ABSTRACT
DEVELOPING A MECHANICAL MODEL OF A SUCTION FEEDER
Suction feeding is a common feeding mode in macroscopic aquatic organisms and the dominant feeding mode in fish. In contrast, microscopic aquatic organisms do not use suction feeding. In fact, the smallest known suction feeders are fish larvae and bladderwort, a genus of carnivorous plants that catches zooplankton in underwater traps, both of which have gapes around 200 microns in diameter. Experimental and theoretical studies of suction feeding have shown that the ability to generate a steep spatial pressure gradient correlates strongly with capture success. Those studies also show that suction feeding is essentially an inertial process and therefore will be effective only if viscous fluid forces can be neglected, which is as long as the gape is large enough and the suction flow (i.e. the negative pressure gradient near the mouth) fast enough to minimize the relative effects of friction. Our current understanding of the hydrodynamics of suction feeding suggests that suction feeding is not effective in small organisms. In fact, both mathematical models of suction feeding, and experimental observations of larval fish suggest that their gape of 200 microns is near the lower size limit of suction feeding and that their suction flows generated by a 0.2 kPa pressure differential are too weak to ensure prey capture. In this project, we explored the lower size limit of suction feeding by characterizing the suction flows of bladderwort and salamanders and using the data collected to develop a robotic model of a suction feeder.
Fatima Hidalgo May 2018
DEVELOPING A MECHANICAL MODEL OF A SUCTION FEEDER
by Fatima Hidalgo
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Biology in the College of Science and Mathematics California State University, Fresno May 2018 APPROVED For the Department of Biology:
We, the undersigned, certify that the thesis of the following student meets the required standards of scholarship, format, and style of the university and the student's graduate degree program for the awarding of the master's degree.
Fatima Hidalgo Thesis Author
Ulrike Müller (Chair) Biology
Otto Berg Chemistry
Stephen Deban University of South Florida, Integrative Biology
For the University Graduate Committee:
Dean, Division of Graduate Studies AUTHORIZATION FOR REPRODUCTION OF MASTER’S THESIS
X I grant permission for the reproduction of this thesis in part or in its entirety without further authorization from me, on the condition that the person or agency requesting reproduction absorbs the cost and provides proper acknowledgment of authorship.
Permission to reproduce this thesis in part or in its entirety must be obtained from me.
Signature of thesis author: ACKNOWLEDGMENTS I would like to thank my advisor and mentor Dr. Ulrike Müller for not letting me quit and knowing where I could focus for my project. I would also like to thank Dr. Otto Berg and Dr. Stephen Deban for the support given as committee members and during the research process. I would also like to thank my fellow lab mates; Ray Kabir, Nolan Avery, Andrea Aparicio Ramirez, Max Hall and Mohammed Shaik. I would also like to acknowledge the help provided by the Deban lab at University of South Florida, especially Dr. Charlotte Stinson. I would like to recognize the support of my family, friends and shipmates that have been by my side during this journey. Lastly, I would like to acknowledge the funding from Bridges to Doctorate program, National Science Foundation, and Sally Casanova Pre-Doctoral Scholarship that has allowed me to present this research and supported me.
TABLE OF CONTENTS Page
LIST OF TABLES ...... vi
LIST OF FIGURES ...... vii
INTRODUCTION ...... 1
Background ...... 1
Suction Feeding in Organisms ...... 1
Morphology of Suction Feeders ...... 3
Aims and Objectives ...... 15
METHODS AND MATERIALS ...... 17
Husbandry ...... 17
Morphological Measurements of Lab Grown Bladderworts ...... 17
Selecting Bladderwort for High Speed Filming ...... 18
High Speed Filming of Feeding Event ...... 19
Data Analysis ...... 20
Building and Operation of a Mechanical Suction Feeder ...... 21
RESULTS ...... 25 Characterizing the Suction Feeding Morphology and Motion of Bladderwort and Salamanders ...... 25 Characterizing the Suction Feeding Flows of Bladderwort and Salamander ...... 28
Building a Mechanical Model of a Suction Feeder ...... 31
DISCUSSION ...... 33
REFERENCES ...... 38
APPENDIX: LITERATURE DATA ...... 44
LIST OF TABLES Page
Table 1 Gape Diameters of Suction Feeders ...... 25
Table 2 Time to Peak Gape (TTPG) of Suction Feeders ...... 27
Table 3 Flow Characterization of Suction Feeders ...... 28
Table 4 Scaling Parameters ...... 32
LIST OF FIGURES
Page
Figure 1. Images showing maximum hyobranchial expansion of aquatic feeding salamanders and their respective clear and stained hyobranchial apparatus...... 5 Figure 2. Diagram of the hyobranchial apparatus of G. porphyriticus...... 6 Figure 3. From Poppinga et al., 2016 – Figure 1. “General bladderwort morphology, depicted exemplarily by U. vulgaris...... 8 Figure 4. From Poppinga et al. (2016) – Figure 2. “Lateral view of different trap types, indicating the position of the trap entrance (te) and of the stalk (st) ...... 9 Figure 5. From Poppinga et al. 2016 – Figure 5. Morphology of the trap body ... 11 Figure 6. Developing flow through entrance of a pipe...... 14 Figure 7. Linear motor controller and power supplies...... 22 Figure 8. Linear motor with slider...... 23 Figure 9. Gantry crane schematic that was sent from Numatic engineering...... 23
Figure 10. Graph showing how body length scales to gape diameter...... 26 Figure 11. Graph showing how body length scales with time to peak gape for U. praelonga, Ambystoma sp., G. porphyriticus, and fish...... 27 Figure 12. Graph showing the scaling coefficient of U. praelonga, Ambystoma sp., and G. porphyriticus...... 29 Figure 13. Still shot from particle tracking video of G, porphyriticus showing path particles traveled...... 29 Figure 14. Still shot of from particle tracking video of Ambystoma sp. showing path particles traveled ...... 30 Figure 15. Still shot of U. praelonga showing path particles traveled ...... 30 Figure 16. Graph showing time to peak gape, peak flow, and gape vs body for bladderwort, fish at different life stages, and salamanders...... 37
INTRODUCTION
Background Organisms have various modes of feeding. Using suction is a mode that various aquatic organisms use to get nutrients and energy. Suction feeding is done by larval fish, adult fish, larval salamanders, adult salamanders, and some carnivorous plants. Suction can be drawn by an organism in various ways. One of the ways is by having a difference between the internal pressure and the environmental pressure. The differential pressure is established through stored elastic energy. The other way that suction can occur is by expanding the internal volume, which can be done by changing the volume of the buccal area. This movement of the buccal area is produced by kinematic energy. Both of these methods exert forces upon the prey and cause them to be entrained with the fluid that is being sucked into the organism’s mouth. In this study we looked at suction feeding of various organisms to aid in designing and building a mechanical model that will simulate suction feeding. The suction feeders we looked at are two species of larval salamander, one species of paedomorphic salamander, and three species of bladderwort.
