Cellular mechanism for selective vertical transmission of an obligate insect symbiont at the bacteriocyte-embryo interface.
ABSTRACT: Many insects are associated with obligate symbiotic bacteria, which are localized in specialized cells called bacteriocytes, vertically transmitted through host generations via ovarial passage, and essential for growth and reproduction of their hosts. Although vertical transmission is pivotal for maintenance of such intimate host-symbiont associations, molecular and cellular mechanisms underlying the process are largely unknown. Here we report a cellular mechanism for vertical transmission of the obligate symbiont Buchnera in the pea aphid Acyrthosiphon pisum. In the aphid body, Buchnera cells are transmitted from maternal bacteriocytes to adjacent blastulae at the ovariole tips in a highly coordinated manner. By making use of symbiont-manipulated strains of A. pisum, we demonstrated that the facultative symbiont Serratia is, unlike Buchnera, not transmitted from maternal bacteriocytes to blastulae, suggesting a specific mechanism for Buchnera transmission. EM observations revealed a series of exo-/endocytotic processes operating at the bacteriocyte-blastula interface: Buchnera cells are exocytosed from the maternal bacteriocyte, temporarily released to the extracellular space, and endocytosed by the posterior syncytial cytoplasm of the blastula. These results suggest that the selective Buchnera transmission is likely attributable to Buchnera-specific exocytosis by the maternal bacteriocyte, whereas both Buchnera and Serratia are nonselectively incorporated by the endocytotic activity of the posterior region of the blastula. The sophisticated cellular mechanism for vertical transmission of Buchnera must have evolved to ensure the obligate host-symbiont association, whereas facultative symbionts like Serratia may coopt the endocytotic component of the mechanism for their entry into the host embryos.
Project description:Many insects host their obligate, maternally transmitted symbiotic bacteria in specialized cells called bacteriocytes. One of the best-studied insect nutritional endosymbioses is that of the aphid and its endosymbiont, Buchnera aphidicola. Aphids and Buchnera are metabolically and developmentally integrated, but the molecular mechanisms underlying Buchnera transmission and coordination with aphid development remain largely unknown. Previous work using electron microscopy to study aphid asexual embryogenesis has revealed that Buchnera transmission involves exocytosis from a maternal bacteriocyte followed by endocytotic uptake by a blastula. While the importance of exo- and endocytic cellular processes for symbiont transmission is clear, the molecular mechanisms that regulate these processes are not known. Here, we shed light on the molecular mechanisms that regulate Buchnera transmission and developmental integration.We present the developmental atlas of ACYPI000536 and ACYPI008904 mRNAs during asexual embryogenesis in the pea aphid, Acyrthosiphon pisum. Immediately before Buchnera invasion, transcripts of both genes were detected by whole-mount in situ hybridization in the posterior syncytial nuclei of late blastula embryos. Following Buchnera invasion, expression of both genes was identified in the region occupied by Buchnera throughout embryogenesis. Notably during Buchnera migration, expression of both genes was not concomitant with the entirety of the bacterial mass but rather expression colocalized with Buchnera in the anterior region of the bacterial mass. In addition, we found that ACYPI000536 was expressed in nuclei at the leading edge of the bacterial mass, joining the bacterial mass in subsequent developmental stages. Finally, quantitative reverse transcription real-time PCR suggested that early in development both transcripts were maternally provisioned to embryos.We venture that ACYPI000536 and ACYPI008904 function as nutrient sensors at the site of symbiont invasion to facilitate TOR-pathway-mediated endocytosis of Buchnera by the aphid blastula. Our data support earlier reports of bacteriocyte determination involving a two-step recruitment process but suggest that the second wave of recruitment occurs earlier than previously described. Finally, our work highlights that bacteriocyte-enriched amino acid transporter paralogs have additionally been retained to play novel developmental roles in both symbiont recruitment and bacteriome development.
