Lateral Line Sensory System Research Articles

Semaphorin7A patterns neural circuitry in the lateral line of the zebrafish

In a developing nervous system, axonal arbors often undergo complex rearrangements before neural circuits attain their final innervation topology. In the lateral line sensory system of the zebrafish, developing sensory axons reorganize their terminal arborization patterns to establish precise neural microcircuits around the mechanosensory hair cells. However, a quantitative understanding of the changes in the sensory arbor morphology and the regulators behind the microcircuit assembly remain enigmatic. Here, we report that Semaphorin7A (Sema7A) acts as an important mediator of these processes. Utilizing a semi-automated three-dimensional neurite tracing methodology and computational techniques, we have identified and quantitatively analyzed distinct topological features that shape the network in wild-type and Sema7A loss-of-function mutants. In contrast to those of wild-type animals, the sensory axons in Sema7A mutants display aberrant arborizations with disorganized network topology and diminished contacts to hair cells. Moreover, ectopic expression of a secreted form of Sema7A by non-hair cells induces chemotropic guidance of sensory axons. Our findings propose that Sema7A likely functions both as a juxtracrine and as a secreted cue to pattern neural circuitry during sensory organ development.

Semaphorin7A patterns neural circuitry in the lateral line of the zebrafish.

In a developing nervous system, axonal arbors often undergo complex rearrangements before neural circuits attain their final innervation topology. In the lateral line sensory system of the zebrafish, developing sensory axons reorganize their terminal arborization patterns to establish precise neural microcircuits around the mechanosensory hair cells. However, a quantitative understanding of the changes in the sensory arbor morphology and the regulators behind the microcircuit assembly remain enigmatic. Here, we report that Semaphorin7A (Sema7A) acts as an important mediator of these processes. Utilizing a semi-automated three-dimensional neurite tracing methodology and computational techniques, we have identified and quantitatively analyzed distinct topological features that shape the network in wild-type and Sema7A loss-of-function mutants. In contrast to those of wild-type animals, the sensory axons in Sema7A mutants display aberrant arborizations with disorganized network topology and diminished contacts to hair cells. Moreover, ectopic expression of a secreted form of Sema7A by non-hair cells induces chemotropic guidance of sensory axons. Our findings propose that Sema7A likely functions both as a juxtracrine and as a secreted cue to pattern neural circuitry during sensory organ development.

Cavefish dorsoventral axis angle during wall swimming: laterality asymmetry

The Astyanax fish exhibits two morphs: an eyed, pigmented surface morph and an eyeless, depigmented cave morph. Previous studies have shown that blind morphs swim nearly parallel to the wall and can sense detailed information about objects by gliding alongside them and sensing changes in the flow field around their body using their lateral line sensory system. Hence, cavefish can build hydrodynamic images of their surroundings. Field observations showed that one of their presumptive prey, mysid shrimp, is predominately found not on the floor, but crawling on the walls. In our study, the angle of the body axis with respect to a vertical wall was measured while fish swam in a tank. Results show that when swimming by a wall, cavefish incline the vertical axis of their body away from the wall. But most significantly, this angle is different when the right side or the left side of their body is oriented towards the wall. Intriguingly, cavefish have a leftward-biased dorso-cranial bend, where the convex side of the head is towards their right side. Other studies have shown behavioral “handedness”. When exhibiting Vibration Attraction Behavior (VAB), cavefish in the field show laterality on the preponderant side they circle to explore a vibrating stimulus. Likewise in larval prey capture (LPC) behavior, larvae strike towards prey preferentially located on one side. Our results support that cavefish also express behavioral lateralization during passive swimming by walls and/or when searching for food that is perched on the walls, such as mysid shrimp.