Suction Feeding in Organisms
Suction Feeding in Adult Fish Suction feeding has been studied extensively in adult fish (Wainwright and Day, 2007; Holzman et al., 2012; Yaniv et al., 2014; Day et al., 2015). Suction feeding in adult fish can be represented as an equation that relates the pressure inside the mouth cavity, to the flow field outside the mouth, until the gills open (Day et al 2007). Adult fish accomplish suction feeding by establishing strong 2 2 unidirectional flow of water into the expanding mouth (Day et al. 2015). Using adult bluegill fish for a study, it was found they reached flow velocities of 5 m/s and a Reynolds number of around 50,000 (Higham et al., 2006). Adult fish also use other strategies, such as ram feeding and jaw protrusion, to supplement suction feeding. Both supplementary strategies allow the fish to get closer to the prey, which brings the prey into the pressure gradient field generated by suction and then accelerates the prey into the fish’s mouth. In adult fish, pressure gradients exerted on prey’s bodies are the strongest predictor of successful prey capture for elusive prey that is swimming or suspended in the water column (Holzman et al., 2012).
Suction Feeding in Larval Fish In contrast to adult fish, much less is known about suction feeding in larval fish. Due to their small body sizes and slow flow speeds, larval fish experience a viscous hydrodynamic regime characterized by low Reynolds numbers, where viscous forces are dominant (China & Holzman, 2014). Experimental and computational studies on larval fish suggest that they operate at Reynolds numbers below 10 (Yaniv et al. 2014, Pekkan et al., 2016) and experience high viscous forces, which reduces their hydrodynamic efficiency (Drost et al., 1988). Due to their poor suction feeding performance, larval fish have a high mortality rate in the first few days post hatching even in areas with a high density of food (Shields, 2001; Holzman et al., 2015). Experimental observations show that larval fish generate weak suction flows at 0.008 m/s (Pekkan et al., 2016) and slow onset flows: larval carp 6mm in length will have a suction feeding event that will last 400 ms (Drost et al, 1988). 3 3 Suction Feeding in Amphibians Suction feeding has also been studied in amphibians. Two studies have been published so far on the flow in suction feeding. One study focuses on suction feeding in tadpoles (Hymenochirus sp.) (Deban and Olson, 2002). It found that tadpoles could capture prey in 4 ms and operated at a Reynolds number of 300. Having a Reynolds number of 300 places them in the intermediate flow regime. The second study describes suction feeding in adult salamanders (Stinson & Deban, 2017). That study found that one of the highest flow velocities was 0.515 m/s. The salamander that produced the highest velocities was an adult aquatic species compared to other species, which were semiaquatic species. So far, we have no flow data on suction feeding in larval and paedomorphic salamanders.
Suction Feeding in Carnivorous Plants Suction feeding also occurs in carnivorous plants, in particular the genus Utricularia (Westermeier et al., 2017). Bladderworts (genus Utricularia) are the largest genus of carnivorous plants with more than 220 species (Poppinga et al., 2016), accounting for more than 40% of all carnivorous plant species (Chormanski & Richards, 2012). Utricularia contains the subgenus Utricularia, the subgenus in which all the bladderwort species of this study are located. So far, studies of Utricularia suction feeding have focused on the mechanics of the trap door rather than on the fluid mechanics. This study will focus on suction feeding in those two understudied groups, larval and paedomorphic salamanders and bladderwort.
Morphology of Suction Feeders
Salamander Habitat and Morphology The habitat of the larval salamanders tends to be in aquatic environments. Gyrinophilus porphyriticus and Desmognathus quadramaculatus are found in the 4 4 gravel beds of the springs in the mountains of North Carolina (Bruce, 1980). The larval period of D. quadramaculatus last 35-48 months (Titus & Larson, 1996), and the larval period of G. porphyriticus is 44-47 months (Bruce, 1980). The snout vent length at metamorphosis of D. quadramaculatus is 36-45 mm. (Tilley & Bernardo, 1993). The snout vent length of larval G. porphyriticus is 19 mm at hatching and grows to 38-40 mm within the first 30 months of their lives (Bruce, 1980). Salamanders in the genus Ambystoma are also commonly known as mole salamanders. There are currently 32 species in that genus which can be found only in North America (AmphibiaWeb, 2018). Larval Ambystoma sp. can be found in ponds and slow streams (Larson, 1996). The snout vent length for larval Ambystoma sp. can range from 50.2 – 61.4 mm (Irschick & Shaffer, 1997). In a study of Ambystoma texanum, it was found that the larval period ranged from 31.4 – 46.7 days depending on if they lived in streams or ponds (Petranka & Sih, 1987).
Salamander Mouth Morphology The morphology of the mouth of larval salamanders is that they have a hyobranchial apparatus that has different percentages of cartilage and bone (see figure 1). Suction feeding of aquatic salamanders is described as the buccal cavity expanding by the depression of the hyobranchial apparatus and the water being sucked in through the mouth and exiting through the gill slits (Deban & Wake, 2000). In the study by Stinson and Deban (2017), a fully aquatic newt had a larger feeding musculature than other semiaquatic newts. The larval hyobranchial apparatus consists of interlinked cartilages forming a four bar linkage (Deban & Wake, 2000) (see figure 2).
5 5
Figure 1. Images showing maximum hyobranchial expansion of aquatic feeding salamanders and their respective clear and stained hyobranchial apparatus. (Stinson and Deban 2017)
6 6
Figure 2. Diagram of the hyobranchial apparatus of G. porphyriticus. Bb1 is the basibranchial 1. Eb1 is the epibranchial 1. Ch is the ceratohyals. Cb1 and Cb2 are the ceratobranchial. (Piatt 1935)
7 7 Salamander Prey Most of the diets of larval salamander consisted of prey that are small enough for them to get through the maximum gape diameter (Deban and Wake, 2000 and references therein). In the study by Dodson and Dodson (1971), they found that the diet of Ambystoma tigrinum larvae consisted mainly of arthropods and mollusks. The diet of larval D. quadramaculatus mainly consisted of insect nymphs (Davic, 1991). According to Bruce (1979), G. porphyriticus larval diet consists of various invertebrates such as arachnids, mayflies, and odonates.
