Sex pheromone recognition and immunolocalization of three pheromone binding proteins in the black cutworm moth Agrotis ipsilon

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Abstract

Insect pheromone binding proteins (PBPs) are believed to solubilize and transport hydrophobic sex pheromones across sensillum lymph to membrane-associated pheromone receptors. To address the molecular mechanisms of PBPs in insect pheromone perception, we undertook a systemic study on the PBPs of the black cutworm Agrotis ipsilon at transcript as well as protein level from tissue distribution and cellular localization to pheromone binding affinity. We cloned three full-length PBP genes AipsPBP1-3 from A. ipsilon antennae, and demonstrated that AipsPBP13 transcripts were highly expressed in male antennae. The electron microscopic examinations revealed at least six types of olfactory sensilla on male and female antenna: trichodea, chaetica, basiconica, coeloconica, squamiformia and Böhm bristles. The immunocytochemistry results demonstrated that AipsPBP1-3 proteins were strongly expressed in the sensillum lymph of the trichoid sensilla of male moth. The binding assays showed that AipsPBP1 had high binding affinities with the major sex pheromone components Z7-12:Ac and Z9-14:Ac among five related chemicals and was clustered together with the long trichoid sensilla-expressing LdisPBPs of Lymantria dispar. AipsPBP2 showed high binding affinities also with Z11-16:Ac. AipsPBP3 displayed a high affinity only with Z11-16:Ac. Our studies provide further detail evidences for the involvement of moth PBPs in pheromone discrimination and selective recognition of specific components of the female sex pheromone blends.

Highlights

► We report the cloning, male antenna expression of PBP genes of Agrotis ipsilon. ► The PBPs are strongly expressed in the sensillum lymph of the sensilla trichodea. ► AipsPBP1 show high binding affinities to Z7-12:Ac and Z9-14:Ac. ► AipsPBP2 show high binding affinities to Z7-12:Ac, Z9-14:Ac and Z11-16:Ac. ► AipsPBP3 specifically binds to the Z11-16:Ac with high binding ability.

Introduction

Lepidoptera insects comprise of the second largest group in insect kingdom (Ando et al., 2004). Many of them are crop pests. They use chemical cures to locate their plant hosts and mates. The first insect sex pheromone bombykol ((E,Z)-10,12-hexadecadienol) was identified in the silkmoth Bombyx mori (Butenandt et al., 1959; Karlson and Butenandt, 1959). Later another antagonist component bombykal ((E,Z)-10,12-hexadecadienal) was identified (Kaissling and Kasang, 1978). In fact, it has been found that most of male moths only attract to a blend of pheromone components produced by female moths in a unique and precise quantitative ratio (Shorey, 1973; Kaissling, 1986; Arn et al., 1992; Schneider, 1992). The sensitivity of male moth to its sex pheromone is extremely high, in the B. mori, for example, even a single bombykol molecule is sufficient to elicit a nerve impulse (Kaissling, 1971; Steinbrecht et al., 1992). The behavior threshold concentrations for the male silkmoth to perceive the pheromone of conspecific females is in the order of 1000 molecules per ml of air (Kaissling, 1987). The high specificity and sensitivity of insect to sex pheromones make them as effective biological control agents for population monitoring and mass trapping of noxious insects in integrated pest management (IPM) programs (McNeil, 1991; Witzgall et al., 2008, 2010). However, the molecular and cellular mechanisms of how sex pheromones are perceived by male moths are still rudimentary.

Species-specific pheromone molecules enter into the sensillum lymph of the pheromone-senstive sensilla trichodea via the multipores of the insect cuticle (Kaissling, 1986; Steinbrecht, 1997). One group of small soluble proteins called odorant binding proteins (OBPs) are believed to be involved in the initial biochemical recognition steps in odorant perception by capturing and transporting pheromone molecules to olfactory receptors (ORs) (Vogt et al., 1985; Pelosi et al., 2006; Leal, 2005; Zhou, 2010). OBPs are presumed to be synthesized by non-neuronal auxiliary cells (trichogen and tormogen cells) of the sensory neurons and secreted into the sensillum lymph with extremely high concentration (up to 10 mM) (Vogt and Riddiford, 1981; Klein, 1987; Steinbrecht et al., 1992). The extremely high concentration of OBPs in insect antennae implies important physiological roles in individual insect chemical communication and survival. Based on their amino acid sequence similarities, the moth OBPs are grouped into three subfamilies: pheromone binding proteins (PBPs) (Vogt and Riddiford, 1981), general odorant binding proteins (GOBP1 and GOBP2) (Vogt et al., 1991) and antennal binding proteins X (ABPX) (Krieger et al., 1996). So far OBP genes are identified from more than 34 insect species of 13 Lepidoptera families, including 44 OBP genes in the B. mori genome (Gong et al., 2009). Usually there are three PBP genes in moth species, which display very high sequence identities (about 55%) within and among species. They with GOBPs belong to a closely related gene cluster and share one common ancestral gene and then diverged by gene duplication events after specialization (Forstner et al., 2006; Gong et al., 2009; Vogt et al., 2002; Robertson et al., 1999; Zhou, 2010). However, recently studies revealed additional PBP genes exist in the Asian corn borer (Allen and Wanner, 2011).

