Discovery of an Insect Neuroactive Helix Ring Peptide from Ant Venom.

Ants are among the most abundant terrestrial invertebrate predators on Earth. To overwhelm their prey, they employ several remarkable behavioral, physiological, and biochemical innovations, including an effective paralytic venom. Ant venoms are thus cocktails of toxins finely tuned to disrupt the physiological systems of insect prey. They have received little attention yet hold great promise for the discovery of novel insecticidal molecules. To identify insect-neurotoxins from ant venoms, we screened the paralytic activity on blowflies of nine synthetic peptides previously characterized in the venom of Tetramorium bicarinatum. We selected peptide U11, a 34-amino acid peptide, for further insecticidal, structural, and pharmacological experiments. Insecticidal assays revealed that U11 is one of the most paralytic peptides ever reported from ant venoms against blowflies and is also capable of paralyzing honeybees. An NMR spectroscopy of U11 uncovered a unique scaffold, featuring a compact triangular ring helix structure stabilized by a single disulfide bond. Pharmacological assays using Drosophila S2 cells demonstrated that U11 is not cytotoxic, but suggest that it may modulate potassium conductance, which structural data seem to corroborate and will be confirmed in a future extended pharmacological investigation. The results described in this paper demonstrate that ant venom is a promising reservoir for the discovery of neuroactive insecticidal peptides.

Bacteriocyte plasticity in pea aphids facing amino acid stress or starvation during development.

An important contributing factor to the evolutionary success of insects is nutritional association with microbial symbionts, which provide the host insects with nutrients lacking in their unbalanced diets. These symbionts are often compartmentalized in specialized cells of the host, the bacteriocytes. Even though bacteriocytes were first described more than a century ago, few studies have explored their dynamics throughout the insect life cycle and in response to environmental stressors. Here, we use the Buchnera aphidicola/pea aphid symbiotic system to study how bacteriocytes are regulated in response to nutritional stress throughout aphid development. Using artificial diets, we analyzed the effects of depletion or excess of phenylalanine or leucine, two amino acids essential for aphid growth and whose biosynthetic pathways are shared between the host and the symbiont. Bacteriocytes responded dynamically to those treatments, while other tissues showed no obvious morphological change. Amino acid depletion resulted in an increase in bacteriocyte numbers, with the extent of the increase depending on the amino acid, while excess either caused a decrease (for leucine) or an increase (for phenylalanine). Only a limited impact on survival and fecundity was observed, suggesting that the adjustment in bacteriocyte (and symbiont) numbers is sufficient to withstand these nutritional challenges. We also studied the impact of more extreme conditions by exposing aphids to a 24 h starvation period at the beginning of nymphal development. This led to a dramatic drop in aphid survival and fecundity and a significant developmental delay. Again, bacteriocytes responded dynamically, with a considerable decrease in number and size, correlated with a decrease in the number of symbionts, which were prematurely degraded by the lysosomal system. This study shows how bacteriocyte dynamics is integrated in the physiology of insects and highlights the high plasticity of these cells.

Aphid BCR4 Structure and Activity Uncover a New Defensin Peptide Superfamily.

Aphids (Hemiptera: Aphidoidea) are among the most detrimental insects for agricultural plants, and their management is a great challenge in agronomical research. A new class of proteins, called Bacteriocyte-specific Cysteine-Rich (BCR) peptides, provides an alternative to chemical insecticides for pest control. BCRs were initially identified in the pea aphid Acyrthosiphon pisum. They are small disulfide bond-rich proteins expressed exclusively in aphid bacteriocytes, the insect cells that host intracellular symbiotic bacteria. Here, we show that one of the A. pisum BCRs, BCR4, displays prominent insecticidal activity against the pea aphid, impairing insect survival and nymphal growth, providing evidence for its potential use as a new biopesticide. Our comparative genomics and phylogenetic analyses indicate that BCRs are restricted to the aphid lineage. The 3D structure of BCR4 reveals that this peptide belongs to an as-yet-unknown structural class of peptides and defines a new superfamily of defensins.

Compartmentalized into Bacteriocytes but Highly Invasive: the Puzzling Case of the Co-Obligate Symbiont Serratia symbiotica in the Aphid Periphyllus lyropictus.