Bladderwort Habitat and Morphology Bladderwort can be found in various nutrient poor habitats around the world. Habitats range from aquatic, terrestrial, to epiphytic. In aquatic habitats, bladderworts are free floating in the water column. The terrestrial bladderworts are found in soil around bogs, and epiphytic bladderworts are found on rocks. Bladderworts do not have roots, instead terrestrial and epiphytic species use rhizoids to anchor themselves (Reifenrath et al., 2006; Poppinga et al., 2016). Bladderworts are considered to have a highly derived morphology (Chormanski & Richards, 2012). The main parts of a bladderwort plant are rhizoids (absent in aquatic species) and stolons. Rhiziods are filaments that form a connection between plants and substrates (Jones & Dolan, 2012). Stolons are lateral-running, branch-like structures to which leaves, and bladders attach (figure 3). The bladders are modified leaf structures that help the plant uptake nutrients from caught prey. Bladders constitute typically 10 to 50% of the total plant biomass (Adamec 2006, 2007, 2010; Friday, 1992; Richards 2001), but number and size of the bladders vary seasonally (Friday, 1992; Guiral & Rougier, 2007; Richards, 2001) and some species, such as U. vulgaris, have dimorphic traps (Friday, 1991; Adamec, 2007; Guiral & Rougier, 2007). Bladderwort plants typically do not 8 8 reach an overall length of 30 cm, but some species, such as U. vulgaris, can form strands that are over 2 m long (Poppinga et al., 2016). The total number of bladders and leaves per strand varies widely between species: U. vulgaris forms dense strands with many hundreds of bladders and leaves per strand (figure 3); in contrast, U. gibba forms very sparse strands (personal observation).
Figure 3. From Poppinga et al., 2016 – Figure 1. “General bladderwort morphology, depicted exemplarily by U. vulgaris. (A) Young plant, resprouting from hibernation. The stolon (sto), leaves (le) and a branching point (bra) are clearly visible. (B) View of a detached leaf foliar shoot node featuring a trap (tra) dimorphism. Note the stolon remnant and a small, morphologically divergent trap. ‘Normal’ traps are dispersed on the pinnate leaves. (C) Detailed view of the leaf base, note the trap stalk (st). (D) Inclined frontal view of a trap. The trap entrance (te) possesses a door, a threshold (th), ‘antennae’ (an) and ‘bristles’ (br). The lateral trap wall (tw) is concave. Hence, the trap has generated underpressure inside and is ready to capture prey. (E) Lateral view of a detached trap, the entrance faces towards the left-hand side. A small prey animal (p), presumably Chydorus spec., grazes algae on the ‘antennae’. Already caught prey is visible inside the trap. The trigger hairs (tr) protrude from the trapdoor.” 9 9 Bladderwort Trap Morphology In Utricularia, there exists three main types of traps, with many aquatic species falling within the U. vulgaris type (Poppinga et al., 2016). A typical vulgaris-type trap comprises a bladder-shaped trap with a single opening that is closed by a trap door. The bladders are made up of a 2 cells thick wall that looks like a small sac, which is roughly circular in shape from a lateral view but flat (loaded) or oval (unloaded) in frontal view (figure 3). The entrance is covered by a door that aids in maintaining a negative pressure. The trigger hairs are located on the door, or they might have no trigger hairs at all just glands (Poppinga et al., 2016). The trigger hairs are small filaments that protrude from the mouth area. In some terrestrial species, the mouth area is also covered by other hair-like structures (Reifenrath et al., 2006). The position of the mouth relative to the stolon varies with species: Poppinga et al. (2016) distinguish three main orientations – basal, terminal, and lateral (figure 4).
Figure 4. From Poppinga et al. (2016) – Figure 2. “Lateral view of different trap types, indicating the position of the trap entrance (te) and of the stalk (st). (A) Basal position (U. circumvoluta). (B) Terminal position (U. bisquamata). (C) Lateral position (U. raynalii). The ventral and dorsal trap parts are indicated. Images modified from Taylor (1989). The genus Utricularia—a taxonomic monograph with kind permission from the Board of Trustees of the Royal Botanic Gardens, Kew.” 10 10
In most species, the trap door is not situated flush with the outer bladder wall, but more or less recessed within a vestibule (figure 5). There is a group of aquatic bladderwort species that share a similar trap morphology, such as U. vulgaris, U. gibba, U. inflata, U. stellaris (Taylor, 1989; Vincent et al., 2011). The traps of these aquatic species have a small vestibule, a nearly circular mouth opening covered by a nearly circular, dome-shaped door (Poppinga et al., 2016). The trap door is a two-cell thick membrane that seals the bladder shut (Poppinga et al., 2016; Reifenrath et al., 2006). The door is attached to the trap along one edge and is seated against a thick threshold along the door’s free edge (Poppinga et al., 2016) (figure 5). The rim of the trap’s mouth possesses several antennae, which have been speculated to help guide prey towards the mouth as they graze on the algae growing on the bladderwort (Meyers & Stricklert, 1979). There is much less known about the trap morphology of terrestrial species. But studies suggest that the mouth opening of terrestrial species is not circular but more slit-shaped, that the trap door is obscured by a dense growth of bristles, and that the throat behind the door is long and narrow compared with the short, wide opening behind the trap door of aquatic species (Poppinga et al., 2016).
Bladderwort Trap Mechanism To load the trap, bladderworts generate a sub-ambient pressure in the traps. This is achieved by the water being pumped out of the bladder through pores that are located all over the bladder. The traps can generate substantial sub-ambient pressures (Sasago & Sibaoka, 1985; Singh et al., 2011; Vincent et al., 2011). Once the set pressure of -16 kPa is achieved the bladderwort is ready to be triggered (Vincent et al., 2011). The door opening is triggered by the prey touching the trigger hairs. Trigger hairs are usually found near the edges of the 11 11
Figure 5. From Poppinga et al. 2016 – Figure 5. “Morphology of the trap body.” (A) Light microscope (LM) image of a longitudinal section of a U. vulgaris trap cut open with a razor blade. The door (td) with its free edge (fe), the threshold (th), trigger hairs (tr), pyriform glands (pg) and the trap wall (tw) are visible. (B) LM image of a 10-mm-thick semi-thin longitudinal section of a U. vulgaris trap, stained with toluidine blue. The trap wall, trapdoor, threshold, spherically headed glands (sg), bifid gland (bg), quadrifid gland (qg) and the velum (ve) can be seen. (C) Scanning electron microscope image of a longitudinal section of a U. vulgaris trap (cut with a razor blade before critical point drying). Among the many structures situated at the trap entrance, especially the pavement epithelium (pe) and the bifid and quadrifid glands covering the inside of the trap are noteworthy. The stalk (st), ‘antennae’ (an) and ‘bristles’ (br) are also visible. (D) Schematic drawing of a longitudinal section of a U. gibba trap. Image modified from Lloyd (1932) (& Canadian Science Publishing or its licensors). Note that in all images, the trapdoor is arranged at an ~908 angle to the threshold surface, which is characteristic for the U. vulgaris trap type.”
