Olfactory neuron responsiveness and pheromone blend preference in hybrids between Ostrinia furnacalis and Ostrinia nubilalis (Lepidoptera: Crambidae)

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Abstract

The olfactory receptor neuron (ORN) and behavioral responses of hybrids between the Asian corn borer (ACB), Ostrinia furnacalis, and the E-strain European corn borer (ECB(E)), Ostrinia nubilalis were examined and compared to the parental populations. In hybrids and both parents, the large-spike-size ORN was capable of responding to all four pheromone components of ACB and ECB, despite differences in which compounds elicited the greatest spike frequency in each population. There was a small-spiking ORN more narrowly tuned to the minor pheromone components in both ACB and ECB(E). In hybrids the homologous small-spiking ORN was tuned primarily to the ECB(E) minor pheromone component, with some responsiveness to the ACB minor component. Both species and all the hybrids had an intermediate spike-size ORN tuned primarily to their common behavioral antagonist. Dominance of responsiveness to the ECB(E) versus the ACB minor pheromone component on the small-spiking ORN may explain the greater tendency of hybrids to fly upwind to the ECB(E) pheromone blend than the ACB blend. This finding points toward a distinct evolutionary role for this ORN in allowing a pheromone shift.

Introduction

The degree of plasticity in the peripheral detection of olfactory stimuli has important implications for understanding how adaptive shifts in such systems occur. For a wide array of insect taxa, relatively broad responsiveness is exhibited by many general odorant olfactory receptor neurons (ORNs), whereas other types of ORNs are more narrowly tuned (Mustaparta, 1975, Priesner, 1979a, Priesner, 1979b, Stensmyr et al., 2001, Olsson et al., 2006, Qiu et al., 2006, Schlief and Wilson, 2007). It has also been discovered in Drosophila that more than one type of odorant receptor (OR) can be expressed on the same ORN (Goldman et al., 2005), increasing the potential breadth of ORN tuning. Pheromone sensitive ORNs are generally assumed to be more narrowly tuned than are ORNs tuned to general odorants. However, responsiveness to multiple compounds per ORN has become apparent in some moth sex pheromone systems when more fully investigated for this property. For example, in the genus Yponomeuta several species have ORNs that respond to multiple compounds including acetates showing considerable variation in chain length and structure (van der Pers, 1982, Löfstedt et al., 1990).

To understand the evolution of sex pheromone systems, in addition to characterizing the breadth of ORN tuning it is also important to describe how such systems can change. One approach to this problem has been to examine the behavior and ORN response profiles of closely related species and their hybrids. Crosses between different moth species have tended not only to show dominance of the olfactory and behavioral characteristics of one species in the hybrids (Gadenne et al., 1997), but also sometimes suggest more complicated epistatic effects (Hansson et al., 1989). The variable penetrance of the parental olfactory characteristics was clear in crosses between Heliothis subflexa × Heliothis virescens (Baker et al., 2006). These F1 crosses exhibited patterns of ORN responsiveness that ranged from intermediate, to being more like either parental species. The pattern of responsiveness also suggested the possibility that different ORs from either parent are expressed in the hybrids.

Hybridization studies are valuable, because they provide evidence of evolutionary changes to the olfactory system that have already occurred. However, from such studies it is inherently difficult to interpret the role of the relevant characters for allowing signal divergence and causing reproductive isolation. Another approach to discern how pheromone systems change is to examine properties of rare individuals that exhibit unusual behavioral responses to novel pheromone blends. This approach has been applied to the 3–5% of “rare” Asian corn borers (ACB), Ostrinia furnacalis, and European corn borers (ECB), Ostrinia nubilalis, that will fly to the pheromones of the opposing species, as well as to their own pheromone (Roelofs et al., 2002, Linn et al., 2003, Linn et al., 2007).

Both species have two behavioral attraction-related ORNs that respond to their own pheromone components (Z)-12-tetradecenyl acetate (Z12-14:OAc) and (E)-12-tetradecenyl acetate (E12-14:OAc) for ACB or (Z)-11-tetradecenyl acetate (Z11-14:OAc) and (E)-11-tetradecenyl acetate (E11-14:OAc) for ECB. The pheromone binding proteins have identical amino acid sequences in ACB and ECB (Willett and Harrison, 1999), and thus are unlikely to affect cross-species ORN responsiveness. Both species also have a third behavioral antagonism-related ORN that responds strongly to (Z)-9-tetradecenyl acetate (Z9-14:OAc), a compound that significantly reduces attraction when co-emitted at very small proportions in a blend (Hansson et al., 1987, Glover et al., 1989, Takanashi et al., 2006). Sensilla from more distal regions of ECB antenna often house only one or two ORNs, but the ORNs in these sensilla are always similar in response characteristics to one of the three ORN types found residing together in more basally located sensilla (Hallberg et al., 1994). A similar phenomenon is found in ACB, in which sensillar classes were found that were differentiable by whether or not all the commonly observed ORN responses were present (Takanashi et al., 2006).