Insect PBPs are believed to solubilize and transport hydrophobic sex pheromones across the sensillum lymph to the membrane-associated pheromone receptors located on the olfactory receptor neurons (ORNs) (Vogt, 1987; Pelosi and Maida, 1990; Prestwich et al., 1995; Breer, 1997; Kaissling, 2009). In situ hybridization and immunocytochemical localization analyses showed that PBPs exclusively or predominantly localized in the lymph of long and short sensilla trichoidea. (Steinbrecht et al., 1992, 1995; Maida et al., 1993; Zhang et al., 2001; Forstner et al., 2006; Groβe-Wilde et al., 2007; Forstner et al., 2009). Differential expressions have been reported between PBPs and between different types of trichoid sensilla. In B. mori, PBP1 expresses in long trichoid sensilla, along with pheromone receptor OR1; but the other PBPs express in short trichoid sensilla that do not express the pheromone receptor (Forstner et al., 2006). Similarly in Manduca sexta, expresses in long trichoid sensilla; however all other PBPs express in other sensilla which are presumably short trichoidea (Nardi et al., 2003). Some PBPs coexpress in the same sensilla. The PBPs of Antheraea polyphemus (Maida et al., 2000) and Lymantria dispar (Vogt et al., 1989) colocalise to long trichoid sensilla.

Some researches revealed that sex pheromones can be detected by heterologously expressed ORs alone without the PBP protein LUSH of Drosophila melanogaster (van der Goes van Naters and Carlson, 2007). However, the pheromone cis-11-vaccenyl acetate was dissolved in dimethyl sulfoxide (DMSO) and the response time to pheromone stimuli usually was in several seconds (Sato et al., 2008; Wicher et al., 2008), which is much slower compared with the fast behavioral reaction time (milliseconds) (Boeckh et al., 1965; Kaissling, 1986). Electrophysiological recording experiments showed that PBPs facilitated the sensitivity of olfactory neurons to the major pheromone component as compared to pheromone alone (Van den Berg and Ziegelberger, 1991). The HEK293 cells expressing the Heliothis virescens OR HvirOR13 showed very little specificity for all the sex pheromone components, however, when applied together with the PBP HvirPBP2, the HvirOR13-expressing cells showed significant sensitivity and specificity to the pheromone (Z)-11-Hexadecenal (Z11-16:Ald), the EC50 values of HvirOR13 alone and HvirOR13/PBP2 complex were 1.2 nM and 0.0002 nM, respectively (Groβe-Wilde et al., 2007). In the silkmoth A. polyphemus, the ApolOR1-expressing cells responded to all three sex pheromones when the pheromones were solubilized by DMSO and also when DMSO was substituted by the PBP ApolPBP2. However, when the sex pheromone concentrations were decreased down to picomolar level and ApolPBP2 was used the ApolOR1-expressing cells only responded to the pheromone (E,Z)-6,11-hexadecadienal (E6,Z11-16Ald) (Forstner et al., 2009). These results indicate that PBPs may selectively solubilize, bind distinct pheromones, transport them to ORs (more precisely pheromone receptors) and enhance the specificity and sensitivity of the olfactory neuronal response to pheromones. The specificity and affinity of many insect PBP proteins with their sex pheromone components have been measured by several different methods, such as polyacrylamide gel electrophoresis (PAGE) experiment with radiolabeled sex pheromones (Vogt and Riddiford, 1981; Prestwich, 1993, 1996), fluorescence competitive binding assays with fluorescence probe (Gu et al., 2011a; He et al., 2010), cold binding assay (Leal et al., 2005; Zhou et al., 2009) and a new developed binding method called two phase binding assays (Zhou et al., 2009; He et al., 2010).

