An investigation into the role of salicin as a deterrent in
specialist moths
Author: Max Louwerens
Student number: 10671765
Supervisor: Dr. Peter Roessingh
Examiner: Dr. Emily Burdfield-Steel
University of Amsterdam
Bachelor Biology
2020
Introduction
The association between plants and phytophagous insects has long been an interesting topic for the studies of speciation and evolutionary ecology (Jermy 1992). A large proportion of phytophagous insects are monophagous; they only feed on a single species or genus of plants (Schoonhoven & van Loon 2002). One of the main proposed reason for this specificity in diet is a response to chemical compounds produced by plants called secondary plant metabolites, and these can either have a positive or negative effect on some of the behaviours of the insects, i.e. act either as stimulants or deterrents. Deterrents are compounds that can be defined as ‘a chemical which inhibits feeding or oviposition when present in a place where insects would, in its absence, feed or oviposit’ (Dethier et al. 1960). Stimulants are chemicals produced by plants that stimulate a feeding or ovipositing response.
Previous research has shown that the more specialised the insect, the more sensitive the insect is to secondary plant metabolites (Bernays et al. 2000). For example, in research of five different beetle species, the more extreme specialists were more deterred by non-hosts (Jermy 1966), and among caterpillars the specialist Spodoptera exempta was more deterred by non-nutrient compounds than the generalist Spodoptera littoralis (Blaney et al. 1996)
Phytophagous insects have highly sensitive and specific chemoreceptors that respond vigorously to these stimuli, because they are indicators of the suitability of the host plant (Knolhoff 2014). If the insect senses the compound, then that indicates that the correct plant has been found, and feeding or oviposition can begin without negative fintness consequences for adults and larvae.
The specificity of the responses to secondary plant metabolites are thought to play a key role in the speciation of specialist phytophagous insects, through potential modifications at the sensory level. These modifications could potentially lead to host-shifts in insects, allowing eventually for the formation of new host-races and species in sympatry. (Menken & Roessingh 1998)
One of the ways in which host-plant shift are thought to occur is due to changes in sensitivity to specific plant secondary metabolites in the lepidopteran’s contact receptors in the medial sensilla styloconica. In Western European Yponomeuta, for instance, several of these host-plant shifts are thought to have taken place, and in some cases multiple shifts to different families have been studied. In the case of the willow ermine moth (Yponomeuta rorrella), two such shifts between families have taken place, first from a relationship with Celastraceae to Rosaceae, and then from Rosaceae to Salicaceae. Y. rorrella feeds on plants of the genus Salix, part of the Salicaceae.
(Chapman 2003) This shift is thought to have taken place due to a loss in sensitivity to the compound salicin, which is a bitter compound known to generate a strong neurological response in the medial styloconica sensilla of non-salix feeding Yponomeuta, which is a strong indicator that salicin is a deterrent for non-salix feeders. (Van Drongelen 1979). Salicin is also shown to be a deterrent to other non-willow feeding lepidopterans, including Heliothis subflexa (Bernays et al. 2000), and the gypsy moth Lymantria dispar (Cook et al. 2003). Food plant recognition in lepidopterous larvae are predominantly governed by the activity of 8 taste neurones present in two styloconica located on each maxilla. These include taste cells that respond to general phagostimulants that increase feeding, but also several cells in these styloconica that when activated by a variety of different stimuli, will cause a deterrent effect, and a decrease in feeding. (Schoonhoven & Van Loon 2002). Salicin is one of the substances that produces this particular response in the lateral sensilla in almost all Yponomeuta species except Y. rorrella, (Van Drongelen 1979) which indicates that only the willow ermine moth is not deterred by the presence of salicin. This could mean that somewhere along the way, sensitivity to this chemical was lost, allowing for the switch from a relationship with Rosaceae
to the Salicaceae. Such a shift is not deemed to be too implausible or require too many adaptational changes (Menken & Roessingh 1998), as there are many micro and macro lepidopteran species that feed on both plant families (Hering 1951).