Dependence on multiple nutritional symbionts that form a metabolic unit has evolved many times in insects. Although it has been postulated that host dependence on these metabolically interconnected symbionts is sustained by their high degree of anatomical integration (these symbionts are often housed in distinct symbiotic cells, the bacteriocytes, assembled into a common symbiotic organ, the bacteriome), the developmental aspects of such multipartner systems have received little attention. Aphids of the subfamilies Chaitophorinae and Lachninae typically harbor disymbiotic systems in which the metabolic capabilities of the ancient obligate symbiont Buchnera aphidicola are complemented by those of a more recently acquired nutritional symbiont, often belonging to the species Serratia symbiotica. Here, we used microscopy approaches to finely characterize the tissue tropism and infection dynamics of the disymbiotic system formed by B. aphidicola and S. symbiotica in the Norway maple aphid Periphyllus lyropictus (Chaitophorinae). Our observations show that, in this aphid, the co-obligate symbiont S. symbiotica exhibits a dual lifestyle: intracellular by being housed in large syncytial bacteriocytes embedded between B. aphidicola-containing bacteriocytes in a well-organized compartmentalization pattern, and extracellular by massively invading the digestive tract and other tissues during embryogenesis. This is the first reported case of an obligate aphid symbiont that is internalized in bacteriocytes but simultaneously adopts an extracellular lifestyle. This unusual infection pattern for an obligate insect symbiont suggests that some bacteriocyte-associated obligate symbionts, despite their integration into a cooperative partnership, still exhibit invasive behavior and escape strict compartmentalization in bacteriocytes. IMPORTANCE Multipartner nutritional endosymbioses have evolved many times in insects. In Chaitophorinae aphids, the eroded metabolic capabilities of the ancient obligate symbiont B. aphidicola are complemented by those of more recently acquired symbionts. Here, we report the atypical case of the co-obligate S. symbiotica symbiont associated with P. lyropictus. This bacterium is compartmentalized into bacteriocytes nested into the ones harboring the more ancient symbiont B. aphidicola, reflecting metabolic convergences between the two symbionts. At the same time, S. symbiotica exhibits highly invasive behavior by colonizing various host tissues, including the digestive tract during embryogenesis. The discovery of this unusual phenotype for a co-obligate symbiont reveals a new face of multipartner nutritional endosymbiosis in insects. In particular, it shows that co-obligate symbionts can retain highly invasive traits and suggests that host dependence on these bacterial partners may evolve prior to their strict compartmentalization into specialized host structures.

Isolation of Insect Bacteriocytes as a Platform for Transcriptomic Analyses.

Over the past few decades, various techniques have been developed and optimized for the accurate measurement of RNA abundance in cells or tissues. These methods have been instrumental in gaining insight in complex systems such as host-symbiont associations. The pea aphid model has recently emerged as a powerful and experimentally tractable system for studying symbiotic relationships and it is the subject of a growing number of molecular studies. Nevertheless, the lack of standardized protocols for the collection of bacteriocytes, the specialized host cells harboring the symbionts, has limited its use. This chapter provides a simple, step-by-step dissection protocol for the rapid isolation of aphid bacteriocytes. We then describe an adapted protocol for efficient extraction and purification of bacteriocyte RNA that can be used for most downstream transcriptomic analyses.

Evolutionary novelty in the apoptotic pathway of aphids.

Apoptosis, a conserved form of programmed cell death, shows interspecies differences that may reflect evolutionary diversification and adaptation, a notion that remains largely untested. Among insects, the most speciose animal group, the apoptotic pathway has only been fully characterized in Drosophila melanogaster, and apoptosis-related proteins have been studied in a few other dipteran and lepidopteran species. Here, we studied the apoptotic pathway in the aphid Acyrthosiphon pisum, an insect pest belonging to the Hemiptera, an earlier-diverging and distantly related order. We combined phylogenetic analyses and conserved domain identification to annotate the apoptotic pathway in A. pisum and found low caspase diversity and a large expansion of its inhibitory part, with 28 inhibitors of apoptosis (IAPs). We analyzed the spatiotemporal expression of a selected set of pea aphid IAPs and showed that they are differentially expressed in different life stages and tissues, suggesting functional diversification. Five IAPs are specifically induced in bacteriocytes, the specialized cells housing symbiotic bacteria, during their cell death. We demonstrated the antiapoptotic role of these five IAPs using heterologous expression in a tractable in vivo model, the Drosophila melanogaster developing eye. Interestingly, IAPs with the strongest antiapoptotic potential contain two BIR and two RING domains, a domain association that has not been observed in any other species. We finally analyzed all available aphid genomes and found that they all show large IAP expansion, with new combinations of protein domains, suggestive of evolutionarily novel aphid-specific functions.

Bis(monoacylglycero)phosphate regulates oxysterol binding protein-related protein 11 dependent sterol trafficking.

