Biology swede midge - SLU

Master project in the Horticultural Science Programme 

2008-02, 20 p (30 ECTS) 

Responses of the swede midge, Contarinia 

nasturtii, to volatile compounds from 

Arabidopsis thaliana and Brassica 

oleracea var. botrytis 

Photo: Linda-Marie Rännbäck 

by 

Johannes Albertsson 

Fakulteten för landskapsplanering, trädgårds- och jordbruksvetenskap 

SLU-Alnarp

PROJECT INFORMATION 

This report is a result of a master thesis (30 ects) carried out at the department of Plant 

Protection Biology within the Horticultural programme at the Swedish University of 

Agricultural Sciences. Tina Boddum and Ylva Hillbur were supervisors and Peter 

Anderson was the examiner. 

2

ABSTRACT 

The swede midge, Contarinia nasturtii, is a serious gall-forming insect pest of most 

cultivated Brassicaceae such as cauliflower and cabbage. Arabidopsis thaliana, also a 

Brassicaceae, is a well-known model plant for which the genome is sequenced and 

several well-characterized mutants and transgenic genotypes are available. Previous 

results indicate that odours from cauliflower attract C. nasturtii. In this study the 

responses of C. nasturtii to volatiles from cauliflower and Arabidopsis were tested in a 

four-arm and a y-tube olfactometer. The results indicate that females are attracted to 

volatiles both from cauliflower and Arabidopsis. Males on the other hand were not 

attracted. This study also suggests improvements for the y-tube as well as a four-arm 

olfactometer setup. 

SAMMANFATTNING 

Kålgallmyggan, Contarinia nasturtii, är en skadegörare på flertalet odlade grödor 

tillhörande familjen Brassicaceae så som blomkål, vitkål och broccoli. Arabidopsis 

thaliana, som också tillhör denna familj, är en viktig modellväxt eftersom dess genom 

helt är kartlagt och att en mängd väldokumenterade mutationer och transgena genotyper 

finns tillgängliga. Tidigare studier har indikerat att C. nasturtii är attraherad av doft från 

blomkål. I det här arbetet undersöker vi, med hjälp av en fyr-arms olfaktometer och en yrörs 

olfaktometer, om C. nasturtii är attraherad av flyktiga ämnen från blomkål och 

Arabidopsis. Resultaten från detta arbete pekar på att honor attraheras av doft från båda 

dessa växter. Hannarna attraherades däremot inte. Detta arbete föreslår också 

modifikationer och förbättringar för y-rörs samt fyr-arms olfaktometrarna. 

3

INTRODUCTION............................................................................................................. 5 

Life cycle ........................................................................................................................ 5 

Host plant finding ........................................................................................................... 5 

Control of C. nasturtii..................................................................................................... 6 

Arabidopsis .....................................................................................................................7 

HYPOTHESIS................................................................................................................... 8 

MATERIALS AND METHODS ..................................................................................... 8 

Insect material.................................................................................................................8 

Plant material ..................................................................................................................9 

Four-arm olfactometer bioassay ..................................................................................... 9 

Y-tube olfactometer bioassay ....................................................................................... 11 

STATISTICS................................................................................................................... 12 

RESULTS ........................................................................................................................ 12 

Four-arm olfactometer .................................................................................................. 12 

Y-tube olfactometer ...................................................................................................... 14 

DISCUSSION .................................................................................................................. 15 

ACKNOWLEDGEMENTS ........................................................................................... 18 

REFERENCES................................................................................................................ 19 

4

INTRODUCTION 

The swede midge, Contarinia nasturtii (Kieffer) (Diptera: Cecidomyiidae) is a serious 

pest on cruciferous crops in many brassica-growing regions (Kikkert et al., 2006). Host 

plants include cruciferous weeds and most cultivated crucifers such as broccoli, 

cauliflower, cabbage and Brussel sprouts (Stokes, 1953; Hallett, 2007). The larva causes 

the damage when it feeds near the growing point of the plant. Symptoms include gall-like 

distortions, deformed plant tissue, and corky brown scars. Adult gall midges are yellow to 

light brown and 1.5-2 mm long. Larvae are white to yellow and 2-2.5 mm long 

(Readshaw, 1966). 