12 12 trap door. When prey touches the trigger hairs, the door collapses inward within 1 millisecond (Vincent et al., 2011). The sub-ambient pressure inside the trap causes water and prey to be sucked into the trap within 1 to 2 milliseconds, reaching flow speeds inside the trap throat of up to 1.5 m/s (Vincent et al., 2011). During the suction event, the walls of the trap bulge outward, releasing the elastic energy stored in the walls to power the suction event (Poppinga et al., 2016). The door closes again within roughly 2 to 10 milliseconds. After the bladder has been sealed again by the door, the bladderwort will start to reset by beginning the pumping cycle again. The traps typically reach maximum sub-ambient pressure and are ready to fire again within 30 minutes, but they continue to pump water out for up to several hours causing the trap walls to become increasingly flat or even concave, but without the pressure inside the trap decreasing further (Sasago & Sibaoka, 1985; Vincent et al., 2011), suggesting that the trap walls are a non- Hookean material or structure.
Bladderwort Predator-prey Interactions The prey that are typically found in bladderwort are a wide variety of aquatic invertebrates and photosynthetic organisms such as algae (Harms, 1999). Wild U. inflata bladders contained cladocerns and rotifers more abundantly, and U. gibba contained most abundantly nematodes, but both contained significantly the same quantities of copepods, and rhizopods (Gordon & Pacheco, 2007). These various zooplanktons all vary in size and shape. Cladocera, also known as water fleas, are an order of zooplankton that inhabit pelagic, littoral, and benthic zones, the size range is 0.2-6.0 mm (Forro et al, 2008). Cladocerans have a hopping pattern when they swim (Allan, 1976). For example, daphnia, which are in the order of cladocera, swims at a rate of 0.74 cm/sec (Allan, 1976). Rotifers are a 13 13 phylum of aquatic microscopic animals that are also found in bladderwort bladders. Rotifers range in size of 0.2-0.6 mm (Allan, 1976). Rotifers use cilia like structures around the mouth to move, at a speed of 600-800 µm/sec, which makes them slower than other zooplankton (Epp & Lewis, 1984). Copepods are small crustaceans that have a size of 0.5-5.0 mm, they use their first set of antennae to establish paddle-like thrusts, reaching speeds of 20 cm/sec (Allan, 1976). In the study by Guiral and Rougier (2007), they found that 50% of the traps they analyzed had at least one prey item, and that occurrence of prey increased as trap size increased. They also say that phytoplankton are incidental captures because something much bigger triggered the trap. Alkhalaf et al. (2008) looked specifically at the phytoplankton found in U. vulgaris and U. australis found in Germany. They found that 44.7% of the traps contained both zooplankton and phytoplankton, 46.6% contained only phytoplankton, and 8.7% were empty. They found a total of 169 species of phytoplankton in the traps they examined. They also found that the breakdown of zooplankton was 60% ciliates, 17% crustacean copepods, 12% cladocerans, 4% rotifera, 4% insect larvae, and 3% of other things like fungal spores and pine pollen. These studies show that bladderwort are generalists, because they will catch anything that is positioned in front of the mouth when a suction event is triggered by the bigger prey.
Fluid Mechanics of Suction Feeding The way in which organisms and objects interact with fluids is often described using Reynolds numbers. Reynolds number is defined as the ratio of inertial to viscous forces acting on an object or organism moving (in) a fluid. Reynolds numbers above 1000 describe a flow regime dominated by inertial forces, Reynolds numbers below 10 describe a flow regime dominated by viscous 14 14 forces, Reynolds numbers between 10 and 1000 describe the so-called intermediate flow regime, in which both inertial and viscous forces play an important role. When water moves through a pipe, then the flow pattern forming at the entrance of the pipe depends on the flow regime. In the viscous (creeping) flow regime (Reynolds number <10), the entrance flow has a nearly semicircular profile; in the inertial flow regime (Reynolds number > 1000) has a flat profile (figure 6).
Figure 6. Developing flow through entrance of a pipe. Red dashed line demarks the area of developed (along the walls; flat velocity profile) versus undeveloped (near the center of the pipe; parabolic velocity profile) flow. Le is the entrance length and is the growth of boundary layer. Adapted from chapter 9 Laminar boundary layers: entry flow in a duct (2009) retrieved from http://www.nptel.ac.in/courses/112104118/31
The flow inside the pipe is dominated by the forming of a so-called boundary layer along the walls of the pipe, in which the flow is considerably slower than the in middle of the pipe. The thickness of the boundary layer is typically defined as the thickness of the water layer from the wall to 99% of the free-stream flow speed (Schlichting & Gersten, 2017). Boundary layer thickness will reduce the diameter of the flow through a pipe, which will reduce the amount of fluid that can pass. This effect of the boundary layer is more pronounced at 15 15 lower Reynolds numbers, when the boundary layer takes up a larger fraction of the pipe diameter than at higher Reynolds numbers.
