Team:KU Leuven/Project/Aphid Background

Synopsis

During a long period of co-evolution, plants and aphids have established a complex interaction. On the one hand, aphids have established complex life cycles involving extensive phenotype plasticity with rapid population growth (Blackman and Eastop, 1984) and a very short generation time (Morrison and Peairs, 1998). On the other hand, plants have also evolved delicate and complicated defence systems comprising constitutive defence traits and defence pathways induced upon aphid attack (Chen, 2008). Breeders and growers are still struggling to find an efficient strategy for aphid control in major crop plants, as the damage can run in the millions. The KU Leuven iGEM project has an answer to this problem. Our project consists of a genetically engineered bacteria that can reduce the aphid damage on a sustainable way by hacking into their communication system.

Basic aphid biology

Aphids are an extremely successful group of insects that can be found throughout the world, with the greatest number of species in temperate regions such as Europe, North America and Asia (Dixon et al., 1987). An individual aphid is small and unnoticeable, however, they can become so numerous that they can damage whole fields or orchards. Their complex life cycles and polymorphisms enables them to exploit their host plants and to respond quickly to changes in their environment (Dixon, 1973). Aphids are small, mostly soft-bodied insects of the super family Aphidoidea (Minks and Harrewijn, 1987). Their body can be reddish, yellow, green, black brown, or almost colourless (Dixon, 1973). About 4400 species of 10 families are known and around 10% of these species are serious pests for agriculture and forestry (Dedryver et al., 2010). Worldwide, the annual crop losses due to aphids are estimated at hundreds of millions of dollars (Morrison and Pears 1998).

How aphids feed on plants

Aphid damage to plants can occur in various ways, mainly by stealing nutrients. Aphids use modified mouthparts, called stylets, to obtain their food by sucking plant juices (Minks and Harrewijn, 1987). Normally, aphids passively feed on phloem sap of their host plants, incidentally they can pierce a xylem vessle. Once a phloem vessel, which is under high pressure, is punctured, the sap is forced into the aphid’s stylet. Phloem sap is rich in sugars, but poor in amino acids, which are essential for growth. Therefore, aphids ingest a very large amount of food in order to acquire a sufficient amount of proteins. The residual digested food, which consists mainly of sugars, is excreted into the exterior in the form of a droplet of honeydew (Dixon, 1973). This sugary liquid is produced through a ‘tail’ at the rear end of the aphid, called a cauda. Most aphids also have paired tubular structures found at the end of the abdomen, called cornicles. Through these cornicles, aphids excrete droplets of a quick-hardening defensive fluid containing triacylglycerols, the cornicle wax. Other defensive compounds, such as alarm pheromones, can also be secreted by the cornicles (Minks and Harrewijn, 1987).

Life cycles

Aphids have a various patterns of complicated life cycles and occur in various types of polymorphisms. These polymorphisms are in relation to two biological characteristics of the aphid life cycle: alternation of sexual and parthenogentic generations and alternation of host plants (Minks and Harrewijn, 1987). Most aphids are parthenogenetic (asexual) and viviparous (give birth to living offsprings) for most of the year, but are also capable of sexual reproduction with the production of eggs during the winter. The annual cycle generally (97% of all species) includes a single sexual generation. These mating females (oviparae) lay eggs, all of which are female. On hatching, each egg gives rise to a wingless offspring that gives birth parthenogenetically to further parthenogenetic females (viviparae). These may be winged or wingless, in response to environmental conditions (Dixon, 1973). Crowding is a particularly strong stimulus to develop aphids of the winged, dispersal stage. Other external factors also play a role in the morph determination: food availability and quality, predators, photoperiod, time of the year and so on (Minks and Harrewijn, 1987).
In addition, aphid species have evolved a wide range of adaptive mechanisms based on the seasonal development of vegetation (Dixon, 1973). Many aphid species are monophagous, meaning they feed on only one plant species. In contrary to the economically important aphids, who have a wider host range and are therefore polyphagous (Blackman and Eastop, 1985). Polyphagous aphids have a primary woody plant during autumn, winter and spring and a secondary herbaceous plant in the summer. During bud burst and leaf development in spring and leaf senescence in autumn, trees are a richer source of food for aphids than in summer, when the leaves are mature. This has resulted in host alternation, in which aphids leave trees in summer and colonize the more recently evolved herbaceous plants that grow and flower throughout summer (Mordvilko, 1928).
In conclusion, aphids can rapidly exploit agricultural landscapes because of their high reproductive potential, endless source of food, dispersal capacities, adaptability to local environment, short generation time and feed on an endless source of plants (Dedryver et al., 2010).

