to my adviso r and thesis sponsor, Dr. Alfred Beulig, who provided me with ideas, sources, and th e motivation to soldier on. Next, I would like to thank Joel Beaver, who helped me crea te, maintain, and care for my test subjects, as well as putting up with my initial unc ertainty in handling equipment. I would also like to thank Dr. Leo Demski, for teaching so many classes related to my field of interest. I would like to thank every biology teach er I have had, for that matter, for making the field so interesting as to draw me to it I would like to thank my mother for urging me to l ook into the natural sciences as an area of interest. I would like to thank my high school social studies teacher, Mr. Gottlieb, for telling his whole class about New Col lege and encouraging us to look into coming here. And last, but certainly not least, I would like to thank my friends, for I frequently feel it is they who have kept me sane during my tim e here.
Table of Contents Acknowledgments.................................... ................................................... .......................ii Abstract........................................... ................................................... .................................iv Introduction....................................... ................................................... ................................4 Methods............................................ ................................................... ...............................14 Results............................................ ................................................... .................................17 Discussion......................................... ................................................... ..............................24 Appendix........................................... ................................................... ................................4 References......................................... ................................................... ..............................26
iv Abstract The sheepshead minnow ( Cyprinodon variegatus variegatus ) is known for being a hardy, social species ideal for study in both the w ild and the laboratory and has been used as a biological standardization in toxicology studi es (Brown & Feldmeth, 1971; Echelle, Echelle & Hill, 1972; Itzkowitz 1974). The specie s' means of marking territory, locomotion, and communication have been studied for over forty years (Barlow, 1961; Echelle, 1973; Itzkowitz, 1979; Kodric-Brown, 1997) A more recent study showed a potential link between environmental stability and activity levels in fishes as the result of a drive to survive (Claireaux & Lefranois, 2007). However, that study did not encompass the Cyprinodontidae, and a definitive ass essment of diurnal activity is lacking in this group. Accordingly, I studied time of day a s an influence upon activity levels in Cyprinodon. Three levels of environmental richness were set up as experimental conditions. Videotapes of fish activity were record ed three times per day morning, afternoon, and evening and the number of times fi sh crossed any line was tallied. A significant difference was found across time of day and levels of environmental richness. No significant interaction between the two variable s was found. This information confirms the importance of each variable, and exper iments involving Cyprinodon variegatus variegatus should control for these factors to ensure consist ency in observations. Alfred Beulig Division of Natural Sciences
1 Introduction Cyprinodon has been a genus in wide use for decade s, especially in toxicology studies and in studies on territoriality and aggres sion. Cyprinodon variegatus variegatus is the species of study in this experiment. George W. Barlow wrote an article entitled Social behavior of the desert pupfish, Cyprinodon macularius in the field and in the aquarium in 1961 for the American Midland Naturalist, in which he describes in detail the assorted behaviors demonstrated among pupfish species another common name for the genus with regard to behavioral differences in the aquarium versus in the wild as t hey mate or vie for dominance. He also describes what to look for in mature fish: When mature the sexes are distinctly dimorphic and dichromatic. The body shape of the male is compressed and arched at the n ape, whereas the shape of the female is rounded (Fig. 1A). In the breeding se ason the body, and the dorsal and anal fins of the male are dark metallic blue; the caudal peduncle, caudal fin, and pectoral fins are yellow-orange. Th e eyes are black, and so are the posterior margins of the dorsal and anal fins. The lower distal portions of the pectoral fins are much darker than the other pa rts of the fins. When the water is clear these males can be seen at distances up to 10 meters. The head and body of the female are yellowish brown with irr egular darker brown blotches; the fins are similarly colored. During th e spawning act the body is paler and an interrupted brown line extending mid-l aterally the length of the body becomes more apparent. Except for the small da rk ocellus found at the
2 posterior base of the dorsal fin in the female, the juvenile and female markings are the same. (Barlow, 1961) Distinguishing gender in immature fish may be diffi cult, and adult fish should be used to avoid any confusion. In observing wild pupfish, he noticed two types of mating patterns. The first was that the males would hold a territory, and that fem ales alone or in groups would move from territory to territory, potentially depositing multiple eggs per male. The females would move in a school, those who were not ready to produce an egg taking to foraging within the territory. A male from a neighboring ter ritory may rush to the side of the female opposite the first male, and spawn with the pair. Raney et al. (1953) reported that two males sometimes spawn together with one female. The second kind of observed reproductive behavior was that several males usually anywhere from one to three would pursue a female without regard for territorial boundaries. According to Barlow, sheepshead minnow also displayed this sort of behavior. While Barlow notes no difference between laborator y and field observations in terms of aggression, he does note that size of the fish is important in determining the likelihood of a fight. He noted also that when plac ed into a new aquarium, males will begin to fight immediately for territory until zone s are established, after which the population will be calmer. Moreover, due to the lim it of space placed upon the fish due to being in a tank Barlow found that in a 30 by 6 0 centimeter tank, at most, four males
