Elsevier

Journal of Insect Physiology

Volume 119, November–December 2019, 103948
Journal of Insect Physiology

Characterization of olfactory sensory neurons in the red clover seed weevil, Protapion trifolii (Coleoptera: Brentidae) and comparison to the closely related species P. fulvipes

https://doi.org/10.1016/j.jinsphys.2019.103948Get rights and content

Highlights

  • Olfactory sensory neurons for clover odors were characterized in Protapion trifolii.

  • Olfactory responses are largely conserved in the congener P. fulvipes.

  • Two neuron classes demonstrated clear response shifts between species.

  • The response shifts may underlie different host specificity of Protapion weevils.

Abstract

Protapion trifolii and P. fulvipes (Coleoptera: Brentidae) are major pests in European clover seed production. Previous studies have reported a high host plant fidelity of these weevils for red and white clover species, respectively, driven by host plant olfactory cues. Given the specific host preferences observed in these weevils, we aimed to elucidate to which extent such selectivity is reflected in their peripheral olfactory systems. Using an electrophysiological approach, we performed the first functional characterisation of olfactory sensory neurons (OSNs) in P. trifolii to a panel of volatile compounds emitted by red clover plants, and compared the results with the reported OSN types of P. fulvipes. Nineteen OSN classes were characterized in P. trifolii, with the majority of these neurons responding strongly to common volatiles released by the host plant. Based on response profiles, eight of these OSN classes have clear matches to OSN classes in P. fulvipes. The OSN colocalisation patterns and antennal frequency of these classes are similar in the two species. Additionally, the responses of these OSNs are generally highly conserved in the two species, with clear response shifts only revealed for two of the OSN classes. These response shifts in combination with additional response dissimilarities for compounds that vary in abundance between red and white clover plants may underlie the species-specific host preferences. Further behavioural and field experiments should focus on these differentially detected compounds to elucidate their potential role in host selection and use in semiochemical-based control of these pests.

Introduction

Clover seed weevils of genus Protapion (Coleoptera: Brentidae) are major pests in European clover seed production, and can cause severe reductions in seed yield (Hansen and Boelt, 2008, Lundin et al., 2012, Lundin et al., 2017). Although adult weevils feed on clover foliage, they usually do not cause significant damage to the crop. Instead, the feeding of the larvae is the major factor reducing the seed yield. Female weevils oviposit into young inflorescences and individual larvae consume up to ten ovules and seeds (Jones, 1950). The supply of clover seeds is vital for the agricultural sector, because clover is used in animal fodder production and provision of green manure (Thorup-Kristensen et al., 2003). Despite being described as one of the major factors reducing the seed yield (Langer and Rohde, 2005, Lundin et al., 2017), there are no established methods for efficient control of Protapion weevils within organic farming. In conventional clover seed production, these weevils can be efficiently controlled using neonicotinoids (Lundin et al., 2012), but these insecticides have a strong negative impact on important pollinators, such as wild bees and bumblebees (Godfray et al., 2014, Rundlöf et al., 2015). Novel methods for management of these pests, which could reduce or eliminate the dependence on pesticides, are thus urgently needed.

Odour-mediated host choice is a crucial component of the reproductive cycle in herbivorous insects. It is also crucial for the final and decisive step governing the level of herbivore attack on crops. Studying the mechanisms underlying host choice, including attraction to and acceptance of host plants, could thus be of considerable interest from a pest management perspective. Characterising the specific host volatiles underlying attraction and host choice could provide specific attractants for improved monitoring or direct control of pest populations, or repellent plants and synthetic blends for reducing attacks of pests (Shrivastava et al., 2010). The role of host volatiles in the Protapion-clover interaction is currently being explored in search for alternative ways to monitor weevils in clover fields. In Sweden, the two closely related species Protapion trifolii L. and Protapion fulvipes Geoff. are the most severe pests in clover seed production. The two species show high host fidelity, with fields of red clover, Trifolium pratense L., dominated by P. trifolii, and fields of white clover, Trifolium repens L., dominated by P. fulvipes (Lundin et al., 2012, Nyabuga et al., 2015). Single-sensillum recordings (SSR) from olfactory sensory neurons (OSNs) of P. fulvipes (previously named Apion fulvipes) revealed a specialised reception of clover volatiles, with individual OSNs responding to a single or a few chemically similar compounds, and with no apparent difference in response pattern between males and females (Andersson et al., 2012a). In addition, feeding and olfactometer experiments have shown that both P. fulvipes and P. trifolii exhibit preference for their regular host when exposed to stimuli from red versus white clover in two-choice bioassays. This preference was abolished upon removal of antennae (Nyabuga et al., 2015). Thus, a combination of physiological and behavioural data suggest that olfactory cues may be a key mechanism facilitating host fidelity in these insects.

