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Mutualism is defined as a beneficial relationship for the associated partners and usually assumes that the symbiont number is controlled. Some trypanosomatid protozoa co-evolve with a bacterial symbiont that divides in coordination with the host in a way that results in its equal distribution between daughter cells. The mechanism that controls this synchrony is largely unknown, and its comprehension might provide clues to understand how eukaryotic cells evolved when acquiring symbionts that later became organelles. Here, we approached this question by studying the effects of inhibitors that affect the host exclusively in two symbiont-bearing trypanosomatids, Strigomonas culicis and Angomonas deanei. We found that inhibiting host protein synthesis using cycloheximide or host DNA replication using aphidicolin did not affect the duplication of bacterial DNA. Although the bacteria had autonomy to duplicate their DNA when host protein synthesis was blocked by cycloheximide, they could not complete cytokinesis. Aphidicolin promoted the inhibition of the trypanosomatid cell cycle in the G1/S phase, leading to symbiont filamentation in S. culicis but not in A. deanei. Treatment with camptothecin blocked the host protozoa cell cycle in the G2 phase and induced the formation of filamentous symbionts in both species. Oryzalin, which affects host microtubule polymerization, blocked trypanosomatid mitosis and abrogated symbiont division. Our results indicate that host factors produced during the cell division cycle are essential for symbiont segregation and may control the bacterial cell number.

Strigomonas culicis (previously referred to as Blastocrithidia culicis) is a monoxenic trypanosomatid harboring a symbiotic bacterium, which maintains an obligatory relationship with the host protozoan. Investigations of the cell cycle in symbiont harboring trypanosomatids suggest that the bacterium divides in coordination with other host cell structures, particularly the nucleus. In this study we used light and electron microscopy followed by three-dimensional reconstruction to characterize the symbiont division during the cell cycle of S. culicis. We observed that during this process, the symbiotic bacterium presents different forms and is found at different positions in relationship to the host cell structures. At the G1/S phase of the protozoan cell cycle, the endosymbiont exhibits a constricted form that appears to elongate, resulting in the bacterium division, which occurs before kinetoplast and nucleus segregation. During cytokinesis, the symbionts are positioned close to each nucleus to ensure that each daughter cell will inherit a single copy of the bacterium. These observations indicated that the association of the bacterium with the protozoan nucleus coordinates the cell cycle in both organisms.

RATIONALE: Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) of tissues became popular in the last decade. Consequently, adapting sample preparation methods for different materials turned to be a pivotal step for a successful analysis, due to the requirement of samples slices of 12-20 µm thickness. However, preparing thin sections compatible with MALDI-IMS for unusual samples is challenging, as existing histological protocols may not be suitable, thus requiring new methods. Açaí (Euterpe oleracea Mart.) seed is an example of a challenging material due to its toughness and resistance to crack. Therefore, our goal was to develop a methodology to obtain thin (<20 µm) and entire longitudinal sections of açaí seeds for MALDI-IMS analysis. METHODS: Different strategies were evaluated for obtaining thin cuts of seeds, being the combination of the following steps the most suitable option: (i) softening of seeds by water immersion for 24 h; (ii) longitudinal cut of seeds to obtain half-seeds using a razor blade and a hammer; (iii) half-seeds imbibition in gelatin; (iv) sectioning using a cryostat at -20 °C to obtain samples with <20 µm thickness; and (v) collection of samples in an indium tin oxide coated glass slide covered by a double-face copper tape to avoid sample wrapping and ensure adhesion after unfreezing. (iv) storage of samples in a -80 °C freezer, if necessary. RESULTS: As a result, this adapted sample preparation method enabled the analysis of açaí seeds by MALDI-IMS providing spatial distribution of carbohydrates in the endosperm. CONCLUSIONS: The adaptations developed for sample preparation will help investigate the metabolic and physiological properties of açaí seeds in future studies.

We investigated changes in the phenolic profile and antioxidant properties in the extracts of developing seeds of acai (Euterpe oleracea). Four developmental stages were evaluated, with earlier stages displaying higher antioxidant activity and polyphenols content, while mass spectrometry analysis identified procyanidins (PCs) as the major components of the extracts in all stages. B-type PCs varied from dimers to decamers, with A-type linkages in a smaller number. Extracted PCs decreased in average length from 20.5 to 10.1 along seed development. PC composition indicated that (−)-epicatechin corresponded to over 95% of extension units in all stages, while (+)-catechin presence as the starter unit increased from 42 to 78.8% during seed development. This variation was correlated to the abundance of key enzymes for PC biosynthesis during seed development. This study is the first to report PC content and composition variations during acai ́seed development, which can contribute to studies on the plant's physiology and biotechnological applications.

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RationaleMatrix‐assisted laser desorption/ionization imaging mass spectrometry (MALDI‐IMS) of tissues became popular in the last decade. Consequently, adapting sample preparation methods for different materials turned out to be a pivotal step for successful analysis due to the requirement of sample slices of 12–20 μm thickness. However, acquiring thin sections compatible with MALDI‐IMS for unusual samples is challenging, as existing histological protocols may not be suitable, thus requiring new methods. Açaí (Euterpe oleracea Mart.) seed is an example of a challenging material due to its toughness and resistance to crack, therefore our goal was to develop a methodology to obtain thin (12–20 μm) and entire sections of açaí seeds for MALDI‐IMS analysis.MethodsDifferent strategies were evaluated for obtaining thin sections of seeds, and the combination of the following steps was found to be the most suitable option: (i) softening of seeds by water immersion for 24 h; (ii) transversal cut of seeds to obtain half‐seeds using a razor blade and a hammer; (iii) half‐seeds imbibition in gelatin; (iv) samples sectioning using a cryostat at −20°C to obtain samples with 12–20 μm thickness; (v) collection of samples in an indium tin oxide‐coated glass slide covered by double‐sided copper tape to avoid sample wrapping and ensure adhesion after unfreezing; and (vi) storage of samples in a −80°C freezer, if necessary.ResultsThis adapted sample preparation method enabled the analysis of açaí seeds by MALDI‐IMS, providing spatial distribution of carbohydrates in the endosperm.ConclusionsThe adaptations developed for sample preparation will help investigate the metabolic and physiological properties of açaí seeds in future studies.