Life cycle 

C. nasturtii overwinters in the soil as larva in spherical cocoons (Readshaw, 1966). At the 

end of May or beginning of June the larvae start to pupate and emerge. After emergence 

the adults mate and the female starts to search for a suitable host plant for oviposition 

(Madsen & Hansen, 2006). The female lays about 100 white eggs in clusters of 2-50 that 

hatch after a few days. The larvae feed on the plant tissue for 4-10 days and then leave 

the plant by falling down to the soil. Depending on temperature, humidity and day length 

the larvae either pupate to develop the next generation, or enter diapause (Readshaw, 

1966). In Sweden there are three overlapping generations each year (Jönsson et al., 

2007). 

Host plant finding 

Insects use a predictable sequence of behaviour when selecting a host plant 

(Schoonhoven et al., 2005). The sequence usually begins with random flying or walking 

and ends with the acceptance of the host plant. As the sequence progresses the intensity 

and number of cues from the host plant increase. Host location of phytophagous insects is 

mediated by a combination of visual and olfactory cues (Schoonhoven et al., 2005). The 

relative importance of the two varies between species and distance to the plant. Currently 

there is no clear evidence that C. nasturtii uses plant volatiles to mediate host finding. 

However, in a previous study, C. nasturtii males responded strongly to plant volatiles 

5

from cauliflower (Möllerström, unpublished data). Mated females were also attracted to 

cauliflower volatiles but not as strongly as males. Host plant volatiles have also been 

shown to play an important role in host finding for several other Cecidomyiidae species 

e.g. Dasinuera tetensi (Crook & Mordue, 1999), Dasinuera mali (Galanihe & Harris, 

1997), Dasinurea brassicae (Pettersson, 1976) and Sitodiplosis mosellana (Birkett et al., 

2004). In crucifers and related plant families glucosinolates are thought to serve as a first 

line of defence against a variety of invading insects (Schoonhoven et al., 2005). 

Hydrolysis of glucosinolates, forming volatile isothiocyanates, takes place at low rate 

during normal catabolism but increase rapidly when plant tissues are ruptured or 

wounded (Schoonhoven et al., 2005). These compounds are not only acting as defence 

compounds, they also play a key role in host selection for several crucifer specialists like 

the cabbage aphid, Brevicoryne brassicae (Nottingham et al, 1991), and the seed weevil, 

Ceutorhynchus assimilis (Blight et al., 1995). Additional, not yet identified compounds in 

crucifers may also play an important role in host plant selection for insects (Alan & 

Renwick, 2002). 

Control of C. nasturtii 

C. nasturtii is hard to control due to its short lifecycle and many generations. Insecticides, 

like pyrethroids can be applied to control them, but there are also many other cultural 

strategies available to lower the damage (Jönsson et al., 2007). The Swedish Board of 

Agriculture recommend the following measures; at least three year crop rotation, no rape 

seed next to cabbage fields, and as long a distance as possible (several hundred meters) 

between early and late batches of cabbage (Jönsson et al, 2007). Another culture strategy 

to decrease C. nasturtii damage is to use exclusion fences. An experiment made with 

these fences in Switzerland reduced damage caused by C. nasturtii significantly (Wyss & 

Daniel, 2004). Since the sex pheromone of C. nasturtii is identified there is also a 

possibility to develop an easy-to-use monitoring system (Hillbur et al., 2005). 

Other control methods, including resistant cultivars and transgenic plants can and may 

be explored in the future (Wu et al., 2006). Resistance to insects in crop cultivars is 

caused either by biochemical or morphological features (Diarisso et al., 1998). One 

example of a morphological feature is the resistance of some sorghum cultivars to the 

6

sorghum midge, Stenodiplosis sorghicola. In these genotypes the spikelets are closed 

when the female sorghum midges are active and oviposition is thus prevented (Diarisso et 

al., 1998). The resistance of several wheat cultivars to the wheat midge, Sitodiplosis 

mosellana is however caused by biochemical features. When attacked by the larvae the 

resistant cultivars react by increasing the production of phenolic acids in combination 

with a local hypersensitive reaction on the seed surface (McKenzie, 2002). The defence 

mechanism reduces survival up to 99 % of the first-instar wheat midges (Ganehiarachichi 

& Harris, 2007). An investigation on host plant susceptibility to C. nasturtii showed 

differences in susceptibility both between various cruciferous species and between 

cultivars (Hallett, 2007). The cause of the differences is not yet understood, but the fact 

that differences exist suggests possibilities to find genotypes conferring resistance to C. 

nasturtii. 

