Analysis of the Defence Mechanisms of Brassica Plants Against White Cabbage Butterfly

Since the concept of coevolution has been presented, there have been many discoveries of species interacting over time, adapting to one another within their respective ecosystem. This can lead to what is known as an “evolutionary arms race”, a term coined to signify when multiple species evolve together because of the interactions between them. A key example of an evolutionary arms race would be between a predator and its prey; the prey must evolve to better be able to out-manoeuvre its predator, this is to say not get caught and potentially eaten, while the predator must evolve to better hunt its prey than its competition. In the case of herbivory, the prey, normally being plants, must evolve to better be able to defend themselves, since their very limited movement nullifies their possibility of avoiding its predators. A plant’s only chance of deterring herbivores is via defensive mechanisms e.g. chemical secretion.

Chemical compound secretion is a common defensive adaptation recognized in various species of plants, with many producing toxic chemicals. In the case of the Brassica genus, the toxins they produce can make them inedible for many herbivores. This type of defense has worked against many insect species but has resulted in several other herbivorous insect species to have specialized on these plants, e.g. the white cabbage butterfly (Pieris Rapae). They have evolved to be better able to resist the toxins produced. In this case, the specialized insect-herbivore species can detect the related volatile chemical blends secreted and use them to its advantage as well. Based on genetic examination, we are able to safely estimate that these adaptations have evolved many millions of years ago (will be denoted as mya), and we are also able to constate that some developed directly in response to these changes, a case of coevolution. The analytically determined data, obtained via advanced genomics, has allowed us to make an accurate estimation regarding the appearance of counter adaptations to toxins following their apparition in the host-plants physiology.

The Brassicaceae family, also known as the Cruciferae or mustard family, is comprised of 338 genera, being represented by about 3700 species (Britannica). Of all these genera, our focus will be on the Brassica genus, inside which exists multiple species that are of agricultural as well as economical importance around the globe. It has come to the attention of many cultivators of these crops that they are the preferred host plant for a few insect herbivore species. This has led to rather vigorous scientific examinations of the relationships between many of these insect-considered-pest species and their host plants. Research in this field looks to investigate exactly what makes these specific plant species suitable to the insect herbivores of their environment, since Brassica species are known to contain toxic chemicals, that can have severe negative impacts on its predators. In more advanced studies, researchers have made discoveries to help explain what makes these specific plants the preferred host for multiple insect species, which has resulted in some having evolved to oviposition and feed exclusively on them.

The relationship between white cabbage butterfly (P. rapae) and various Brassica species has been thoroughly investigated, in some cases even to the genomic degree. It is the advancements in the fields of genomics, notably genomic mapping and the ability to place the appearance of new alleles in a species genome that has allowed us to make accurate predictions when researching the matter. In turn, the better understanding of the genetic sequences contained within the DNA of these species allows us to better understand exactly which proteins and enzymes are coded by which specific segments, which in the end results in the various chemical compounds found inside and/or secreted by the insect herbivore and its host plant. By comparing the genomes of related species, sharing common ancestry, we are able to estimate timeframes for divergence due to shifts in allele frequencies.

The large majority of the Brassicales order, including the economically and nutritionally important Brassica crops, such as oilseed rape (Brassica napus) and cabbage (Brassica oleracea) contain Glucosinolates, which are secondary metabolites well-known for their role in plant resistance to insects, pathogens as well as their cancer-preventive properties (Halkier). Glucosinolates aren’t toxic on their own, but once they have received tissue damage, they can be converted into various toxins via hydrolysis reactions normally involving myrominase enzymes (Richard Hopkins). These 2 substances normally occur in separate parts of the plant, but when a portion of the plant is being crushed e.g. chewed, they can be joined together, combining and then breaking down to form a variety of toxins. Some of these toxins include isothiocyanates, and nitriles, among many others. These can result in pungent smells, and some substances such as horseradish and mustard, this is why the Brassicaceae family is also known as the mustard family. It must be noted however, that it is possible for intact glucisonolates ingested by some insect herbivores to confer some resistance to the product, via host plant specialization and sequestration of the chemical(s) (Hopkins et al.). Some other common factors taking part in the reaction include pH of the environment, the concentrations of certain proteins, the presence of specific ions and the side chains present in the original compounds, to name a few. This means that there is a large number of variables that are interchangeable in the equation, since usually the environment is the mouth of the insect, and there are a lot of differences in between and even within species, due to other variable factors such as nutrition, physiological state, etc.

