Rapid conduction in myelinated nerves keeps distant parts of large organisms in timely communication. It is thus surprising to find myelination in some very small organisms. Calanoid copepods, while sharing similar body plans, are evenly divided between myelinate and amyelinate taxa. In seeking the selective advantage of myelin in these small animals, representatives from both taxa were subjected to a brief hydrodynamic stimulus that elicited an escape response. The copepods differed significantly in their ability to localize the stimulus: amyelinate copepods escaped in the general direction of their original swim orientation, often ending up closer to the stimulus. However, myelinate species turned away from the stimulus and distanced themselves from it, irrespective of their original orientation. We suggest that faster impulse conduction of myelinated axons leads to better precision in the timing and processing of sensory information, thus allowing myelinate copepods to better localize stimuli and respond appropriately.
Because predator-avoidance strategies, such as escape responses, are under strong selective pressure in the ‘arms race’ between predator and prey, they have provided good models for investigating the linkages between evolution, ecology, behavior and physiology (Card, 2012; Herberholz and Marquart, 2012). Adaptations for high-speed nerve impulse conduction are ubiquitous features of the neural circuits mediating such behavior (e.g. Hartline and Colman, 2007). Exceptional escape behavior has evolved in pelagic planktonic copepods, which have no place to hide from diverse and numerous predators. Escapes – elicited by small mechanosensory cues and characterized by short latencies, high accelerations and maximum swim speeds – appear to be mediated through a giant fiber system, which promotes rapid impulse conduction (Alcaraz and Strickler, 1988; Boxshall, 1992; Lenz and Hartline, 1999; Park, 1966; Yen et al., 1992). In addition to giant fibers, some calanoid copepods have evolved to have myelin (Davis et al., 1999; Hartline and Colman, 2007), raising the question of how higher conduction speed could be an advantage in such small organisms (<10 mm).
Historically, the benefits of myelin have been deduced from studies on isolated nerve (Bullock and Horridge, 1965). Direct comparisons between myelinate and amyelinate organisms have been lacking. The biological distances and morphological differences between organisms like myelinate frogs and amyelinate squids preclude such behavioral comparisons. Our study minimizes confounding factors by using copepods that are closely related, occupy similar ecological niches, share an evolutionary path and are similar in their body plan (Hutchinson, 1961; Huys and Boxshall, 1991). While calanoid copepods can be divided into two large groups based on their myelin (Davis et al., 1999; Lenz et al., 2000), their physiology, anatomy and behavior are similar. The musculature of the swimming legs and the force output during the escape are similar (Boxshall, 1992; Hartline et al., 1999; Lenz and Hartline, 1999; Lenz et al., 2000), as is the organization of their nervous system, including the innervation of the escape circuitry (Lenz et al., 2000; Lowe, 1935; Park, 1966; Wilson and Hartline, 2011). Both possess highly sensitive antennular mechanoreceptors that are similar in structure (Weatherby and Lenz, 2000; Yen et al., 1992); and both use either antennular sweeps or a backward rotation to redirect their escape swim (Gill, 1986; Gill and Crips, 1985; Lenz et al., 2004; Park, 1966).
Both myelinate and amyelinate calanoid copepods are targets of high-speed attacks from predators such as planktivorous fish and chaetognaths (Coughlin and Strickler, 1990; Gemmell et al., 2013; Holzman et al., 2007; Wainwright et al., 2001, 2007). While copepods that possess myelin may react faster when evading predators (Davis et al., 1999; Lenz, 2012; Lenz et al., 2000), the predicted difference in response time is less than 1 ms in small species (≤1 mm) (Bradley et al., 2013; Waggett and Buskey, 2008). Nevertheless, species possessing myelin inhabit environments with higher predation risk (Lenz, 2012) forcing the question: might myelin confer other behavioral advantages beyond a saving in response time? We addressed this experimentally by comparing escape behaviors of copepods of similar size, morphology and ecology, but differing in the presence or absence of a myelinated nervous system.