Sublethal effects of methylmercury on lateral line sensory and ion-regulatory functions in zebrafish embryos

Methylmercury can interfere with the normal functioning of the nervous system, causing a variety of behavioral and physiological changes in fish. However, the influence of MeHg on the lateral line sensory and ion-regulatory functions of fish is not clear. Zebrafish embryos were utilized as a model to address this question. After exposure to water-borne MeHg (5, 10, 50, or 100 ppb) for 96 h (4–100 h post-fertilization), the survival rate declined by ca. 50 % at 100 ppb. However, MeHg at sublethal concentrations delayed hatching and decreased heart rates and body length. As to effects on the lateral line sensory system, MeHg at ≥10 ppb decreased the number of hair cells and impaired hair bundles and Ca2+-mediated mechanical transduction. As to ion regulation, MeHg at ≥10 ppb decreased the densities of skin stem cells and ionocytes, leading to declines in ion (Na+, K+, and Ca2+) contents and H+/NH4+ excretion levels. A gene expression analysis also revealed declines in messenger RNA levels of several ion-regulatory genes (ncc2b, trpv6v1a, trpv5/6, ncx1b, and rhcg1). This study demonstrated that the lateral line sensory and ion regulatory functions of fish are extremely sensitive to MeHg.

Tail Beat Synchronization during Schooling Requires a Functional Posterior Lateral Line System in Giant Danios, Devario aequipinnatus.

Swimming in schools has long been hypothesized to allow fish to save energy. Fish must exploit the energy from the wakes of their neighbors for maximum energy savings, a feat that requires them to both synchronize their tail movements and stay in certain positions relative to their neighbors. To maintain position in a school, we know that fish use multiple sensory systems, mainly their visual and flow sensing lateral line system. However, how fish synchronize their swimming movements in a school is still not well understood. Here, we test the hypothesis that this synchronization may depend on functional differences in the two branches of the lateral line sensory system that detects water movements close to the fish's body. The anterior branch, located on the head, encounters largely undisturbed free-stream flow, while the posterior branch, located on the trunk and tail, encounters flow that has been affected strongly by the tail movement. Thus, we hypothesize that the anterior branch may be more important for regulating position within the school, while the posterior branch may be more important for synchronizing tail movements. Our study examines functional differences in the anterior and posterior lateral line in the structure and tail synchronization of fish schools. We used a widely available aquarium fish that schools, the giant danio, Devario equipinnatus. Fish swam in a large circular tank where stereoscopic videos recordings were used to reconstruct the 3D position of each individual within the school and to track tail kinematics to quantify synchronization. For one fish in each school, we ablated using cobalt chloride either the anterior region only, the posterior region only, or the entire lateral line system. We observed that ablating any region of the lateral line system causes fish to swim in a "box" or parallel swimming formation, which was different from the diamond formation observed in normal fish. Ablating only the anterior region did not substantially reduce tail beat synchronization but ablating only the posterior region caused fish to stop synchronizing their tail beats, largely because the tail beat frequency increased dramatically. Thus, the anterior and posterior lateral line system appears to have different behavioral functions in fish. Most importantly, we showed that the posterior lateral line system played a major role in determining tail beat synchrony in schooling fish. Without synchronization, swimming efficiency decreases, which can have an impact on the fitness of the individual fish and group.

Developmental integration between the lateral line sensory system and the craniofacial complex in a natural population

Non-traditional model systems can provide insight to the developmental and genetic basis of fundamental biological processes. One such process is the origin and patterning of the facial skeleton. Here, we present evidence that the intramembranous bones comprising the circumorbital complex in a teleost model are marked by the positions of large mechanosensory structures, canal neuromasts. Neuromasts comprise the lateral line system, and provide pressure and proprioceptive information to enable a number of key behaviors, such as predator avoidance, foraging, schooling and shoaling. Canal neuromasts appear to set the stage for facial patterning, since they colocalize to ossification sites destined to give rise to the six constituent bones of the circumorbital complex. Here, we demonstrate that closely related surface-dwelling morphs demonstrate an invariant pattern of development that gives rise to a symmetric and stereotypical circumorbital complex. In cavefish, however, this pattern is frequently altered - giving rise to ossification centers that are set closer, or more distant, from one another. Moreover, these alterations in position can vary across the left-right axis. Interestingly, in situations where two ossification centers are more closely set towards one another, they result in an inter-bone fusion (synostotic) event. Cavefish demonstrate at least three forms of cranial asymmetry, and we discuss how facial bone fusions may impact global craniofacial shape. These three-dimensional changes may prefigure, or facilitate, the lateral swimming preferences observed in cavefish. Moreover, the ‘predictive’ nature of circumorbital bone fusion will enable deeper insight to the connection between the earliest developmental events, and adult bone patterning and morphology. Collectively, the Mexican tetra is poised to provide new insight to the developmental basis for craniofacial aberrations shared deeply among the vertebrate subphylum.

