A discrete neuropeptide difference between two hybridizing grasshopper subspecies

Blackwell Publishing LtdOxford, UKBIJBiological Journal of the Linnean Society0024-4066© 2007 The Linnean Society of London? 2007 914 541548 Original Articles NEUROPEPTIDE DIFFERENCE BETWEEN GRASSHOPPER SUBSPECIES S. ROTH ET AL . Biological Journal of the Linnean Society, 2007, 91, 541–548. With 3 figures A discrete neuropeptide difference between two hybridizing grasshopper subspecies STEFFEN ROTH1*, GÜNTER KÖHLER2, KLAUS REINHARDT3,4 and REINHARD PREDEL1,5 Saxon Academy of Sciences, Research Group Jena, Erbertstrasse1, 07743 Jena, Germany Institute of Ecology, Friedrich-Schiller University of Jena, Dornburger Strasse 159, 07743 Jena, Germany 3 School of Biological Sciences, The University of Leeds, Leeds LS2 9JT, UK 4 Department of Animal and Plant Sciences, The University of Sheffield, Sheffield S10 2TN, UK 5 Institute of General Zoology, Friedrich-Schiller University of Jena, Erbertstrasse1, 07743 Jena, Germany 2 Received 22 November 2005; accepted for publication 10 August 2006 Population divergence can be detected by the divergence of functional and neutral characters. Under some circumstances, it is desirable to have available a character that is discretely expressed in either of the diverging genomes, rather than the evaluation of qualitative variation of continuous characters. In the present study, we investigated mass peaks of peptide hormones in a model system of population divergence, the hybrid zone of two Chorthippus parallelus subspecies in the French–Spanish Pyrenees. Mass spectra from neuroendocrine tissues have previously been identified as species-specific and may have a sufficient resolution to vary at the subspecies level. For the first time, we succeeded in the detection of a subspecies-specific expression of neuropeptides collected from single individuals. Mass spectra sampled from populations across the C. parallelus grasshopper hybrid zone indicated neuropeptide identity between the sexes and within sample sites. The distribution of a single distinct but variable peptide signal, however, very closely followed the cline of the hybrid zone as derived from the mean variation in several continuous characters. The identity of this peptide in populations from the northern Pyrenees and central Europe supports a neuropeptide differentiation of preglacial origin. The observed differentiation in the peptide profile of the two subspecies demonstrates that a peptidomic approach may be a promising perspective to reconstruct reproductive isolation in an insect hybrid zone. © 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 91, 541–548. ADDITIONAL KEYWORDS: Chorthippus – insect – mass spectrometry – Orthoptera – perisympathetic organ. INTRODUCTION Reproductive isolation is the first crucial step in speciation and is achieved via prezygotic and postzygotic barriers. Postzygotic isolation is likely to originate from epistatic interaction of many loci (Coyne & Orr, 2004). Such postzygotic isolation can be studied by crossing populations varying in genetic relatedness and measuring the degree of hybrid disadvantage, such as testis sterility (Virdee & Hewitt, 1994; Tregenza, Pritchard & Butlin, 2002) or reduced olfactory responses to the host plant (Linn et al., 2004). Less related populations are more likely to have diverged *Corresponding author. E-mail: genetically and therefore are more likely to possess a larger degree of negative epistasis. An important facet in searching for loci that lead, or add, to negative interactions of genomes is the identification of discretely expressed characters in either of two diverging populations, rather than quantitative variation of continuous characters which is more difficult to locate in the genome. We investigated the suitability of a potentially very powerful tool to identify discrete characters, the mass spectrometric analysis of neuropeptides from single insect specimens. Neuropeptides are structurally and functionally the most diverse group of messenger molecules and a considerable number of them can be released into the haemolymph and act as hormones (Strand, 1999). Organs in which such peptide hor- © 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 91, 541–548 541 1 542 S. ROTH ET AL. neurohemal organs of the different tagmata (head, thorax, abdomen) (Predel & Eckert, 2000; Predel et al., 2004a), we indeed found a distinct difference between the two subspecies. This indicates that neuropeptides may suitably serve as a subspecies marker, which is also supported by preliminary data of a cross mating. Because the mass spectrometric approach to detect the differentiation is fast, reproducible, and needs only single neurohemal organs, we present our findings as a tool that may be of interest to researchers studying hybridization in insects. MATERIAL AND METHODS INSECTS AND SAMPLE SITES Chorthippus parallelus parallelus (Cpp) and C. p. erythropus (Cpe) were sampled along a hybrid transect across the Col de la Quillane in the eastern Pyrenees. The geographical locations of the sample sites are provided in Butlin & Hewitt (1985a, b), whose abbreviations we also adopted (Fig. 1) and Köhler et al. (2007). The abbreviations of the sample sites are as follows: BEL, Bellver, Eyne; QMT1, Quillane Mike Transect (between Matemale and la Llagone); MA, Matemale; RT, Real Turnoff (between Puyvalador and Formiguères); PU, Puyvalador; PS, Puyvalador South; ARG, Argouzeles. In July and August of 2002, adult grasshoppers of both subspecies of C. parallelus were sampled from the study plots and transferred to Jena/Germany for investigation. Grasshoppers were caged with Cocksfoot grass (Dactylis glomerata) as food. Additionally, we investigated individuals from Jena/Germany (September 2002), which are genetically closely related to individuals from the north of the hybrid zone. We also collected egg pods from population crosses Cpe × Cpp and Cpp × Cpe. However, after hibernation under laboratory conditions, only one nymph of an Eyne female × PU male crosshatched and was brought to adulthood. The neuropeptides of this single female were nevertheless analysed because it represented a cross of populations from either end of the hybrid zone, which in nature is a very unlikely cross. DISSECTION OF NEUROHEMAL ORGANS AND SAMPLE PREPARATION FOR MASS SPECTROMETRY Decapitated insects were dissected under a stereomicroscope. Parts of the nervous system and adjacent neurohemal release sites were made visible by removal of other tissues, and flushed with insect saline. Neurohemal organs were dissected rapidly using fine scissors and transferred to a sample plate for matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. On the sam- © 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 91, 541–548 mones are stored until release are called neurohemal organs. The major neurohemal organs o (...truncated)