The Development of Copepod Capture Strategies in Juvenile Squid, Loligo opalescens Donald S. Chen William F. Gilly September 22, 1994 I Abstract: Juvenile squid, Loligo opalescens, face a great challenge in catching quick, elusive copepods. The mastery of copepod capture, an acquired ability, develops progressively past a basic attack strategy, through various strategies which facilitate capture, namely the "arm-intercept" and the "tentacular grab." Squid raised exclusively on easily captured Artem ia nauplii and introduced to a copepod diet 40 days after hatching could not master copepod capture and died, presumably of starvation. II. Introduction: Juvenile squid are active predators with high metabolic rates sustained by consuming as much as 35-80% of their body weight each day (Hurley, 1976). Little is known about the life history of juvenile squid, such as where they are found or what they eat-one can naturally speculate that they prey upon copepods, among other species, to obtain their nourishment. In captivity, hatchling squid vigorously hunt and attack copepods (Hurley, 1976), a major component of marine plankton, and it is likely that copepods are a main food source in the wild as well. Because copepods are small, erratically moving creatures with an extremely quick escape response triggered by the motion of nearby predators (Yen & Fields, 1992), capture of these organisms is a definite challenge for a newly hatched squid. Thus, we may ask, what performance modifications occur in squid, as their motor capabilities develop after birth, so that they may be more successful in copepod captures. As Hurley (1976) described, young L. opalescens, in their attacks on Artemia nauplii, copepods, and larval fish, display three distinct sequential attack behaviors: attention, positioning, and seizure. This paper builds upon this earlier work, through a more in depth study of attacks on copepods, a major prey type which is the same as those squid hatchlings encounter in the wild, and by focusing on the ontogeny of this complex behavior. Hanlon and Hixon (1983) characterized a wave of mortality during the first two weeks post-hatching, due to squid that fail to properly initiate feeding behavior. This work focuses on prey capture behavior after this initial period of mortality, by squid which have presumably acquired the most basic skills to feed. Development of copepod prey capture behavior was examined through the simultaneous rearing of two experimental groups. Whereas copepods were fed to the one group of squid shortly after birth, only slow, easily captured Artemia nauplii were fed to the other group of squid from birth until day 40. On day 40, Artemia feeding ceased, and these squid were thereafter fed copepods only. This experimental design allowed us not only to observe the developing prey capture techniques in squid exposed to copepods from birth, but also to compare the success and quality of copepod attacks between these squid and the non-challenged, Artemia-fed squid. Study of the impaired attacks by Artemia-fed squid provided additional insight into the nature of successful copepod captures. Results from these two experiments suggest that first, copepod-capture is an acquired ability developing through a progression of attack strategies, and second, a window of opportunity exists early in life for squid to master copepod capture. Results of this study may be relevant to foraging behavior in the field, and thereby be important to fisheries biology. In addition, findings from this study will serve to guide approaches to squid mariculture in the optimal design of feeding regimens. Finally, the characterization of specific behaviors revealed at certain ages in this study may provide a valuable model system for developmental neurobiology studies in relation to experience¬ dependent modification of behavior. IIi. Matenals anò Memoûs: Culture: Five egg cases of the California market squid, species Loligo opalescens were held at the Monterey Bay Aquarium in each of two identical 320 liter tanks. Each of the tanks, labelled tank two and tank three, represented an experimental group, hereafter referred to as group two and group three, respectively. These black cylindrical tanks possess gently sloping conical bottoms and measure one meter in diameter at the top. Tanks were exposed to natural daylight that entered the room, and overhead fluorescent lights illuminated the tanks between 6 a.m. and 6 p.m. daily. Except for periods of maintenance and feeding, the tanks were kept partially covered with a black plastic sheet. Each morning, all dead squid were siphoned out of the tank, and the daily mortality count was tabulated. Detritus was also siphoned off the bottom and organic debris skimmed from the surface at this time. Flow-through water was provided to the tanks from a temperature-controlled reservoir (-300 liters volume) fed with natural seawater passed through a 10 micron filter, and water recirculated into the tanks. Egg cases were held at 14-15°C. until hatching, after which the temperature was increased by approximately 0.6 °C. per day until 17- 18°C. was reached. Once the hatching commenced, the eggs were removed after several days, and day one post-hatching was assigned to the day of most significant hatching