Effects of Toluene on the Development of the Inner Ear and Lateral Line Sensory System of Zebrafish

ObjectiveThe aim of this study was to explore the ototoxicity of toluene in the early development of zebrafish embryos/larvae. MethodsZebrafish were utilized to explore the ototoxicity of toluene. Locomotion analysis, immunofluorescence, and qPCR were used to understand the phenotypes and molecular mechanisms of toluene ototoxicity. ResultsThe results demonstrated that at 2 mmol/L, toluene induced zebrafish larvae death at 120 hours post fertilization (hpf) at a rate of 25.79% and inhibited the rate of hatching at 72 hpf. Furthermore, toluene exposure inhibited the distance travelled and average swimming velocity of zebrafish larvae while increasing the frequency of movements. As shown by fluorescence staining of hair cells, toluene inhibited the formation of lateral line neuromasts and middle line 1 (Ml1) neuromasts in 3 days post fertilization larvae in a concentration-dependent manner. Toluene altered the expression level of genes involved in ear development/function in zebrafish, among which the mRNA levels of cd164l2, tekt3, and pcsk5a were upregulated, while the level of otofb was downregulated, according to the qPCR results. ConclusionThis study indicated that toluene may affect the development of both the inner ear and lateral line systems in zebrafish, while the lateral line system may be more sensitive to toluene than the inner ear.

Variation in the Sensory Space of Three-spined Stickleback Populations.

The peripheral sensory systems, whose morphological attributes help determine the acquisition of distinct types of information, provide a means to quantitatively compare multiple modalities of a species' sensory ecology. We used morphological metrics to characterize multiple sensory modalities-the visual, olfactory, and mechanosensory lateral line sensory systems-for Gasterosteus aculeatus, the three-spined stickleback, to compare how sensory systems vary in animals that evolve in different ecological conditions. We hypothesized that the dimensions of sensory organs and correlations among sensory systems vary in populations adapted to marine and freshwater environments, and have diverged further among freshwater lake-dwelling populations. Our results showed that among environments, fish differed in which senses are relatively elaborated or reduced. When controlling for body length, littoral fish had larger eyes, more neuromasts, and smaller olfactory tissue area than pelagic or marine populations. We also found differences in the direction and magnitude of correlations among sensory systems for populations even within the same habitat type. Our data suggest that populations take different trajectories in how visual, olfactory, and lateral line systems respond to their environment. For the populations we studied, sensory modalities do not conform in a predictable way to the ecological categories we assigned.

Interface Deformable, Thermally Sensitive Hydrogel–Elastomer Hybrid Fiber for Versatile Underwater Sensing

AbstractUnderwater sensing plays a vital role in perceiving various hydrodynamic stimuli for underwater operations, while fishes evolve an adaptable, durable, and multifunctional lateral line sensory system to feel mechanical deformations from nearly all sources as well as water temperature changes. Such perfect integration of multiple functions into one biological system poses a great challenge for artificial soft sensors. Here, by constructing a stretchable and water‐proof core‐cladding hydrogel–elastomer hybrid optical fiber, nearly all the underwater sensations of fish lateral lines can be realized with unprecedented sensing stability. High‐refractive‐index salt, LiBr, is introduced to the hydrogel core to enable long‐range light propagation with a low loss coefficient (≈0.32 dB cm−1), and the dissimilar yet tightly adhered hydrogel–elastomer interface is readily deformable, contributing to the ultrasensitive optical response to subtle environmental stimulations, induced by either motions, hydrostatic pressure variations, ultrasonic/audible sound waves, or water flows. Moreover, the optical loss of the hybrid fiber is linearly responsive to wide temperature changes (5–70 °C), caused by the altered light scattering from hydrogel chain clustering. The present elastomer–hydrogel hybrid optical fiber offers a new designing strategy in developing next‐generation underwater stretchable ray‐optic sensors.

Water Waves to Sound Waves: Using Zebrafish to Explore Hair Cell Biology.

Although perhaps best known for their use in developmental studies, over the last couple of decades, zebrafish have become increasingly popular model organisms for investigating auditory system function and disease. Like mammals, zebrafish possess inner ear mechanosensory hair cells required for hearing, as well as superficial hair cells of the lateral line sensory system, which mediate detection of directional water flow. Complementing mammalian studies, zebrafish have been used to gain significant insights into many facets of hair cell biology, including mechanotransduction and synaptic physiology as well as mechanisms of both hereditary and acquired hair cell dysfunction. Here, we provide an overview of this literature, highlighting some of the particular advantages of using zebrafish to investigate hearing and hearing loss.