MATERIALS AND METHODS
Escapes were analyzed for five small copepod species: two myelinates [Bestiolina similis (Sewell 1914) and Parvocalanus crassirostris (F. Dahl 1894)] and three amyelinates [Acartia tonsa Dana 1849, Eurytemora affinis (Poppe 1880) and Centropages hamatus (Lilljeborg 1853)]. Multiple developmental stages were tested [nauplii: stages NI–NVI; copepodites: CI–CIII and CVI (CVI: E. affinis, P. crassirostris only)]. Animals ranged in total length from 80–100 µm for early nauplii to 1.4 mm for E. affinis (stage CVI).
The two myelinate species were collected from Kaneohe Bay, Island of Oahu, HI, USA (21°26.3′N, 157°47.0′W), two amyelinate species (E. affinis and C. hamatus) were collected in Frenchman Bay, ME, USA (44°25.7′N, 66°11.8′W), and the fifth species (amyelinate: A. tonsa) was collected from the channel in Port Aransas, TX, USA (27°50.0′N, 96°3.7′W). The behavioral experiments on the species collected in Maine and Texas were done at the Marine Science Institute (Texas), and the two species collected in Hawaii were tested at Békésy Laboratory (Hawaii) using duplicate behavioral set-ups (Bradley et al., 2013). Eggs were collected from adult copepods and grown in cultures as described previously (Bradley et al., 2013). For the behavioral experiments, sets of individuals were transferred into the experimental chamber (1.25×1.25×4.5 cm) at densities of 7–15 individuals per ml for nauplii and 1.5–3 individuals per ml for copepodites. A larger experimental chamber (4.5×4.5×6 cm) was used for adult (stage CVI) E. affinis.
Escapes from a fluid disturbance and the swim sequence just prior to the stimulus were recorded on high-speed digital video (500 frames s−1) in 3D. The optical set-up included prisms and beam splitters to obtain front and side views on each frame simultaneously (Bradley et al., 2013; Strickler, 1998). Thus, a single camera (Photron FastCam 10K series or Kodak Motioncorder SR-3000) was used to obtain paired high-resolution images of individual copepods (x/z front view, y/z side view) (Bradley et al., 2013).
The hydromechanical stimulus was abrupt, in contrast to siphon flows and other stimuli that are characterized by slowly rising deformation rates (Burdick et al., 2007; Fields and Yen, 1996; Strickler and Balazsi, 2007; Titelman and Kiørboe, 2003). A 3 mm-diameter inert plastic sphere was attached to a stiff rod mounted to a piezoelectric pusher (DSM LPA 100 Dynamic Structures) and controlled by a pulse trigger that displaced the sphere downward by 35 μm in 0.5 ms, and then returned to its initial position 60 ms later (Bradley et al., 2013). The sphere was positioned in the upper quarter of the optical vessel, such that the majority of free-swimming copepods were oriented towards the stimulus.
More than 800 escape sequences were analyzed. The copepods' body-axis orientation just prior to the stimulus trigger was recorded, as was the direction of its subsequent escape jump. Initial orientation and escape direction were categorized using a simple classification system: towards the sphere or away from it, according to the angle relative to the horizontal plane. In a few cases, the copepod's swim/escape direction was perpendicular to the stimulus (±5 deg from horizontal), and these were excluded from the analysis (fewer than 5% of observations). The data were analyzed independently by three authors, with all working on data from both groups. One author analyzed one pair, and two authors analyzed the three remaining species (one myelinate and two amyelinate species) twice.
Results for each group (myelinate versus amyelinate) were combined and tested using the Fisher exact test (two-tailed) to compare swimming behavior of the two groups. Nauplii and copepodites were tested separately. Specific comparisons included: (1) swim direction prior to the stimulus; (2) whether the escape direction was in the direction of the original swim (original swim direction versus reverse direction); and (3) whether escapes were directed towards or away from the stimulus (towards sphere versus away from sphere). The 2 ms temporal resolution did not allow for a quantitative comparison of reaction times between myelinate and amyelinate species as responses occurred within one or two video frames. Maximum swim speeds were computed on a subset of escapes for B. similis and A. tonsa from the change in the copepod's x, y and z positions between video frames as described previously (Bradley et al., 2013). Data for P. crassirostris, E. affinis, C. hamatus and adult A. tonsa were obtained from the literature (Bradley et al., 2013).