3 would hold territory at the tank's bottom in a disp lay of dominance, and that fish without territories either remain in vegetational thickets or remain at the water's surface. Of further note is an observation that the holding of territories in natural habitats seemed to fluctuate during the day. During the afte rnoon, Barlow observed that large male pupfish would sometimes stray from their terri tories to search for food, whereas during the early morning most of the fish would gat her in the deeper part of the pools. Unfortunately, these notes do not extend to aquariu m-based observation. However, he does observe that the territory of a tank's dominan t male can be shifted by gradually shifting where food is put into the aquarium. The l arger males control the largest territories and hold them throughout mating season. The above factors are of particular note because t hey could influence activity levels in an inverse manner based upon the amount of food available: less food means more activity. Barlow's study suggests that in expe rimentation an equal number of males and females in each experimental condition should b e sought, and he cautioned against misidentifying juveniles and females. Moreover, foo d should be placed into the aquariums at the same location every time the fish are to be fed. Anthony A. Echelle (1973) built upon Barlow's wor k in a study on the Red River pupfish. Territorial behavioral attributes describe d therein apply to sheepshead minnow as well. He noted color differentiation in males and female s around the eyes themselves when they are not spawning, and this should make id entifying males or females easier. More specifically, blackening of the eye area was s een as a sign of increased aggression in females and non-breeding males.
4 Spawning seemed more common at certain times of th e day based upon the season. During the winter, spawning was usually obs erved in the afternoon, as opposed to warmer months seeing a shift into mid-morning times especially during the summer. There is a threshold temperature before the fish wi ll begin to spawn, but if temperatures permit, spawning may take place throughout an entir e day. The temperature range at which spawning was observed was between 13 34o C. Thus it can be seen that temperature is an importa nt variable in studying breeding behavior in the laboratory and should be kept const ant within ranges noted. Murray Itzkowitz (1974) studied C yprinodon variegatus variegatus in its natural habitat, a small four to five acre pond. He believe d that while one could study the behaviors associated with dominance in fish in an a quarium, natural habitat should not be ignored. To that end, he observed the behavior of m ales within an entire community of Cyprinodon variegatus variegatus to determine whether it was different from that exhibited in a dyad within a tank. This study also examined the inter-species effect of other fish species on Cyprinodon variegatus variegatus but this is beyond the scope of the present experiment. Itzkowitz found that in a natural habit, conspeci fic intruders would trigger a protection behavior and lead to chasing. However, i nterruption influenced a spawning pair to reduce the number of offspring produced and always caused the female to spend less time in the male's territory than she would ha ve otherwise. Instead, spawning took place in shorter but more frequent time frames. Thi s is important information because it anticipates that an environment with more competiti on for territory will increase the amount of roving done on the part of females.
5 In another study, Itzkowitz (1979) examined the ef fect of competition on territory size. He noted that acquisition of territory is bal anced by competition. Guaranteeing that defending an area will have a pay-off requires the area to be high-yield. This same desire for high-yield territory leads to vigorous competit ion, causing such territories to be smaller than an area of less competition. Itzkowitz set off to test the hypothesis that anim als may make tactical adjustments in antagonistic behavior with regard to habitat qua lity. One reason he chose the pupfish is that they maintain long-term territories with obvio us ability to demonstrate preference. He defined habitat enrichment in terms of homogeno us versus heterogeneous sand coverage and tested for resource-holding potential of territorial males for enriched versus non-enriched areas.. In homogenous habitat, where s and covered the entire tank bottom, the dominant male roamed the whole tank, his path i ntersecting with the subordinate fish at times. In heterogeneous habitat, however, where sand only covered half the tank's bottom, superior and inferior areas were establishe d, as females like to mate in the sand; the dominant male kept the subordinates spatially s eparated from himself and his median point was closer to the sand area than theirs. Whe n multiple fish intruded into the territory, the dominant male relinquished control o ver his more inferior territory space to maximize his control over the superior space that being the sand area. These results suggest that a dominant male might b e more likely to claim the richest territory amidst an impoverished habitat. T his would increase activity levels within a generally poor habitat, as the dominant ma le or males will have only a few spots of somewhat better quality from the rest of the hab itat, and will be pressed to defend it from intruders.