Another possible control method is the use of transgenic plants. The best example of 

this method is the engineered crop plants expressing Bacillus thuringiensis (Bt). Bt crops 

exhibit resistance to major insect pest in both corn and cotton and in 2004 such crops 

were grown on more than 22 million hectares (Ferry et al., 2006). Recent studies have 

also demonstrated that genetic transformation can be used to alter the emissions of a plant 

and render it more attractive to beneficial arthropods (Kappers et al., 2005; Schnee et al., 

2006). 

Arabidopsis 

Arabidopsis thaliana belongs to the Brassicaceae family, which includes around 3350 

species worldwide (Koch et al., 2001). It is an important model plant for plant sciences 

and potentially also for ecological research (Schoonhoven et al., 2005). The genome has 

been fully sequenced and well-characterized mutants and transgenic genotypes are 

available. C. nasturtii host plants, like cauliflower and oilseed rape, are the closest 

agricultural relatives to Arabidopsis (Kliebenstein et al., 2001; Mitchell-Olds, 2001). In 

response to herbivores, Arabidopsis use similar transduction pathways as other crucifer 

plants, both when activating induced direct and indirect defence (Schoonhoven et al., 

2005). 

7

HYPOTHESIS 

The properties of Arabidopsis and the indication of C. nasturtii attraction to host plant 

volatiles suggest a possibility for a new approach when searching for host plant volatiles. 

The aim of this study is therefore to verify the response of C. nasturtii to cauliflower and 

to test our hypothesis that plant volatiles from Arabidopsis attract C. nasturtii males and 

females. To test our hypothesis a bioassay suitable for testing C. nasturtii attraction to 

host plant volatiles had to be developed and evaluated. 

MATERIALS AND METHODS 

Insect material 

All the C. nasturtii used in the experiments originated from Switzerland. A C. nasturtii 

population had been kept at the Department of Crop Science, SLU Alnarp, since August 

2004. A new batch of C. nasturtii was incorporated into the rearing system in September 

2007 by collecting infested cauliflower plants from a field in Wädenswil, Switzerland. 

The infested plants were packed in plastic bags and transported to Sweden by car. In 

Alnarp, all plants were placed in large plastic boxes filled with 5-7 cm peat substrate. The 

boxes were placed in a climate chamber (25°C, 70% RH, LD 18:6 h) and covered with a 

white cotton cloth. Fourteen days after collection of the plant material the first midges 

emerged from the substrate. Some midges were used for experiments but most were used 

to infest new cauliflowers plants for the rearing. 

All midges were reared in ventilated glass cages (30 x 30 x 35 cm) on cauliflower, 

Brassica oleracea var. botrytis, in the climate chamber described above. Every third day 

a new pot with cauliflower plants, with 6-9 true leaves, was placed in the cages and 

midges were allowed to mate and oviposit on the plants. The infested plants were then 

removed, replaced with a new batch of plants, and incubated under the same climate 

condition as described above. Infested plants were misted every day with tap water. 

Approximately two weeks after oviposition, the larvae left the plants and pupated in the 

pot substrate. One-day prior to emergence, 17 to 18 days after oviposition, all the 

8

aboveground parts of the cauliflower plants were cut off. The pots with substrate and 

pupae were then placed in a cage where midges were allowed to emerge. 

Males, one day old, and mated females were used in the bioassays. The females had 

been allowed to mate during the day before the bioassay. If some females exposed their 

ovipositor in the afternoon, indicating calling behaviour (Harris, & Foster, 1999), they 

were considered as unmated and discarded. 

Plant material 

In all bioassays with plant material, cauliflower plants (Brassica oleracea var. botrytis 

‘Vito’) with 7-10 true leaves, or 5-6 weeks old Arabidopsis (Arabidopsis thaliana 

‘Columbia’) served as odour sources. Cauliflower plants were grown two and two in pots 

(13 cm diam, 8 cm height) in a substrate consisting of 92% peat and gravel. Arabidopsis 

plants were grown two and two in pots (6 x 6 cm width, 5 cm height) in the same 

substrate. All pots were placed in a climate chamber (20°C, 60 % RH, and LD 16:8 h, 

250 μmol m -2 s -1 ) and treated with Steinernema feltiae, to control sciarid flies. In 

bioassays with plant material one cauliflower pot or two Arabidopsis pots were used. To 

minimize the volatiles released from the soil surface and the pot itself, they were covered 

with aluminium foil. 