Canal neuromast position prefigures developmental patterning of the suborbital bone series in Astyanax cave- and surface-dwelling fish

Developmental patterning is a complex biological phenomenon, involving integrated cellular and molecular signaling across diverse tissues. In Astyanax cavefish, the lateral line sensory system is dramatically expanded in a region of the cranium marked by significant bone abnormalities. This system provides the opportunity to understand how facial bone patterning can become altered through sensory system changes. Here we investigate a classic postulation that mechanosensory receptor neuromasts seed intramembranous facial bones in aquatic vertebrates. Using an in vivo staining procedure across individual life history, we observed infraorbital canal neuromasts serving as sites of ossification for suborbital bones. The manner in which cavefish departed from the stereotypical and symmetrical canal neuromast patterns of closely-related surface-dwelling fish were associated with specific changes to the suborbital bone complex. For instance, bony fusion, rarely observed in surface fish, was associated with shorter distances between canal neuromasts in cavefish, suggesting that closer canal neuromasts result in bony fusions. Additionally, cavefish lacking the sixth suborbital bone (SO6) uniformly lacked the associated (sixth) canal neuromast. This study suggests that patterning of canal neuromasts may impact spatial position of suborbital bones across development. The absence of an eye and subsequent orbital collapse in cavefish appears to influence positional information normally inherent to the infraorbital canal. These alterations result in coordinated changes to adult neuromast and bone structures. This work highlights complex interactions between visual, sensory and bony tissues during development that explain certain abnormal craniofacial features in cavefish.

Lateral line placodes of aquatic vertebrates are evolutionarily conserved in mammals.

ABSTRACTPlacodes are focal thickenings of the surface ectoderm which, together with neural crest, generate the peripheral nervous system of the vertebrate head. Here we examine how, in embryonic mice, apoptosis contributes to the remodelling of the primordial posterior placodal area (PPA) into physically separated otic and epibranchial placodes. Using pharmacological inhibition of apoptosis-associated caspases, we find evidence that apoptosis eliminates hitherto undiscovered rudiments of the lateral line sensory system which, in fish and aquatic amphibia, serves to detect movements, pressure changes or electric fields in the surrounding water. Our results refute the evolutionary theory, valid for more than a century that the whole lateral line was completely lost in amniotes. Instead, those parts of the PPA which, under experimental conditions, escape apoptosis have retained the developmental potential to produce lateral line placodes and the primordia of neuromasts that represent the major functional units of the mechanosensory lateral line system.

Design of a 3-D Printed, Modular Lateral Line Sensory System for Hydrodynamic Force Estimation

AbstractThis paper presents a sensory system that is biologically inspired by the lateral line sensory system found in fish. This artificial lateral line system provides sensory information to be used in vehicle control algorithms, both to reduce model complexity and to measure hydrodynamic disturbances. The system presented in this paper is a modular implementation that can fit around a vehicle without requiring modifications to the hull. The design and manufacturing processes are presented in detail along with considerations for sensor placement and port spacing. An algorithm for calculating the hydrodynamic forces acting on the surface of a vehicle is derived and experimentally validated. An underwater motion capture system and strain sensors are used to calculate a reference hydrodynamic force that compares favorably with the hydrodynamic force calculated by the lateral line system.

Cranial asymmetry arises later in the life history of the blind Mexican cavefish, Astyanax mexicanus.

As a consequence of adaptation to the cave environment, the blind Mexican cavefish, Astyanax mexicanus, has evolved several cranial aberrations including changes to bone sizes, shapes and presence of numerous lateral asymmetries. Prior studies of cranial asymmetry in cavefish focused strictly on adult specimens. Thus, the extent to which these asymmetries emerge in adulthood, or earlier in the life history of cavefish, was unknown. We performed a geometric morphometric analysis of shape variation in the chondrocranium and osteocranium across life history in two distinct cavefish populations and surface-dwelling fish. The cartilaginous skull in juveniles was bilaterally symmetric and chondrocranial shape was conserved in all three populations. In contrast, bony skull shapes segregated into significantly distinct groups in adults. Cavefish demonstrated significant asymmetry for the bones surrounding the collapsed eye orbit, and the opercle bone posterior to the eye orbit. Interestingly, we discovered that cavefish also exhibit directional “bends” in skull shape, almost always biased to the left. In sum, this work reveals that asymmetric craniofacial aberrations emerge later in the cavefish life history. These abnormalities may mirror asymmetries in the lateral line sensory system, reflect a ‘handedness’ in cavefish swimming behavior, or evolve through neutral processes.