6 Kodric-Brown (1977) examined the breeding system i n Cyprinodon (sp.) to try to determine the factors which contribute to a male's reproductive success and therefore understand the selective pressures responsible for the development of the territorial mating system. Though her colleagues have given qua litative reports on the success of a pupfish's breeding, she carried out a quantitative analysis on the cause of variation in success. She established the pupfish mating system as simil ar to a lek in broader categories; that is, success among males is related to population density and habitat quality, but is mostly based on male-male interacti on and females choosing their mates. Moreover, like in a lek, reproductive success can b e quantified due to smaller, isolated groups. She established two areas of study: Area I and Area II. Area I was an embankment lined with crevices, boul ders, and scattered pebbles, providing topographical diversity and was defende d by single, large males, composing an area in which roughly 5% of the spawning observe d took place. Area II was composed of a series of isolated rocks scattered on the bottom of the pond of a depth near the maximum at which pupfish t ypically form their territories. Nonetheless, a number of males defended each rock t o be found here, and approximately 35% of spawning occurred here. Kodric-Brown's results determine that a larger num ber of territories existed in Area I (53) despite lesser numbers of fish per rock Area II had 16 territories. This supports the notion that the deeper area is inferio r territory. Moreover, when KodricBrown was making her observations during the summer she noted that activity levels changed with temperature to reflect the diurnal tem perature cycle, and that the fish were
7 most active when the sun was approaching mid-mornin g for water's daily temperature maximum. During breeding season, territories shrank and bec ame more numerous, with there being more males than there were breeding territori es, meaning that all territories were strenously defended. Only the largest males could h old territory in Area I and even Area II saw fierce competition. When males could not fin d territories, they took to patrolling and defending areas which would have previously bee n considered unsuitable as a breeding habitat. Areas of greater topographical co mplexity saw the most use as territories overall, meaning that an area of high e nrichment will be favored for breeding grounds. She also mentioned satellite males: smaller male s which did not defend a territory but instead hovered nearby and spawned on the territories of larger fish than themselves. This made them a frequent target for ch asing, although the satellite could never be chased off for good. It is also these sate llite males which will interrupt a dominant male during spawning in an attempt to enga ge in courtship of their own. Satellite males may also sometimes entice small fem ales by bringing them to a spawning site. She also noticed that females showed a preference for depositing their eggs on bare rock. To that end, even though more males were gathered in Area I, and Area II had fewer territories present, the majority (86%) of eg g laying took place in Area II despite a significant difference in the number of courtships taking place; Area I had all the mating, and all the spawning took place in Area II. Kodric-Brown (1986) focused on satellite males and their breeding tactics in
8 Cyprinodon pecosensis the Pecos River pupfish She also introduced sne akers, who sneak-spawn by maintaining the female coloration and occasionally spawning on territories. She established a theory that pupfish have a prima ry and superior means of spawning, which is the normal attempt to control ad vantageous territory. Given that this is the primary method, it tends to be the most succ essful at reproducing in mass. The other two methods satellites and sneakers are e xplained by the fact that most male reproductive systems will develop alternative means of passing on genes nonetheless. The most frequent type of variant consists of oppor tunistic and optional behaviors, usually triggered by a change in social or ecologic al environment. Satellite and sneaking tactics are therefore secondary means of spawning, and these methods tend to have a lower rate of reproductive success. However, fish w hich resort to these secondary tactics tend to be those which are simply smaller or someho w otherwise disadvantaged compared to the dominant males in the group, and so it is their only means of having even a few mates. Moreover, holding a territory req uires a lot of energy, and the secondary methods do not. Sneak-spawning is the tactic which requires the le ast energy and is usually reserved for inferior males without the ability to invest energy into territorial or satellite behavior. They do not take on coloration and mainta in the appearance of female fish. Their only hopes of mating are to join a spawning p air in a territory or to spawn with females off the breeding grounds. Due to the low en ergy output to mating output ratio, Kodric-Brown considered this to be the most opportu nistic of the three mating strategies. Satellite males tend to be more complex in behavio r. They take on the same
9 coloration as territorial males and establish a pe rch several centimeters above a territory, where they wait. As opportunity presents itself, they will swim down to intercept females entering the territory and spawn with them. This frequently results in chasing by the territorial fish, but the satellite male is not likely to leave the vicinity of the territory except to forage for food during peri ods of low mating activity. They do not engage in antagonistic behavior, and so the territo rial fish seems content to only chase them out but not beyond. In general, satellites wil l spawn with both fewer and smaller females, diminishing their chances of reproductive success. Kodric-Brown sought to elucidate social and ecolog ical situations that would inspire secondary breeding systems. She was interes ted in environmental factors that might affect breeding systems due to resource avail ability. Consequently, she manipulated population density, the availability of substrate in which females will lay eggs, and habitat size. Although Kodric-Brown used the Pecos river pupfish C. pecosensis behavior across the genus is similar enough to variegatus to warrant comparison. Her results note that a male's choice of breeding tactics should be affected by the energy he can potentially invest in reproductive activities and h is expected reproductive success, both of which should be influenced by the intensity of comp etition for access to females and oviposition sites. The intensity of competition is determined by the number and size of adult males, the number of active females, and the availability of oviposition substrate. She found that the territorial breeding system was favored when either population density was high or when breeding substrate was sca rce, with breeding substrate tending to be the more important factor. As long as a terri tory system was favored, then ratios of
