Strepsipteran Xenos Vesparum Rossi
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Expression of the Pupal Determinant broad during Metamorphic and Neotenic Development of the Strepsipteran Xenos vesparum Rossi Deniz F. Erezyilmaz1*., Alex Hayward2., Yan Huang1, Jordi Paps3, Zoltan Acs4, Juan A. Delgado5, Francisco Collantes5, Jeyaraney Kathirithamby3 1 Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, United States of America, 2 Science for Life Laboratory, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala Biomedical Centre, Uppsala, Sweden, 3 Department of Zoology, University of Oxford, Oxford, United Kingdom, 4 Kaposvar University, Faculty of Animal Science, Kaposvar, Hungary, 5 Departamento de Zoologia, Facultad de Biologia, Universidad de Murcia, Murcia, Spain Abstract Derived members of the endoparasitic order Strepsiptera have acquired an extreme form of sexual dimorphism whereby males undergo metamorphosis and exist as free-living adults while females remain larviform, reaching sexual maturity within their hosts. Expression of the transcription factor, broad (br) has been shown to be required for pupal development in insects in which both sexes progress through metamorphosis. A surge of br expression appears in the last larval instar, as the epidermis begins pupal development. Here we ask if br is also up-regulated in the last larval instar of male Xenos vesparum Rossi (Stylopidae), and whether such expression is lost in neotenic larviform females. We clone three isoforms of br from X. vesparum (Xv’br), and show that they share greatest similarity to the Z1, Z3 and Z4 isoforms of other insect species. By monitoring Xv’br expression throughout development, we detect elevated levels of total br expression and the Xv’Z1, Xv’Z3, and Xv’Z4 isoforms in the last larval instar of males, but not females. By focusing on Xv’br expression in individual samples, we show that the levels of Xv’BTB and Xv’Z3 in the last larval instar of males are bimodal, with some males expressing 3X greater levels of Xv’br than fourth instar femlaes. Taken together, these data suggest that neoteny (and endoparasitism) in females of Strepsiptera Stylopidia could be linked to the suppression of pupal determination. Our work identifies a difference in metamorphic gene expression that is associated with neoteny, and thus provides insights into the relationship between metamorphic and neotenic development. Citation: Erezyilmaz DF, Hayward A, Huang Y, Paps J, Acs Z, et al. (2014) Expression of the Pupal Determinant broad during Metamorphic and Neotenic Development of the Strepsipteran Xenos vesparum Rossi. PLoS ONE 9(4): e93614. doi:10.1371/journal.pone.0093614 Editor: Daniel Doucet, Natural Resources Canada, Canada Received December 27, 2013; Accepted March 7, 2014; Published April 7, 2014 Copyright: ß 2014 Erezyilmaz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Leverhulme Trust Research Grant to JK (no. No. F/08 502/G) (http://www.leverhulme.ac.uk), Stony Brook University startup funds to DE (http://www. stonybrook.edu). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] . These authors contributed equally to this work. Introduction phosis are also used to regulate other types of life history polymorphisms. In terms of both diversity and abundance, the insects are one of Some of the most dramatic switches in insect form are found in the most successful animal classes [1]. Many groups within the the holometabolous order Strepsiptera (Figure 1). All strepsipter- insects are distinguished by sharp life history transitions that occur ans are endoparasites, and switches between forms occur at at molts. Such life history switches occur between two fully transitions between the free-living and endoparasitic stages. differentiated states that are tailored to exploit different resources, Female Strepsiptera produce their young by hemocoelous to cope with environmental changes, or to subdivide labor [2]. vivipary, and produce 1st instar larvae that are specialized for The most common life history polymorphism among the insects is host seeking that have rudimentary eyes, thoracic limbs, and a complete metamorphosis, which is the central trait that defines the sclerotized cuticle. Upon finding a host, the first switch in group Holometabola, which includes butterflies, beetles, bees and differentiated form occurs as the 1st instar larva molts to an flies. In the Holometabola, metamorphosis between the larval endoparasitic, apodous larva with a soft cuticle [3]. In the early form and the winged, sexually mature adult form occurs over two branching