For a great variety of insect herbivores, the mixture of toxins resulting from the breakdown of glucosinolates has proven to be too much for their metabolisms to handle, to the point of even being lethal in various situations. The specific combinations of toxins released can trigger different responses in predators feeding on the plant tissue. There have already been over 120 different glucosinolates identified, the majority of which have been observed being present in the Brassicaceae family species (Fahey). This diversity in glucosinolates contained within Brassicales is the greatest, in comparison to known plants of any other order. This great diversity has occurred due to gene duplications, which are a very important source of genetic diversity in a most plant taxa. The fact that these species have had an abundance of time (tens of millions of years) is what has allowed for these various gene duplication events to occur (Edger et al). The glucosinolates can directly affect the insect herbivore in multiple ways, via 2 general categories that encompass the rest: antixenosis and antibiosis.

Antixenosis results in the non-preference treatment of the selected plant(s), the insect herbivore of focus can even learn to completely avoid the plant. Antibiosis means the plant must be consumed or at least contacted physically, allowing induced chemically mechanisms to have their effect. Sometimes it is this contact that will be the sensory stimulus required to induce these chemicals. This will result in an overall negative effect on the predatory herbivore. An example of antibiosis would be the products of the glucosinoluate-myrocinase reaction interfering with the absorption of other valuable nutrients in the insect gut. These and other antinutritional, usually toxic, by-products make these species unsuitable for most insect species. It must be noted that some of these products of the hydrolysis reaction may also be beneficial to other species such as ours, due to their anticarcinogenic effects, it is all a matter of dosage. Too much can also cause harm. For example, there is increased chance of developing goitrin because of a cruciferous-rich diet, though it remains relatively rare even in populations that consume moderate amounts of cruciferous vegetables (Bischoffet al.).

A 2012 study uncovered that in Arabdosis thaliana, a species in the Brassicales order, the production of its preferred suite of glucosinolates can increase photosynthetic requirements by up to at least 15%. In other scenarios, this requirement can be more or less, depending on the specific combination of glucosinolates produced, as well as the metabolism related requirements of the plant itself. These findings suggest that the maintenance of these defensive chemicals is highly costly, but the plants still invest in them. This implies that these chemical defenses are of great importance. When predators are present, they can significantly increase the plants fitness, by keeping numerous would be predators away. Glucosinolates are usually synthesized at low concentrations, but their synthesis can be induced via numerous signaling pathways, for example the jasmonate signal pathway. Jasmonic acid (JA) is a key phytohormone for inducing plant defensive mechanisms. A study investigated what effect the treatment of cabbage plants with JA would affect oviposition by two Pieris species, including P. rapae. It was observed that both species laid significantly fewer eggs on the leaves of JA-treated plants in comparison to control herbivores plants (Bruinsma et al.). Herbivores can sense when defensive compounds have been induced in plants, generalist species will generally respond by avoiding these plants. This signals poor suitability as a host plant. (Schoonhoven). This creates less inter-specific competition for the species that have specialized to feed on these specific plant species.

Many of these signaling pathways are put in motion when the plant is under herbivorous attack (Bekaert et al.). In plants, fitness is positively correlated with an increase in seed production, and in the wild there is a finite source of sunlight that can be used for photosynthesis. Plants have evolved to use this resource, as well as the others in their respective environment, in the most optimal of ways, usually producing hundreds or thousands of seeds during the appropriate season. Therefore, producing these compounds becomes defective for the plant if there are no predators to use them against, or at least none that are harmed/deterred by the chemicals. This type of defensive mechanism can prove to be even more costly to the plant if its predators are able to incorporate the toxin into their body and use them as their own defensive mechanism, which is what occurs when an organism evolves to be able to sequester the toxin(s) itself, for example. They will be unwillingly aiding their predator, potentially harming their own fitness, all while allocating more resources to the creation of these chemical compounds.

The defensive glucosinolates, which were a key innovation in the Brassicales order, evolved around 90 million years ago (mya), and have been diversifying since then (Wheat CW). This is due to natural selection taking place over time. Given the multiple ecosystems in which Brassicas spread their presence, there is a wide range of herbivores looking to take advantage of the various species of the diverse taxa. These individual species all have their own specific weaknesses to certain types of glucosinolates, and therefore the species that displayed the right suite of defensive compounds, relative to their community, were less susceptible than their counterparts to detrimental and sometimes devastating herbivory damage. The development and evolution of these defensive chemical compounds have however also led to the development of counteradaptations to them by multiple insect herbivore species, including the butterfly species of interest, Pieris rapae. Within 10 million years of the innovation of glucosinolates in the Brassicales order, the Pierinae, a sub family of Pieridae order responded with development of the nitrile-specific protein (NSP) and was able to successfully colonize the Brassicales between 60-75 mya, at which point Pierinae diversification rates began substantially increasing (Wheat CW).