The genus Yponomeuta (Lepidoptera, Ditrysia), is particularly suitable to study host shifts. It is a relatively small genus of the family Yponomeutidae (Menken et al. 1992). and contains some 30 species, of which 9 well-defined species occur in Western Europe (Povel 1984, Ulenberg 1998). Of these, six are monophagous on shrubs of the families Celastraceae, Rosaceae, and Salicaceae (Menken et al. 1992). Almost all of the described taxa of Yponomeuta feed exclusively on plants of the Celastraceae family, leading to the belief that the present affiliation of Yponomeuta evolved from an ancestral association with Celastraceae through allopatric speciation, but was able to shift to other food general through sympatric speciation and host-plant shifts (Menken et al. 1992)
Another potential consequence of a host-plant shift is that, given enough time, instead of simply losings sensitivity to a specific deterrent, the previous deterrent could actually become a
phagostimulant, where the presence of the chemical actually stimulates feeding and oviposition. has for instance been described in Pieris butterflies, some of whom are now stimulated by the presence of glucosinolates (Chapman 2003). It is an open question if this has also happend in Yponomeuta. This bachelor project aims to further explore the relationship between Yponomeuta and salicin, using a series of behavioural experiments. The first aim of this research is to explore whether salicin is indeed a deterrent of non-salix feeding Yponomeuta in behavioural experiments. These will be performed using the spindle moth (Y. cagnagella), and the hawthorn moth (Y. padella). Yponomeuta cagnagella feeds on spindle (Euonymus europeaus), which is part of the Celastraceae, and Y. padella feeds on hawthorn (Crateagus sp.) and blackthorn (Prunus spinosa), which are part of the Rosaceae family. This project also aims to explore the possibility that Y. rorrella is potentially stimulated by salicin, rather than just not being deterred by the presence of the chemical.
Previous electrophysiological research by van Drongelen (1979) and Chapman (2003) has indicated that salicin evokes stong sensory responses in non-willow feeders, but that it does not have a huge effect on the willow-feeding moth Y. rorrella. From this, it is predicted that both larval Y. cagnagella and Y. padella will be deterred from feeding in response to salicin. The second part of the project will focus on the effect of salicin on the feeding behaviour of Y. rorrella. The expectations are that this moth will not be deterred but stimulated to feed by the presence of salicin.
Materials and Methods
The insects
Insects were collected as caterpillars in the field. Y. cagnagella was collected from the Amstelpark (Amsterdam, 52.329628, 4.893155), and Park Cronesteyn in Leiden (52.139167, 4.497222). Y.
padellus was collected from hawthorn also in the Amstelpark. Yponomeuta rorrella was collected
from willows in the Amstelpark and the Grebbeberg (Rhenen, 51.95064, 5.60107).
Rearing
The caterpillars were reared in petri dishes and were fed fresh leaves from their host plants. Populations were kept in the fridge for 24 hours at a time, then allowed to feed for 6 hours, before being returned to the fridge. This was done mainly to slow down the development of the caterpillars to ensure a long-lasting and steady supply needed for testing. When the caterpillars reached the fourth instar (around 1-1.5cm in length) they were ready for testing, and were taken out of the fridge until pupation.
Feeding experiments
Leaf discs of 12mm diameter were made from fresh host leaves and put in the centre of a petri dish on top of a piece of filter paper slightly wetted with distilled water (Roessingh 2006). Before the start of the feeding experiments, the caterpillars used were starved for two hours, after which they were placed on top of the leaf disc and allowed to feed for four hours. Leaf discs were either treated or untreated with a 10 µm droplet of 10mM salicin. This concentration of salicin was chosen because it is the same concentration used in the neurological experiments by van Drongelen,
The same experiments were also attempted using leaves extracted in 70% ethanol, so as to extract some of the secondary plant chemicals and create a ‘half-acceptable’ substrate for the caterpillars by decreasing the baseline palatability of the substrate. For experiments with Y. cagnagellus, spindle leaves were extracted in 70% EtOH for 24 hours (Roessingh 2006). Extraction times for Crataegus and Salix leaves in order to reduce palatability were not readily found in the literature, so a series of extraction times were tested. For Crataegus, an extraction time of 3 hours was used, a suitable extraction time for Salix leaves was not found.
