Phenotypic Plasticity of The Pupal Color of Mycalesis Mineus

Phenotypic plasticity (PP) is the ability of an organism with the same genotype to produce a range of phenotypes in response to varying environments. PP results from the interaction of the gene with the environmental cues, so under PP concept, nature and nurture cannot be separated from each other. Many factors can act as a cue or stimuli to initiate PP and can originate internally (E.g. pathogen presence etc.) or externally (E.g. photoperiod etc.). Cues tend to be harmless stimuli (like photoperiod) without any direct effect on the individual but induce adaptive plasticity by predicting future environmental conditions, whereas harmful agents like toxins are considered as stimuli, though the division between these two is blurred. Phenotypic plasticity has been shown to be adaptive for the organisms expressing it. The environment is always changing and all living things are susceptible to the effects of abiotic and biotic factors. The only way for an individual to adapt to a changing environment is by changing its phenotype. This has been shown in a variety of organisms and studied extensively in insects. Butterflies having a shorter generation time and found in a diverse seasonal environment are also phenotypically plastic for various traits, such as wing coloration, body size, and pupal coloration.

However, the studies of PP especially on the early stages ( for example, pupal coloration) of tropical butterflies are comparatively less to the temperate butterflies. Studies have shown that most of the swallowtails exhibit a plastic trait for dimorphic pupal coloration (green and brown), whereas pink and green pupae are reported in Danaus chrysippus. In nature, the environment is heterogeneous depending upon the seasons, for example in one of the seasons there are lots of greenery (grasses, leaves etc) and in another season the greenery is very less. As the color of the pupation substrate (the background) is a decisive factor for the survival of the immobile pupa, i.e. a green pupa can match the green background very effectively and remain undetectable from the predators, whereas the brown coloration can be advantageous where the greenery is less or on the substrates which are not green (for example, brown tree trunk, soil and dead brown leaf). Thus, this color dimorphism seasonally varying heterogeneous background helps them to survive their critical stage of life. Though there is a good amount of research work on environmental factors affecting the phenotypes, the genetic basis of plasticity remains less explored. In my thesis, I will address tropical butterfly pupal color plasticity and how it is related to environmental factors like different pupation substrate (grasses), as well as its underlying genetics.

Project 1. Genetic basis of the pupal color plasticity in Mycalesis mineus

Overview: Pupal color variation does not completely depend upon the environmental factors. Many studies have been shown the heritable nature of this phenomenon in different swallowtail butterflies like P. zelicaon, P. polyxenes and so on. Offspring produced by artificial selection of selected line is more similar in color to their parents. Some studies suggested that the pupal color plasticity is inherited as a threshold trait. According to this model, the ability of a larva to produce green or brown color pupa is very much depended upon an underlying phenotypic variable which is heritable. Larva which is very sensitive towards green coloration can be less affected by the brown color-inducing environmental cues and become a green pupa and vice versa. However, the strength of the inducing stimuli is also an important factor along with the sensitivity and it usually varies among population and species as the ecological conditions are not uniform in every place. A tropical Nymphalidae butterfly Mycalesis mineus, exhibit pupal plasticity in the form of green and brown coloration. Studies have shown that in low relative humidity condition they produce 20% brown and 80% green pupae whereas in higher relative humidity condition (i.e. 85% RH) they are almost exclusively green. In tested laboratory conditions they have also shown that green pupae can form under all kinds of environmental cues and substrates but brown pupal coloration are exclusively confined to particular conditions such as, they do not form on leaf substrate (exclusively on off leaf substrate, i.e. anywhere except the leaf substrate) and their number are significantly low at high RH. My question: Is “sensitivity” of the larvae to produce a particular color is heritable in Mycalesis mineus butterfly?

Hypothesis 1: If the “sensitivity” is heritable, then selected lines of green and brown pupae should produce a higher proportion of green and brown pupae respectively in subsequent generations.

Hypothesis 2: Green pupae from the off-leaf substrate will tend to produce more green pupae on off-leaf in subsequent generations compared to green pupae from leaf substrate. Methodology: I am conducting a series of artificial selection experiments. For my “Hypothesis 1”, I am using a larval growth chamber in which the relative humidity is fixed at 60%, where we are getting a significant number of brown pupae along with usual green pupae. Brown and green pupae will be segregated. Adults eclosing from brown and green pupae will be reared separately. Every generation will be filtered for pupal color with respect to lineage (green or brown). Experiment shall run for at least ten generations. Work done and works in progress: For “Hypothesis 1”, initially I started with 25 brown pupae. I got 15 adults (F0) and the larvae produced by them are very less and subsequently, I got no brown but all green pupae in next (F1) generation. Currently, I’m having a large number of eggs from the stock and I want to repeat the experiment with a large sample size. For “Hypothesis 2”, I have started the experiment by putting a single wandering stage larva in an empty round cage. For the first round, I put 6 larvae, each one inside a round cage of similar height and structure and put those cages inside the 60% RH growth chamber. I got 3 green pupae formed on the net of the cages and other 3 larvae died. I am going to test it again with a large sample size.

Project 2. Effect of different host plant on pupal color plasticity. Overview: Pupation substrate structure, color, smoothness, and texture have been shown to affect the pupal color plasticity. Previous studies on Mycalesis mineus pupal color polymorphism was done on maize plants. As maize is not found in natural wild condition for them to pupate, I want to check for the pupation substrate patterns for commonly available grasses. Some of the local grasses have already been identified from flower seed morphology (example- Eleusine indica, Paspalum scrobiculatum, Oplismenus sp., Axonopus sp.). Other grasses to be used for comparison are Ragi and Wheat. Some of these grasses grow horizontally whereas some grow vertically. Their leaves and stems are different from each other in various aspects like shape, structure, smoothness etc. The distance from the soil to the pupation substrates may also have some effect on the coloration as the soil color is different from the green leaves or stems. So, for this project my hypothesis is, the grasses having branches close to the soil (horizontally growing close to the ground) will have more number of brown pupae than the grasses growing vertically. Work done and work in progress : Identification of some of the wild grasses has been done. Currently, I’m trying to grow the grasses in laboratory condition.

Future plan: 1. Carrying out the artificial selection experiment on pupal color dimorphism of Danus chrysippus. 2. Looking for the evolutionary route of the pupal color plasticity from the butterfly phylogeny. Work in progress: A literature review on butterfly pupal color plasticity is currently under process. Course work: I have completed two courses (BIO4202 and BIO4207) in the 1st semester. My SGPA for 1st semester is 8.