Project description:Symbioses between animals and microbes are often described as mutualistic, but are subject to tradeoffs that may manifest as shifts in host and symbiont metabolism, cellular processes, or symbiont density. In pea aphids, the bacterial symbiont Buchnera is confined to specialized aphid cells called bacteriocytes, where it produces essential amino acids needed by hosts. This relationship is dynamic; Buchnera titer varies within individual aphids and among different clonal aphid lineages, and is affected by environmental and host genetic factors. We examined how host genotypic variation relates to host and symbiont function among seven aphid clones differing in Buchnera titer. We found that bacteriocyte gene expression varies among individual aphids and among aphid clones, and that Buchnera gene expression changes in response. By comparing hosts with low and high Buchnera titer, we found that aphids and Buchnera oppositely regulate genes underlying amino acid biosynthesis and cell growth. In high-titer hosts, both bacteriocytes and symbionts show elevated expression of genes underlying energy metabolism. Several eukaryotic cell signaling pathways are differentially expressed in bacteriocytes of low- versus high-titer hosts: Cell-growth pathways are up-regulated in low-titer genotypes, while membrane trafficking, lysosomal processes, and mechanistic target of rapamycin (mTOR) and cytokine pathways are up-regulated in high-titer genotypes. Specific Buchnera functions are up-regulated within different bacteriocyte environments, with genes underlying flagellar body secretion and flagellar assembly overexpressed in low- and high-titer hosts, respectively. Overall, our results reveal allowances and demands made by both host and symbiont engaged in a metabolic "tug-of-war."
Project description:BACKGROUND:Obligate intracellular symbionts of insects are metabolically and developmentally integrated with their hosts. Typically, reproduction fails in many insect nutritional endosymbioses when host insects are cured of their bacterial symbionts, and yet remarkably little is known about the processes that developmentally integrate host and symbiont. Here in the best studied insect obligate intracellular symbiosis, that of the pea aphid, Acyrthosiphon pisum, with the gammaproteobacterium Buchnera aphidicola, we tracked the expression and localization of amino acid transporter ApGLNT1 gene products during asexual embryogenesis. Recently being characterized as a glutamine transporter, ApGLNT1 has been proposed to be a key regulator of amino acid biosynthesis in A. pisum bacteriocytes. To determine when this important mediator of the symbiosis becomes expressed in aphid embryonic bacteriocytes, we applied whole-mount in situ hybridization and fluorescent immunostaining with a specific anti-ApGLNT1 antibody to detect the temporal and spatial expression of ApGLNT1 gene products during asexual embryogenesis. RESULTS:During embryogenesis, ApGLNT1 mRNA and protein localize to the follicular epithelium that surrounds parthenogenetic viviparous embryos, where we speculate that it functions to supply developing embryos with glutamine from maternal hemolymph. Unexpectedly, in the embryonic bacteriome ApGLNT1 protein does not localize to the membrane of bacteriocytes, a pattern that leads us to conclude that the regulation of amino acid metabolism in the embryonic bacteriome mechanistically differs from that in the maternal bacteriome. Paralleling our earlier report of punctate cytoplasmic localization of ApGLNT1 in maternal bacteriocytes, we find ApGLNT1 protein localizing as cytoplasmic puncta throughout development in association with Buchnera. CONCLUSIONS:Our work that documents ontogenetic shifts in the localization of ApGLNT1 protein in the host bacteriome demonstrates that maternal and embryonic bacteriomes are not equivalent. Significantly, the persistent punctate cytoplasmic localization of ApGLNT1 in association with Buchnera in embryos prior to bacteriocyte formation and later in both embryonic and maternal bacteriomes suggests that ApGLNT1 plays multiple roles in this symbiosis, roles that include amino acid transport and possibly nutrient sensing.
Project description:Bacteriocytes set the stage for some of the most intimate interactions between animal and bacterial cells. In all bacteriocyte possessing systems studied so far, de novo formation of bacteriocytes occurs only once in the host development, at the time of symbiosis establishment. Here, we present the free-living symbiotic flatworm Paracatenula galateia and its intracellular, sulfur-oxidizing bacteria as a system with previously undescribed strategies of bacteriocyte formation and bacterial symbiont transmission. Using thymidine analogue S-phase labeling and immunohistochemistry, we show that all somatic cells in adult worms - including bacteriocytes - originate exclusively from aposymbiotic stem cells (neoblasts). The continued bacteriocyte formation from aposymbiotic stem cells in adult animals represents a previously undescribed strategy of symbiosis maintenance and makes P. galateia a unique system to study bacteriocyte differentiation and development. We also provide morphological and immunohistochemical evidence that P. galateia reproduces by asexual fragmentation and regeneration (paratomy) and, thereby, vertically transmits numerous symbiont-containing bacteriocytes to its asexual progeny. Our data support the earlier reported hypothesis that the symbiont population is subjected to reduced bottleneck effects. This would justify both the codiversification between Paracatenula hosts and their Candidatus Riegeria symbionts, and the slow evolutionary rates observed for several symbiont genes.