Sawfishes stealth revealed using computational fluid dynamics.

Detailed computational fluid dynamics simulations for the rostrum of three species of sawfish (Pristidae) revealed that negligible turbulent flow is generated from all rostra during lateral swipe prey manipulation and swimming. These results suggest that sawfishes are effective stealth hunters that may not be detected by their teleost prey's lateral line sensory system during pursuits. Moreover, during lateral swipes, the rostra were found to induce little velocity into the surrounding fluid. Consistent with previous data of sawfish feeding behaviour, these data indicate that the rostrum is therefore unlikely to be used to stir up the bottom to uncover benthic prey. Whilst swimming with the rostrum inclined at a small angle to the horizontal, the coefficient of drag of the rostrum is relatively low and the coefficient of lift is zero.

SS-31 peptide enables mitochondrial targeting drug delivery: a promising therapeutic alteration to prevent hair cell damage from aminoglycosides

Aminoglycoside-induced hearing loss stems from damage or loss of mechanosensory hair cells in the inner ear. Intrinsic mitochondrial cell death pathway plays a key role in that cellular dysfunction for which no proven effective therapies against oto-toxicities exist. Therefore, the aim of the present study was to develop a new mitochondrial targeting drug delivery system (DDS) that provided improved protection from gentamicin. Particularly, SS-31 peptide-conjugated geranylgeranylacetone (GGA) loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles were constructed successfully via emulsion-solvent evaporation method. The zebrafish lateral line sensory system was used as an in vivo evaluating platform to investigate the protective efficiency against gentamicin. SS-31 modification significantly reduced the activity of mechanoelectrical transduction (MET) channel and gentamicin uptake in zebrafish lateral line hair cells. As expected, SS-31 conjugated nanoparticles showed mitochondrial specific accumulation in hair cells when compared with unconjugated formulations. Furthermore, intracellular SS-31 modified PLGA NPs slightly enhanced mitochondrial membrane potential (MMP, ΔΨm) and then returned to a steady-state, indicating their effect on the respiratory chain complexes in mitochondria. GGA loaded SS-31 conjugated nanoparticles demonstrated the most favorable hair cells survivals against gentamicin when compared with unconjugated groups whereas blank formulations failed to exhibit potency, indicating that the efficiency was attributed to drug delivery of GGA. These results suggest that our constructed mitochondria-targeting PLGA based DDS have potential application in protecting hair cells from ototoxic agents.

Fishlike rheotaxis

Fish rheotaxis, or alignment into flow currents, results from intertwined sensory, neural and actuation mechanisms, all coupled with hydrodynamics to produce a behaviour that is critical for upstream migration and position holding in oncoming flows. Among several sensory modalities, the lateral-line sensory system is thought to play a major role in the fish ability to sense minute water motions in their vicinity and, thus, in their rheotactic behaviour. Here, we propose a theoretical model consisting of a fishlike body equipped with lateral pressure sensors in oncoming uniform flows. We compute the optimal sensor locations that maximize the sensory output. Our results confirm recent experimental findings that correlate the layout of the lateral-line sensors with the distribution of hydrodynamic information at the fish surface. We then examine the behavioural response of the fishlike model as a function of its orientation and swimming speed relative to the background flow. Our working hypothesis is that fish respond to sensory information by adjusting their orientation according to the perceived difference in pressure. We find that, as in fish rheotaxis, the fishlike body responds by aligning into the oncoming flow. These findings may have significant implications on understanding the interplay between the sensory output and fish behaviour.

The invisible fish: hydrodynamic constraints for predator-prey interaction in fossil fish Saurichthys compared to recent actinopterygians.