10 territorial males and satellites remained consisten t regardless of amount of substrate present. The number of territorial and satellite fi sh was directly proportional to the amount of breeding substrate present. This suggests that as competition gets fiercer, both mating strategies are affected. Moreover, satellite s were more likely to associate themselves with a territorial male not of larger si ze than themselves, and who were reproductively successful whilst holding a large te rritory. This allowed them to be rather parasitic in nature, as they disrupted spawnings an d stole copulations from territorial males. However, a dominance hierarchy became favored at l ow population density, featured no satellite breeders, and was characteriz ed in the broad sense by an unequal amount of breeding substrate being split amongst a small number of dominant breeding males, constantly shifting territorial boundaries, and a high variation in spawning success. In a series of environments where oviposition subs trate is not scarce and population density is not excessive, Kodric-Brown's first study dictates that topography should play a part in spawning behavior and general activity levels. From her second study, it can be concluded that even in instances w here males cannot claim territory, it can be normally expected that dominant males who have t o compete for territory with sneakers and satellites will still contribute to th e recording of activity levels. It seems likely that a dominance hierarchy will emerge in th e habitat without an abundance of topographic abundance, resulting in a great deal of movement and accompanying raises in activity levels. Dunson etal (1998) studied the effect of salinity on growth and reproduction of Cyprinodon variegatus. They noted that sheepshead minnow populations were less
11 abundant in fresh water climates. While this has be en noted by prior researchers, it was usually assumed to be the work of nonnative or pred atory species. However, they tested specifically for salinity levels and found that wit hout the cations present in salt water, reproduction and growth of the individual was sever ely limited. If the lack of salt in the fresh water had health effects, they found it diffi cult to confirm based upon only being able to carry out their experiment for sixty days. The salinity level at which growth stunting ceased was approximately equivalent to 1 ppt, although more than that remains recommend ed. The impact of this particular study is clear: salinity should be maintained at a level at least equivalent to brackish, if not outright salt water. The goal of this experiment is to reproduce the pa tterns in activity levels put forward by previous researchers and to determine wh ether an interaction of environmental richness and time of day affect the a ctivity levels of Cyprinodon variegatus (sheepshead minnow) to a significant degree.
12 Methods Subjects: 42 fish of unsorted sex were received from the Gul f Coast Research Laboratory on 2/16/12. They were hatched on 8/17/11, making th em approximately 6 months old at the start of testing. We received them in bags with salinity measurements of 15 ppt (parts per thousand) and used a drip acclimatization metho d to adjust them to 20 ppt for the purposes of being put in the tanks used for the exp eriment. They were put into tanks for initial storage purposes, 14 to each of the three t anks. On 2/21/12 they were sorted and separated to as close to a 50/50 ratio as could be discerned for juveniles and females have similar coloration (Barlow, 1961). Genders wer e later determined, and the fish were sorted into groups of 4 males and 6 females per tan k, still with 12 to spare. Materials: 1) Three 55 gallon tanks Internal measurements: 48 inches long, 12 inches w ide and 18 inches high Indicators: black tape used for zone purposes Lengthwise: indicator every 12 inches Width-wise: indicator every 4 inches Heightwise: indicator every 4.5 inches
13 Low enrichment tank Normal enrichment tank
14 High enrichment tank 2) Miscellaneous: Roll of black tape Ruler Scissors Video camera Experimental Protocol: To determine the effects of time and enrichment in activity levels, three tanks were utilized. One was marked poor enrichment, an d consisted of two stones with a single piece of plastic vegetation. The normal en richment tank possessed a coral structure, two pieces of vegetation, and a number o f stones. The enhanced enrichment tank possessed two coral structures, three pieces o f vegetation, and enough rock structures to cover the tank floor, designed to max imize the compatibility between sheepshead minnow and habitat. Zones were marked us ing evenly spaced pieces of tape
15 with four zones along the length and height axises, and three across width,. During recording times, the number of times all fish cross ed between zones were counted to give total activity levels. Salinity levels across all t anks was 20 ppt, feeding was regular at every 3 days, filters were cleaned every three days and temperature was uniform across all tanks (25o C + 2). Barlow's (1961) ethogram of Cyprinodon macularius behaviors is reprinted in Appendix I as a reference for discussion. Experimental Design: The experimental hypothesis states that an interac tion exists between time of day and enrichment levels. The dimensions of the 55 gallon tanks being used f or this experiment are on the inside roughly 48 inches long, 12 inches wide and 18 inches high, making for 122 by 30.84 centimeter tanks, which was enough room for t he planned five males and five females. Three times per day 10:00 a.m, 1:30 p.m, and 5:0 0 p.m., or 3hr : 30min intervals where schedules conflict thirty seconds of footag e was recorded of each tank for a total of four and a half minutes per day. The independent variables being tested are time of day and environmental richness. The dependent variable being recorded is the number of lines crossed, or activities. Data was collected daily du ring a one-month period, yielding six sets of five days apiece, totaling 30 days. Data we re subjected to a Two-way Analysis of Variance (ANOVA) test to determine effects of three levels of enrichment and three times of day on activity levels.