Project description:Symbiosis often entails the emergence of novel adaptive traits in organisms. Microbial symbionts are indispensable for diverse insects via provisioning of essential nutrients, wherein novel host cells and organs for harboring the microbes, called bacteriocytes and bacteriomes, have evolved repeatedly. Molecular and developmental mechanisms underpinning the emergence of novel symbiotic cells and organs comprise an unsolved question in evolutionary developmental biology. Here, we report that a conserved homeotic gene, Ultrabithorax, plays a pivotal role in the bacteriocyte differentiation in a hemipteran insect Nysius plebeius. During embryonic development, six pairs of aggregated presumptive bacteriocytes appear on both sides of six abdominal segments, incorporate the symbiotic bacteria at the stage of germband retraction, and fuse into a pair of lateral bacteriomes at the stage of germband flip, where bacteriocyte-associated Ultrabithorax expression coincides with the symbiont infection process. Suppression of Ultrabithorax expression by maternal RNA interference results in disappearance of the bacteriocytes and the symbiont localization therein, suggesting that Ultrabithorax is involved in differentiation of the host cells for symbiosis. Suppression of other homeotic genes abdominal-A and Antennapedia disturbs integrity and positioning of the bacteriomes, affecting the configuration of the host organs for symbiosis. Our findings unveil the molecular and developmental mechanisms underlying the bacteriocyte differentiation, which may have evolved either via cooption of the transcription factors for inducing the novel symbiotic cells, or via revival of the developmental pathway for the bacteriocytes that had existed in the ancestral hemipterans.
Project description:Aphids possess bacteriocytes, cells specifically differentiated to harbor obligatory mutualistic bacteria of the genus Buchnera, which have lost many genes that are essential for common bacterial functions. To understand the host's role in maintaining the symbiotic relationship, bacteriocytes were isolated from the pea aphid, Acyrthosiphon pisum, and the host transcriptome was investigated by using EST analysis and real-time quantitative RT-PCR. A number of genes were highly expressed specifically in the bacteriocyte, including (i) genes for amino acid metabolism, including those for biosynthesis of amino acids that Buchnera cannot produce, and those for utilization of amino acids that Buchnera can synthesize; (ii) genes related to transport, including genes for mitochondrial transporters and a gene encoding Rab, a G protein that regulates vesicular transport; and (iii) genes for putative lysozymes that degrade bacterial cell walls. Significant up-regulation of i clearly indicated that the bacteriocyte is involved in the exchange of amino acids between the host aphid and Buchnera, the key metabolic process in the symbiotic system. Conspicuously high expression of ii and iii shed light on previously unknown aspects of the host-Buchnera interactions in the symbiotic system.
Project description:Whiteflies possess bacterial symbionts Candidatus Portiera aleyrodidium that are housed in specialized cells called bacteriocytes and are faithfully transmitted via the ovary to insect offspring. In one whitefly species studied previously, Bemisia tabaci MEAM1, transmission is mediated by somatic inheritance of bacteriocytes, with a single bacteriocyte transferred to each oocyte and persisting through embryogenesis to the next generation. Here, we investigate the mode of bacteriocyte transmission in two whitefly species, B. tabaci MED, the sister species of MEAM1, and the phylogenetically distant species Trialeurodes vaporariorum. Microsatellite analysis supported by microscopical studies demonstrates that B. tabaci MED bacteriocytes are genetically different from other somatic cells and persist through embryogenesis, as for MEAM1, but T. vaporariorum bacteriocytes are genetically identical to other somatic cells of the insect, likely mediated by the degradation of maternal bacteriocytes in the embryo. These two alternative modes of transmission provide a first demonstration among insect symbioses that the cellular processes underlying vertical transmission of bacterial symbionts can diversify among related host species associated with a single lineage of symbiotic bacteria.
