Description

The rapid closure of Mimosa’s leaves in response to mechanical disturbances such as controlled rop is believed to be a defense tactic to reduce predation risk. The Mimosa Habituation apparatus allows for such controlled falls for habituation training. Each disturbance causes the mimosa leaflets to fold along the stem, and droop downwards.

The apparatus consists of an acrylic container mounted with to a marked steel rail. A soft foam base lies underneath. Individual potted plants can be elevated to the 15-cm height mark and allowed to drop by sliding along the rail. A shallow depression in the foam base prevents bouncing at impact.

Mimosa pudica was first identified in Brazil. It is a perennial shrub which is now considered a pantropical invasive weed.  Many plant species show two types of movements of the anatomical parts. Nyctinastic movements, also known as sleeping movements, occur in response to changes in visible light. Seismonastic movements occur in response to other stimuli like mechanical, thermal, chemical or electrical (Sanberg 1976).  Mimosa pudica shows both types of movements in the form of leaflet closure and petiole dropping. Wallace researched the environmental factors related to seismonastic movements of Mimosa pudica. He found that the optimum sensitivity is seen at a temperature of 40^oC with an apparent response still shown within the range of 14^oC to 60^oC, with no change in sensitivity seen due to the relative humidity in the environment.  Under artificial lightning, in a 14-hour light: 10-hour dark cycle, the sensitivity is highest near 5 AM and lowest at 7 PM. The sensitivity of the leaves also decreases with age.

Mimosa pudica show inducible defenses which are described as behavioral responses initiated only in the presence of a threat. Such behaviors when used by the plant at appropriate times provide greater resistance to attack from herbivores and increase chances of survival. Mimosa pudica temporarily closes its leaves when touched. The time the leaves remain closed is referred to as the hiding time (Reed-Guy et al., 2017). It is believed that the leaf folding behavior shown by Mimosa pudica reduces predation risk by scaring away predators due to rapid closing movement of the leaves, decrease visibility of the plant due to closed leaves, decrease surface area exposed to predators and making thorns more visible to the predators (Jensen et al., 2011). Such defense responses inflict energy costs required to reopen the leaflets and also opportunity costs as this causes the photosynthetic rate of the plant to decrease by up to 40% which represents a substantial cost associated with such antipredator behavior (Reed-Guy et al. 2017). Plants decide to forage based on the availability and distribution of resources. Optimal foraging theory states that an organism faces trade-offs during foraging and optimal strategy will be naturally selected during foraging that maximizes fitness by balancing risk and reward. When plants are in a poor quality environment, they will put more effort into foraging which will expose the plants to a higher risk of predation to gain resources as compared to the plants in a good quality environment (Simon et al., 2016).

Habituation is the simplest form of learning which refers to decrease in response to a repeated stimulus. Habituation can be either short-term or long-term depending upon the length of recall. Mimosa pudica shows habituation behavior by folding its leaflets initially, but when they are repeatedly disturbed physically, the plant learns to ignore the stimuli. Plants growing in environments that require a lot of energy show more marked leaf-folding habituation which can last even for a month if the plant is left undisturbed. Such persistent behavioral change of long-lasting habituation is similar to the habituation observed in animals.  Learning of habituation response allows the individuals to pay attention to important stimuli and save energy by ignoring harmless or irrelevant stimuli (Gagliano et al., 2014).

A decrease in the sensitivity of the plant to respond can be due to sensory adaptation or effector fatigue that must be ruled out. Sensory adaptation occurs due to intense periods of stimulation of sensory organs causing a decrease in responsiveness. To rule out sensory adaptation, a longer inter-trial interval should be selected between stimuli. A test trial can also be used in which habituation is tested after training with a long enough time interval between training and the test trail. Effector fatigue is the inability of the effector response mechanisms to function properly. A test trial is given to the subject using a second stimulus that triggers the target response. If then, the reintroduction of the original stimulus produces a response, effector fatigue can be ruled out. This procedure is referred to as dishabituation which is used as a control in most of the habituation experiments (Abramson et al., 2016).

Habituation was first described in 1887 by George and Elizabeth Peckham in a publication on the ‘Mental Powers of Spiders’ (Christoffersen 1997). In plants, the first habituation experiment was reported in 1873 by Pfeffer using Mimosa pudica. Pfeffer used repeated mechanical stimulation of leaflets, which initially caused leaflets to close and petiole to drop but eventually, the leaflets stopped responding and stayed open despite continuous stimulation. Later on, in 1906, Bose conducted another study using Mimosa which confirmed the findings of Pfeffer. Bose also used electrical stimulation along with mechanical stimulation, both of which resulted in the habituation of leaflets. He also demonstrated that leaflet closure could be deployed again if sufficient rest period is allowed in between stimulation. Holmes and Gruenberg in 1965 then studied Mimosa habituation in the context of stimuli discrimination. They used a drop of water and finger touch as stimuli to show that the plant can discriminate between the two stimuli because the leaflets close due to finger touch after they have habituated to water drops. Also, the leaflet closure was not due to fatigue otherwise it would not have responded to stimuli from finger touch (Abramson et al., 2016).