To further explore the sensory basis for host choice in clover seed weevils, we performed an electrophysiological characterisation of OSNs in P. trifolii, for comparison with the previously reported OSN types of P. fulvipes (Andersson et al., 2012a). In the present study, we used the same SSR approach, together with a similar but slightly expanded array of synthetic test stimuli, as in Andersson et al. (2012a). Given the selective host preference observed for P. fulvipes and P. trifolii in field surveys and laboratory assays (Nyabuga et al., 2015), we set out to study to what extent, if any, these different preferences were reflected in the respective OSN arrays of the two species. We aimed to explore how these weevil species might differ in their tuning or frequency of OSNs to enable them to distinguish the scent of T. pratense versus T. repens. Thus, the objectives of this study were (i) to characterise the OSNs of P. trifolii, (ii) to compare the OSN classes of P. trifolii and P. fulvipes in search for interspecific differences in the olfactory response that could explain the specific host preferences in these species, and (iii) provide suggestions for specific host volatiles that could be candidates for host differentiation and pest control.

Section snippets

Insects

Weevils were collected using a sweep net in organic red clover fields in southern Sweden during May-June (2012–2014). They were then kept in insect rearing net cages (30 × 30 × 30 cm, DP1000, BugDorm, Mega View, Thaichung, Taiwan) in a climate chamber at 20 ± 3 °C, 65% relative humidity, and a photoperiod of 16:8 (L:D) until the experiment. Insects were regularly provided with fresh red clover twigs to feed on, which were sprinkled with water once a day.

Scanning electron microscopy (SEM)

SEM pictures of the antennae of P.

Scanning electron microscopy

The antennae of P. trifolii are morphologically similar in the two sexes and also similar to the antennae of P. fulvipes (Andersson et al., 2012a). The antennae have a length that ranges from 700 to 800 μm, and they consist of two basal segments, the scape and the pedicel, followed by nine additional segments constituting the flagellum. The last three segments form a club of approximately 200 μm length and 90 μm maximal diameter, which is where all of the olfactory sensilla targeted in this

Discussion

In this study, we characterised olfactory sensory neurons on the antenna of the red clover seed weevil P. trifolii based on their responses to clover-associated synthetic volatiles. One of the main objectives was to identify volatiles that may be important for the host-seeking behaviour of this specialist herbivore, and which potentially may be useful for semiochemical-based control. In parallel, the aim was to perform comparisons with the previously characterised array of OSNs in the closely

Acknowledgements

This work was funded by the Swedish Farmers' Foundation for Agricultural Research (H1033099 and H1333073), SLU EkoForsk program from the Swedish University of Agricultural Sciences, and the Linnaeus initiative grant “Insect Chemical Ecology, Ethology and Evolution” IC-E3 provided by The Swedish Research Council, FORMAS and the Swedish University of Agricutltural Sciences. M.N.A. acknowledges FORMAS (217-2014-689 and 2018-01444) for funding.

The authors declare no competing interests.

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  • 1

    Current address: Laboratory MIVEGEC (IRD, CNRS, Univ. of Montpellier), FR-34394 Montpellier, France.

    2

    Current address: University of Embu, Department of Biological Sciences, P.O. Box 6 – 60100, Embu, Kenya.

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