bioRxiv preprint doi: https://doi.org/10.1101/789172; this version posted October 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
1 FEEDING STATUS INFLUENCES ADULT VINE WEEVIL (COLEOPTERA:
2 CURCULIONIDAE) BEHAVIOUR TO CONSPECIFIC VOLATILES
3
4 RUNNING TITLE: FEEDING STATUS IMPACTS VINE WEEVIL AGGREGATION
5
6 JOE M. ROBERTS 1,2 * · JHAMAN KUNDUN 1 · TOM W. POPE 1
7
8 Corresponding author (*): [email protected] / [email protected]
9
10 1 Centre for Integrated Pest Management, Department of Crop and Environment Sciences, Harper
11 Adams University, Newport, Shropshire, TF10 8NB, United Kingdom
12
13 2 Centre for Applied Entomology and Parasitology, School of Life Sciences, Huxley Building, Keele
14 University, Keele, Staffordshire, ST5 5BG, United Kingdom
15
16 ACKNOWLEDGEMENTS
17 The authors thank Dr Andrew Beacham for help in preparing Figure 1. This work was funded by
18 AHDB Horticulture [Project number HNS 195].
19 bioRxiv preprint doi: https://doi.org/10.1101/789172; this version posted October 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
20 ABSTRACT
21 Vine weevil, Otiorhynchus sulcatus F. (Coleoptera: Curculionidae), is widely considered to be one of
22 the most economically important pests of soft-fruit and ornamental crops. The chemical ecology of vine
23 weevil has been extensively studied in the pursuit of a semiochemical lure to improve monitoring tool
24 sensitivity/reliability and develop novel control methods. Although vine weevil adults exhibit a strong
25 tendency to aggregate, the mechanism underlying this behaviour has not, to date, been identified. It is
26 notable, however, that previous studies have not considered the impact of feeding status on vine weevil
27 aggregation behaviour. To investigate the importance of feeding status on aggregation behavior, this
28 study recorded the responses of satiated and starved vine weevil adults to the odour of starved
29 conspecifics. Satiated adults exhibited a preference for the odour of conspecifics while starved vine
30 weevils exhibited no such preference. Therefore, this study provides evidence to support the hypothesis
31 that feeding status is an important factor for vine weevil aggregation.
32
33 KEY WORDS
34 Otiorhynchus sulcatus; Aggregation; Olfactometry, Soft fruit, Ornamentals, Monitoring
35
36 INTRODUCTION
37 Vine weevil, Otiorhynchus sulcatus F. (Coleoptera: Curculionidae), is one of the most
38 economically important pests of soft-fruit and ornamental crops (van Tol, Bruck, Griepink & De Kogel,
39 2012). Adults are flightless and deposit their eggs into cracks in the soil or growing medium at night
40 (Smith, 1932), where, upon hatching, the larvae complete four to nine molts prior to pupation (Masaki
41 & Ohto, 1995). Crop damage and economic losses are the result of feeding on plant roots, corns and
42 rhizomes by larvae and on leaves by adults (Moorhouse, Charnley & Gillespie, 1992). Control of vine
43 weevil adults has typically relied on foliar applications of pyrethroid insecticides while control of larval
44 stages has been through incorporation of slow release granules of neonicotinoid insecticides (Karley,
45 Shepherd, Hall, McLaren & Johnson, 2012). However, larval control has now shifted in many situations
46 toward replacing these insecticides with entomopathogenic nematodes and fungi with some success
47 (Ansari, Shah & Butt, 2008).
48 The chemical ecology of vine weevil has been extensively studied in the pursuit of a
49 semiochemical lure to improve monitoring tool sensitivity/reliability and develop novel control methods, bioRxiv preprint doi: https://doi.org/10.1101/789172; this version posted October 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
50 such as autodissemination of entomopathogenic fungi (Pope et al., 2018). As vine weevil reproduce via
51 thelytokous parthenogenesis (Lundmark, 2010), and are subsequently all female, they do not produce
52 a sex pheromone (van Tol et al., 2012). Although vine weevil does not produce a sex pheromone, it is
53 thought an aggregation pheromone may exist as adult vine weevils exhibit aggregation behaviour and
54 a preference for volatiles originating from conspecifics in olfactometer experiments (Pickett, Bartlett,
55 Buxton, Wadhams & Woodcock, 1996; Kakizaki 2001; Tol et al., 2004; Nakamuta et al., 2005).
56 However, studies so far completed focus only on the behavioural response of starved individuals. To
57 address this gap in our understanding of vine weevil behavior we investigated the effect of feeding
58 status on the behavioural responses of vine weevil adults to the odour produced by conspecifics.
59
60 MATERIALS AND METHODS
61 Insect cultures
62 Adult vine weevils, Otiohynchus sulcatus F., were collected from commercial strawberry crops
63 grown in Shropshire and Staffordshire (UK) during the summer in 2017. Recovered vine weevils were
64 maintained on branches of yew, Taxus baccata (L.), and moist paper towels inside insect cages (47.5
65 x 47.5 x 47.5 cm) (Bugdorm, MegaView, Taiwan) placed in a controlled environment room (20 °C; 60%
66 RH; L:D 16:8 h) (Fitotron, Weiss Technik, Ebbw Vale, Wales).
67
68 Y-tube olfactometer bioassays
69 Behavioural responses of starved and satiated adult vine weevils were recorded to the volatiles
70 from 40 starved conspecifics that had been starved for 24 hours prior to the start of the experiment. Air
71 passing through the olfactometer was charcoal filtered air. The Y-tube olfactometer was made of glass
72 (Sci-Glass Consultancy, UK) based upon the design used by van Tol, Visser & Sabelis (2002) with a
73 stem length of 120 mm, arm length 190 mm and an internal diameter 18 mm. Prior to use the
74 olfactometer was ‘smoke-tested’ for airflow visualisation to ensure odour fields were discrete. The
75 airflow in each arm was 600 ml/min and conspecifics acting as an odour source were held in a 500 ml
76 Dreschel bottle (Sci-Glass Consultancy, UK) for at least two hours before an experiment began, an
77 empty Dreschel bottle was used for the control.