The black cutworm moth Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae), is a destructive pest of many crops (Rings et al., 1974; Clement et al., 1982).The female sex pheromone blend of the Chinese strain of A. ipsilon moth comprises of five main components: (Z)-7-dodecenyl acetate (Z7-12:Ac) (40.5%), (Z)-9-tetradecenyl acetate (Z9-14:Ac) (13.2%), (Z)-11-hexadecenyl acetate (Z11-16:Ac) (14.9%), (Z)-5-decenyl acetate (Z5-10:Ac) (14.3%) and (Z)-8-dodecenyl acetate (Z8-12:Ac) (17.2%) (Xiang et al., 2009). The two principal pheromone components Z7-12:Ac and Z9-14:Ac in a mixture of 3:1 were necessary and sufficient to attract the male A. ipsilon moths. The third pheromone component Z11-16:Ac was a synergist when it was added to the original two-component blend with the ratio as 3:1:1 (Xiang et al., 2009). Both long and short trichoid sensilla of A. ipsilon respond to sex pheromone components (Renou et al., 1996). The receptor neuron in the long trichoid sensella responds mainly to Z7-12:Ac, while the neurons in the short trichoid sensilla mainly respond to Z5-10:Ac, Z8-12:Ac and Z9-14:Ac (Renou et al., 1996).

In present study, we report the full-length of the two PBP genes AipsPBP1 and AipsPBP2 of A. ipsilon and, in additional, a novel PBP gene AipsPBP3 for the first time. To address the molecular recognition of the A. ipsilon PBPs and examine their involvement in pheromone perception, the tissue distributions of AipsPBP13 transcripts were measured by qRT-PCR, the different types and fine structures of A. ipsilon sensilla were characterized, and the specific sensillum location of the AipsPBP1–3 proteins was investigated by immunocytochemistry techniques. The pheromone binding abilities of the recombinant AipsPBP1–3 proteins to the sex pheromone components of A. ipsilon were measured using fluorescence competitive binding assays and two-phase binding assays. These studies suggest AipsPBP1 and AipsPBP2 as the carriers for the major sex pheromone component of A. ipsilon. Our systematic studies provide further detailed evidences for the involvement of insect PBPs in insect semiochemical perception and possible pheromone recognition.

Section snippets

Sex pheromones and general odorants

The five sex pheromone components of A. ipsilon (Z)-7-dodecenyl acetate (Z7-12:Ac), (Z)-9-tetradecenyl acetate (Z9-14:Ac), (Z)-11-hexadecenyl acetate (Z11-16:Ac), (Z)-5-decenyl acetate (Z5-10:Ac) and (Z)-8-dodecenyl acetate (Z8-12:Ac) were purchased from Bedoukian (Bedoukian Research, Danbury, USA). The purity of the pheromones (>98%) was quantified using GC on a HP 6980 series GC system (Hewlett Packard, USA). Dodecane, Tetradecane, Hexeadecane, Tetradecanol, Hexadecanol were purchased from

Full-length A. ipsilon PBP genes

Three PBP cDNA fragments were obtained by PCR using two pairs of degenerate primers. The remaining 5′ and 3′ end of the PBP genes were amplified by the RACE PCR strategy. The sequences obtained from the 5′- and 3′-RACE PCR were assembled with the original PBP fragments to generate the full-length PBP genes and named as AipsPBP1, AipsPBP2 and AipsPBP3 (GenBank accession number: JQ822240, JQ822241 and JQ822242, respectively). The AipsPBP13 contains an open reading frame (ORF) of 513 bp, 498 bp

Discussion

We have obtained three full-length pheromone binding protein genes (AipsPBP1-3) of A. ipsilon moth. The partial genes encoding for AipsPBP1 and AipsPBP2 were reported previously (Picimbon and Gadenne, 2002; Abraham et al., 2005), but AipsPBP3 is a novel PBP gene of the A. ipsilon. Sequence alignment showed relatively high sequence identities (about 40%) with each other, supporting the notion that the moth PBPs share one common ancestral PBP gene and then diverged by gene duplication events

Acknowledgments

This work was supported by the China National “973” Basic Research Program (2012CB114104), the National Natural Science Foundation of China (31071694, 31272048 and 31171858). JJ Zhou and YJ Zhang acknowledge the financial support from the Royal Society, UK for the international joint project between China and UK (31111130203; JP100849) and BBSRC International Partnering Award (BB/J020281). SH Gu thanks the Royal Society, UK for the financial support to work at Rothamsted Research, United

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