Aims and Objectives Body size has important consequences for motion in fluids, including feeding. Many aquatic organisms capture prey by suction, and the mechanics of suction feeding are well understood in large animals (> 10 cm). Yet the smallest suction feeders (≤ 3 mm) present a fluid-dynamic puzzle: current models predict that suction on this scale is ineffective. This study focused on two groups suction feeders that are so far understudied: bladderwort and larval or paedomorphic salamanders. This study is part of a larger project that aims to define the boundaries of suction feeding by identifying its fundamental physical limits, and to explore the position of existing suction feeders within these limits. We used high-speed flow visualization to quantify suction flows on live organisms to develop a robotic model that will be used in future studies to explore capture mechanics both inside and outside the performance envelope of biological suction feeders. Aim 1: Characterizing the suction feeding morphology and motion of bladderwort and salamanders Objective 1.1: Determine gape diameter for three species of bladderwort (U. australis, U. gibba, U. praelonga) and three species of salamander (G. porphyriticus, D. quadramaculatus, and Ambystoma sp.). These data will feed into Aim 2. Objective 1.2: Determine time to peak gape for three species of bladderwort (U. australis, U. gibba, U. praelonga) and three species of salamander (G. 16 16 porphyriticus, D. quadramaculatus, and Ambystoma sp.). These data will feed into Aim 2. Hypothesis 1.1: Gape diameter scales with body size in the same way as in fish. Hypothesis 1.2: Time to peak gape scales with body size in the same way as in fish. Aim 2: Characterizing the suction feeding flows of bladderwort and salamander. Objective 2: Determine flow pattern and flow speed (time to peak flow speed, peak flow speed) for one species of bladderwort and two species of salamander. Hypothesis 2: Peak flow speed scales with gape size. Aim 3: Build a mechanical model of a suction feeder: Objective 3: Use fluid dynamics scaling laws to design and build a mechanical suction feeder. Hypothesis 3: This aim does not have a corresponding hypothesis.
METHODS AND MATERIALS
Husbandry
Bladderwort Bladderwort are maintained inside the lab in a fish tank. The fish tank has a layer of sphagnum as the substrate in the bottom. The sphagnum is kept from floating away by placing mesh on top of it. The mesh is kept in place by small rocks placed in various locations around the edge. Distilled water will be used to fill the fish tank to a predetermined level. A pH meter was used to determine a proper acidity level of the tank. Bladderwort was then placed evenly along the bottom of the tank, ensuring an even spread of the plants.
Salamander The larval salamanders were kept in a temperature-controlled room on the University of South Florida campus. The temperature was maintained around 18C. The Desmognathus and Gyrinophilus were kept in plastic deli containers with perforated lids. Each container had a paper towel and artificial spring water that was changed out weekly. They were fed bloodworms when they would not be used for imaging that week. The Ambystoma were kept in a fish aquarium that was kept at room temperature. All procedures for the salamanders were approved by the Institutional Animal Care and Use Committee of the University of South Florida.
Morphological Measurements of Lab Grown Bladderworts Using lab cultivated bladderwort, different measurements were taken of various parts of the plant. Using a ruler, the distance between the nodes on the 18 18 stolon were measured, it was labeled as the internode length. The diameter of the stolon was measured using a digital caliper. Bladderwort gape length was calculated by taking measurements on frontal and lateral images of the bladders taken from a microscope (Olympus stereo dissection microscope). Bladder gape shape was also observed from those images. Bladders were placed in agar filled petri dishes with a slide micro ruler to calibrate distances. Images were taken with a microscope and using computer software (ImageJ) different data points were calculated. U. australis, U. gibba, and U. praelonga had a separate set of data collected. This data was then used to calculate the scaling coefficients of each species. Scaling coefficients are calculated by graphing log transformed measurements of body length vs gape length, then fitting a linear regression to the log-transformed data; the slope of the line is the scaling coefficient. A slope of 1 indicates isometric growth, a slope different from 1 indicates allometric growth.
Selecting Bladderwort for High Speed Filming When selecting bladderwort for filming a high-speed trapping event a few requirements need to be met like, shape of the bladder, age of the bladder, and position of the stalk. Shape of the bladder has to be concave which indicates water has been pumped out of it and it’s ready to trigger. Age of the bladderwort is an important requirement for the trapping event to even occur. For Utricularia australis, the age of the bladder is determined by its position along the stolon. The bladders that can be used are located on the stolon between the older bladders closer to the flowering end and the younger one closer to the growing end. On U. praelonga, it is harder to tell the age of the bladder by its location on the stolon. The U. praelonga bladders that I used are grown in a greenhouse as a potted plant. It is harder to determine the age of U. praelonga bladders because the stolons are 19 19 tangled within the dirt. The position of the stalk is important because it will determine the way the bladder is attached to the wire and the way it will sit inside the cuvette.
High Speed Filming of Feeding Event
Bladderwort Feeding Event For the filming of a feeding event a high-speed camera is required. We used a Phantom v12.1 digital high-speed camera for visualizing a suction feeding event of a bladderwort. A bladderwort bladder that is ready to trigger was chosen from a sample taken from the greenhouse located on the Fresno State campus. A bladder is ready to be triggered when it is concave. When the appropriate bladder was chosen, it was carefully cut and removed from the stolon. Then the bladder was attached to a thin wire using a gel type super glue. The wire with the attached bladder was placed in a glass cuvette that has a very small amount of tacky gum at the bottom to hold the wire upright. Once the bladder was aligned properly inside the cuvette with the gape aimed toward the top of the cuvette. The cuvette was then filled with distilled water and placed on the platform. Two drops from a syringe of 10-micron microspheres were added to the cuvette. Microspheres are supplied from Phosphorex and come in a 1% solid suspension (10mg/ml). The microspheres are polystyrene with a density of 1.06 g/cm3. A three-axis platform was located in front of the high-speed camera, on the opposite side of the camera, a LED light is placed to better illuminate the cuvette and bladder. Using Phantom Camera Control application to view the live video feed, the camera was moved to focus the bladder on the computer screen. Proper focus is achieved when the microspheres on the bladder surface have a clear, sharp outline. Once everything was in proper focus, a cat hair that was attached to a mechanical micro- 20 20 manipulator, was used to trigger the bladder by hitting the trigger hairs or hitting the door. As the suction event was observed, a remote-control trigger of the camera is pressed to save the video. Suction feeding events were recorded at a frame rate ranging from 6270 fps to 28000 fps.
Salamander Feeding Event A larval salamander was placed in an imaging chamber so that the salamander could be imaged in a lateral view with an Edgertronic SC1 with a Nikon camera lens. A laser was aimed into the aquarium, perpendicular to the camera. We used two different lasers, the first one was a 500mW green light and the second laser we used was a 1W red light. The water that was used to fill the aquarium was artificial spring water which was then seeded with glass sphere particles. In order for the suction feeding event to be filmed we had to make the salamander perform a suction event within the field of view of the camera. We triggered this by attaching a bloodworm to a small wire and placing it in front of the salamander’s mouth when it was aligned perpendicular to the camera. The suction feeding events were filmed at 1000 fps for G. porphyriticus and 3000 fps for Ambystoma sp. For the D. quadramaculatus I was not able to get any videos, for the G. porphyriticus I was able to get four videos of two organisms, and for the Ambystoma sp. I was able to get five videos for two organisms. The video was then analyzed using ImageJ. The analysis is done by tracking particles through the feeding event and calculating the distance each particle traveled.