16 It is understood that all three tanks are environm entally impoverished compared to field conditions, and that observations of activity levels are arbitrary. However, as the same bias applies across all conditions, it is cont rolled for. Furthermore, the environmental conditions used in this experiment ar e more relevant to conditions of aquariums than to the wild.
17 Results While conducting recordings over the course of a m onth, technical difficulties caused the loss of information regarding the time o f day at which almost half of the data was recorded. The remaining 18 days were organized into six blocks of three days apiece. Recorded within each box is the total numbe r of activities lines being crossed expressed across those three days. For the raw dat a collected for this experiment, see Appendix I. These data were then organized into nine cells to be subjected to a two way ANOVA test. Table 1. Number of activities of Cyprinodon on three types o f environmental richness three times per day. Eighteen days total used to re cord data. Each number within a cell represents three days of recordings. Time of Day Enrichment Morning Afternoon Evening Mean Standard Deviation Low 143 287 199 263 256 188 198 230 247 186 237 203 235 204 268 210 259 228 224.5 36.12 Normal 206 197 134 226 217 189 146 134 107 164 186 146 281 158 226 239 235 207 188.778 45.6 High 234 232 144 213 221 216 125 251 162 195 168 209 226 253 265 217 234 230 210.833 38.29 Mean 209.167 183 231.944
18 Standard Deviation 40.75 42.44 28.4 Grand Mean 208.037 42.15 Table 2. ANOVA Summary of data results. E x T is the interac tion between enrichment and time of day. Source SS df MS F P Enrichment 11695.81 2 5847.91 4.78 0.0131* Time 21594.48 2 10797.24 8.82 0.0006** E x T 5788.97 4 1447.24 1.18 0.3324 Error 55066.67 45 1223.7 Total 94145.93 53 *p < 0.05 **p < 0.01 It can be concluded that both time and environmen t show statistically significant differences in activity levels beyond the 0.05 leve l. The interaction of the conditions, however, is not statistically significant, as it is likely to occur naturally (P = 0.3324) and experienced sufficient variance as to make results unreliable.
19 A graph of the means for morning, afternoon, and e vening with collapsing over enrichment is presented here. The evening is signif icantly different from both the afternoon and morning (F(2,45) = 8.82; P < 0.001). This graph measures activity levels across environ ments, without concern for time of day. The group with normal enrichment is signifi cantly different from either the high Morning Afternoon Evening 0 50 100 150 200 250TimeTime of DayActivity Levels Low Normal High 170 180 190 200 210 220 230Environmental RichnessRichnessActivity Levels
20 enrichment or low enrichment tank (F(2,45) = 4.78; P =0.01). This graph is a representation of the interaction of environmental richness with time of day. Interaction graphs can be interpreted based on the degree to which their lines intersect. Parallel lines are going to have very li ttle interaction. While not perfectly parallel, the overlap present is not enough to make it likely to be statistically significant. The implication herein is that while environmental richness and time of the day both carry weight with regards to Cyprinodon variegatus life cycle, these are wholly independent factors and that there are no unique va lues that are significantly above or below each of the means of the two factors. LowMiddleHigh 0 50 100 150 200 250Enrichment on Time Effects MorningAfternoonEveningEnrichmentMean
21 Discussion The effects of time of day and of environmental qu ality and habitat on pupfish activity levels as described by prior researchers h as been studied, observed, and documented. Data was collected which confirmed tha t a poorer environment leads to greater territoriality, and that Cyprinodon will be most active in the mornings after they have rested and moved into a cluster and again in the evenings, when they seek food. Many of the concerns I had whilst researching this topic were addressed. Sorting the fish into males and females according to the gu idelines given by Barlow was easier to do than it initially seemed. When watching recordin gs of the fish to get a count of activities, I could usually tell immediately if the animal I was focusing on was a male or female, and when it behaved in a manner consistent with the descriptions provided by the same, I found that it helped ease data recording. Certain control elements, such as food being put i nto the tank at the same spot each time the fish were fed, temperature remaining constant, and salinity being sufficient were handled easily. I always fed the fish myself a nd put the food in in the middle of the right half of the tanks. The tanks were indoors, so temperature was a constant. Moreover, spawning was observed at multiple periods of the da y, meaning that the temperature throughout the day was permissible, and thus played no part in the results. And lastly, salinity when the fish were put into the tanks was kept in brackish water range, about 25 ppt. Given that salt water maintains an average of 35 ppt, this number is closer to salt than the minimum considered safe for Dunson. The fierce competition for territory in the low en richment environment led to a few recorded interruptions of mating, similar to an incident described in Itzkowitz' paper