Cauliflower plants for insect rearing were grown two and two in pots (13 cm diam, 8 

cm height) in a substrate consisting of 92% peat and gravel in a greenhouse with a 

temperature of 18-25°C and 10 hours of additional light (kb 1141, 400 W). 

Four-arm olfactometer bioassay 

The four-arm olfactometerer was based on the six-arm olfactometer described by 

Turlings et al. (2004). It consisted of a four–armed central choice chamber with one 

insect trapping bulb connected to each arm (Figure 1). Air was pumped into the 

olfactometer through two 250 ml gas-wash bottles, one with granulated activated 

charcoal and one with distilled water. After the gas-wash bottles, the inflow of air was 

divided into four lines of Teflon tubing via four flowmeters (BA-4AR, Kytölä, Muurame, 

Finland) to the test plants. The test plants were enclosed in polyethylene cooking bags 

9

(45 x 55 cm, Toppits, Melitta) and connected via Teflon tubes to each olfactometer arm. 

To avoid changes in air pressure a vacuum pump (Micro pump NMP 30 KNDC, 12 V, 

KNF Neuberger, Germany) was connected to the insect release point. The flow of 

incoming air was 0.9 l/min in each of the four Teflon tubes and the flow at the release 

point was 3.6 l/min. To get a uniform illumination and eliminate any visual distractions, 

the central choice chamber was covered with a white cotton cloth, placed 35 cm above 

the chamber and 35 cm to the sides. The olfactometer was illuminated with a mercury 

lamp (Tungsram 9L, HgMIF, 400/DM) placed 1.2 meter above the central choice 

Figure 1. The four-arm olfactometer 

bioasssay setup. Photo: Linda-Marie 

Rännbäck & Thilda Nilsson. 

chamber. After each experiment, all glassware was 

heated for 8 hours in 350 °C and all Teflon tubing 

and connections were washed once with 70 % 

ethanol to remove any odour contamination. The 

olfactometer was completely rebuilt after 10 days. 

The entire setup was disconnected and connections 

were, if spare ones existed, replaced with new ones. 

Tubing and all connections were placed over night 

in a box with 70 % ethanol. To get a more uniform 

illumination a white cylinder (50 cm diam and 20 

cm high) was placed around the central choice 

chamber. 

To evaluate the four-arm olfactometer setup, two 

experiments were made. In the first experiment, C. 

nasturtii males were tested against four empty bags. 

This experiment was made both with the initial and 

the rebuilt setup. In a second experiment, C. 

nasturtii males were tested against one bag, 

containing a cauliflower pot and three empty bags. 

The second experiment was only made using the 

rebuilt setup. In both experiments males were 

10

eleased simultaneously using a glass tube with removable cotton plugs at each end. All 

experiments lasted for two hours and were made between 10 pm to 2 am. Insects were 

only tested once. 

Y-tube olfactometer bioassay 

The y-tube olfactometer consisted of a y-shaped glass tube with an entry arm (14 cm 

long, 22 mm i.d) and two side arms (14 cm long, 22 mm i.d.). Both arms of the y-tube 

were connected to a polyethylene cooking bag (45 x 55 cm, Toppits, Melitta) which 

contained the odour source. Air was pumped into the olfactometer through two 250 ml 

gas-wash bottles, one with granulated activated charcoal and one with distilled water. 

After the gas-wash bottles the inflow of air (0.6 l/min) was divided into two lines of 

Teflon tubing via two flowmeters (BA-4AR, Kytölä, Muurame, Finland) to the bags. 

Visual cues were excluded in the olfactometer setup by placing the y-tube in a box (40 x 

40 x 40 cm) covered with a white cotton cloth. The olfactometer was illuminated with a 

lamp (Massive, 906609, 400 W) placed behind the cloth and 30 cm in front of the two 

side arms. 

A single C. nasturtii male was introduced into the tube and observed until he made a 

Figure 2. The female y-tube olfactometer bioassay 

setup. The male setup did not have the insect trapping 

bulbs. 

choice or until 5 min had elapsed. Males 

that did not choose a side arm within 5 

min were recorded as ‘no choice’. When 

half of the males had been tested in each 

batch the y-tube and the odour source 

was turned to avoid any directional 

effects. To test if C. nasturtii males were 

attracted to cauliflower or Arabidopsis, 

the attraction of a bag containing a 

cauliflower pot or a Arabidopsis pot was 

compared to the attraction of an empty 

bag. To evaluate the y-tube olfactometer 

11

setup a control experiment was made with one empty bag connected to each side arm. All 

the experiments with males were made between 10 am and 1 pm. 