activity. Feeding: Squid hatchlings were fed once daily with either Artemia nauplii enriched with algae and Super Selco (a nutrient medium rich in lipids, fatty acids, and vitamins; produced by Artemia Systems N.V., Belgium), or with live marine plankton, obtained by conventional surface tows in Monterey Bay, that consisted primarily of small, very quick, Acartia sp. copepods, although larger Calanoid sp. copepods, chaetognaths, and an assortment of crustacean larvae were also present in significant numbers. Group two hatchlings (those in tank two) were fed marine plankton when available, and Artemia nauplii when plankton was not available. Group three hatchlings were fed only Artemia nauplii until day 40, when their diet was switched permanently to wild plankton. Fig. 1 details the feeding regimen for each tank and presents daily mortality counts. Two waves of mortality occur over days 1-12 and 25-35 in both groups, and this is commonly seen in squid rearing operations (Hanlon & Hixon, 1983). The large numbers of deaths in Group three starting on day 40 is discussed below. Filming: Feeding behavior was recorded with a Canon Al Digital Hi8 camcorder fitted with a f 1 Vivatar close up lens and mounted on a tripod. The camcorder filmed at 30 frames per second with a shutter speed of 1/60 second. Floodlights provided supplemental lighting. With the camcorder elevated on the tripod and turned downward, filming occurreò at an angle àmost perpendicular to me surface oi me water. » ên mummateò squid displaying attack behavior were selectively tracked while filming until the squid either successfully caught the prey item, stopped attacking, or moved out of the focal range of the camcorder. Each filming session lasted approximately half an hour. Due to the fluctuating availability of copepods, attacks on copepods were not filmed on every day. Frame-by-frame analysis of the data was undertaken using a Sony 9700 Hi 8 editing deck and a Sony Trinitron video monitor. Total attack distance and maximum attack speed were measured using a caliper precise to .001 inch and normalized to the mantle length of the attacking squid. Attack distance refers to the apparent straight-line distance between the tip of the squids' arms and the copepod on the last frame just before the squid's arms began to open up to initiate the actual attack. Maximum, or peak, attack speed was measured on the monitor as the greatest distance travelled by the squid between two consecutive frames during the attack lunge. Peak speed was usually attained within four frames and represents the final approach of the squid, for after this burst of speed, either the squid closed its arms upon the copepod in the culmination of a successful attack, or the copepod escaped from the squid (an unsuccessful attack). Attacks in which the squid was obviously moving vertically to a significant extent were not included in the analysis. Direction of attack and copepod escape direction were also recorded when they could be unambiguously determined by the orientation of the copepod antennae. IV. Results: The Basic Attack Sequence: In the most general attack strategy observed (Fig. 2), the squid first fix their attention upon and orient towards the potential copepod prey, pointing their arms in a cone directly at the copepod. The squid actively maintains this posture through rhythmic jetting and presumably fin control to remain in a fixed position relative to the prey. As the actual attack commences, the squid opens its arms over a period ranging from one to four video frames (33 msec/frame) and then swiftly jets forward at the prey item. An attack ends successfully when the squid snaps its arms shut, having captured the prey, or, unsuccessfully when the copepod responds to the attack with a quick escape response and eludes the squid. This strategy, the first type of attack observed in the youngest squid, appears to be the most general strategy. Squid utilized this strategy to attack not only copepods, but also Artemia and other prey species. Attacks on all species were attempted from all directions, but a preferred direction of attacks on copepods appeared to be head-on, i.e. towards the anterior head region of the copepod. This tendency was revealed by a variation in the basic attack that can be described as "circling" behavior (Fig. 3), in which a squid pursuing a copepod would move angularly, rotating around the copepod and apparently trying to attain the head-on position. During this process, the copepod often would react to the squid's approach by jumping forward at an angle, propelling it out of the squid's lane of approach. In response, the squid would approach the copepod and repeat the rotation behavior in another attempt to position itself in front of the copepod. This sequence of action and reaction often repeated itself many times. Such movements would appear to be an adaptive behavior by the squid, because the head-on positioning lends to the copepod jumping partially towards the attacking squid in a fairly stereotyped escape response. Although the exact direction taken by the copepod may be unpredictable, the possibilities are basically limited to a solid angle in which the squid is waiting. This would greatly increase the probability of interception, and circling