Around 32 mya, the evolution of the Brassicaceae family, which contains all Brassica species, had come into existence and began diversifying itself. This plant family contains the greatest diversity of glucosinolates. The diversification of Pierinae is however not associated with the origin of Brassicales alone, but with the origin of the indolic glucosinolates they developed (Edgar et al.). Nitrile-specifier proteins (NSP) are structurally different compared to any known detoxifying enzymes. Only Pierinae butterflies possess these proteins. NSP shifts the hydrolysis of glucosinolate to nitriles instead of toxic substances such as isothiocyanates, which is why it is considered a key innovation in evolutionary history of Pieridae. NSP seems to have arisen via a process of gene duplication from a sequence of unknown function, that remains widespread in insect species (Fischer et al.).

In a study focusing on this matter, a phylogenomic tree was generated representing glucosinolate diversity as well as Brassica species richness, which were both mapped so that the original evolutionary points for new glucosinolate groups and their shifts in diversification rates could be identified. Two whole genome duplication events of Brassicales were uncovered, with their exact spot on the phylogenetic tree still not accurately pinpointed, due to the complexity as well as specificity of the matter. With more research we should be able to uncover more precise timeframes. However, this information proved enough to push towards significant discoveries and formulate new predictions. Using the already sequenced genomes of species in the genus Pieris, researchers began to study the activity of identified NSP, when put in contact with the glucosinolate-myrosinase reaction (Edger et al.).

Since it is known that the diversity of glucosinolates is greatest in the Brassicacea family, and that this diversity is due to ancient genomic duplications, it was then investigated whether the origination of this butterfly detoxification mechanism had developed in response to the appearance of glucosinolates. It would be logical to assume that the appearance and diversification of NSP has reflected the appearance and increase in diversity/complexity the evolutionary history it shares with glucosinolates, its counterpart.

Instead of being present only as redundant copies, the genetic duplicates of glucosinolate core pathways have specific substrates as well as products that are regulated by each their own individual transcription factors. The use of new amino acid substrates is what permitted the production of new classes of defensive compounds that could be used in the fight against insect herbivores such as butterflies. The diversification of the Brassicacea family coincides with the diversification of glucosinolate defensive compounds. These compounds then elicited an evolutionary response in some of the insect herbivores of the given environments, leading them to adapt and take advantage of their toxicity. By being the one of the select few species able of feeding on Brassicas without being harmed, an abundant, relatively untouched prey species became available. Some of these formerly toxic compounds even resulted in becoming oviposition and feeding attractants for species such as Pieris rapae (Edger et al.).

Pieris rapae is unaffected by either the breakdown products of glucosinolates or the associated proteinase inhibitors. Since most insect herbivores aren’t capable of ingesting Brassica species into their diet without suffering the consequences, they usually avoid them. This has allowed the Brassicaceae family to spread and thrive around the globe, with certain exceptions due to unfavorable weather, in some distinct geographic regions. Many studies have been conducted on the response of P. rapae to Brassica plant defenses, confirming that glucosinolates are not an effective defensive agent against them (AGRAWAL and KURASHIGE). This has resulted in P. rapae also having populations around the globe, following the range spread of its host plant, whom prefers a temperate climate. It is during the larval stage that the feeding on Brassica tissue is most abundant. The larvae prefer to feed on foliage but have been reported to have burrowed and ate other parts of the plants such as the heads of the broccoli and cabbage (CAPINERA).

The development of nitrile-specifier proteins by P. rapae is what set the stage for an extensive case of coevolution between them and their host-plant. The Pierina butterflies have undergone contemporaneous radiation in response to the increase in diversity that occurred in Brassicas (Bekaert et al.). P. rapae larvae, when consuming Brassica tissue, are able to redirect the normally occurring hydrolysis reaction of glucosinolate which is catalyzed by myrosinase. When doing this, the products of the equation are changed, from the formation of isothiocyanates, which are toxins, to the formation of harmless nitriles that are then excreted in the frass of the larvae. The nitrile-specific proteins found in the gut of the larvae are what allows this chemical reaction redirecting to happen.

Molecular analyses have permitted scientists to date the origin of the Pieridae family in comparison to that of the Brassicales order and help them realize that the significant diversification in the Pieridae family following the diversification of the Brassicales is an example of an evolutionary arms race. Brassicas developed glucosinolates to be able to effectively defend themselves against insect various species of herbivores, but then some species, such as P. rapae, developed various NSPs in order to be able to counter the various glucosinolate toxic products (Hopkins et al). It must be noted that P. rapae do not sequester these toxins, they simply change their chemistry and excrete them. P. rapae contain two activated nitrile-specifier coding genes and based on genetic research of other insect-herbivores filling similar niches in their environment over a long period of time, we have determined that it is the feeding on glucosinolates that gives the NSP gene sequences their value (Edger et al.).