order of Strepsiptera, the Mengenillidae, both male and molts, the molt to the pupal stage and the final molt to the adult female larvae emerge from the host to form puparia, and stage. This strategy allows the larval stages to specialize in feeding eventually produce free-living male and wingless neotenic female and growth, while the adult form specializes in dispersal and adults. However in the more derived Stylopiformia, females reproduction. Much research has focused on the regulation of exhibit an extreme form of neoteny, and remain endoparasitic metamorphosis in holometabolous insects. Little is known, within the host, existing as a ‘‘bag of eggs’’ [4], [5]. In Xenos however, of whether the mechanisms used to regulate metamor- vesparum Rossi, a parasite of paper wasps that is a member of Stylopidia, male 4th instar larvae molt to the pupal and adult PLOS ONE | www.plosone.org 1 April 2014 | Volume 9 | Issue 4 | e93614 Dimorphic Expression of broad stages. Upon completion of adult development, the fully differen- to the larva- pupa transition in holometabolous insects [16]–[18]. tiated free-living male emerges from the endoparasitic puparium Loss of br expression at this stage results in larval-adult hybrids in [4],[6]. X. vesparum females do not form a puparium after the 4th the flour beetle Tribolium castaneum (Herbst) [11]–[13]. in the instar. Instead they remain larviform, and lack eyes, mouthparts, silkmoth Bombyx mori (L.) [9] and in the lacewing Chrysopa perla,(L.) antennae, wings and external genitalia as adults. During the final a fairly primitive holometabolous insect [11]. br null mutants in larval instar, the head and anterior thoracic region of the female is Drosophila melanogaster Meigen fail to enter metamorphosis alto- extruded through the host cuticle, and sclerotizes to form the gether [8]. chephalothorax. This structure facilitates mating and the exodus of The br locus is complex, and produces multiple transcripts 1st instar larvae [4]–[6]. Therefore, two modifications of typical through alternative splicing of an N-terminal Broad-Tramtrack- holometabolous insect life history are found in Strepsiptera: 1) the Bric-a-brac (BTB)-containing core region to one of several C2H2 switch between the free-living 1st instar larva and the apodous zinc finger-containing C-terminal sequences. The Broad gene is endoparasitic larval form that lives within the host, and 2) the loss best characterized in D. melanogaster, where alternative splicing of a switch between the apodous, larval stage and the pupal stage produces many different transcripts, with all splice products in female Stylopidia. containing one of four zinc finger types Z1–Z4 [19],[20]. The four Two hormones, juvenile hormone (JH) and ecdysone, regulate zinc finger isoforms have varied expression levels and temporal transitions between instars and progression through metamorpho- profiles in different tissues ([20]–[22]. Each isoform in D. sis. The timing of molts is triggered by the steroid hormone melanogaster binds DNA and regulates metamorphosis-specific ecdysone, which is released in temporally regulated pulses. The expression of target genes [22]–[24]. presence of JH during the larval stages prevents progression to the Here we propose that the appearance of neotenic females in pupal stage, while the presence of JH at the pupal stage prevents Strepsiptera is linked to the modification of pathways that underlie progression to the adult stage [7]. A number of functional studies pupal determination. To test this hypothesis, we isolate broad have established that JH-dependent switches exert their effect sequences from the male and female X. vesparum, which exhibits through expression of the JH-effector, broad (br) [8]–[15]. extreme sexual dimorphism. We examine the expression of total Expression of high levels of br is restricted by JH and ecdysone Xv’br and of three br isoforms, and ask: 1) whether Xv’br expression Figure 1. Life cycle of Xenos vesparum within its paper wasp host, Polistes dominula. A free-living male (top left) mates with the endoparasitic neotenic female via her extruded cephalothorax, which protrudes through the host cuticle (top right). The female produces first instar larvae that exit through the brood canal in the extruded cephalothorax. The free-living 1st instar larvae (center) seek a host within paper wasp nests. Upon entering a host, the 1st instars molt to apodous endoparasitic second instar larvae with a soft cuticle. The second instar larvae molt two additional times. Male 4th instar larvae (bottom left) molt to form a pupa,