Data Analysis The feeding event videos were then analyzed to find the speed of the microspheres that entered the bladder during the suction event. The analysis is done by taking the coordinates of a single particle through different sequential 21 21 frames and figuring out the distance it traveled. These videos were also used to measure the time for maximum gape, time to maximum speed and to visualize the flow pattern of the bladderwort. The feeding event videos for salamanders were also analyzed using ImageJ. The position of the microspheres in a frame were saved as X-Y coordinate points. The coordinates were transferred to an excel sheet were the distances traveled was calculated. Using the distance traveled and knowing the frame rate, the flow speed was calculated. The peak gape was calculated using ImageJ tools to measure distance between two points, the top jaw and lower jaw. The time to peak gape was calculated using the frame number when peak gape was reached and the frame rate of the video. To find the scaling coefficients between the different groups of data such as gape diameter versus body length it has to be graphed, and the slope of the line of best fit has to be calculated. To graph the data, it has to be log transformed first. When that has been done it is graphed on a standard X-Y axis. Then using the trend line feature on excel, the slope is calculated.
Building and Operation of a Mechanical Suction Feeder Using scaling laws, a mechanical model was designed. The model is scaled up from the different specifications of the live bladderwort. The multiplication factor that will be used for the mechanical model is 200 times. Using the Reynolds number calculated from the measurements of a live bladderwort, the viscosity of the liquid media was chosen, and the linear motor was chosen. A linear motor that would meet the required specifications was chosen from Linmot, a linear motor manufacturer located in Elkhorn, Wisconsin. The motor, model PS01-23x160- R20, has the max velocity of 6 m/s, and a peak force of 86N (data sheet from 22 22
Linmot). The motor velocity and time to peak will be controlled through a motor controller that was also purchased through Allied electric (figure 7). The motor controller will be controlled remotely from a computer.
Figure 7. Linear motor controller and power supplies.
The motor has a slider that will give motion to an attached piston (figure 8). The piston was manufactured from stainless steel rod in the Fresno State Science department metal workshop. Mineral oil then will be used as the media to scale up the viscosity from water. Kinematic viscosity of the mineral would have to be 70 cSt according to the scaling calculations. We purchased a 55-gallon drum of mineral oil “Crystal plus food grade oil 350fg” from STE Oil Company, Inc. located in San Marcos, Texas. This mineral oil met the standards that are required for this part of the experiment. Part of the mineral oil from the drum was placed inside a 50-gallon fish tank. The whole robot will then be submerged in the mineral oil that is in the fish tank by a gantry that is constructed from 80/20 aluminum extrusions. Gantry material was purchased from Numatic engineering and designed by Muller research group (figure 9). 23 23
Figure 8. Linear motor with slider.
Figure 9. Gantry crane schematic that was sent from Numatic engineering.
24 24
Using the motor controller, the piston will then be able to draw suction through a pipe that will simulate a suction feeding event. Air bubbles produced by an air stone will generate bubbles that allow us to visualize the flow. I used a 38mm x 38mm x 152mm aquarium air stone. A 4.5 W air pump from Penn-Plax, model silent air X4, was used to supply the air stone through a 2-valve manifold. The air stone produces 1.4 mm diameter air bubbles that stay suspended in the mineral oil.
RESULTS
Characterizing the Suction Feeding Morphology and Motion of Bladderwort and Salamanders
Gape Diameter In bladderwort, the average gape diameter that was found for U. australis was 0.54 mm (SD=0.13), it ranged from 0.216 mm to 0.971 mm (n=200). The average gape diameter for U. gibba was 0.21 mm (SD=0.07) and ranged from 0.06 mm to 0.71 mm (n=2179). The average gape diameter of U. praelonga was 0.67 mm (SD=0.08) and the range was 0.47 mm to 0.82 mm (n=20) (see table 1). In salamander, the average gape diameter for G. porphyriticus was 2.54 mm (SD=1.6), and the range was 1.74 mm to 3.46 mm (n=4). The average gape diameter for Ambystoma sp. was 6.29 mm (SD=1.6) and the range was 4.88 mm to 8.28 mm (n=5) (see table 1). There are no results for D. quadramaculatus, we had 5 organisms, but they never showed any interest in any of the food offered.
Table 1
Gape Diameters of Suction Feeders Avg Gape Diameter Gape Species Name (mm) diameter (mm) U. australis 0.54 0.216 - 0.971 U. gibba 0.21 0.06 - 0.71 U. praelonga 0.69 0.627 - 0.76 G. porphyriticus 2.54 1.74 - 3.46 Ambystoma sp. 6.29 4.88 - 8.28 26 26
We hypothesized that gape diameter scales with body size in the same way as in fish. Scaling coefficients are the slope of the line of best fit for each species data. The scaling coefficients of the salamanders and fish appear to be higher than those of the three bladderwort species in this study (see figure 10).
Figure 10. Graph showing how body length scales to gape diameter. Fish data were collected from literature (Higham et al. 2006, Ferry-Graham et al., 2003, Van Wassenbergh et al. 2005).
Time to Peak Gape In bladderwort, the average time to peak gape for U. praelonga was 0.41 ms (n=11) and the range was 0.15 ms to 0.8 ms (SD=0.24). The average time to peak gape for U. gibba was 0.7 ms (SD=0.3) (Brown, 2016). The average time to peak gape for U. australis was 1 ms (SD=0.6) (Brown, 2016). (see table 2) In the salamander, the average time to peak gape for G. porphyriticus was 7.42 ms (SD=2.32) and had a range of 3.96 ms to 8.91 ms (n=4). The average time 27 27 to peak gape for Ambystoma sp. was 23.2 ms (SD=6.53) and the range was 15 ms to 32 ms (n=5) (see table 2).
Table 2
Time to Peak Gape (TTPG) of Suction Feeders Species Name Avg TTPG (ms) TTPG (ms) U. australis 1 N/A U. gibba 0.7 N/A U. praelonga 0.41 0.15 – 0.8 G. porphyriticus 7.42 3.96 – 8.91 Ambystoma sp. 23.2 15 - 23
We hypothesized that time to peak gape scales with body size in the same way as in fish. We found that U. praelonga, G. porphyriticus, and Ambystoma sp. Fell along the same line when plotted together (figure 11).