behavior clearly plays an important role in the more developed attack strategies discussed below. In general, the basic attack strategy appeared most successful when the squid lunged at the copepod from a modest distance with adequate speed. Attacks from distances above one mantle length almost always failed, regardless of attack speed. As shown in Fig. 4, essentially all of the filmed successful attacks (days 15-42) occur within a range of less than 1.0 ML distance. Data in Fig. 4 also show that peak attack speed is strongly correlated to total attack distance. Thus, very fast attacks are not launched from short distances. Squid of each age group initiate successful captures over a wide range of distances, but, as described below, some specialized strategies develop that facilitate successful captures from both short and long distances. As Figs. 5A-C show, the correlation between peak attack speed and attack distance exists in both successful attacks and unsuccessful attempts at all times in this study. The strength of the correlation appears to increase somewhat with age; however, a quantitative analysis has not been carried out. Figures 6A-C show, for each age group, histograms of successful copepod captures grouped by distance (Group two squid). In Fig. 6A, successful captures in the youngest squid (days 15-23) occurred from shorter distances, between 0.1 and 0.4 ML. Such captures occurred when the squid attacked the copepod from a short distance, using an adequate amount of thrust. During this earlier time period, the squid employed predominantly the basic attack strategy described above. As shown in Fig. 6B, day 26 to day 35 squid captured copepods more successfully throughout a broader range of distances and speeds. While the captures in the lower range can be attributed to the basic attack strategy observed in the younger squid, the striking increase in captures from greater distances may be due to a newly developed behavior. The appearance of these long range captures coincides with the appearance of a specialized attack strategy to be discussed later, the "arm-intercept.' In the oldest squid, days 41-42, successful captures were concentrated below 0.4 ML distance, although some still occurred at greater distances (Fig. 6C), and probably involved arm-intercepts. The apparent improvement in short-distance attack performance in these oldest squid may be attributed to the development of yet another strategy to be discussed later, the "tentacular grab." The development of new strategies such as the arm- intercept and the tentacular grab may allow the squid to explore greater ranges of attack distances (and speed). Figures 7A-C show complementary data for unsuccessful attempts by squid of each age group. In each case, the attack distance distributions for unsuccessful attacks is skewed towards long distances in comparison with data in Figs. 6A-C, but differences in the three unsuccessful distributions also exist. The youngest squid in the study appear to attack from two distance ranges, the primary one below 1.0 ML and a secondary one above 1.2 ML (Fig. 7A). Squid in the intermediate age group also display attacks in the same range below 1.0 ML (Fig. 7B). Attacks from greater distances were not observed. Older, day 42 squid (Fig. 7C) similarly show a similar distribution of attempts predominantly below 1.0 ML. Specialized Strategies: Squid utilize characteristically different attacks at different ages. Speed adjustments alone cannot account for the changing patterns of attack distance distribution; rather, these changing patterns reflect the development of specific prey capture strategies in squid. Here we present two such strategies. In the intermediate-aged and in the oldest squid, two specific attack strategies were observed: the arm-intercept and the tentacular grab. In the arm-intercept, (Fig. 8), the squid first positions itself in front of the copepod, most often through the circling behavior described above. Having postured itself head-on to the copepod, the squid then opens its arms up very wide. Next, keeping its arms wide open, the squid jets at the copepod, The strong jet of the squid triggers the copepod's escape response and the copepod projects forward at some angle. Because the squid's arms are wide open, the copepod may, during its escape attempt, run into the squid's arms to be captured A distance/speed plot of attacks unambiguous arm-intercepts reveals that such attacks usually occur from relatively large distances and speeds. (20.6 ML distance and - 0.3 ML/frame speed). Successful arm intercepts were first observed on day 29 and occur in both the intermediate and the oldest squid groups, but their earliest developmental 10 appearance is not precisely known. In the tentacular grab (Fig. 9), the squid must first smoothly maneuver itself quite close to the copepod (so as not to trigger the copepod’s escape response). When it is in position, the squid rapidly extends it long feeding tentacles (appendages longer than the arms) within one to two video frames (33 msec/frame), grabs the prey, retracts the tentacles, and then wrestles the copepod into submission with its arms. During this procedure, most of the movement occurs in the tentacles--the body moves only slightly. Most tentacular