Results
Effects of salicin on feeding of non-specialists on fresh leaf discs
On fresh leaf discs untreated with salicin, Yponomeuta cagnagella larvae were eating a mean of 62% of the leaf disc (n=140, standard deviation = 20.266), when salicin was added, no feeding was observed (figure 1). Yponomeuta padella larvae were eating a mean of 38.35% of the fresh leaf disc in 4 hours (n=54, standard deviation = 18.612). When salicin was added, no feeding was observed for
Y. padella (figure 2)
Figure 1 – Box plot showing the differences in the percentage of the fresh leaf disc eaten by Y. cagnagella without treatment (n=140) and with treatment (n=60) of the leaf with 10µl of 10mM salicin When fresh leaf discs were treated with salicin, no feeding was observed.
Figure 2 – Box plot showing the differences in the percentage of the fresh leaf disc eaten by Y. padella without treatment (n=54) and with treatment (n=27) of the leaf with 10µl of 10mM salicin. When leaf discs were treated with salicin, no feeding was observed
Y. padella Pe rc en tag e eate n Pe rc en tag e eate n Y. cagnagella
Effects of salicin on feeding of non-specialists with a half-acceptable substrate
On a substrate with medium acceptability for the caterpillars, a very limited amount of feeding was observed. After extraction of the leaf discs in alcohol, Y. cagnagella consumed a mean average of 20.25% of the leaf disc, with more than half of the leaf discs left uneaten (median = 0). When treated with salicin, no feeding was observed (figure 3)
Figure 3 – Box plot showing the differences in the percentage of the extracted leaf disc eaten by Y. cagnagella without treatment (n=59) and with treatment (n=25) of the extracted leaf with 10µl of 10mM salicin. When salicin was added, no feeding was observed
Figure 4 - Box plot showing the differences in the percentage of the extracted leaf disc eaten by Y. padella without treatment (n=24) and with treatment (n=27) of the extracted leaf with 10µl of 10mM salicin. When salicin was added, no feeding was observed.
Y. cagnagella Y. padella Pe rc en tag e eate n Pe rc en tag e eate n
Y. padella also saw a limited amount of feeding. The larvae consumed a mean average of 7.19% of
the extracted leaf disc (n=24, standard deviation = 5.71), when treated with salicin, no feeding was observed (figure 4)
Effects of salicin on feeding of a willow-feeding specialist moth
On fresh leaf discs untreated with salicin, Y. rorrella larvae were eating a mean average of 43.9% of the leaf disc (n=39, standard deviation = 27.262). When salicin was added, the larvae consumed a mean average of 44.9% of the leaf disc (n=42, standard deviation = 27.177) (figure 5)
Figure 5 - Box plot showing the differences in the percentage of the fresh leaf disc eaten by Y. rorrella with treatment (n=42) and without treatment (n=39) with 10µl of 10mM Salicin. No significant differences in feeding exist between the two treatments.
No means was found to achieve a half-acceptable substrate for Y. rorrella.
The link to the data repository which contains all the photographs and measurements used in this project can be found at: https://doi.org/10.5281/zenodo.3999198
Y. rorrella P er ce nt ag e e at en Treatment
Discussion
This project aimed to explore the feeding behaviour of specialist moths, and their reaction to a normally bitter deterrent, salicin. Three specialist moths were fed leaf discs comprising of their host plant, with and without added salicin. The effects of salicin on feeding were further explored by trying to create a half-acceptable substrate for the caterpillars by extracting the leaves in alcohol The results clearly show that non-willow feeders were deterred by salicin, both on extracted and non-extracted leaf discs. This is in line with the expectations set out by the electrophysiological experiments conducted by van Drongelen (1979), which showed that salicin caused an increased electrophysiological response in moths that didn’t feed on willows, which indicated that salicin could be a deterrent for these moths. These experiments carried out by van Drongelen and others often focus solely on the electrophysiological and neurological responses that these deterrents illicit, but very little research was available on whether this was also true when tested in a behavioural setting. The experiments with Y. rorrella clearly showed that unlike Y. cagnagella and Y. padella, the willow-feeding moths were not deterred from willow-feeding by the addition of salicin to the host leaves.