In the female y-tube olfactometer setup an insect-trapping bulb was connected to each 

side arm (Figure 2). The modification was made to be able to run experiments during 

several hours. The females were released simultaneously by placing a glass tube, with 20- 

25 females, in the entry arm. To avoid females from escaping the glass tube was covered 

with an insect net at the end facing outwards. The attraction of C. nasturtii females to 

cauliflower or Arabidopsis was tested by comparing a bag containing cauliflower or 

Arabidopsis to an empty one. All the experiments with females lasted for 16 hours and 

started 5 pm. The light was on between 5 pm and 8 pm. After each experiment, all 

glassware was heated for 8 hours in 350 °C and all Teflon tubing and connections were 

washed once with 70 % ethanol to remove any odour contamination. 

STATISTICS 

Only those insects that made a choice were included in the statistical analyses. To test the 

difference between the arms in both the y-tube olfactometer and the four-arm 

olfactometer a chi square test was performed on the data from each bioassay (Zar, 1999). 

RESULTS 

Four-arm olfactometer 

In both the initial and the rebuilt four-arm olfactometer setup, most responding males 

made the choice within the first five minutes of the bioassay. In the initial control 

experiment there was a significant difference between the arms (chi 2 = 23.59, p < 0.001). 

There was still a significant difference between the arms when the experiment was 

repeated in the rebuilt setup (chi 2 = 12.79, p < 0.01) although another arm was preferred 

(Figure 3B). When cauliflower was tested against three controls, significantly more males 

(chi 2 = 7.91, p < 0.05) choose one of the controls (Figure 3C). Only 8 out of 54 males 

chose the cauliflower arm. The sample size and percentages of responding C. nasturtii in 

the four-arm olfactometer bioassays are presented in Table 1. 

12

Table 1. Sex, sample size (N) and percentages of responding C. nasturtii for the four-arm olfactometer 

bioassay. 

Bioassay (number of arms) Sex N Responding (%) Figure 3 

Initial Control (4) male 141 79 A 

Rebuilt Control (4) male 29 66 B 

Rebuilt Control (3) - Cauliflower (1) male 54 85 C 

A 

Number of males 

B 


C 


60 

50 

40 

30 

20 

10 

0 

12 

10 

8 

6 

4 

2 

0 

20 

18 

16 

14 

12 

10 

8 

6 

4 

2 

0 

Control Control Control Control 

Control Control Control Control 

Cauliflower Control Control Control 

Figure 3. Response of male C. nasturtii in the four-arm olfactometer bioassay. A) Initial control, chi 2 = 

23.59, p < 0.001, B) Rebuilt control, chi 2 = 12.79, p < 0.01, C) Rebuilt setup with one cauliflower arm and 

three control arms, chi 2 = 7.91, p < 0.05. 

13

A 


B 


C 


Y-tube olfactometer 

Almost all males (90 %) that made a choice made it within the first two minutes. The 

majority of the responding males flew into the side arms whereas nearly all responding 

females walked. The sample size and percentages of responding C. nasturtii in the y-tube 

olfactometer bioassays are presented in Table 2. 

Table 2. Sex, sample size (N) and percentages of responding C. nasturtii for the y-tube olfactometer 

bioassay. 

Bioassay Sex N Responding (%) Figure 4 

Control - Control male 20 90 A 

Cauliflower - Control male 61 85 B 

Arabidopsis - Control male 51 78 C 

Cauliflower - Control female 62 90 D 

Arabidopsis - Control female 37 86 E 

15 

10 

5 

0 

30 

25 

20 

15 

10 

5 

0 

30 

25 

20 

15 

10 

5 

0 

Control Control 

Arabidopsis Control 

Cauliflower Control 

D 

Number of females 

E 

Number of females 

25 

20 

15 

10 

5 

0 

40 

30 

20 

10 

0 

Arabidopsis Control 

Cauliflower Control 

Figure 4. Response of C. nasturtii in the y-tube olfactometer bioassay. A) Control-control (male), chi 

14 

2 = 

0.22, p > 0.05, B) Arabidopsis-control (male), chi 2 = 1.6, p > 0.05, C) Cauliflower-control (male), chi 2 = 0, 

p > 0.05, D) Arabidopsis-control (female), chi 2 = 3.13, p > 0.05. E) Cauliflower-control (female), chi 2 = 

2.57, p > 0.05.