grabs occur within 0.4 ML distance and below 0.2 ML/frame speed. Tentacular grab attempts were first observed in intermediate-aged squid, but successful captures using this strategy were not observed until days 41 and 42. Copepod Capture by Artemia-raised Squid: Results described thus far were obtained on squid that had been exposed to copepods as the predominant food source from birth, and mortality in this group was extremely low from day 40 until the end of the study (day 53) as indicated in Fig. 1. On the other hand, Group three squid were reared from birth on only enriched Artem ia nauplii and subjected to a complete switch to copepod prey items on day 40. Coinciding with this switch to plankton, the death count accelerated to more than 30 mortalities per day. Of the 142 squid present on day 40, only six survived to day 53. During the period of copepod exposure, the Group three squid clearly recognized the copepods as potential prey items and made large numbers of attacks. These attacks were studied on days 41-43 and found to be launched almost exclusively from distances greater than 0.5 ML (Fig. 10) at speeds appropriate for this range (data not illus.). 11 Despite this, the physical attack sequence differed from that of the plankton reared squid, however, and very few attacks were successful. When faced with copepods, Group three squid did not appear to carefully position themselves before attacking or to engage in normal circling behavior to achieve the head-on position. Comparison of Fig. 10 with Fig. 7C indicates that the distribution of unsuccessful attack distances in Group three squid is skewed towards long distances relative to those of the equivalent age group of plankton-fed Group two squid. Moreover, Group three squid showed no attempts whatsoever from the close (short distance) range below 0.4 ML, over which the day 42 Group two squid were highly successful. Instead, the 41-43 day old Artemia-raised squid are more similar to the youngest group two squid studied (15-21 days) in that both displayed a high percentage of long-distance failures (Fig. 10 vs. Fig. 7A). V. Discussion: In this discussion, two points shall be addressed: first, copepod capture is an ability that must be acquired through experience; and second, squid acquire such an ability through a progressive development of increasingly complex strategies. Acquisition of Prey Capture Behavior-A Window of Opportunity: Proof that experience is critical in the ontogeny of copepod capture is most evident in a comparison of mortality counts between group two and group three squid. Before we develop this point, let us first interpret the mortality data. As food type was the only variable manipulated in the comparison between group two and group three squid, significant differences in mortality between the two groups can be attributed to diet- 12 dependent factors. All other factors, environmental and handling factors included, were identical in the two groups. Invariably, "noise" in the mortality records emerges during such an experiment that makes detailed comparisons between groups difficult, and replicate experiments can help separate "signal" from noise. Results of the prey-item switch described in this report were confirmed in a previous experiment carried out one year earlier. Before squid can acquire specific strategies for copepod attacks, they must possess the basic ability to attack and feed during the first 1-2 weeks of life. The initial wave of mortality between day one and day 13 in both Groups two and three most likely is due to squid that perished from starvation because they did not make the transition from internal yolk nourishment to feeding by developing successful prey capture (Hanlon & Hixon, 1983). Thus, those squid that survived the first mortality wave must possess the fundamental attack skills described in this paper as the "basic attack." We are presently studying this two-week period in order to reveal the progression of development of this behavior necessary for survival. After the initial wave of mortalities, the subsequent period of low mortality in both tanks indicates that nearly all the squid were capturing and feeding upon prey. Between days 26 and 39, Group two squid experienced another wave of mortalities, possibly due to a change in the feeding regimen. Because of increased copepod availability starting on day 20, these squid were fed copepods exclusively for 11 days. It is possible that certain Group two squid had not developed the ability to catch the fast copepods and had been surviving on the regular provisions of Artemia nauplii during the first 20 days. Faced 13 with an extended period of time where copepods were the predominant, if not exclusive prey item available in the tank, these squid may have perished from malnourishment. This explanation would be consistent with the interpretation, to be discussed below, of the extremely high mortality experienced by Group three squid after the introduction of a copepod diet. After day 39, Group two squid exhibited minimal deaths until day 53 when the study was terminated. An indication of the importance of experience in mastering prey capture is evident in the mortality data for Group three squid. The dramatic increase in mortality rates that occurred after