Massive horizontal gene transfer and the evolution of nematomorph-driven behavioral manipulation of mantids.

To complete their life cycle, a wide range of parasites must manipulate the behavior of their hosts.1 This manipulation is a well-known example of the "extended phenotype,2" where genes in one organism have phenotypic effects on another organism. Recent studies have explored the parasite genes responsible for such manipulation of host behavior, including the potential molecular mechanisms.3,4 However, little is known about how parasites have acquired the genes involved in manipulating phylogenetically distinct hosts.4 In a fascinating example of the extended phenotype, nematomorph parasites have evolved the ability to induce their terrestrial insect hosts to enter bodies of water, where the parasite then reproduces. Here, we comprehensively analyzed nematomorphs and their mantid hosts, focusing on the transcriptomic changes associated with host manipulations and sequence similarity between host and parasite genes to test molecular mimicry. The nematomorph's transcriptome changed during host manipulation, whereas no distinct changes were found in mantids. We then discovered numerous possible host-derived genes in nematomorphs, and these genes were frequently up-regulated during host manipulation. Our findings suggest a possible general role of horizontal gene transfer (HGT) in the molecular mechanisms of host manipulation, as well as in the genome evolution of manipulative parasites. The evidence of HGT between multicellular eukaryotes remains scarce but is increasing and, therefore, elucidating its mechanisms will advance our understanding of the enduring influence of HGT on the evolution of the web of life.

Regulation and remodeling of microbial symbiosis in insect metamorphosis.

Many insects are dependent on microbial mutualists, which are often harbored in specialized symbiotic organs. Upon metamorphosis, insect organs are drastically reorganized. What mechanism regulates the remodeling of the symbiotic organ upon metamorphosis? How does it affect the microbial symbiont therein? Here, we addressed these fundamental issues of symbiosis by experimentally manipulating insect metamorphosis. The stinkbug Plautia stali possesses a midgut symbiotic organ wherein an essential bacterial symbiont resides. By RNAi of master regulator genes for metamorphosis, Kr-h1 over nymphal traits and E93 over adult traits, we generated precocious adults and supernumerary nymphs of P. stali, thereby disentangling the effects of metamorphosis, growth level, developmental stage, and other factors on the symbiotic system. Upon metamorphosis, the symbiotic organ of P. stali was transformed from nymph type to adult type. The supernumerary nymphs and the precocious adults, respectively, developed nymph-type and adult-type symbiotic organs not only morphologically but also transcriptomically, uncovering that metamorphic remodeling of the symbiotic organ is under the control of the MEKRE93 pathway. Transcriptomic, cytological, and biochemical analyses unveiled that the structural and transcriptomic remodeling of the symbiotic organ toward adult emergence underpins its functional extension to food digestion in addition to the original role of symbiont retention for essential nutrient production. Notably, we found that the symbiotic bacteria in the adult-type symbiotic organ up-regulated genes for production of sulfur-containing essential amino acids, methionine and cysteine, that are rich in eggs and sperm, uncovering adult-specific symbiont functioning for host reproduction and highlighting intricate host-symbiont interactions associated with insect metamorphosis.

Mechanisms Underpinning Morphogenesis of a Symbiotic Organ Specialized for Hosting an Indispensable Microbial Symbiont in Stinkbugs.

Microbial mutualists are pivotal for insect adaptation, which often entails the evolution of elaborate organs for symbiosis. Addressing what mechanisms underpin the development of such organs is of evolutionary interest. Here, we investigated the stinkbug Plautia stali, whose posterior midgut is transformed into a specialized symbiotic organ. Despite being a simple tube in newborns, it developed numerous crypts in four rows, whose inner cavity hosts a specific bacterial symbiont, during the 1st to 2nd nymphal instar stages. Visualization of dividing cells revealed that active cell proliferation was coincident with the crypt formation, although spatial patterns of the proliferating cells did not reflect the crypt arrangement. Visualization of visceral muscles in the midgut, consisting of circular muscles and longitudinal muscles, uncovered that, strikingly, circular muscles exhibited a characteristic arrangement running between the crypts specifically in the symbiotic organ. Even in the early 1st instar stage, when no crypts were seen, two rows of epithelial areas delineated by bifurcated circular muscles were identified. In the 2nd instar stage, crossing muscle fibers appeared and connected the adjacent circular muscles, whereby the midgut epithelium was divided into four rows of crypt-to-be areas. The crypt formation proceeded even in aposymbiotic nymphs, revealing the autonomous nature of the crypt development. We propose a mechanistic model of crypt formation wherein the spatial arrangement of muscle fibers and the proliferation of epithelial cells underpin the formation of crypts as midgut evaginations. IMPORTANCE Diverse organisms are associated with microbial mutualists, in which specialized host organs often develop for retaining the microbial partners. In light of the origin of evolutionary novelties, it is important to understand what mechanisms underpin the elaborate morphogenesis of such symbiotic organs, which must have been shaped through interactions with the microbial symbionts. Using the stinkbug Plautia stali as a model, we demonstrated that visceral muscular patterning and proliferation of intestinal epithelial cells during the early nymphal stages are involved in the formation of numerous symbiont-harboring crypts arranged in four rows in the posterior midgut to constitute the symbiotic organ. Strikingly, the crypt formation occurred normally even in symbiont-free nymphs, revealing that the crypt development proceeds autonomously. These findings suggest that the crypt formation is deeply implemented into the normal development of P. stali, which must reflect the considerably ancient evolutionary origin of the midgut symbiotic organ in stinkbugs.

Morphogenesis and development of midgut symbiotic organ of the stinkbug Plautia stali (Hemiptera: Pentatomidae).

Diverse insects are intimately associated with microbial symbionts, which play a variety of biological roles in their adaptation to and survival in the natural environment. Such insects often possess specialized organs for hosting the microbial symbionts. What developmental processes and mechanisms underlie the formation of the host organs for microbial symbiosis is of fundamental biological interest but poorly understood. Here we investigate the morphogenesis of the midgut symbiotic organ and the process of symbiont colonization therein during the developmental course of the stinkbug Plautia stali. Upon hatching, the midgut is a simple and smooth tube. Subsequently, symbiont colonization to the posterior midgut occurs, and thickening and folding of the midgut epithelium proceed during the first instar period. By the second instar, rudimentary crypts have formed, and their inner cavities are colonized by the symbiotic bacteria. From the second instar to the fourth instar, while the alimentary tract grows and the posterior midgut is established as the symbiotic organ with numerous crypts, the anterior midgut and the posterior midgut are structurally and functionally isolated by a strong constriction in the middle. By the early fifth instar, the midgut symbiotic organ attains the maximal length, but toward the mid fifth instar, the basal region of each crypt starts to constrict and narrow, which deforms the midgut symbiotic organ as a whole into a shorter, thicker and twisted shape. By the late fifth instar to adulthood, the crypts are constricted off, by which the symbiotic bacteria are confined in the crypt cavities and isolated from the midgut main tract, and concurrently, the strong midgut constriction in the middle becomes loose and open, by which the food flow from the anterior midgut to the posterior midgut recovers. This study provides the most detailed and comprehensive descriptions ever reported on the morphogenesis of the symbiotic organ and the process of symbiont colonization in an obligatory insect-bacterium gut symbiotic system. Considering that P. stali is recently emerging as a useful model system for experimentally studying the intimate insect-microbe gut symbiosis, the knowledge obtained in this study establishes the foundation for the further development of this research field.