It has been predicted that this evolved detoxification mechanism resulted in the adaptive radiation of multiple herbivore lineages, including that of P. rapae. NSP activity in the larvae of P. rapae matches the distribution of specific glucosinolate suites in their host plants. Through the use of 5 separate temporal estimates, it was determined that NSP evolved shortly after the evolution of the Brassicales, which saw them obtain glucosinolates as defensive chemical compounds. This led to the diversity in number of species found in the Pierinae order, in comparison to related clades. Several molecular datasets were used to create accurate estimations of when Pieridae evolved in relation to their host plants. Comparative genome analysis of glucosinolate-myrosinase systems in Brassica species have led scientists to realize that evolution of the system is still ongoing, in response to continued pressure created by the specialized herbivores. There are many variables that can be factored into the equation, e.g. the variety of enzymes that can take part in the reactions, the allelic variations or the relative concentrations of required chemicals, which all have an effect on the herbivores as well. Some of the genes essential to glucosinolate production have been identified to have undergone gene duplications as of recently. In response to this, P. rapae along with other herbivores that feed on these plants must adapt (Wheat).

Brassica plants are united by their ability to produce various defensive phytochemicals, however multiple species, such as P. rapae, were able to adapt and are not only be able to consume these plants without suffering the consequences but are now also able to use the secreted chemical volatile compounds released by the plant as cues. These cues can be especially important when it comes host selection for oviposition, the act of laying eggs. Female choice in oviposition site is a crucial factor, since choosing the better host will have a severe impact on the number of larvae that will be able to reach maturity, and eventually mate for themselves. Therefore, oviposition by egg laying females has a crucial impact on her overall genetic fitness. Glucobrassicin, one of the various secreted chemicals, was identified as the most effective stimulant in cabbage (Brassica oliracea.) when it comes to stimulating oviposition.

The nutrition of the plant can also have an effect on the released volatile chemical compounds. This is because each suite of glucosinolates requires specific ratios of nutrients, which must be available for the plant to use. For example, it has been noticed that nitrogen fertilization has resulted in increased rates of oviposition by P. rapae on mustard and cabbage plants, certainly due to the fact that nitrogen is a requirement for the creation of proteins (van Loon et al.). A great deal more research is required before we know which ratios are preferred by each species in the field, and in what ratios the nutrients are available given their specific environment. Certain ratios might make a plant more/less prone to oviposition and consummation, but other variables such as behaviour and the physiology of the herbivore also play a major role in this situation.

The larvae of this species are small and not very mobile, they tend to stay on the 1 leaf where they hatched, unless they eat it all of course. The larvae require about 15 days before they have consumed enough food, and grown enough in size to enter pupation, which will last another 11 or so days, if it occurs during the summer, before they enter their final life cycle which is that of an adult butterfly. Adults normally live around 3 weeks, during this time the female can produce 300 to 400 eggs. Adults feed on the nectar of many plant species (CAPINERA).

The mobility and energy reserves of first instar larvae are what limits the larvae from finding a suitable host plant on their own. This is what makes host-plant selection so important in P. rapae. Initially, the female will sense some of the volatile chemicals emitted by a host-plant of preference, and then upon contact with some of the plant tissue itself will be able to further analyse the chemical composition of the tissue. They are capable of this because of the tarsal taste sensilla they possess, and it was shown that their preference was for compounds eliciting the highest activity in the glucosinolate-sensory receptor cells (van Loon et al.).

Plant breeders as well as the agricultural sector dealing with Brassica species are particularly interested in the chemical defenses of their crop plants, and the degree of resistance they have against herbivores and diseases. Glucosinolates, a significant deterrent towards most insect species, can now also act as both oviposition and feeding stimulants in more than 25 specialized insect species (Hopkins). In humans, some of the products from the glucosinolate breakdown reaction are found to be enjoyable condiments to our meals, for example horseradish. For the cultivators of many Brassica species, keeping the neighbourhood P. rapae butterfly population away can prove crucial. Since its host plant is no longer effective in doing so, and actually attracts the pest species, research into control methods is of utmost importance.

The problem is, most control programs come with their own potential side effects, for example the use of pesticides might lead to more resistant strains of adapted P. rapae. The introduction of P. rapae parasites is also another option that can have negative side effects towards native species, if the introduced parasites manage to adapt to feeding on them as well. The more we understand about the mechanisms of both the plant and the butterfly species, the greater our odds of finding a solution to an issue many cultivators of Brassica species are facing. It took these species millions of years to evolve towards the state they are now at. We have figured out many of their complex mechanisms in relatively short time. Many more are yet to be discovered, but complexity like this is to be expected from millions of years of coevolution between interacting species.