the switch to a copepod food source can be attributed to an inability to catch copepods rather than to lack of prey recognition, as evidenced by the many failed attempts (Fig. 10). Total mortality during this period vastly exceeded not only that of Group two during the same time period (days 41-52), but also that suffered by either Group two or three squid during their initial introduction to prey directly after hatching. These comparisons suggest that after 40 days, the squid's ability to master copepod capture is greatly impaired. We hypothesize that squid deprived of exposure to their natural food source of copepods for several weeks after birth fail to receive the developmental cues and experience necessary for the ontogeny of copepod capture. Copepod capture is an ability that must be experientially acquired within the first forty days, more likely earlier than later. The low rate of survival after switching foods refutes the idea that copepod capture may be simply an innate ability or a developmentally programmed skill. An additional strong indication that copepod capture is an acquired ability can be found in the 14 improvements in capture frequency and refinements in technique between younger and older Group two squid. Analysis of Group three squid attacks on copepods (days 41-43) reveals that these squid resemble younger (day 15-23), Group two squid in their fast, long distance attacks. While such long distance attacks were not observed in Group two squid after day 26, they predominate in Group three squid at day 41. Long distance attack attempts probably reflect either a lack of agility that prohibits a close approach of the copepod without triggering its escape response, or the inability to properly time reactions to the erratic, fast moving prey. Thus, these older, but inexperienced squid appear to be developmentally stunted at a level more or less similar to that of Group two, day 15-23 squid exposed to copepods shortly after hatching. Exceptions do occur. Out of the 142 Group three squid alive on day 40 when copepods were introduced, six had survived the crash 12 days later. Presumably these squid possessed the ability to catch copepods. Despite the lack of exposure to copepods, these six may have developed such an ability independently, by some mechanism other than experience with copepod attacks, or they may have been extraordinarily flexible in their ability to acquire the new skills necessary for copepod capture at day 40. Progression of Strategies During the Period of Acquisition: Having established that copepod capture is an acquired ability, we now move on to examine the progressive development of this ability. First, the correlation between attack distance and peak attack speed strongly suggests that a squid can accurately gauge the distance between itself and the prey target, and then adjust its attack speed accordingly. 15 Such a correlation roughly forms a 45 degree line as shown in Fig. 4. Points significantly below this 45 degree line are indicative of a slow approach that would not be expected to be successful unless coupled to a special strategy, such as the tentacular grab. The paucity of points above this line implies that squid can accurately adjust attack speed to match attack distance, and that they do not use more speed than is necessary. The fact that a correlation exists in successful and unsuccessful attempts at all age groups studied indicates that the ability to gauge distance and adjust attack speed accordingly either is inborn or develops before day 15. Careful observation of the earliest feeding attempts by squid on copepods would serve to distinguish these possibilities and to reveal the ontogeny of the basic attack pattern. This work is presently in progress. For the youngest squid in the study (days 15 to 23), the dominant strategy was the basic attack. Although the successes only appeared to occur when the squid attacked from short distances using sufficient speeds, many squid still attacked from above 1.0 ML, a distance beyond which no successful captures were observed. The almost complete disappearance by day 26 of attacks in this ineffective, long distance range takes on a developmental significance as a means by which squid apparently improve their chances of copepod capture. Such long, futile attacks were apparently selected against. Whether the squid displaying these attacks died or modified their behaviors is not clear. During days 26-35, squid expanded their effective attack range through the use of a new, specialized attack strategy. The regular appearance of successful captures originating from above 0.5 ML distance marks this development, and the appearance of the bimodal distribution evident in Fig. 6B is consistent with the idea that squid in this age group 16 employed more than one strategy. The basic attack strategy most likely accounts for the captures at distances less than 0.4 ML, whereas the more recently acquired arm-intercept at least partially accounts for the improved performance in the longer distance range. Arm intercepts, which were still practiced by day 41-42 squid, largely account for the existence of captures at greater distances in the oldest squid studied, but in this group, successful attacks were most prominent in a region of very