By 1960’s, enough studies had been conducted to form an operational definition of habituation, so in 1966 Thompson and Spencer defined habituation based on the list of nine behavioral characteristics that are till date used as a framework for future studies. The list of characteristics have been confirmed by most of the studies and have been expanded over the years by many researchers (Gagliano et al., 2018, Rankin et al., 2009).

Applewhite in 1972 studied whether the training variables that are known to influence habituation in animals also influence habituation in Mimosa. He used detached leaflets placed in water for the experiment along with using a dishabituation control. More recently, Gagliano et al., investigated short-term and long-term habituation along with effects of light intensity on the leaflet closure response of habituation in Mimosa pudica. The results of the experiment confirmed the findings of Holmes and Grunberg.

The apparatus consists of a marked vertical steel railing fixed to a foam base. A clear acrylic vessel is mounted onto the steel railing using variable hangers in which potted plants will be placed during the experiment. The foam base is cut out and hollowed in the center making a shallow depression for the acrylic vessel to drop into from a height by sliding along the steel railing.

Set up a growth room approximately 5.30 m² in size for the experiments. Make three compartments in the room using black plastic sheets. The training trials will be conducted in the middle compartment, and the test trials will be conducted in the side compartments. In each of the side compartments fit fluorescent lights, one compartment with low-intensity lights (LL) while the other compartment with high-intensity lights (HL). Measure light intensity just above the plants in each compartment before starting the experiment. Grow each individual Mimosa pudica plant with similar heights (6-8 cm) in a 10 cm round acrylic pot with a standard mixture of loamy soil and organic compost with a 1:1 of volume. Fertilize and water the plants as per requirement. Maintain a 12-h light: 12-h dark cycle, 60%-70% relative humidity and 21-24^oC temperature.

Habituation Training

Randomly assign individual potted plants to either HL or LL conditions. Leave them undisturbed for 5 days until training day.

Training 1

Place a potted plant in the acrylic vessel and drop it from a set height of 15 cm in the morning and then repeat the drop from same height 8 hours later. Repeat this for all the plants from each of the side compartment.

Training 2

Place a potted plant in the acrylic vessel and drop it from a set height of 15 cm. Repeat this procedure 60 times with an interval of 5 to 10 seconds between each drop. Repeat another set of 60 drops, for a total of seven times consecutively one after the other in a single day with increasing intervals between each train of 60 drops. Repeat this for all the plants from each of the side compartment.

Dishabituation Test

Place the individual plant in a close fitted foam container that is attached to a shaker plate. Turn on the shaker plate and set it to 250 revolutions per minute for 5 seconds. Observe the leaf folding behavior.

Test for the Short‑term memory of the habituated leaf‑folding response

Test each of the trained plants from both side compartments six days after 1-day training using 60 consecutive drops from a set height of 15 cm.

Test for Long‑term memory in Mimosa exposed to a new environment

Transfer plants from the side compartment with LL to the compartment with HL and vice versa. Test the plants after 28 days using complete training protocol mentioned above under training 2.

After each drop, for each individual plant select three random leaves. Measure the breadth of the leaf (mm) from tip-to-tip using digital calipers. Take an average of the three measurements.  The response is then represented as the maximum leaf breadth immediately after the drop relative to the maximum leaf breadth before the drop. The observations should be taken in quick succession from the three leaves both before and after the drops.

Mimosa pudica is an ideal plant for habituation experiments as they are easy to maintain, significant information is available about its natural history, and they have a visible leaflet closing response to external stimuli (Abramson et al., 2016). Experiments related to habituation are generally easy to perform as they require little equipment.

Mimosa pudica has a long recovery time of up to 15 minutes which can be problematic as several training variables like inter-stimulus time interval and the time period between a response, and its outcome must be very short for any association to be formed. This can also produce a hindrance when comparing animal and plant behavioral studies and techniques as animal responses are much quicker than responses in plants. Researchers need to develop an automatic way of presentation of stimuli as well as the recording of the response to further develop the field of research related to plant learning. It is crucial to know the rate, length and temporal pattern of the target response before studying habituation. A control group must be used that will be subject to the training situation, but no habituation training will be given (Abramson et al., 2016).

Some researchers do argue that Mimosa has a neural capacity for learning behavior. If this is agreed upon, then further studies will broaden understanding of the nervous system and clarify mechanisms involved in Mimosa’s response. One advantage of using Mimosa for studying hormonal secretion induced by the nervous system is that the cells are relatively large, accessible and excitable as compared to the small size and inaccessibility of the cells involved. This can provide new opportunities for studying interactions between electrical and chemical mechanisms of control.

Studies related to plant learning that involves habituation are an excellent way of collecting data to compare them with animals as habituation is universally observed in the animal kingdom. Habituation although considered simplest form of learning, shares many properties of complex learning such as the ability to recover from a response over time, forming new behavior patterns and improved performance over repeated sessions. These properties and sensitivity to training variables like intensity, frequency, and pattern of stimulation can be further investigated in plants.