22 (1974). The female promptly departed, and the male chased the intruder out of his territory. When this occurred, however, it created a brief rise in the average activity level of the tank as fish crossed multiple zones to get a round an aggressive male. Moreover, the females themselves do a lot of roaming after the in cident, usually biting and chasing any individuals who cross their path. With regard to territories, another tendency was n oteworthy: the smaller, less dominant fish usually found a spot within the tank and seemed content to remain sequestered there for a long while. Itzkowitz (1979 ) demonstrated that a homogenous sand layer on the tank bottom would cause the domin ant male to roam the whole tank, his path sometimes intersecting with subordinates. I ob served this happening in all tanks, and given that the dominant male usually tried to contr ol an area near the center of the tank, it seemed that the smaller fish were limited to remain ing near the substrate and in a corner amidst several rocks. And yet the sum of those smal l territories could sometimes consist of half the tank. That the locations the small fish chose to make their own were so consistent speaks to Itzkowitz' statement that pupf ish could retain a territory very well. Yet, his assertion that fish will adjust to changin g factors also holds true. In the high enrichment tank, while the smaller fish were still the least likely to contribute to activity levels, they did seem more inclined to roam the ent irety of the tank floor amidst the large amounts of rock cover. Meanwhile, the dominant male would usually be quit e preoccupied with fending off intruders, performing a large number of the act ivities recorded particularly in the low enrichment tank. By comparison, the dominant ma les present in the high enrichment tank only chased on occasion and never seemed to ho ld a distinctive territory. Relatively
23 speaking, they contributed very little versus those dominant males in the other two environments. This may be the heart of the reason t hat a significant difference was found between the low enrichment tank and the normal enri chment tank, in addition to having a larger overall activity count than the high enrichm ent tank as well. Kodric-Brown's experiment raises an interesting no te, which is that of elevation. The substrate used for this experiment was largely uniform in elevation, but she noted a large degree of competition amidst the rockier, mor e varied, and more shallow Area I. All territories were very small due to males attempting to hold choice pieces of rock. By comparison, the deeper, sandier, and less varied Ar ea II saw less competition, but much larger territories. This distinctly denotes that ro ckier terrain is favored, but if such was the case, it might initially seem to make my own result s contradictory to this, based on the lack of competition. This is actually not so. A spe cific aspect of Kodric-Brown's observations is that even with small territories, m ales would eventually settle in and patrol a given area or territory. In an environment with more competition, that territory is going to be smaller. However, this does not mean th at a smaller territory is going to result in a lesser activity count. By virtue of the smalle r territory being contested, chasing and patrolling were more frequent. On the topic of elevation, Itzkowitz noted that in the mornings most pupfish will gather in the deeper end of a pool, even the domina nt male. The smaller pupfish in my experiments tended to stay in one area, particularl y close to the substrate. Since deeper water is considered inferior from a pupfish perspec tive, it is possible that the smaller fish are taking deep territory on the fringes of the t ank. During the process of making the recordings, a pat tern was almost recognizable in
24 the way data sets were turning out. The number of r ecorded activities would be higher for the low enrichment group, and lower for the midday recording. As more data was taken, the more noticeable it became. Unfortunately, the part of this experiment which w as supposed to make it unique did not turn out. With the emerging pattern, it was determined that the experimental hypothesis relating times of day at which activit y levels at different enrichment conditions would stand out was not verified. For instance, if the effects of drugs were being tested on pupfish a common experiment the n the investigator would be aware of the possible effects the environmental complexit y and times of data collection might have upon his results. During the recording of data, a portion of it had to be discarded, as noted previously in the Observations section. The graph s howing interaction was not completely parallel. This might be worth further st udy, as the end of the graph seemed to be on the verge of overlap. Running data felt like an interaction was being revealed. Like with Dunson's salinity experiment, I feel if I had more time, I would have liked to see if a larger sample size would be sufficient to bring abo ut the interaction which felt as though it should have been present. If interaction were fo und, it might signify in Cyprinodon variegatus variegatus an ability to take cues from light and respond as appropriate within the environment.