Figure 11. Graph showing how body length scales with time to peak gape for U. praelonga, Ambystoma sp., G. porphyriticus, and fish. 28 28 Characterizing the Suction Feeding Flows of Bladderwort and Salamander
Peak Flow Speed The flow pattern of one bladderwort and two species of salamanders is used to determine the time it takes to reach peak flow speed, and the peak flow speed that was reached. Using the high-speed videos, I analyzed the sequence using ImageJ. The average time to peak speed for U. praelonga was 0.78 ms (SD=0.58) and ranged from 0.3 ms to 2.08 ms (n=8). The average peak speed of U. praelonga was 0.39 m/s (SD=0.23) and ranged from 0.09 m/s to 0.89 m/s (n=8) (see table 3). In salamanders, the time to peak speed for G. porphyriticus was 6.1 ms (SD=2.26) and the range was 3.96 m/s to 8.91 ms (n=4). Average peak speed for G. porphyriticus was 0.69 m/s (SD=0.51) and the range was 0.1 m/s to 1.36 m/s (n=4). The average time to peak speed for Ambystoma sp. was 22.8 ms (SD=2.95) and the range was 18 ms to 25 ms (n=5). Average peak speed was 1.1 m/s (SD=0.25) and the range was 0.76 m/s to 1.46 m/s (n=5) (see table 3).
Table 3
Flow Characterization of Suction Feeders Species Avg Time to Peak Speed Avg Peak Speed (m/s) (ms) U. praelonga 0.78 0.39 G. porphyriticus 6.1 0.69 Ambystoma sp. 22.8 1.1
We hypothesized that peak flow speed scaled with gape size. We found that peak flow speed scaled with gape along the same line for U. praelonga, Ambystoma sp., and G. porphyriticus. The slopes of the lines of best fit are 29 29 different for each of the species, but due to the low n number, significance can’t be determined (see figure 12). Still shots showing the paths particles traveled can be seen in figures 13-15.
Figure 12. Graph showing the scaling coefficient of U. praelonga, Ambystoma sp., and G. porphyriticus.
Figure 13. Still shot from particle tracking video of G, porphyriticus showing path particles traveled. 30 30
Figure 14. Still shot of from particle tracking video of Ambystoma sp. showing path particles traveled
Figure 15. Still shot of U. praelonga showing path particles traveled 31 31 Building a Mechanical Model of a Suction Feeder Using the data collected from the first two aims we had the information to calculate the Reynolds number (Re). To calculate the Re number we needed the kinematic viscosity, diameter of the opening, and velocity of the fluid.
푢퐿 푅푒 = 푣
Where u is fluid velocity, L is the hydraulic diameter, and v is the kinematic viscosity. For bladderwort using the peak flow speed which was 0.39 m/s, the hydraulic diameter which is the gape diameter was 0.69 mm, and the kinematic viscosity of water is 1.0 mm2/s. The Re number for bladderwort was calculated as 269.1. We did the same calculation for G. porphyriticus and the Re number was 1752.6. The Re number for Ambystoma sp. was calculated as 6919. In order to see a feeding event, we needed it to be slowed down. To see a slower feeding event, we built a scaled up mechanical model. To scale up we used the Reynolds number (Re). We wanted the feeding event in the model to last 1 second. We also scaled up the gape diameter to better visualize the flow patterns. According to the calculations (see table 4) we needed to purchase a motor that could produce speeds that ranged from 130 mm/s to 380 mm/s. We looked at Linmot, a linear motion company, because their website states they produce linear motor with speeds of up to 7.3 m/s and accelerations of over 780 m/s2. We chose a linear motor that has a speed of 6 m/s, which satisfies the velocity requirements needed. The accelerations needed to reach peak speed at the specified time (see table 4) the linear motor would need to range from 134 mm/s2 to 87000 mm/s2, which is under the manufacturer specifications.
32 32 Table 4
Scaling Parameters Species Flow speed Gape dia. k. viscosity Re TTPS [mm/s] [mm] [mm2/s] [s] U. praelonga 390 0.69 1 270 0.00078 G. porphyriticus 690 2.54 1 1800 0.0061 Ambystoma sp. 1100 6.29 1 7000 0.0228 Mechanical model 134 138 68.5 270 1 of bladderwort Mechanical model 894 138 68.5 1800 0.15 of G. porphyriticus Mechanical model 3475 138 68.5 7000 0.04 of Ambystoma sp.
DISCUSSION
The aims of this study were to develop a mechanical model of a suction feeder that can be used to study the fluid dynamics of suction feeding in bladderworts and larval salamanders. Concerning the first aim of this study – characterization of the suction feeding morphology and kinematics as relevant to building the model – we found that the gape diameter of suction feeders ranges from 0.2 mm (larval fish, tadpoles, bladderwort) (Hernandez 2000; Deban and Olson, 2002; Westermeier et al., 2017) to 20 mm (blue gill sunfish) (Ferry-Graham et al., 2003). Concerning the second aim of this study – characterization of the flow in organisms as relevant to determine the scaling of the model (Reynolds number – we found that peak flow speeds ranged from 0.3 mm/s (Pekkan et al, 2016) to 6.8 m/s (Higham et al., 2006). These values correspond to a range of Reynolds numbers from 0.6 (fish larva, Pekkan et al., 2016) to 80,000 (large-mouth bass; Higham et al., 2006). Also relevant for the design of a mechanical model is the time to peak gape, for which experimental data range from 3ms (bladderwort, Westermeier et al., 2017) to 67 ms (adult salamander, Stinson and Deban, 2017) in suction feeders. Compared with the aforementioned literature data, the values for the focal organisms of this study (bladderworts, larval salamanders) range more narrowly from 0.2 to 8 mm for gape diameter, 0.4 to 1.1 m/s for peak flow speed, 1 to 23 ms for time to peak gape, and 80 to 7000 for Reynolds number. With values for Reynolds number ranging across 2 orders of magnitude for our focal organisms and across 3 orders of magnitude if we include literature values for adult fish, a mechanical model of a suction feeders is required to accommodate a wide range of 34 34 flow conditions. This accommodation can be achieved in the model design of this study by altering the speed of the piston, which affects flow speed. It could also be achieved by using mineral oil with a lower viscosity or by increasing gape diameter. The first option is readily feasible and can be used to change Reynolds numbers by an order of magnitude because the viscosity of suitable mineral oils ranges over more than two orders of magnitude at room temperature from values close to water (10 cST, water: 1 cST) to very viscous (>1000 cST). In the current design, mineral oil viscosity could be lowered by less than an order of magnitude from 70 to roughly 10 cST; larger drops in viscosity might be achieved by switching back to water (viscosity: 1 cST). The second option – altering gape diameter – is limited by tank size and the piston design; in the current design, gape diameter is easy to reduce by an order of magnitude, but it can be increased by only a factor of 2 before wall effects from the tank might affect the flow (Rayner and Thomas, 1991). A further limitation is