short distances and low speeds. Coinciding with this shift in attack parameters is the first appearance of successful tentacular grabs (day 41), which may serve to allow squid to capture copepods with less effort, shooting their tentacles out rather than propelling their whole bodies, This pattern is very similar to that employed by adult squid (Kier, 1982). It is noteworthy that in the tentacular-based short distance attacks observed in day 41-42 squid, these animals achieve significantly slower peak attack speeds than do younger squid. It is likely that such slow attacks require far less energy expenditure than high speed alternatives, but the extent of savings in relation to the total metabolic requirements of the growing squid remains to be determined. Because of significant growth of animals during the course this study, measurements of attack distance and speed relative to mantle length are obviously dependent on the size of the attacking squid. As a significant size variation exists within age groups, and because neither individual nor mean lengths were determined for animals in either group, the effects of mantle size on the analyses presented here cannot be rigorously assessed. One might argue that if attack distances and speeds were plotted on absolute scales, the apparent shifts in the character of attacks between age groups would disappear. Several points argue against this scenario, however. First, in comparing successful attacks between the youngest squid and the intermediate-aged squid, the use of absolute scales would further accentuate the differences in successful attack character evident in Figs. 6A and 6B due to the more successful exploitation of a long distance strategy by the larger, intermediate-aged squid Similarly, absolute comparisons of successes between intermediate-aged and the oldest squid could be argued to lead to a convergence of attack distances in the histograms presented in Figs. 6B and 60. However, the shapes of the histograms for these two groups appear to be decidedly different, and a simple adjustment of the curves by a constant factor to compensate for average growth would not render the two histograms equivalent. Finally, unique specialized capture strategies observed to emerge in specific age groups can account for improved performance at the appropriate distance and serve to distinguish each age group from the others. The arm-intercept technique that allows squid to capture copepods from greater distances first appeared in day 29 squid and coincided with the increase in successful long distance attacks in the intermediate-aged squid. Similarly, the oldest squid (day 41-42)--the only age group to display the low distance, low speed tentacular grab strategy--exhibited more successful attacks at shorter distances The observation of these characteristically unique attack strategies occurring in the two age groups opposes the argument that attacks by the intermediate-aged and by the oldest squid differ only relative to the attacking squid's mantle length. Juvenile squid raised on copepods demonstrate a progressive development of complex, acquired prey capture behaviors. Squid raised on Artemia nauplii and then 18 switched to a copepod diet demonstrate a lack of such acquired abilities, as evidenced by a severe mortality rate after the switch. When considering the results of this study, one must keep in mind that copepods are but one of many potential prey species in the wild. While this study may provide insight into development in the field, the complete picture is surely much more complex as juvenile squid most likely feed on a multitude of species and may have specific strategies for each species. Just as Artemia fed squid appear handicapped in prey capture ability when introduced to copepods, the relatively competent squid raised on copepods may likewise be impaired if they were to be introduced to the wild. Such knowledge would be important if, in the future, squid reared in tanks were released into the wild, for tracking studies or for attempts at repopulation, for example. For mariculture, results of this study reflect on the advisability of certain abrupt changes in feeding regimen, Neurobiologist may be able to capitalize on behaviorally impaired organisms, such as the Group three squid, to search for neurological signs of the behavioral deficit in these organisms. Finally, the notion of experience-based modules of behavioral development may be a factor to consider in designing neurobiological experiments. Acknowledgements: We would especially like to thank Gilbert Van Dykhuizen and the Monterey Bay Aquarium for their assistance and the use of their facilities in rearing the squid, and for their valuable input into this study. 