25 Appendix I. Meandering .The female swims slowly in midwater or near the surface, changing course frequently and rather aimlessly. Swimming mo vements seem to be slightly exaggerated. The median fins are held against the b ody. Nuzzling .-The male swims directly under the female while sh e meanders. His body is tilted up about 300, and he maintains a position in which the top of his head is just below, or in contact with, the abdominal region of the fem ale (Fig. 1D). (The abdominal region here includes the ventral surface from directly bel ow the pectoral fins to just posterior to the vent.) The median fins of the male are folded. Contacting .-As the female moves slowly over the bottom the ma le stays beside her, and her head is usually just in front of his. Often the y are actually touching. In the early stages of the mating the male usually contacts the female anteriorly in the region of her pectoral fin base with his snout. As mating proceed s, the male contacts the female progressively more posteriorly. Eventually, the are a of contact is almost restricted to the region of the vent. During most of this time the do rsal fin is folded. Movements of the male which are directed at keeping station beside t he female are called contacting. Side to side contacts are not separated from snout to side contacts. Tilting .-While swimming slowly over the bottom the female tilts her body toward the bottom, head down, at an angle of about 450. The do rsal fin is normally raised at this time. Tilting is the beginning movement of nipping. Nipping.-From the tilted position, the female opens her mouth, presses it against the bott om, and normally takes up a mouthful of the substrate (Fig. 1B). Then the body is droppe d down against the bottom. When
26 horizontal, the female either immediately spits out the substrate, or swims forward a short distance, stops, and then expels it. The female app ears to turn the substrate over in her mouth. The dorsal fin is held open. The female may nip two or three times in quick succession; each successive nip after the first one is initiated from the horizontal position. Females of another toothcarp, Cyprinodon variegatus were seen by Raney et al. (1953) to pick up objects from the bottom when encountered by males, and this action was interpreted as food-seeking. In the roach, Leuciscu s rutilus (L.), and in the goldfish, Carassius auratus (L.), the female evidently signal s the moment of spawning by snapping at some object as if feeding (Fabricius, 1959). Halting .-The female stops swimming after nipping. Usually her vent is close to the substrate so, that the long axis of the body is til ted up slightly anteriorly (the male assumes a corresponding angle). The dorsal fin is s till spread. Sidling .-The male swims forward and laterally against the female. The male's dorsal fin is folded or half open. The region supporting the anal fin of the male is thrust against the posterior line of the abdomen of the female. The bo dy of the male is tipped out of the median plane only slightly, or not at all. In this maneuver the male cannot swim by means of trunk undulations and must therefore rely on scu lling movements of the free pectoral fins and of the caudal fin. This rapid sculling imp arts a quivering appearance to the male. S-shaping .-Seen from above, the body of the pupfish forms a gentle "S" (Fig. 1C). At this time the male and female norrnally lie side by side on the bottom in parallel S-shapes. The curvature is more pronounced in the male. The h ead and the anal region of the male are directed toward the female, and the anal fin of the male is extended in her direction. The dorsal fin of the male is spread, and sometimes is bent slightly toward the female. In
27 the female the dorsal fin is spread maximally. More over, her vent is pressed against the bottom and her caudal fin beats rapidly, but with a very small amplitude. Wrapping .-While both fish are S-shaped the male wraps his a nal fin around the posterior line of the female's belly (Fig. 1C). The male's an al fin forms a crude cup under the vent of the female. The extended dorsal fin was sometime s, bent toward the female, but it was never seen to enfold her. Jerking .-While still S-shaped the head is jerked toward th e side opposite that to which it is already directed, thus initiating a wave of cont raction which passes down the body reversing the direction of the S-shape. In the fema le one egg is extruded by this flexure, and the male presumably emits sperm at this moment. Patrolling .-The male in his territory swims straight ahead in spurts of about 30 to 50 cm with the dorsal, anal and pectoral fins folded back The lateral beats of the orange caudal fin contrast against the bright blue body. At the e nd of each spurt the male stops abruptly by throwing the colorful pectoral fins forward toge ther in a quick movement that catches the eye. Then the male stands momentarily with the median fins raised, and the pectoral fins beating alternately and rapidly. The caudal peduncle is frequently flexed to one side in the intention swim posture. Then he swims off ag ain in a new direction. In this manner the male pupfish continually crisscrosses his terri tory. Facing .-Males approach one another head on and usually pa use momentarily face to face about one body length apart. The median fins are sp read. Eyeing .-Two male stand momentarily eye to eye facing in o pposite directions, bodies parallel, and about one half body length separating them. The median fins are spread. Arching .-The body is bent into a C-shape with the concave side toward the other fish. The