RESULTS AND DISCUSSION
Prior to the stimulus trigger, the majority of copepodites and nauplii were oriented upward, toward the stimulus (80% and 72%, respectively), with no difference in orientation between myelinate and amyelinate groups for either nauplii or copepodites (P>0.8, Fisher exact test). All five species and their developmental stages responded with an escape to the hydromechanical stimulus out to a distance of 5 mm, with the probability of a response decreasing with distance. Maximum escape speeds scaled to body length were similar in myelinate and amyelinate taxa in the five species (Fig. 1), adding to the set of shared characteristics that make calanoids especially suitable for myelin-related behavioral comparison.
The escape response of copepodites often includes an initial re-orientation of the body axis just prior to the power stroke by the pereiopods: either a backward rotation of the prosome by urosome elevation or an asymmetrical sweep of the antennules (Burdick et al., 2007; Buskey et al., 2002; Lenz et al., 2004; Park, 1966; Strickler, 1975; Strickler and Bal, 1973). In the present set-up, with the stimulus coming from above, when copepodites were oriented away from the sphere, i.e. down, the tendency to re-orient at the beginning of the escape contributed to the observation that ca. 25% of the escapes involved a reversal in direction (Fig. 2A) that was directed towards the sphere, and this percentage was over 50% in one amyelinate species (E. affinis; Fig. 2A).
Dramatic differences between myelinate and amyelinate copepodites with respect to the direction of the escape were observed when the original swim direction was oriented towards the sphere (Fig. 2B). The majority of myelinate copepodites executed a strong turn and directed their escapes away from the sphere, while the escape direction of the amyelinate ones was mostly towards the sphere (Fig. 2B). Thus, the myelinate copepods were far more likely to reverse the direction of their escape than the amyelinate ones (P<0.0001, Fisher exact test; Table 1). Furthermore, regardless of original orientation, myelinate copepodites were significantly more likely to direct their escape away from the stimulus than amyelinate ones (P<0.0001, Fisher exact test; Table 1). Given that the myelinate copepods in this study (adult length ∼0.5 mm) were smaller than the amyelinate ones (adult length ∼1 mm), this result is contrary to the prediction that larger copepods would be better at localizing a stimulus than smaller ones (Kiørboe and Visser, 1999).
Nauplii use a different set of appendages for swimming, and while they can change swim direction, their escapes are mostly forward (Borg et al., 2012; Bradley et al., 2013; Lenz et al., 2015). Thus, not surprisingly, escapes of nauplii of both myelinate and amyelinate taxa were primarily in the direction of their original orientation (Fig. 2C,D). After completing an escape, nauplii were usually closer to the sphere when their original orientation was towards it, and farther away when they were oriented away. Over 70% of nauplii kept the same escape direction as their original orientation, and there was no difference between myelinate and amyelinate nauplii (P=0.67, Fisher exact test; Table 1). Interestingly, in the second statistical analysis, which compared the direction of the initial escape with respect to the stimulus location, we found a significant difference between myelinate and amyelinate nauplii (0.05>P>0.01, Fisher exact test; Table 1). This difference was opposite from findings for the copepodites: a larger number of nauplii (58%) in the myelinate group directed their escapes towards the stimulus compared with the amyelinate nauplii (46%; Table 1). Further studies are needed to understand the reasons underlying this difference in behavior.
The data have focused on whether the copepod escaped toward or away from the source of the hydrodynamic disturbance. Two alternative hypotheses need to be considered. First, suppose the myelinate copepods have a greater tendency to turn when stimulated. In this case, with 80% of copepodites oriented toward the stimulus to start with, there would be a greater percentage of myelinates, having turned, to escape away from it. The evidence against this is that for copepodites initially facing away from the stimulus, the turning tendency was the same or greater for the amyelinate species. Second, suppose that startled myelinate copepods preferentially escape downward. With the stimulus located above the copepod in our set-up, a downward tendency would coincide with an escape directed away from the stimulus. Thus, we tested the myelinate copepodites (B. similis and P. crassirostris) using a non-directional hydromechanical stimulus (a sphere tapping the side of the observation chamber). Under these conditions, escapes (n=43) were directed upward (49%), downward (35%) and sideways (16%).