Damage to plants and solutions

Aphid damage to plants may be caused directly, by nutrient drain, or indirectly by several mechanisms. These indirect mechanisms include plant virus transmission through the aphids’ stylet. Injection of saliva containing phytotoxic components has a disastrous effect on plant growth and harmful fungi growth on the aphids’ honeydew hinders photosynthetic activity. Common damage symptoms on plants are decreased growth rates, mottled leaves, yellowing, stunted growth, curled leaves, gall formation and low yields and death (Dixon, 1973). Different methods can be used to control an aphid infestation. For many crops, insecticides provide a simple and effective strategy for aphid control. However, the application of such chemicals is not desirable in the long term, because of the development of insecticide resistance and the potential negative effects on the population dynamics of other beneficial insects. Aphids are vulnerable to many kinds of predators, including ladybird beetles and parasitoid wasps, and parasites and these can also be used to biologically control aphids (Minks and Harrewijn, 1987).

Economical important crops

Aphids represent the most important group of insect pests in agriculture in temperate Europe. They can infest a wide range of arable and horticultural crops in a very short period of time, which is why farmers are advised to treat this aphid pest problem from the very first symptom. The most widespread species in Europe are: green peach aphid (Myzus persicae), Bird cherry-oat aphid (Rhopalosiphum padi), potato aphid (Macrosiphum euphorbiae) and Black bean aphid (Aphis fabae).
In general, aphids reduce crop yield and/or quality, which lead to significant economical losses. 
The major crops infected are wheat, barley, sugar beet, potatoes, lettuce, and beans. The damage caused by aphids depends on the type, for instance the black bean aphid causes damage by direct feeding whereas the green peach aphid causes the most damage by being a virus vector for more than 120 viruses (Rothamsted insect survey).
The average percentage loss caused by different aphid species is variable, ranging from 4,4% on potatoes, since these plants are rather resistant to aphids, compared to 46-43% on field beans. In 1979, aphids caused a potential economic loss of £70 x 106 in Britain. In the US, aphids have an economic impact of more than one-quarter billion dollars in an outbreak year (Larsson et al., 2005).

Economic treshold & injury level

When does an infestation become economically viable to control?
An economic threshold is the insect's population level or extent of crop damage at which the value of the crop destroyed exceeds the cost of controlling the pest. In other words, it indicates the number of insects that should trigger pest management action. If a pest population is growing as the season progresses, growth rates are predicted and the economic threshold is set below the economic injury level (Agnote; Mickan et al., 2006). 
Various economic thresholds have been suggested for different aphids, e.g. 2 - 3 aphids/main stem at full bloom or 8 aphids/main stem at green boll stage for the potato aphid, 12 - 15 aphids/stem prior to soft dough for the bird cherry-oat aphid (Saskatchewan Ministry of Agriculture). As the grains of cereal plants form and mature, they pass through the clear liquid stage, then become milky, followed by soft and hard dough stages and eventually as a dry grain suitable for grain harvesting (Agnote; Mickan et al., 2006). 

Crop loss must however be distinguished from economic loss: the type of crop and production level depends on aphids. For example, ornamental crops for aesthetic purposes are more easily rejected and thus lost than of other crop types eg. food crops. Whereas loss of these food crops could represent a higher economic loss et al., 2010).