However, in order to try and fully investigate the effect of salicin on Y. rorrella, the possibility that salicin is a stimulant was also investigated. In order to do this, the willow leaves needed to be
extracted in 70% ethanol. This was done in order to extract some of the salicin already present in the willow leaves, and to create a substrate that was less acceptable to the caterpillars. If such a
substrate were to be created, the potential stimulating effects of adding salicin can be more easily determined. Unfortunately, this turned out to be difficult. No specific times for extraction in alcohol were found in the literature, except for the 24 hours given by Roessingh et al. (2006) for Y.
cagnagella. The correct extraction time had to be found in a process of trial and error. Extraction
time over three hours led to the leaf discs shrivelling up in the four-hour feeding period, making it impossible to analyse the surface area due to the compromised structural integrity of the disc. The experiments were all undertaken at home rather than in a climate chamber due to the Covid-19 pandemic, which meant that there was no way to control the humidity. These lower levels of ambient humidity were probably partly a reason that the leaf discs could not survive the extraction process structurally. Another problem that of the three species, Y. rorrella was by far the most difficult to find. This led to a situation where for a long time, only two populations of Y. rorrella were available for testing. This resulted in not only the necessity to re-use every individual caterpillar multiple times, but also a much shorter timeframe to be able to successfully conduct tests.
After these experiments were completed there was unfortunately not enough time and not enough correctly sized individuals left to continue trying to find a suitable ‘half-acceptable’ substrate using alcohol extraction. However, it was clear from the results of these tests that adding additional salicin to fresh willow leaves does not significantly deter Y. rorrella from feeding.
In conclusion, this project has shown in several behavioural experiments that salicin acts as a deterrent to Y. cagnagella and Y. padella, both of whom do not feed on Salicaceae. Adding
additional salicin to willow leaves does not significantly impact the feeding rates of Y. rorrella, while a clear effect was found on the other two species. Together with the available electrophysiological evidence this indicates that Y. rorrella has lost its sensitivity to salicin, which Y. cagnagella and Y.
padella still possess. This supports the hypothesis formulated by Menken and others (Menken et al,
1992) that the host-shift from Rosaceae to Salicaceae could well have been facilitated by this loss of sensitivity. It’s still unclear as to whether salicin is a stimulant for willow-feeding specialists, and more research is needed to fully explore the potential stimulant nature of salicin. This be done by correctly creating a half-acceptable substrate by extracting willow leaves in alcohol and feeding these extracted leaves with and without salicin treatment to the moths. Another possibility for further research is to try and disable or otherwise incapacitate the taste senses of the caterpillar. This can be done by ablation, where the palps or antennae are removed to investigate what effect this has on the caterpillar’s behaviour. (De Boer 2006) With modern technologies such as CRISPR-CAS or RNAi, it might be possible to create caterpillars without functioning deterrent cells. If after the removal of these cells the behaviour changes significantly it could provide evidence both for the role of salicin or any other specific chemical, and to give us more insight into the senses of the caterpillars. The results of this research also could indicate that secondary plant metabolites might also be available for use in the agricultural industry. With the rise of the use in synthetic pesticides leading to ever greater problems and costs, using other forms of pest control can be useful. Many plants all over the world produce secondary plant metabolites that act as deterrents to a variety of insects, and trying to use these compounds to create less harmful and more cost-efficient means of pest control could be advantageous (Adeyemi 2010).
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