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.

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.

Alternative splicing of CNOT7 diversifies CCR4-NOT functions.

The CCR4-associated factor CAF1, also called CNOT7, is a catalytic subunit of the CCR4-NOT complex, which has been implicated in all aspects of the mRNA life cycle, from mRNA synthesis in the nucleus to degradation in the cytoplasm. In human cells, alternative splicing of the CNOT7 gene yields a second CNOT7 transcript leading to the formation of a shorter protein, CNOT7 variant 2 (CNOT7v2). Biochemical characterization indicates that CNOT7v2 interacts with CCR4-NOT subunits, although it does not bind to BTG proteins. We report that CNOT7v2 displays a distinct expression profile in human tissues, as well as a nuclear sub-cellular localization compared to CNOT7v1. Despite a conserved DEDD nuclease domain, CNOT7v2 is unable to degrade a poly(A) tail in vitro and preferentially associates with the protein arginine methyltransferase PRMT1 to regulate its activity. Using both in vitro and in cellulo systems, we have also demonstrated that CNOT7v2 regulates the inclusion of CD44 variable exons. Altogether, our findings suggest a preferential involvement of CNOT7v2 in nuclear processes, such as arginine methylation and alternative splicing, rather than mRNA turnover. These observations illustrate how the integration of a splicing variant inside CCR4-NOT can diversify its cell- and tissue-specific functions.

A specific anti-citrullinated protein antibody profile identifies a group of rheumatoid arthritis patients with a toll-like receptor 4-mediated disease.

BACKGROUND: Increased expression of toll-like receptor 4 (TLR4) and its endogenous ligands, is characteristic of rheumatoid arthritis (RA) synovitis. In this study, we evaluated how these TLR4 ligands may drive pathogenic processes and whether the fine profiling of anti-citrullinated protein antibodies (ACPA) based on their target specificity might provide a simple means to predict therapeutic benefit when neutralizing TLR4 in this disease. METHODS: The capacity of RA synovial fluids (RASF) to stimulate cytokine production in monocytes from patients with RA was analyzed by ELISA. The presence of TLR4 activators in RASF was determined by measuring the levels of ACPA, ACPA subtypes with reactivity to specific citrullinated peptides and other TLR4 ligands. Neutralization of TLR4 signaling was investigated using NI-0101, a therapeutic antibody that targets TLR4. RESULTS: RASF exhibited a heterogeneous capacity to induce production of proinflammatory cytokines by monocytes isolated from patients with RA. Such cytokine responses were significantly modified by TLR4 blockade achieved using NI-0101. The analysis of the content of RASF and matched sera demonstrated that ACPA fine specificities in patient samples predict cellular response to anti-TLR4 exposure in vitro. CONCLUSION: TLR4 represents a possible therapeutic target in RA. Our study demonstrates that TLR4 inhibition in an ex vivo model of RA pathogenesis can significantly modulate cytokine release and does so in specific subgroups of RA patient-derived samples. It also suggests that ACPA fine profiling has the potential to identify RA patients with a predominantly TLR4-driven pathotype that could be used to predict preferential response to TLR4 antagonism.

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.

Monochromatic aberrations as a function of age, from childhood to advanced age.

PURPOSE: To describe monochromatic optical aberrations of the eye as a function of age. METHODS: One hundred fourteen subjects with a spherical equivalent within +/-3.50 D from emmetropia, corrected visual acuity of 20/40 or better, and normal findings in an ophthalmic examination were enrolled. The mean age was 43.2 +/- 24.5 years (range, 5.7-82.3). Monochromatic optical aberrations were measured with a Hartmann-Shack wavefront sensor after pharmacological dilation and cycloplegia. RESULTS: For a 5-mm pupil and for third- to seventh-, third-, fourth-, and fifth- to seventh-order aberrations, as well as for coma and spherical aberrations, the root mean square (RMS) error as a function of age was modeled by a second-order polynomial regression. It decreased progressively through childhood, adolescence, and early adulthood; reached a minimum level during the fourth decade of life, then increased progressively with age, to age 82. For a 5-mm pupil, the mean modulation transfer function (MTF) was reduced in both the child-teenage (5-20 years; n = 29) and the elderly (61-82 years; n = 37) groups versus the middle-aged adult group (41-60 years; n = 24; P < 0.05). In young adults (21-40 years; n = 23) and elderly subjects, the MTF curves were very close and almost superimposed at spatial frequencies higher than 38 cyc/deg. CONCLUSIONS: Aberrations of the whole eye were objectively measured from early childhood to an advanced age, and the relationship between monochromatic aberrations and age has been shown to fit a quadratic model. The results suggest that the definition of emmetropization should be broadened to include the reduction of higher order aberrations.

Objective measurement of optical aberrations in myopic eyes.

PURPOSE: To determine whether the degree of myopia affects the optical quality of the retinal image after appropriate correction, monochromatic aberrations of the human eye were measured as a function of the degree of myopia. METHODS: Using a modified Hartmann-Shack method, a population of 27 myopic (up to -9.25 D) and 7 emmetropic optometry students (18-32 years) were objectively evaluated before and after pupil dilation. We then identified for each subject the most influential aberration types, and we calculated the profile of wavefront aberration. To study the behavior of aberration as a function of the degree of myopia, we determined the maximum value of aberration as well as the root mean square (rms) value. RESULTS: It turned out that aberration increases with the refractive error in a quasi-linear relationship for pupil diameters of 5 and 9 mm. This result is valid for both rms and maximum values. CONCLUSIONS: We objectively showed that optical quality decreases as myopia increases and as the pupil gets larger. Coma is more frequent in high myopia, and spherical aberration occurs more frequently for dilated pupils.