Copepodites of the myelinate species had the ability to detect the location of the stimulus and react appropriately; this difference was not observed in nauplii. Early developmental stages (nauplii) have less well developed antennular sensory systems (Boxshall and Huys, 1998), they use the antennule for locomotion (Lenz et al., 2015), they are less sensitive to hydromechanical stimuli (Bradley et al., 2013), and the nauplii of myelinate species possess much less myelin (Wilson and Hartline, 2011). Thus, it is not surprising that the differences between myelinate and amyelinate species are not present in nauplii, but become apparent in the copepodite form.
How might this orientation to the stimulus be achieved by myelinate copepods but not by amyelinate ones? Calanoid copepods detect hydromechanical signals with highly sensitive mechanosensory setae on their appendages and elsewhere on their bodies (Lenz and Hartline, 2014; Strickler and Bal, 1973; Yen et al., 1992). The signal used in our experiments, being an abrupt near-field water displacement, would arrive at about the same time at all setae distributed over the antennules, appendages and body. However, the signal strength (i.e. water deformation) would differ, as it decays as a fourth power with distance from the source. With nerve cells starting to fire at some threshold depolarization, dependent on the strength and rise-time of the deformation, neurons innervating sensory hairs closer to the stimulus would fire earlier than those farther away. Sequential activation of spatially separated sensors is a well-established mechanism for decoding directional cues (Barlow and Levick, 1965; Hager and Kirchner, 2014; Knudsen and Konishi, 1979). However, inherent variability in conduction speeds (ca. 20%) within any particular axon will affect the ability to differentiate small differences in timing (Wang et al., 2008). In such a case, the faster spikes in the myelinated axons would provide more precise temporal separation of the signals arriving at the central nervous system. In addition, higher conduction speed allows more time to integrate multiple sensors or make more sophisticated calculations before deciding on a response. In neuronal processing, ‘microseconds matter’ (Carr and MacLeod, 2010), as precision in those microseconds is essential for sensory localization and accurate motor control (Calvin, 1983; Hager and Kirchner, 2014). Myelin is an innovation capable of precision-tuning of microsecond-scale timing (Seidl et al., 2010).
Escapes mediated by giant fibers are fast and are used in responses that require minimal processing (Herberholz and Marquart, 2012). Our behavioral results suggest that the added computational accuracy and processing capabilities conferred by myelin are sufficient to localize the abrupt stimulus more precisely to orient the escape away from the attack. The experimental results demonstrate a key advantage of myelin, which might have been a factor in its evolution. While this myelin advantage allows for more sophisticated processing, escapes elicited with longer delays have less need for high-speed processing. In these cases, one would predict that stimuli are localized accurately, regardless of the state of myelinization, a hypothesis for future testing.
We would like to thank Hinano Akaka, Ted Murphy and Brian Kodama for technical assistance and construction of apparatus, and Cammie Hyatt for culturing assistance. This is the University of Hawaii at Manoa School of Ocean and Earth Science and Technology contribution number 9921.
Conceived and designed experimental set-up and study: E.J.B., J.R.S., P.H.L., D.K.H. Performed experiments in Texas and Hawaii: E.J.B., C.J.B., J.R.S., D.K.H., P.H.L. Analyzed the data: E.J.B., C.J.B., P.H.L. Wrote the manuscript: E.J.B., J.R.S., D.K.H., P.H.L.
Financial support was provided by the National Science Foundation (OCE 04-51376; OCE 12-35549 to P.H.L. and D.K.H.; and OCE 04-52159 to E.J.B.). The views expressed herein are those of the authors and do not reflect the views of NSF or any of its sub-agencies.
Data are available through the Biological & Chemical Oceanography Data Management Office (BCO-DMO) under project: The drive to survive: copepods vs. ichthyoplankton (http://www.bco-dmo.org/project/562097).
The authors declare no competing or financial interests.