28 median fins are spread. A sculling action of the ca udal fin and the pectoral fin on the convex side imparts a slight quivering appearance t o the fish. Tail-beating .-The flexure of the peduncle appears to be rever sed from the arched position so that the caudal fin, apparently folded, is first directed away from the other fish. Then the spread caudal fin is beaten back tow ard the opponent, often causing the head of the fish delivering the beat to swing away. This movement requires further observation. Charging .-The median fins are folded, the mouth is opened, and the fish darts forward toward another fish. Circling .-From the arched position two fish often start to circle at high speed while maintaining the head to tail orientation. It seems that each fish attempts simultaneously to charge into the flank of the other and to dodge the charge of the opponent by turning. Fleeing .-The fish swims away from the attacking fish at hi gh speed with the median fins folded. Flight is toward the surface in the absence of cover, but if plants are present the fish will sometimes take refuge among them. Escaping .-The fish darts toward the bottom and into the mul ch, algae, or plants there, and then lies motionless. Cowles (1934) observed this b ehavior in free living pupfish which he tried to capture. Digging .-While standing over the bottom, with head and bod y tilted downward about 450, the fish moves ahead, i.e., obliquely downward while opening the mouth. The mouth contacts the bottom; substrate is taken into the mo uth; the fish rebounds; the body is tilted up into the normal horizontal position as the fish moves forward slightly; and the substrate is expelled through the mouth. The substr ate is often expelled immediately at
29 the end of the rebound. Plowing .-This movement starts just like digging, but when the substrate is taken into the mouth, the fish then drops the body down against th e bottom and swims forward through vigorous beating of the tail. At the same time, the pectoral fins beat forward resisting the thrust of the caudal fin. The fish briefly swims in place expelling bottom material both forward and backward. Then the pectoral fins cease to resist the tail propulsion, and the fish moves forward expelling the mouthful of substr ate. After this the pupfish commonly returns to the excavated spot and digs there in the normal manner. According to the description by Raney et al. (1953), Cyprinodon vari egatus plows in the same way. Plowing in Cyprinodon seems similar to the movement s in cichlid fishes which have been called digging by Baerends and Baerends-van Ro on (1950). (Barlow, 1961)
30 II. Block I (Three days of data) Time of Day Environmental Richness Morning Afternoon Evening Low 143 198 235 Medium 206 146 281 High 234 125 226 Block II (Three days of data) Time of Day Environmental Richness Morning Afternoon Evening Low 287 230 204 Medium 197 134 158 High 232 251 253 Block III (Three days of data) Time of Day Environmental Richness Morning Afternoon Evening Low 199 247 268 Medium 134 107 226 High 144 162 265 Block IV (Three days of data) Time of Day Environmental Richness Morning Afternoon Evening Low 263 186 210 Medium 226 164 239 High 213 195 217 Block V (Three days of data) Time of Day Environmental Richness Morning Afternoon Evening
31 Low 256 237 259 Medium 217 186 224 High 221 168 235 Block VI (Three days of data) Time of Day Environmental Richness Morning Afternoon Evening Low 198 203 228 Medium 189 146 207 High 216 209 230
32 References Barlow, G. W. (1961). Social behavior of the desert pupfish, cyprinodon macularius, in the field and in the aquarium. American Midland Naturalist 65 (2), 339-359. Retrieved from http://www.jstor.org/stable/2422959 Claireaux, G., & Lefranois, C. (2007). Linking env ironmental variability and fish performance: Integration through the concept of sc ope for activity. Philosophical Transactions: Biological Sciences 362 (1487), 2031-2041. Retrieved from http://www.jstor.org/stable/20210003 Dunson, W. A., Paradaise, C. J., & Dunson, D. B. (1 998). Inhibitory effect of low salinity on growth and reproduction of the estuarine sheeps head minnow, cyprinodon variegatus. Copeia 1998 (1), 235-239. Retrieved from http://www.jstor.org/stable/pdfplus/1447727.pdf Echelle, A. A. (1973). Behavior of the pupfish, cyp rinodon rubrofluviatilis. American Society of Ichthyologists and Herpetologists (ASIH ) 1973 (1), 68-76 Retrieved from http://www.jstor.org/stable/1442359 Itzkowitz M. (1974). The effects of other fish on the reproductive behavior of the male cyprinodon variegatus (pisces: cyprinodontidae). Behaviour 48 (1), 1-22. Retrieved from http://www.jstor.org/stable/4533559
33 Itzkowitz M. (1979). Territorial tactics and habi tat quality. The American Naturalist 114 (4), 585-590. Retrieved from http://www.jstor.org/stable/2460373 Kodric-Brown, A. (1977). Reproductive success and t he evolution of breeding territories in pupfish (cyprinodon). Society for the Study of Evolution 31 (4), 750-766. Retrieved from http://www.jstor.org/stable/2407437 Kodric-Brown, A. (1986). Satellites and sneakers: O pportunistic male breeding tactics in pupfish (cyprinodon pecosensis). Behavioral Ecology and Sociobiology 19 (6), 425432. Retrieved from http://www.jstor.org/stable/4599979 Martin, B. A., & Saiki, M. K. (2005). Relation of d esert pupfish abundance to selected environmental variables in natural and manmade hab itats in the salton sea basin Environmental Biology of Fishes 73 (1), 97-107. Retrieved from http://www.springerlink.com/content/g6563075l26032n 0/fulltext.pdf Raney, E. C., R. H. Backus, R. W. Crawford, & Robin s C. R. (1953). Reproductive behavior in Cyprinodon variegatus Lacepede, in Flo rida. Zoologica, 38:97-104.