Chlamydomonas reinhardtii is a model alga of increasing interest as a cell factory for the production of valuable compounds, including therapeutic proteins and bioactive metabolites. Expression of foreign genes in the chloroplast is particularly advantageous as: (i) accumulation of product in this sub-cellular compartment minimises potential toxicity to the rest of the cell; (ii) genes can integrate at specific loci of the chloroplast genome (plastome) by homologous recombination; (iii) the high ploidy of the plastome and the high-level expression of chloroplast genes can be exploited to achieve levels of recombinant protein as high as 5% total cell protein; (iv) the lack of any gene silencing mechanisms in the chloroplast ensures stable expression of transgenes. However, the generation of C. reinhardtii chloroplast transformants requires efficient methods of selection, and ideally methods for subsequent marker removal. Additionally, the use of reporter genes is critical to achieving a comprehensive understanding of gene expression, thereby informing experimental design for recombinant applications. This review discusses currently available selection and reporter systems for chloroplast engineering in C. reinhardtii, as well as those used for chloroplast engineering in higher plants and other microalgae, and looks to the future in terms of possible new markers and reporters that will further advance the C. reinhardtii chloroplast as an expression platform.

The chloroplast of microalgae such as Chlamydomonas reinhardtii represents an attractive chassis for light-driven production of novel recombinant proteins and metabolites. Methods for the introduction and expression of transgenes in the chloroplast genome (=plastome) of C. reinhardtii are well-established and over 100 different proteins have been successfully produced. However, in almost all reported cases the complexity of the genetic engineering is low, and typically involves introduction into the plastome of just a single transgene together with a selectable marker. In order to exploit fully the potential of the algal chassis it is necessary to establish methods for multigenic engineering in which many transgenes can be stably incorporated into the plastome. This would allow the synthesis of multi-subunit proteins and the introduction into the chloroplast of whole new metabolic pathways. In this short communication we report a proof-of-concept study involving both a combinatorial and serial approach, with the goal of synthesizing five different test proteins in the C. reinhardtii chloroplast. Analysis of the various transgenic lines confirmed the successful integration of the transgenes and accumulation of the gene products. However, the work also highlights an issue of genetic instability when using the same untranslated region for each of the transgenes. Our findings therefore help to define appropriate strategies for robust multigenic engineering of the algal chloroplast.

The inappropriate use of antibiotics leads to antibiotic resistance, which reduces their efficacy. The education of undergraduates is likely to influence their practices. Assessing awareness is critical in the general effort to confront the spread of antibiotic resistance. This cross-sectional investigation was carried out using the questionnaire “Antibiotic resistance: Multi-country public awareness” developed by the World Health Organization. Students from different backgrounds at the Central University participated in the study (n = 733). The survey comprised five sections: demographics, knowledge, usage, sources of information, and attitudes. The rate of correct answers was 64.88%; differences were detected between programs of study (p < 0.001); effect size analysis showed that these differences cannot be considered large. Individuals from applied sciences scored higher than their counterparts from social studies. Mostly, interviewees were knowledgeable about usage, but mistakenly associated antibiotics with conditions such as cold/flu or viral illnesses; also, they associated antibiotic resistance with the patient and not with bacteria. Despite these misconceptions, positive attitudes were registered overall, and students generally adhered to common practices. They cited doctors/nurses and teachers as sources of information. As a consequence, it is recommended to develop courses that address deficient knowledge regarding antibiotic resistance, especially for individuals affiliated to social disciplines.

This pilot investigation aimed at studying the feasibility of using a low dose (0.2%) of dietary microalgae as a means of improving intestinal morphometry, body weight, and selected meat quality parameters in broilers. A total of 72 one-day-old ROSS 308 male chicks were randomly separated into four groups; three experimental pens in which the birds were fed with biomass from Tysochrysis lutea, Tetraselmis chuii, and Porphyridium cruentum over 30 days and a control group. T. chuii and P. cruentum had a positive effect with regard to body weight. In treated animals, duodenal and ileal sections showed characteristic tall and thin villi, with serrated surfaces and goblet cell differentiation. In both sections, values of the villus-height-to-crypt-depth ratio were increased by microalgae ingestion. The thawing weight loss of fillets was reduced in T. chuii-fed animals. The positive effects exerted by T. chuii and P. cruentum on intestinal architecture were associated with the improved body weight. Arguably, these outcomes exhibit the potential of using these species to enhance growth performance in broiler chickens by promoting gut homeostasis and thus nutrient absorption.

Public concern over drug resistance has led to governmental regulations banning the use of antibiotics as growth promoters, stimulating interest in developing complementary strategies to maintain animal production, mitigate infections, and enhance muscle characteristics and quality parameters, especially in meat-producing animals. Probiotics are recognized as a potential strategy for improving growth, primarily by promoting intestinal homeostasis. These microorganisms are suggested to modulate gut microbiota, preserving their ecosystem and influencing secondary metabolite production, which can directly or indirectly regulate skeletal muscle metabolism by influencing the expression of key muscle-related genes and the activity of various signaling factors. Several studies have documented the potential benefits of various strains of Bacillus, Enterococcus, and members of the Lactobacillaceae family on muscle characteristics. These studies have shown that probiotics not only modulated myogenic factors but also influenced proteins and enzymes involved in signaling pathways related to carbon metabolism, inflammatory response, mitochondrial dynamics, and antioxidant activity. These effects have been associated with improvements in meat quality parameters and enhanced growth performance. This manuscript seeks to present a brief overview of the impact of probiotic supplementation on muscle health and the quality of meat in broiler chickens.

There are no studies reporting the effects of Salmonella enterica subsp. enterica serovar Infantis (S. Infantis) on intestinal architecture and immunoglobulin serum levels in chickens. Here, we measured these parameters and hypothesized whether probiotic administration could modulate the observed outcomes. Two-hundred 1-day-old COBB 500 male chicks were allocated into four groups: (I) the control, (II) the group treated with L. fermentum, (III) the group exposed to S. Infantis, and (IV) the group inoculated with both bacteria. At 11 days post infection, blood was gathered from animals which were then euthanized, and samples from the small intestine were collected. Intestinal conditions, as well as IgA and IgM serum levels, were assessed. S. Infantis reduced villus-height-to-crypt-depth (VH:CD) ratios in duodenal, jejunal, and ileal sections compared to control conditions, although no differences were found regarding the number of goblet cells, muc-2 expression, and immunoglobulin concentration. L. fermentum improved intestinal measurements compared to the control; this effect was also evidenced in birds infected with S. Infantis. IgM serum levels augmented in response to the probiotic in infected animals. Certainly, the application of L. fermentum elicited positive outcomes in S. Infantis-challenged chickens and thus must be considered for developing novel treatments designed to reduce unwanted infections.