19 VI. Figure Legends: Figure 1. Daily Mortality Count. This chart details the mortalities in Group two and Group three squid. The feeding regimen for both groups of squid is indicated by the horizontal bars directly beneath the X-axis. Note the dramatic increase in mortality in Group three after the switch to a copepod diet on day 40. Figure 2. The Basic Attack Sequence. Squid utilized this most general strategy, the first to be observed in the youngest squid, in attacks on both copepods and Artemia nauplii. Figure 3. "Circling" Behavior. Before lunging at the prey item, squid often maneuvered angularly to obtain position in front of the copepod prey. As the squid repositioned itself in response to the copepod's escapes, its overall motion appeared circular. Figuie 4. Successful Copepod Attacks Grouped by Age. This scatter plot shows the distribution of successful copepod captures by Group two squid along axes of total attack distance and peak attack speed. Note the correlation between total attack distance and peak attack speed, as revealed by the distribution of points along the 45 degree line. Figure 5A. Successful and Unsuccessful Attacks by the Youngest Squid. 5B. Successful and Unsuccessful Attacks by the Intermediate-Aged Squid. 5C. Successful and Unsuccessful Attacks by the Oldest Squid. A comparison of these scatter plots reveals that, with age, the distribution of attacks on copepods is shifted towards shorter distances and lower speeds. Note, once again, the apparent correlation between attack distance and attack speed in both successful and unsuccessful attacks. Figure 6A. Successful Captures, Group 2, Days 15-23. 6B. Successful Captures, Group 2, Days 26-35. 6C. Successful Captures, Group 2, Days 41-42. These histograms of successful captures reflect the changing character of successful copepod captures. Refinements in strategy allow the intermediate-aged squid to expand their effective copepod capture range, and also facilitate captures by the oldest squid from shorter distances. Figure 7A. Unsuccessful Attacks, Group 2, Days 15-21. 7B. Unsuccessful Attacks, Group 2, Day 33. 7C. Unsuccessful Attacks, Group 2, Day 42. These histograms depict the attack distance distributions in unsuccessful attacks. The upper range of attack distances present in days 15-21 squid has almost completely disappeared in the older squid. 20 Figure 8. The Arm-Intercept. First observed in day 29 squid, this strategy allows squid to capture copepods from greater distances. Figure 9. The Tentacular Grab. A low distance, low speed attack strategy, the tentacular grab, observed in day 41-42 squid, may facilitate copepod captures at reduced energy expenditures. Figure 10. Attacks on Copepods, Group 3, Days 41-43. The presence of ineffectual, long distance attacks in these Artemia-raised squid, and the similarity in attack distance distribution between these and the younger, day 15-21 squid (Fig. 7A) may reflect developmentally impaired prey capture abilities that resulted from a lack of exposure to fast moving copepod prey early in life. 21 VII. References Cited: Hanlon, R.T. and Hixon, R.F., 1983. Laboratory maintenance and culture of octopuses and Loliginid squids in Culture of Marine Invertebrates, C.J. Berg, Jr., ed., Hutchinson Ross, Stroudsburg, Penn, pp. 44-61. Hurley, A.C., 1976. Feeding behavior, food consumption, growth, and respiration of the squid Loligo opalescens raised in the laboratory. Fisheries Bulletin, 74(1): 176- 182. Kier, W.M., 1982. The functional morphology of the musculature of squid (Loliginidae) arms and tentacles. Journal of Morphology, 172: 179-192. Yen, J. and Fields, D.M., 1992. Escape responses of Acartia hudsonica (Copepoda) nauplii from the flow field of Temora longicornis (Copepoda). Arch. Hydrobiol. Beih., 36: 123-134. 22 29 09 O 0e 92 — 8 o statakakakaka- Seo 1o lequin 54 5582 JOE The Basic Attack Sequence (1) Squid postures at copepod (3) Arms open wider (5) Arms close: squid catches copepod Fig. 2 (2) Arms begin to open (4) Squid lunges at copepod "Circling" Behavior (1) Squid moves to achieve position in front of copepod (3) Squid moves laterally to regain position in front of copepod S L 9 (2) Copepod leaps away (4) Copepod jumps to new position (6) Copepod moves (8) Copepod moves (9) Squid reacts, etc. Fig. 3 (5) Squid once again maneuvers in front of copepod (7) Squid reacts S . 89 8 oo * DX 9 (ue/) peeds yeny eed o X X X X X XX X( XX XX X X X X oo (ue) peeds yoeny yeed . T 0 5 0 — X Xn XX — ooo O uen) peeds yoeny yeed 0 — X X XXX XXX X oo 8 eny 0 Successful Captures, Group 2, Days 15-23 C - k taava- Attack Distance (ML) Figure 64 Successful Captures, Group 2, Days 26-35 9 O — 8 8 Attack Distance (ML) Figure 6B 10 4 0 Successful Captures, Group 2, Days 41-42 a taatai 8 Attack Distance (ML) Figure 60 Unsuccessful Attacks, Group 2, Days 15-21 20 18 14 5 10 O L oooo--ood-onooo addaadad-------- aaaa Attack Distance (ML) Figure 74 20 14 Unsuccessful Attacks, Group 2, Day 33 N oooo--ooo-oonooo - - ------addodooi ooooooo Attack Distance (ML) Figure 20 Unsuccessful Attacks, Group 2, Day 42 0 ooo--oood-oonooo — — — — — — — — — oooodoo o v di di di di di di oi Attack Distance (ML) Figure 70 The Tentacular Grab 5 Fig. 8 (1) Squid postures near copepod (2) Squid arms begin to open (3) Squid arms open; tentacles begin to extend (4) Tentacles fully extend and grab copepod (5) Tentacles retract; copepod captured The Arm-Intercept Fig. 9 (1) Squid circles to achieve position in front of copepod (2) Squid arms begin to open S (3) Arms open wide (4) Squid lunges (a), copepod makes escape attempt (b) but runs into squid's arms (5) Squid grabs copepod-successful capture Figure 10 Attacks on Copepods, Group 3, Days 41-43 20 18 Note: All attacks were unsuccessful 5 10 ooo--oood-ooooo —————— — —— oooooo div oi ddoicioioi Attack Distance (ML)