There were no significant differences between the arms when two controls – empty 

arms – were compared (chi 2 = 0.22, p > 0.05; Figure 4A). When males were given the 

choice between cauliflower and control exactly 50 % of the males chose the control arm 

(chi 2 = 0, p > 0.05 ; Figure 4B). More males chose the arm with Arabidopsis than the 

control arm (Figure 4C), but the difference was not significant (chi 2 = 1.6, p > 0.05). 

Two y-tube olfactometer bioassays were made with females. In the first, females were 

given the choice between cauliflower and control. Cauliflower showed a tendency to be 

more attractive than control (Figure 4D), but the difference was not significant (chi 2 = 

2.57, p > 0.05). In the last bioassay, Arabidopsis attracted far more females than the 

control, 21 out of 32 (Figure 4E). However, the difference was not large enough to be 

significant (chi 2 = 3.13, p > 0.05). 

DISCUSSION 

The results from this study suggest that C. nasturtii females are attracted to plant volatiles 

from both cauliflower and Arabidopsis. A new approach for investigating C. nasturtii - 

host plant interactions could thus be developed. The method would test the attraction of 

C. nasturtii to transgenic or mutant Arabidopsis lacking certain volatile compounds. It 

would not only give the information about C. nasturtii attractiveness to a mutant or 

genetically modified Arabidopsis, but also hopefully the gene responsible for the 

attraction. This information could in a longer perspective be used for ecological studies 

and/or for traditional/transgenic breeding purposes (Turlings & Ton, 2006). In contrast to 

the females, the males did not show any clear attraction to either cauliflower or 

Arabidopsis. 

Initially, a four-arm olfactometer was used to test the behavioural response of C. 

nasturtii. However, the control bioassays showed significant difference between the arms 

both before and after the setup had been rebuilt. Therefore a decision was taken, two 

months into the study, to discard the four-arm olfactometer and instead use a y-tube 

olfactometer. The y-tube olfactometer is more time consuming to use because the insects 

are released one by one. This was not a problem when working with males since their 

response time was very short (see result). The females one the other hand had a much 

15

longer response time (data not presented). To be able to test the females within the time 

frame of this study a modification with insect trapping bulbs connected to each side arm 

was made. The modification made it possible to release females simultaneously without 

constant supervision. 

Different types of olfactometers have been used in several experiments to test insect 

behaviour (Pettersson, 1976; Turlings et al., 2004). The reason why the C. nasturtii males 

significantly chose one arm in our four-arm olfactometer setup is not yet clarified. One 

possible explanation could be that the plastic connections between the Teflon tubing were 

contaminated with some repellent or attractant. However, the Teflon tubing and the 

connections were cleaned with 70 % ethanol between each test, so the compounds must 

then be resistant to such treatment. Another explanation could be visual cues. However, 

the light intensity was measured several times with a luxmeter (Gossen, 1.70-291, 

Germany) to verify the uniformity and no difference in light intensity was seen between 

the four arms. 

To get the four-arm olfactometer to work properly the following improvements are 

proposed; replace the polyethylene cooking bags with glass cylinders, use press-fit 

connections when connecting the glass cylinder with Teflon tubing, replace plastic 

connections between Teflon tubing with Teflon connections, use a light bulb as Turlings 

et al (2004) to get a more uniform illumination, and place the setup in a climate chamber 

with the possibility to have constant background illumination, temperature, and humidity. 

In the y-tube olfactometer setup used in this study, C. nasturtii males did not show any 

behavioural response to plant volatiles from cauliflowers. The result is contradictory to 

results by Möllerström (unpublished data), where C. nasturtii males flew significantly 

more to arms containing cauliflower plants in a four-arm olfactometer. Since the variety 

of the cauliflower is unknown in Möllerstöms study, it is not possible to rule out 

differences in volatile composition between the varieties. It is also possible that the 

difference between the studies is due to other reasons such as contamination of the setup. 

However, since we didn’t use any females in the y-tube olfactometer prior to the male 

bioassays and made several control bioassays, it is unlikely that our setup was 

contaminated. 