the actuator driving the piston, which can move the piston with a top speed of 6 m/s, limiting Reynolds numbers that can be achieved with the current design to 12,000. To justify using the robot across a wide range of Reynolds numbers and be able to claim that this range is representative of the range of suction feeders found in nature, I combined the experimental data from this study with data from the literature. This combined data set revealed that salamanders scaled the same as fish when looking at body length vs gape diameter, with larval salamander data filling the gap between the larval/juvenile fish data and the adult vertebrate data (Figure 16). Bladderworts scale differently from vertebrates because of their completely different body plan. When looking at current literature of suction feeding organisms, a study by Yaniv et al. (2014) says that organisms with smaller mouth diameters produce 35 35 weak suction flows. When the three bladderwort and two salamander species of this study are graphed together the adult salamander and fish data, it turns out that bladderworts generate suction flows that are two orders of magnitude higher than larval fish (Brown, 2016; Pekkan et al., 2016; Westermeier et al., 2017). The peak flow speed of bladderworts are higher than expected for their gape diameter, if we assume that bladderwort follow the same scaling relationship of flow with gape as vertebrates. While peak flow speeds of fish and salamanders all fall in line with each other, bladderworts don’t follow the same line; they have the smallest gape diameter of all four organism groups, yet their peak flow is in the same order of magnitude as salamanders. In summary, the morphological differences between bladderworts and vertebrates continue into the kinematic and flow parameters – time to peak gape and peak flow speed. Again, the larval salamander flow data from this study fill the gap in the fish data, but bladderwort flow speeds are much higher than would be expected for their gape size. Time to peak gape varies little across the more than two orders of magnitude in body length for vertebrates, ranging from 10 to 70 ms. In contrast, bladderworts have times to peak gape that are an order of magnitude smaller than vertebrates (around 1 ms). The combined data set from this study and the literature suggests that Hypothesis 1 (gape scales with body size as in fish) and Hypothesis 2 (peak flow scales with size as in fish) should be accepted for salamanders and rejected for bladderwort. Bladderwort have a lower time to peak gape, and a higher peak flow speed for their body length compared with vertebrate suction feeders. These findings do not preclude us from using the robot to model both bladderworts and vertebrate suction feeders (Aim 3). These findings, however, do show that the mechanical model will need to be programmed differently for these 36 36 two organism groups, with bladderworts requiring relatively high piston speeds and accelerations to model these relatively small organisms. I conclude that a mechanical model designed to represent bladderworts is likely suitable also to model larval vertebrates because bladderworts place higher demands (higher piston speeds) on the design than larval vertebrates of the same size. In fact, the design chosen in this study is able to model adult suction feeders, such as adult Ambystoma sp. For the third aim we had no hypothesis, but as previously shown, we achieved the aim of building a mechanical model that can model a wide range of different suction feeders. This mechanical model is useful not only to replicate existing suction feeders, but also to ‘fill gaps’ and explore areas in the parameter space that are not occupied by existing organisms. Our literature study showed that there are considerable gaps in the parameter space (see figure 16). One way that these gaps can be explored is by manipulating the different variables (gape diameter, piston speed, time to peak flow) of the mechanical model. Furthermore, the mechanical model allows us to separate the effects of different variables by manipulating each variable individually, then study interactions by manipulating these variables in groups. So, this model can be used to better understand existing organisms not only by replicating them in a scaled-up model that allows us to look at phenomena in, for example, higher spatial or temporal resolution. But mechanical models also allow us to explore functional morphology and mechanisms by modelling counterfactual designs and comparing their performance with actual designs. Mechanical models can map parameter spaces quickly and are possibly faster at mapping parameter spaces than computational models (Koehl, 2003).
37 37
Figure 16. Graph showing time to peak gape, peak flow, and gape vs body for bladderwort, fish at different life stages, and salamanders. The data collected from the literature are included in the Appendix.
One of the important limitations of this mechanical model is that it does not model any supplementary strategies used by animal suction feeders to aide in their suction feeding, such as jaw protrusion. It also does not model gape expansion but instead assumes that the time scale of mouth opening is much smaller than the time scale of flow generation. This assumption is probably valid in bladderwort, whose time to peak gape is extremely short. However, in vertebrates, time to peak gape is much longer. A future model design should include a mechanism that models an increasing gape. Overall, this model is entirely suitable to model bladderwort suction feeding. To model vertebrate suction feeding, the basic design specifications are suitable, but a specific mouth design might need to be developed to simulate mouth opening and jaw protrusion.
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APPENDIX: LITERATURE DATA 45 45
Organism Reference Body Size TTPG Peak flow Gape [mm] [ms] [m/s] [mm] Micropterus Higham et 174.3 22.4 6.8 26 salmoides al. (2006) Lepomis Higham et 152.3 28.3 5 13 macrochirus al. (2006) Lepomis Ferry- 200 22 22.2 macrochirus Graham et al. (2003) Danio rerio Pekkan et 4 10 0.003 0.125 al. (2016) Danio rerio Hernandez 8 10 0.0009 0.7 (2000) Danio rerio Hernandez 10 10 0.0012 0.8 (2000) Danio rerio Hernandez 20 10 0.0054 1.05 (2000) Danio rerio Hernandez 4.5 0.15 (2000) Danio rerio Hernandez 5.5 0.17 (2000) Danio rerio Hernandez 6 0.25 (2000) Utricularia gibba Brown 0.9 0.9 0.2 0.2 (2016) Utricularia Brown 2 0.9 0.4 0.5 australis (2016) Pleurodeles waltl Stinson & 73.4 44 0.28 4.46 Deban (2017) Notophthalmus Stinson & 46.1 67 0.2 3.24 viridescens Deban (2017) Triturus Stinson & 73 38 0.26 3.59 dobrogicus Deban (2017)
46 46 Cynops cyanurus Stinson & 57.5 38 0.21 4.16 Deban (2017) Paramesotriton Stinson & 74.2 28 0.52 5.1 labiatus Deban (2017) Ambystoma sp. 80 22 1.14 5 Micropterus Higham et 243.5 26.3 salmoides al. (2006) Lepomis Higham et 103.5 31.3 macrochirus al. (2006) Micropterus Sanford & 249.6 35.2 salmoides Wainwright (2002)