Research groups

Due to the high impact of aphids on crop damage, several research groups have been trying to tackle this problem by the use of biological pest control.
Rothamsted research group is currently investigating the use of genetically modified crops that produce E-beta-Farnesene in order to repel aphids. They are carrying out a two year experiment, the Rothamsted wheat trial.
Other research groups we worked together with is Biobest and pcfruit. Biobest offers a whole series of parasitoids and predators against aphids for the purpose of biological control of pests. Biobest green lab investigates the relationship between plant and aphid in search of a method to protect plants from this pest. Pcfruit has a similar mission, but more focused on fruit trees. The advice that Biobest and pc fruit provides is very well appreciated amongst the farmers.
APPEAL, an organisation that stands for ‘Assessment and valuation of Pest suppression Potential through biological control in European Agricultural Landscapes’, is controlling aphids by providing natural enemies in order to create a natural ecosystem service has an immense economic value.

Chemical control of aphids - aphicide

Several factors need to be taken into consideration when choosing a good aphicide. An aphicide should be selectively toxic to aphids so that predators and parasitoids of aphids, as well as pollinating insects, are not killed in the process. This is important especially in fruit growing. Systemic action of an aphicide is essential, in order to reduce the number of treatments, since aphids can colonise a plant almost continuously, in order to reduce the number of treatments. This means that application of the aphicide could be on the foliage, in the soil or to the seed, and would still be able to carry out its function. Residual activity is favoured to treat crops that are often re-infested by aphids but this is not favoured for food crops, as the aphicide should be decomposed into harmless compounds before harvest. In order to prevent transmission of plant viruses, aphicides should act rapidly. Besides this aphicides should not harm the plant any further; low phytotoxicity (Schepers, A., 1989). It is difficult for one chemical compound to comply with all these requirements and on top of that, the induction of aphid resistance should be avoided. Pest resistance to a pesticide is commonly managed through pesticide rotation, which involves alternating among pesticide classes with different modes of action to delay the onset of or mitigate existing pest resistance. The use of pesticides has many disadvantages to which the BanAphids should provide a solution.

Ladybug has found an aphid

Prey localization

Predatory and parasitoid insects have a specialized sensory nervous system to detect their prey (Hatano et al., 2008). They use volatiles produced by the herbivores, reliable but at low concentration, or those produced by the plant to locate their prey. These latter are easily detectable, but are less reliable. To overcome the reliablility-detectability problem predators and parasitoids focus on the responses on stimuli created by specific interactions between the herbivore and its plant (Vet & Dicke, 1992). In response to an aphid attack, plants modify their volatile emissions and these changes are detected by aphid enemies (Du et al., 1998). Host selection occurs in three phases: habitat localization, host localization and host acceptance (Vinson, 1976). Aphid natural enemies must first locate the aphid habitat, the host plant where aphids are present. Therefore, plant-derived volatiles are used, since evidence of aphid damage is acquired. One of the important herbivore-induced plant volatiles that are used by predators is methyl salicylate (MeS) (Zu & Park, 2005). The secretion of MeS is induced by the feeding of aphids on the plant (Birkett et al., 2000). Following habitat localization, natural enemies use short range physical (colour, shape, movement of aphid) and chemical cues to search for a suitable herbivore on the host plant (Hatano et al., 2008). One of the chemical cues used by natural enemies is aphid honeydew. Mostly, the natural enemies need physical contact with honeydew to change their behavior (Ide et al., 2007). In addition to aphid honeydew, the aphid alarm pheromone, (E)-β-Farnesene (EBF) is also an important semiochemicals in aphid localization. It is released in the cornicle secretions of many aphid species (Franscis et al., 2005) to alert surrounding aphids of the presence of natural enemies (Kunert et al., 2005). Detection of these short range chemical cues leads not to the aphid directly, but only indicates the presence, improving prey detection of the predators and parasitoids. Once an aphid is located, natural enemies have to recognize it as a potential prey before they attack it. For host recognition, nonvolatile chemical cues are important, in particular contact with the cornicle wax on the surface of the aphids. Predators use their antennae or their mouthparts to recognize the prey. Parasitoids use probing to assess host quality before oviposition (Hatano et al., 2008).