Bis(Monoacylglycero) Phosphate (BMP) is a unique phospholipid localized in late endosomes, a critical cellular compartment in low density lipoprotein (LDL)-cholesterol metabolism. In previous work, we demonstrated the important role of BMP in the regulation of macrophage cholesterol homeostasis. BMP exerts a protective role against the pro-apoptotic effect of oxidized LDL (oxLDL) by reducing the production of deleterious oxysterols. As the intracellular sterol traffic in macrophages is in part regulated by oxysterol binding protein (OSBP) and OSBP-related proteins (ORPs), we investigated the role of ORP11, localized at the Golgi-late endosomes interface, in the BMP-mediated protection from oxLDL/oxysterol cytotoxicity. Stably silencing of ORP11 in mouse RAW264.7 macrophages via a shRNA lentiviruses system had no effect on BMP production. However, ORP11 knockdown abrogated the protective action of BMP against oxLDL induced apoptosis. In oxLDL treated control cells, BMP enrichment was associated with reduced generation of 7-oxysterols, while these oxysterol species were abundant in the ORP11 knock-down cells. Of note, BMP enrichment in ORP11 knock-down cells was associated with a drastic increase in free cholesterol and linked to a decrease of cholesterol efflux. The expression of ATP-binding cassette-transporter G1 (ABCG1) was also reduced in the ORP11 knock-down cells. These observations demonstrate a cooperative function of OPR11 and BMP, in intracellular cholesterol trafficking in cultured macrophages. We suggest that BMP favors the egress of cholesterol from late endosomes via an ORP11-dependent mechanism, resulting in a reduced production of cytotoxic 7-oxysterols.

Bacteriocyte Reprogramming to Cope With Nutritional Stress in a Phloem Sap Feeding Hemipteran, the Pea Aphid Acyrthosiphon pisum.

Nutritional symbioses play a central role in the ability of insects to thrive on unbalanced diets and in ensuring their evolutionary success. A genomic model for nutritional symbiosis comprises the hemipteran Acyrthosiphon pisum, and the gamma-3-proteobacterium, Buchnera aphidicola, with genomes encoding highly integrated metabolic pathways. A. pisum feeds exclusively on plant phloem sap, a nutritionally unbalanced diet highly variable in composition, thus raising the question of how this symbiotic system responds to nutritional stress. We addressed this by combining transcriptomic, phenotypic and life history trait analyses to determine the organismal impact of deprivation of tyrosine and phenylalanine. These two aromatic amino acids are essential for aphid development, are synthesized in a metabolic pathway for which the aphid host and the endosymbiont are interdependent, and their concentration can be highly variable in plant phloem sap. We found that this nutritional challenge does not have major phenotypic effects on the pea aphid, except for a limited weight reduction and a 2-day delay in onset of nymph laying. Transcriptomic analyses through aphid development showed a prominent response in bacteriocytes (the core symbiotic tissue which houses the symbionts), but not in gut, thus highlighting the role of bacteriocytes as major modulators of this homeostasis. This response does not involve a direct regulation of tyrosine and phenylalanine biosynthetic pathway and transporter genes. Instead, we observed an extensive transcriptional reprogramming of the bacteriocyte with a rapid down-regulation of genes encoding sugar transporters and genes required for sugar metabolism. Consistently, we observed continued overexpression of the A. pisum homolog of RRAD, a small GTPase implicated in repressing aerobic glycolysis. In addition, we found increased transcription of genes involved in proliferation, cell size control and signaling. We experimentally confirmed the significance of these gene expression changes detecting an increase in bacteriocyte number and cell size in vivo under tyrosine and phenylalanine depletion. Our results support a central role of bacteriocytes in the aphid response to amino acid deprivation: their transcriptional and cellular responses fine-tune host physiology providing the host insect with an effective way to cope with the challenges posed by the variability in composition of phloem sap.

Bacteriocyte cell death in the pea aphid/Buchnera symbiotic system.