There are two ECB strains. The E-strain (ECB(E)) utilizes a blend of 99% E11-14:OAc and 1% Z11-14:OAc in its blend, whereas the Z-strain (ECB(Z)) uses a reverse 97%:3% blend of Z11- and E11-14:OAc (Kochansky et al., 1975, Glover et al., 1987). Most other species in the genus use blends of Z11- and E11-14:OAc as components of their pheromone blends (Ishikawa et al., 1999). Because ACB is the only species in the genus to use Z12- and E12-14:OAc as pheromone components, it is likely that a shift occurred recently in its lineage from a pheromone blend consisting of Z11- and E11-14:OAc to one using Z12- and E12-14:OAc. The ACB pheromone blend consists of two isomers at relatively similar concentrations, although the composition is variable (Klun et al., 1980, Ando et al., 1980), as is the range of pheromone blends that will elicit behavioral attraction of ACB males in the field (Boo and Park, 1998) and wind-tunnel (Linn et al., 2007).

The ORNs of ACB and ECB regularly respond to the pheromone components of the opposing species, but specific changes in patterns of ORN responsiveness are linked to cross-species behavioral attraction of the ‘rare males’ (Domingue et al., 2007a, Domingue et al., 2007b). In normal ECB E-strain males, ACB components elicited responses from both of the neurons involved in attraction. On the ORN associated with the major pheromone component, E11-14:OAc, there were responses to Z12- and E12-14:OAc in all males, including the rare males. On the minor pheromone-component ORN tuned to Z11-14:OAc of the ECB(E) males, relatively lower spike frequency responses also occurred in response to the Z12-14:OAc. However, in the rare ECB(E) males that also flew to the ACB blend, the strength of the ORN response to Z12-14:OAc was reduced (Domingue et al., 2007b). This pattern is consistent with a hypothesis that the relative firing rates of the two relevant neurons serves as a critical factor for promoting upwind flight. In rare ECB(E) males this firing ratio is similar whether it occurs in response to the ECB E-strain blend, in which E11-14:OAc is the major component, or to the ACB blend, in which Z12-14:OAc is the major component.

The converse situation was also examined, where ACB rare males also fly to ECB(E) or ECB(Z) blends (Domingue et al., 2007a). The ORNs of normal ACB males responded to ECB attraction-related components, but the behaviorally antagonism-related ORN normally associated with Z9-14:OAc also responded to the ECB component Z11-14:OAc (Takanashi et al., 2006, Domingue et al., 2007a). It has been proposed that such responsiveness to ECB components on the behaviorally antagonism-related ORN prevents upwind flight to ECB blends (Takanashi et al., 2006). This property of ACB males may serve as a premating isolating mechanism that precludes mating attempts between ACB males and females of ECB or other Ostrinia that emit pheromone blends containing Z11- and E11-14:OAc. Further supporting this hypothesis, we found that the behavioral antagonism-related ORN of rare ACB males did not respond to Z11- or E11-14:OAc (Domingue et al., 2007a). Thus, normal ACB males experience the imposition of antagonistic ORN input from ECB pheromone components, which is not exhibited by ACB rare males.

The results of these studies suggested distinct changes to the peripheral olfactory system that may have been at work at the beginning (relative firing rates) and end (antagonist imposition) of a shift from a pheromone system using Z11- and E11-14:OAc to result in the ACB system using Z12- and E12-14:OAc. Perhaps, as currently observed in the rare ECB(E) males, there was some change in an ORN associated with the minor pheromone component (Domingue et al., 2007b). Subsequently, reproductive isolation of the new population would have been achieved by the imposition of antagonism to the pheromone blend of the ancestral population (Domingue et al., 2007a). In the current study, we performed hybridization studies between the ACB and ECB(E) to determine whether the above scenario might be consistent with the genetic differences between the species that are observable in the hybrids, or if other changes in the olfactory system can be inferred to have been required for such a shift.

Section snippets

Insects

ECB(E) male moths were obtained from a colony of the bivoltine E-strain of ECB that has been maintained in the laboratory of W.L. Roelofs as previously described (Roelofs et al., 1985). ACB male moths were obtained from a subset of those described in a previous study (Linn et al., 2007). The colony of the ACB originated from Jin Kyo Jung, National Institute of Crop Sciences, South Korea. Moths were maintained at 25 °C, and 16:8 L:D photoperiod as previously described for ECB (Roelofs et al., 1985

Results

The ORN response profiles of the hybrids exhibited characteristics of both parental types (Fig. 1). A large-spike-size ORN responded strongly to Z12-, E12-, and E11-14:OAc as in the parental populations. A small-spike-size ORN was stimulated by E12-14:OAc as it was in the ACB males, and also by Z11-14:OAc as it was in ECB E-strain males. The hybrids and both parental species had one type of ORN that was highly responsive to Z9-14:OAc. The reciprocal F1 crosses responded to the same compounds on

Discussion

There are several apparent homologies in the peripheral olfactory systems of ACB and ECB(E) that are also observed in the hybrids. Despite differences in the relative amplitudes of the antagonistic versus the large-spike-size ORNs, spike size relationships remain generally similar across all three populations (Table 1). We observed that there is a large-spike-size ORN that tends to be most broadly tuned, an intermediate spike-sized ORN that is primarily responsive to the behavioral antagonist

Acknowledgments

We thank Kathy Poole for help in maintaining the colonies. The project was funded by NSF IBN #034340, to WLR and TCB. Andy Myrick wrote the Labview program used for analyzing spike amplitude.

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