Project description:Endosymbiotic associations constitute a driving force in the ecological and evolutionary diversification of metazoan organisms. Little is known about whether and how symbiotic cells are coordinated according to host physiology. Here, we use the nutritional symbiosis between the insect pest, Acyrthosiphon pisum, and its obligate symbiont, Buchnera aphidicola, as a model system. We have developed a novel approach for unculturable bacteria, based on flow cytometry, and used this method to estimate the absolute numbers of symbionts at key stages of aphid life. The endosymbiont population increases exponentially throughout nymphal development, showing a growing rate which has never been characterized by indirect molecular techniques. Using histology and imaging techniques, we have shown that the endosymbiont-bearing cells (bacteriocytes) increase significantly in number and size during the nymphal development, and clustering in the insect abdomen. Once adulthood is reached and the laying period has begun, the dynamics of symbiont and host cells is reversed: the number of endosymbionts decreases progressively and the bacteriocyte structure degenerates during insect aging. In summary, these results show a coordination of the cellular dynamics between bacteriocytes and primary symbionts and reveal a fine-tuning of aphid symbiotic cells to the nutritional demand imposed by the host physiology throughout development.
Project description:Endosymbiotic associations have played a major role in evolution. However, the molecular basis for the biochemical interdependence of these associations remains poorly understood. The aphid-Buchnera endosymbiosis provides a powerful system to elucidate how these symbioses are regulated. In aphids, the supply of essential amino acids depends on an ancient nutritional symbiotic association with the gamma-proteobacterium Buchnera aphidicola. Buchnera cells are densely packed in specialized aphid bacteriocyte cells. Here we confirm that five putative amino acid transporters are highly expressed and/or highly enriched in Acyrthosiphon pisum bacteriocyte tissues. When expressed in Xenopus laevis oocytes, two bacteriocyte amino acid transporters displayed significant levels of glutamine uptake, with transporter ACYPI001018, LOC100159667 (named here as Acyrthosiphon pisum glutamine transporter 1, ApGLNT1) functioning as the most active glutamine transporter. Transporter ApGLNT1 has narrow substrate selectivity, with high glutamine and low arginine transport capacity. Notably, ApGLNT1 has high binding affinity for arginine, and arginine acts as a competitive inhibitor for glutamine transport. Using immunocytochemistry, we show that ApGLNT1 is localized predominantly to the bacteriocyte plasma membrane, a location consistent with the transport of glutamine from A. pisum hemolymph to the bacteriocyte cytoplasm. On the basis of functional transport data and localization, we propose a substrate feedback inhibition model in which the accumulation of the essential amino acid arginine in A. pisum hemolymph reduces the transport of the precursor glutamine into bacteriocytes, thereby regulating amino acid biosynthesis in the bacteriocyte. Structural similarities in the arrangement of hosts and symbionts across endosymbiotic systems suggest that substrate feedback inhibition may be mechanistically important in other endosymbioses.
Project description:Bacteriocytes are insect cells harboring symbiotic bacteria that are required by their insect host and are transmitted vertically via the female ovary . In most insect groups, the bacteria are released from the bacteriocytes and transferred to the ovary [2, 3], but in whiteflies, maternal bacteriocytes migrate to each egg [4-6], where they have been reported to lyse, releasing the symbionts . To investigate bacteriocyte inheritance in whiteflies further, we applied microsatellite genotyping and genomic analysis to a genetically diverse population of Bemisia tabaci, and we observed the fate of the bacteriocyte in embryos. Surprisingly, the microsatellite profile of the bacteriocytes was uniform, and insect cross experiments demonstrated that the bacteriocytes have a stable genotype that differs from the genotype of the insect head (which lacks bacteriocytes). Comparative genomic analysis indicates that genomes of the bacteriocyte and whitefly head are distinct. Interestingly, the bacterioyte genome contains the canonical arthropod telomere repeats TTAGG, and the bacteriocytes express telomere maintenance genes that may underlie cellular immortality in animal cells . Microscopy observations confirmed that a single bacteriocyte transmitted to each egg is retained and divides once just before egg hatch, yielding two bacteriocytes in the neonate insect. These data demonstrate the maternal inheritance of an absolutely required somatic insect cell, violating the developmental separation of germline and soma [8, 9]. Future investigation on the mechanism and phylogenetic distribution of maternally inherited bacteriocytes will shed light on the developmental origins and evolutionary diversification of bacteriocytes  and the processes underlying cellular immortality .