Symbiotic associations play a pivotal role in multicellular life by facilitating acquisition of new traits and expanding the ecological capabilities of organisms. In insects that are obligatorily dependent on intracellular bacterial symbionts, novel host cells (bacteriocytes) or organs (bacteriomes) have evolved for harboring beneficial microbial partners. The processes regulating the cellular life cycle of these endosymbiont-bearing cells, such as the cell-death mechanisms controlling their fate and elimination in response to host physiology, are fundamental questions in the biology of symbiosis. Here we report the discovery of a cell-death process involved in the degeneration of bacteriocytes in the hemipteran insect Acyrthosiphon pisum This process is activated progressively throughout aphid adulthood and exhibits morphological features distinct from known cell-death pathways. By combining electron microscopy, immunohistochemistry, and molecular analyses, we demonstrated that the initial event of bacteriocyte cell death is the cytoplasmic accumulation of nonautophagic vacuoles, followed by a sequence of cellular stress responses including the formation of autophagosomes in intervacuolar spaces, activation of reactive oxygen species, and Buchnera endosymbiont degradation by the lysosomal system. We showed that this multistep cell-death process originates from the endoplasmic reticulum, an organelle exhibiting a unique reticular network organization spread throughout the entire cytoplasm and surrounding Buchnera aphidicola endosymbionts. Our findings provide insights into the cellular and molecular processes that coordinate eukaryotic host and endosymbiont homeostasis and death in a symbiotic system and shed light on previously unknown aspects of bacteriocyte biological functioning.

Disruption of phenylalanine hydroxylase reduces adult lifespan and fecundity, and impairs embryonic development in parthenogenetic pea aphids.

Phenylalanine hydroxylase (PAH) is a key tyrosine-biosynthetic enzyme involved in neurological and melanin-associated physiological processes. Despite extensive investigations in holometabolous insects, a PAH contribution to insect embryonic development has never been demonstrated. Here, we have characterized, for the first time, the PAH gene in a hemimetabolous insect, the aphid Acyrthosiphon pisum. Phylogenetic and sequence analyses confirmed that ApPAH is closely related to metazoan PAH, exhibiting the typical ACT regulatory and catalytic domains. Temporal expression patterns suggest that ApPAH has an important role in aphid developmental physiology, its mRNA levels peaking at the end of embryonic development. We used parental dsApPAH treatment to generate successful knockdown in aphid embryos and to study its developmental role. ApPAH inactivation shortens the adult aphid lifespan and considerably affects fecundity by diminishing the number of nymphs laid and impairing embryonic development, with newborn nymphs exhibiting severe morphological defects. Using single nymph HPLC analyses, we demonstrated a significant tyrosine deficiency and a consistent accumulation of the upstream tyrosine precursor, phenylalanine, in defective nymphs, thus confirming the RNAi-mediated disruption of PAH activity. This study provides first insights into the role of PAH in hemimetabolous insects and demonstrates that this metabolic gene is essential for insect embryonic development.

Direct flow cytometry measurements reveal a fine-tuning of symbiotic cell dynamics according to the host developmental needs in aphid symbiosis.

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.

New insight into the RNA interference response against cathepsin-L gene in the pea aphid, Acyrthosiphon pisum: molting or gut phenotypes specifically induced by injection or feeding treatments.

RNA interference (RNAi) has been widely and successfully used for gene inactivation in insects, including aphids, where dsRNA administration can be performed either by feeding or microinjection. However, several aspects related to the aphid response to RNAi, as well as the influence of the administration method on tissue response, or the mixed success to observe phenotypes specific to the gene targeted, are still unclear in this insect group. In the present study, we made the first direct comparison of two administration methods (injection or feeding) for delivery of dsRNA targeting the cathepsin-L gene in the pea aphid, Acyrthosiphon pisum. In order to maximize the possibility of discovering specific phenotypes, the effect of the treatment was analyzed in single individual aphids at the level of five body compartments: the bacteriocytes, the gut, the embryonic chains, the head and the remaining body carcass. Our analysis revealed that gene expression knockdown effect in each single body compartment was dependent on the administration method used, and allowed us to discover new functions for the cathepsin-L gene in aphids. Injection of cathepsin-L dsRNA was much more effective on carcass and head, inducing body morphology alterations, and suggesting a novel role of this gene in the molting of these insects. Administration by feeding provoked cathepsin-L knockdown in the gut and specific gut epithelial cell alteration, therefore allowing a better characterization of tissue specific role of this gene in aphids.

Tyrosine pathway regulation is host-mediated in the pea aphid symbiosis during late embryonic and early larval development.