β-Farnesene

Generally, aphid populations are regulated by natural enemies such as ladybird beetles and parasitoid wasps (Minks and Harrewijn, 1978). For many species of aphids, avoidance of predators involves the release of an alarm pheromone, generally (E)-β-farnesene (EβF). EβF is a volatile sesquiterpene released from the aphid’s cornicles when attacked by its natural enemies (Dixon 1998). Therefore, EβF functions as a direct repellent of the aphids and acts as an attractant of the aphid’s natural predators (Hatano et al., 2008).
The high oxidation rate of EβF has hampered the application of EβF to crop plants against aphids (Lambers and Schepers 1978). We get around this problem by using E. coli to produce the aphids’ own alarm pheromone (EβF) and use them directly on the plants. Besides using the aphid communication, we also want to attract aphid predators such as ladybugs to make sure the aphids are thoroughly removed.

Methyl Salicylate

When plants are attacked by herbivore insects, they emit a volatile signal that is dependent on the type of insect (Vlot et al., 2008). Herbivore-induced plant volatiles mediate relationships between plants and insects through the attraction of natural enemies and the repulsion of herbivores (Turlings and Ton, 2006). Manipulation of herbivore-induced plant volatiles can be used to concentrate and increase populations of natural enemies in a specific place, such as crop fields, or to repel pests from crop plants (Khan et al., 2008). Methyl salicylate (MeS) is a phenolic compound that is released when plants are attacked by herbivore insects (Reymond and Farmer, 1998). Upon the activation of the salicylic acid pathway, liquid MeS concentration increases within a plant, the volatile form is subsequently released in high concentrations (Vlot et al., 2008). Furthermore, MeS itself also induces the salicylic acid pathway and can therefore elicit responses in neighbouring healthy plants (Heidel and Baldwin, 2004). MeS is also involved in plant defence, it functions as an activator of defence-related genes.

Green peach aphid

In our experiments, we used Myzus persicae or the green peach aphid, a small generalist aphid that can be found worldwide. The green peach aphid attacks plants in the field as well as in greenhouses. Their primary overwintering host is Prunus sp. (peach trees and their hybrids). However, in the summer, they leave their primary host to feed on a wide range of plants including vegetable crops, weeds and ornamentals.
Temperature greatly influences the lifecycle of the green peach aphid, especially cold winters. Aphids overwinter in the egg stage on Prunus trees. As temperatures warm, the aphid may go through multiple generations while still on the tree, but as densities increase, winged adults are produced. These winged forms colonize nearby plants by depositing daughters on one plant and then moving on to the next plant. Each daughter begins asexual reproduction (parthenogenesis) by rapidly giving birth to new daughters, which often already have developing daughter inside them ready to emerge in a few days. As densities increase on the newly colonized plants, and as the plants deteriorate, new winged forms are produced. This cycle repeats as long as the weather is favourable. Eventually, as day length begins to shorten and temperatures change, the winged females search for Prunus trees to colonize. Females arrive on the overwintering locations first and give birth to wingless egg laying forms (oviparae), which mate with winged males.

Adalia bipunctata

In our experiments we used the two-spotted ladybug. Adalia bipunctata, commonly known as the two-spotted ladybug, is a carnivorous beetle of the family Coccinellidae that is found in western and central Europe and North America. The two-spotted ladybeetle feed on aphids and other small insects. The wing covers of adult two-spotted lady beetles are bright-red with one black spot on each side (Marshall, 2000). Its life cycle starts with bright yellow eggs and ladybugs pass through four larval stages. The larvae and the adults are predators. The larvae of older ages move actively on the whole plant for the search of food and also migrate to other plants. The adult individuals keep not far from preys where they mate and lay the eggs. Therefore, ladybugs are frequently used in ornamentals, fruit and vegetable crops as beneficial to control aphids, for example in greenhouses.

Macrolophus pygmaeus

In our experiments, we also used Macrolophus pygmaeus as aphid predator. Macrolophus pygmaeus is a bright green and long-legged predatory. After the birth of the nymphs, they pass through five mobile stages. During the first stages nymphs are yellowish green, but older nymphs are bright green as the adults. In the last two stages the growth of the wings can be seen. Macrolophus predates several pest insects aphids, whiteflies, spider mites and moth eggs. Adult and nymphs search actively for their prey, insert their sucking mouthparts and suck out the contents. Macrolophus pygmaeus is very common in the Mediterranean, therefore it is mostly used as biological control in greenhouses.

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