ABSTRACTRecent pike-like predatory fishes attack prey animals by a quick strike out of rest or slow movement. This fast-start behaviour includes a preparatory, a propulsive and a final phase, and the latter is crucial for the success of the attack. To prevent prey from escape, predators tend to minimise the duration of the interaction and the disturbance caused to surrounding water in order to not be detected by the prey's lateral line sensory system. We compared the hydrodynamic properties of the earliest fossil representative of the pike-like morphotype, the Triassic actinopterygian Saurichthys, with several recent pike-like predators by means of computational fluid dynamics (CFD). Rainbow trout has been used as a control example of a fish with a generalist body shape. Our results show that flow disturbance produced by Saurichthys was low and similar to that of the recent forms Belone and Lepisosteus, thus indicative of an effective ambush predator. Drag coefficients are low for all these fishes, but also for trout, which is a good swimmer over longer distances but generates considerable disturbance of flow. Second-highest flow disturbance values are calculated for Esox, which compensates the large disturbance with its extremely high acceleration performance (i.e. attacks at high speeds) and the derived teleostean protrusible mouth that allows prey catching from longer distances compared to the other fishes. We show CFD modelling to be a useful tool for palaeobiological reconstruction of fossil fishes, as it allows quantification of impacts of body morphology on a hypothesised lifestyle.

Zebrafish prion protein PrP2 controls collective migration process during lateral line sensory system development.

Prion protein is involved in severe neurodegenerative disorders but its physiological role is still in debate due to an absence of major developmental defects in knockout mice. Previous reports in zebrafish indicate that the two prion genes, PrP1 and PrP2, are both involved in several steps of embryonic development thus providing a unique route to discover prion protein function. Here we investigate the role of PrP2 during development of a mechano-sensory system, the posterior lateral line, using morpholino knockdown and PrP2 targeted inactivation. We confirm the efficiency of the translation blocking morpholino at the protein level. Development of the posterior lateral line is altered in PrP2 morphants, including nerve axonal outgrowth and primordium migration defects. Reduced neuromast deposition was observed in PrP2 morphants as well as in PrP2−/− mutants. Rosette formation defects were observed in PrP2 morphants, strongly suggesting an abnormal primordium organization and reflecting loss of cell cohesion during migration of the primordium. In addition, the adherens junction proteins, E-cadherin and ß-catenin, were mis-localized after reduction of PrP2 expression and thus contribute to the primordium disorganization. Consequently, hair cell differentiation and number were affected and this resulted in reduced functional neuromasts. At later developmental stages, myelination of the posterior lateral line nerve was altered. Altogether, our study reports an essential role of PrP2 in collective migration process of the primordium and in neuromast formation, further implicating a role for prion protein in cell adhesion.

Testing Biological Hypotheses with Embodied Robots: Adaptations, Accidents, and By-Products in the Evolution of Vertebrates

Evolutionary robotics allows biologists to test hypotheses about extinct animals. We modeled some of the first vertebrates, jawless fishes, in order to study the evolution of the trait after which vertebrates are named: vertebrae. We tested the hypothesis that vertebrae are an adaptation for enhanced feeding and fleeing performance. We created a population of autonomous embodied robots, Preyro, in which the number of vertebrae, N, were free to evolve. In addition, two other traits, the span of the caudal fin, b, and the predator detection threshold, ζ, a proxy for the lateral line sensory system, were also allowed to evolve. These three traits were chosen because they evolved early in vertebrates, are all potentially important in feeding and fleeing, and vary in form among species. Preyro took on individual identities in a given generation as defined by the population’s six diploid genotypes, Gi. Each Gi was a 3-tuple, with each element an integer specifying N, b, and, ζ. The small size of the population allowed for genetic drift to operate in concert with random mutation and mating; the presence of these mechanisms of chance provided an opportunity for N to evolve by accident. The presence of three evolvable traits provided an opportunity for direct selection on b and/or ζ to evolve N as a by-product linked trait correlation. In selection trials, different Gi embodied in Preyro attempted to feed at a light source and then flee to avoid a predator robot in pursuit. The fitness of each Gi was calculated from five different types of performance: speed, acceleration, distance to the light, distance to the predator, and the number of predator escapes initiated. In each generation, we measured the selection differential, the selection gradient, the strength of chance, and the indirect correlation selection gradient. These metrics allowed us to understand the relative contributions of the three mechanisms: direct selection, chance, and indirect selection.