78 All bioassays were done in darkness under controlled environmental conditions (20 °C; 60 %
79 RH; 16:8 h L:D). Prior to use in a bioassay, vine weevils were either starved for a minimum of 24 hours bioRxiv preprint doi: https://doi.org/10.1101/789172; this version posted October 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
80 or collected from their culture cages and used immediately. Groups of 40 vine weevils (starved or
81 satiated) were introduced into the olfactometer via a release chamber (100 mm diameter; Sci-Glass
82 Consultancy, UK). The experiment was replicated four times with fresh individuals. Each replicate lasted
83 for a maximum of 20 min and the number of weevils reaching the end of each arm during this time was
84 recorded. Odour source positions were alternated between replicates to eliminate directional bias. After
85 each pair of odour sources had been tested glassware was cleaned by rinsing with warm water followed
86 by HPLC-grade acetone (Sigma Aldrich, UK), then baked in a glassware oven at 120 °C for 15 min.
87
88 Statistical analyses
89 All statistical analyses were performed using R (Version 3.5-3; R Core Team, 2019). Y-tube
90 olfactometer bioassay data was analysed using an exact binomial test against the null hypothesis that
91 the number of vine weevils reaching the end of either olfactometer arm had a 50:50 distribution. Prior
92 to statistical analysis, replicated results from each feeding status tested were pooled and non-
93 responding individuals were excluded.
94
95 RESULTS
96 Satiated vine weevil adults exhibited a preference for the Y-tube olfactometer arm containing
97 air blown over 40 starved conspecifics with 74 % of responding individuals choosing this arm over the
98 clean air control arm (P < 0.001) while starved adults exhibited no such preference with 44 % choosing
99 the arm containing conspecific volatiles (Fig. 1).
100
101 DISCUSSION 102 During daylight hours, vine weevil adults are often found aggregating within refuges close to
103 their host plants. Understanding vine weevil aggregation has been the subject of extensive research,
104 with results suggesting that this behaviour is likely to be mediated by volatile cues originating either
105 from the frass produced by conspecifics (Pickett et al., 1996; van Tol, Visser & Sabelis, 2004) or the
106 conspecifics themselves (Kakizaki 2001; Nakamuta, van Tol & Visser, 2005). Previous work, however,
107 has largely focussed on the behavioural response of starved weevils toward conspecific volatiles, which
108 is likely to be an unrealistic situation as vine weevil adults aggregate at dawn after feeding (Moorhouse
109 et al., 1992). bioRxiv preprint doi: https://doi.org/10.1101/789172; this version posted October 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
110 It is possible that any positive behavioural response to the frass produced by conspecifics by
111 starved vine weevils is a response toward plant volatiles from undigested plant material within the frass
112 rather than to the weevils themselves. Vine weevil frass is known to contain volatile organic compounds
113 (VOCs) commonly emitted by plants (e.g. β-caryophyllene, linalool, eugenol, etc.) (Karley et al., 2012),
114 many of which have been implicated in host-plant selection in previous studies (Karley et al., 2012; van
115 Tol et al., 2012). In the study presented here, conspecifics used as an odour source were starved for
116 24 hours prior to the start of the experiment to avoid the production of frass. In this way satiated weevils
117 responding to the odour of conspecifics were doing so in response to volatile cues produced directly
118 from the weevils rather than a mixture of cues produced by weevils and their frass. As starved vine
119 weevils showed no preference for conspecific volatiles in this study, it seems likely that the positive
120 behavioural responses of starved weevils to conspecifics reported in previous studies was due to the
121 presence of frass volatiles (Pickett et al., 1996; Tol et al., 2004). As satiated vine weevil adults exhibited
122 a preference for conspecific volatiles in the absence of frass in this study there is, for the first time,
123 evidence of a positive behavioural response to volatiles produced directly by conspecifics.
124 This study highlights the importance of feeding status to vine weevil behavioural responses and
125 provides new information that may help inform development of new monitoring tools for management
126 strategies. Enhancing our understanding of how feeding status influences aggregation could lead to the
127 identification of an aggregation pheromone by providing optimum experimental conditions for
128 pheromone collection and analysis.
129
130 CONFLICT OF INTEREST
131 The authors declare that they have no conflict of interest.
132
133 AUTHOR CONTRIBUTION
134 • JMR and TWP conceived research.
135 • JK conducted experiments.
136 • JMR analysed data and conducted statistical analyses.
137 • JMR, JK and TWP wrote the manuscript.
138 • TWP secured funding.
139 • All authors read and approved the manuscript. bioRxiv preprint doi: https://doi.org/10.1101/789172; this version posted October 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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198
199 200 Figure 1. Behavioural response of adult vine weevil when presented with a choice between two odours (n = 160). The feeding status of the vine weevils being
201 presented with the choice are: (1) starved and (2) satiated. Asterisks indicate a preference that is significantly different from the expected distribution of 50:50
202 using a binomial exact test: *** P < 0.001; N.S. = not significant.