BACKGROUND: Nutritional symbioses play a central role in insects' adaptation to specialized diets and in their evolutionary success. The obligatory symbiosis between the pea aphid, Acyrthosiphon pisum, and the bacterium, Buchnera aphidicola, is no exception as it enables this important agricultural pest insect to develop on a diet exclusively based on plant phloem sap. The symbiotic bacteria provide the host with essential amino acids lacking in its diet but necessary for the rapid embryonic growth seen in the parthenogenetic viviparous reproduction of aphids. The aphid furnishes, in exchange, non-essential amino acids and other important metabolites. Understanding the regulations acting on this integrated metabolic system during the development of this insect is essential in elucidating aphid biology. RESULTS: We used a microarray-based approach to analyse gene expression in the late embryonic and the early larval stages of the pea aphid, characterizing, for the first time, the transcriptional profiles in these developmental phases. Our analyses allowed us to identify key genes in the phenylalanine, tyrosine and dopamine pathways and we identified ACYPI004243, one of the four genes encoding for the aspartate transaminase (E.C. 2.6.1.1), as specifically regulated during development. Indeed, the tyrosine biosynthetic pathway is crucial for the symbiotic metabolism as it is shared between the two partners, all the precursors being produced by B. aphidicola. Our microarray data are supported by HPLC amino acid analyses demonstrating an accumulation of tyrosine at the same developmental stages, with an up-regulation of the tyrosine biosynthetic genes. Tyrosine is also essential for the synthesis of cuticular proteins and it is an important precursor for cuticle maturation: together with the up-regulation of tyrosine biosynthesis, we observed an up-regulation of cuticular genes expression. We were also able to identify some amino acid transporter genes which are essential for the switch over to the late embryonic stages in pea aphid development. CONCLUSIONS: Our data show that, in the development of A. pisum, a specific host gene set regulates the biosynthetic pathways of amino acids, demonstrating how the regulation of gene expression enables an insect to control the production of metabolites crucial for its own development and symbiotic metabolism.

OPA1 links human mitochondrial genome maintenance to mtDNA replication and distribution.

Eukaryotic cells harbor a small multiploid mitochondrial genome, organized in nucleoids spread within the mitochondrial network. Maintenance and distribution of mitochondrial DNA (mtDNA) are essential for energy metabolism, mitochondrial lineage in primordial germ cells, and to prevent mtDNA instability, which leads to many debilitating human diseases. Mounting evidence suggests that the actors of the mitochondrial network dynamics, among which is the intramitochondrial dynamin OPA1, might be involved in these processes. Here, using siRNAs specific to OPA1 alternate spliced exons, we evidenced that silencing of the OPA1 variants including exon 4b leads to mtDNA depletion, secondary to inhibition of mtDNA replication, and to marked alteration of mtDNA distribution in nucleoid and nucleoid distribution throughout the mitochondrial network. We demonstrate that a small hydrophobic 10-kDa peptide generated by cleavage of the OPA1-exon4b isoform is responsible for this process and show that this peptide is embedded in the inner membrane and colocalizes and coimmunoprecipitates with nucleoid components. We propose a novel synthetic model in which a peptide, including two trans-membrane domains derived from the N terminus of the OPA1-exon4b isoform in vertebrates or from its ortholog in lower eukaryotes, might contribute to nucleoid attachment to the inner mitochondrial membrane and promotes mtDNA replication and distribution. Thus, this study places OPA1 as a direct actor in the maintenance of mitochondrial genome integrity.

Structure and dynamics of the operon map of Buchnera aphidicola sp. strain APS.

BACKGROUND: Gene expression regulation is still poorly documented in bacteria with highly reduced genomes. Understanding the evolution and mechanisms underlying the regulation of gene transcription in Buchnera aphidicola, the primary endosymbiont of aphids, is expected both to enhance our understanding of this nutritionally based association and to provide an intriguing case-study of the evolution of gene expression regulation in a reduced bacterial genome. RESULTS: A Bayesian predictor was defined to infer the B. aphidicola transcription units, which were further validated using transcriptomic data and RT-PCR experiments. The characteristics of B. aphidicola predicted transcription units (TUs) were analyzed in order to evaluate the impact of operon map organization on the regulation of gene transcription.On average, B. aphidicola TUs contain more genes than those of E. coli. The global layout of B. aphidicola operon map was mainly shaped by the big reduction and the rearrangements events, which occurred at the early stage of the symbiosis. Our analysis suggests that this operon map may evolve further only by small reorganizations around the frontiers of B. aphidicola TUs, through promoter and/or terminator sequence modifications and/or by pseudogenization events. We also found that the need for specific transcription regulation exerts some pressure on gene conservation, but not on gene assembling in the operon map in Buchnera. Our analysis of the TUs spacing pointed out that a selection pressure is maintained on the length of the intergenic regions between divergent adjacent gene pairs. CONCLUSIONS: B. aphidicola can seemingly only evolve towards a more polycistronic operon map. This implies that gene transcription regulation is probably subject to weak selection pressure in Buchnera conserving operons composed of genes with unrelated functions.