16

Growing conditions for the plants were also different between the studies. In our 

study, the plants used for the y-tube olfactometer bioassays were grown in a climate 

chamber with no occurrence of any herbivore insects. It is well known that plants 

attacked by herbivore insects increase the volatile production due to their induced direct 

and indirect defence (Kessler & Baldwin 2001; Dicke & Van Poecke, 2002). Hence, the 

contradictory results may depend on differences in herbivore damage of the tested plants. 

In other Cecidomyiidae species such as D. tetensi (Crook & Mordue, 1999), D. mali 

(Harris et al. 1996), and D. brassicae (Pettersson, 1976; Williams & Martin, 1986) no 

male attraction was found to host plant volatiles. However, Murchie et al. (1997) caught 

D. brassicae males in traps baited with synthetic secondary volatiles from crucifers. The 

conflicting results from the D. brassicae studies may indicate that factors, not yet known, 

could affect the behavioural response of male gall midges to host plant volatiles. Such 

factors could perhaps be temperature, humidity, time of the day and light conditions since 

these factors affected male mating activity of S. mosellana (Pivnick, 1993). McNeil & 

Brodeur (1995) have also suggested that differences in mating success of Aphidius 

nigripes could depend on changes in atmospheric pressure. The strong D. brassicae male 

response to the traps could also indicate that the synthetic volatiles used as lures, or a 

volatile contamination in the trap, could be a component of the D. brassicae sex 

pheromone. 

Both D. tetensi (Crook & Mordue, 1999) and D. mali (Harris et al. 1996) have 

perennial host plants. Hence, the lack of response to host plant volatiles could be 

explained by the fact that they emerge directly below their host plants (Crook & Mordue, 

1999). However, this argument is not applicable to C. nasturtii since most of its host 

plants are annuals (Stokes, 1953). What benefit would a C. nasturtii male then have of 

detecting host plant volatiles? This question is difficult to answer because there are no 

studies made on C. nasturtii female behaviour after emergence. If the female only mates 

once, like Mayetiola destructor (Harris & Foster, 1999), and stays at the emergence place 

until she is mated, like Contarinia oregonensis (Miller & Borden, 1984), there are no 

obvious benefits for the male to fly to crucifers. However, if the female mates several 

times and/or fly to crucifers directly after emergence males would clearly have a better 

chance to find a mating partner. 

17

Regardless if females are to be tested with a y-tube olfactometer or with a four-arm 

olfactometer optimizations could be made to get better results. Evidently, it is important 

that the females are tested when they are in the right physiological state, hence searching 

for host plants. One factor influencing this behaviour is probably the mating status since 

only mated females responded to host plant volatiles in both D. brassicae (Pettersson, 

1976) and D. tetensi (Crook & Mordue, 1999). Our way to decide if a female is mated or 

not is not very accurate, since a virgin female does not call all the time. Therefore, a more 

accurate method has to be used which guarantee that mating has occurred. Another factor 

that could affect the result is the experimental period. Cecidomyiidae species like S. 

sorghicola only search actively for host plants between 8 am and 11 am (Diarisso et al., 

1998). Hence, tests or observations that establish the time when the C. nasturtii are active 

should be performed. In our bioassays either two cauliflower plants or four Arabidopsis 

were used as an odour source. This study did not investigate the suitability of this number 

of plants, however since dose-response test with other insects have shown that volatile 

concentration affect their choice (Turlings et al., 2004), this is an important area to 

improve. Furthermore the plants used in the bioassays should be illuminated since several 

plants increase the odour release when exposed to increased light intensity (Gouinguene 

& Turlings, 2002). An additional optimization is to use filters that trap volatiles from the 

odour sources during the bioassay. If these filters are analysed they will detect variations 

among bioassays in volatile emissions from the tested plants (Turlings et al., 2004), and 

make the results more valid. If the suggested improvements and optimizations are made 

to the olfactometers and if more females are tested my personal opinion is that more 

convincing results could be achieved. 

ACKNOWLEDGEMENTS 

I would like to thank all the people that helped and inspired me in this project. Special 

thanks to my supervisors Ylva Hillbur and Tina Boddum. You two are probably the most 

positive people I ever met. I would also like to thank Elin Isberg and Linda-Marie 

Rännbäck for practical assistance with bioassays and insect rearing, and Peter Anderson 

for technical support. Finally I would like to thank Alexandra Nikolic and Joakim Ekelöf 

who I forced listen to gall midge chats during the whole study. 

18

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22

Biology swede midge - SLU

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