Coffee berry borer—CBB (Hypothenemus hampei) is a globally important economic pest of coffee (Coffea spp.). Despite current insect control methods for managing CBB, development of future control strategies requires a better understanding of its biology and interaction with its host plant. Towards this objective, we performed de novo CBB genome and transcriptome sequencing, improved CBB genome assembly and predicted 18,765 protein-encoding genes. Using genome and transcriptome data, we annotated the genes associated with chemosensation and found a reduced gene repertoire composed by 67 odorant receptors (ORs), 62 gustatory receptors (GRs), 33 ionotropic receptors (IRs) and 29 odorant-binding proteins (OBPs). In silico transcript abundance analysis of these chemosensory genes revealed expression enrichment in CBB adults compared with larva. Detection of differentially expressed chemosensory genes between males and females is likely associated with differences in host-finding behavior between sexes. Additionally, we discovered male-specific genome content and identified candidate male-specific expressed genes on these scaffolds, suggesting that a Y-like chromosome may be involved in the CBB’s functional haplodiploid mechanism of sex determination.

The metataxonomic diversity and microbial composition of microorganisms during the coffee fermentation process as well as their relationship with coffee quality were determined across 20 farms in the department of Cesar, Colombia, by sampling coffee fruits from Coffea arabica; Var. Castillo General^®, Var. Colombia, and Var. Cenicafé 1. In each farm, the fruits were processed and the fermentation process took place between 10 and 42 h following this. Three samples of mucilage and washed coffee seeds were collected per farm during the fermentation process. The microorganisms present in the mucilage were identified using metataxonomic methods by amplifying the 16S rRNA gene for bacteria and ITS for fungi. The microorganisms’ morphotypes were isolated and identified. The analysis of bacteria allowed for the identification of the following genera: Gluconobacter, Leuconostoc, Acetobacter, Frateuria, Pantoea, Pseudomonas, Tatumella, and Weisella, as well as unclassified enterobacteria; the Lactobacillacea and Secundilactobacillus families were only identified in the Var. Cenicafé 1. For fungi, the top 11 genera and families found included Hanseniaspora, Candida, Meyerozyma, Wickerhamomyces, Pichia, f-Saccharomycodaceae, f-Nectriciae, unclassified fungi, and Saccharomycetaceae, which were only found in Cenicafé 1. A total of 92% of the coffee samples obtained scored between 80.1 and 84.9, indicating “Very Good” coffee (Specialty Coffee Association (SCA) scale). Farms with the longest fermentation times showed better coffee attributes related to acidity, fragrance, and aroma. During coffee fermentation, there is a central microbiome. The differences between the microorganisms’ genera could be influenced by the coffee variety, while the specific conditions of each farm (i.e., altitude and temperature) and its fermentation processes could determine the proportions of and interactions between the microbial groups that favor the sensory characteristics responsible for coffee cup quality.

Mechanistic understanding of interactions in many host-microbe systems, including the honey bee microbiome, is limited by a lack of easy-to-use genome engineering approaches. To this end, we demonstrate a one-step genome engineering approach for making gene deletions and insertions in the chromosomes of honey bee gut bacterial symbionts. Electroporation of linear or non-replicating plasmid DNA containing an antibiotic resistance cassette flanked by regions with homology to a symbiont genome reliably results in chromosomal integration. This lightweight approach does not require expressing any exogenous recombination machinery. The high concentrations of large DNAs with long homology regions needed to make the process efficient can be readily produced using modern DNA synthesis and assembly methods. We use this approach to knock out genes, including genes involved in biofilm formation, and insert fluorescent protein genes into the chromosome of the betaproteobacterial bee gut symbiont Snodgrassella alvi . We are also able to engineer the genomes of multiple strains of S. alvi and another species, Snodgrassella communis , which is found in the bumble bee gut microbiome. Finally, we use the same method to engineer the chromosome of another bee symbiont, Bartonella apis , which is an alphaproteobacterium. As expected, gene knockout in S. alvi using this approach is recA -dependent, suggesting that this straightforward procedure can be applied to other microbes that lack convenient genome engineering methods. IMPORTANCE: Honey bees are ecologically and economically important crop pollinators with bacterial gut symbionts that influence their health. Microbiome-based strategies for studying or improving bee health have utilized wild-type or plasmid-engineered bacteria. We demonstrate that a straightforward, single-step method can be used to insert cassettes and replace genes in the chromosomes of multiple bee gut bacteria. This method can be used for investigating the mechanisms of host-microbe interactions in the bee gut community and stably engineering symbionts that benefit pollinator health.