A functional annotation was obtained using the RefSeq plant protein database as in Minio et al. .Berries from Cabernet Franc grapevines grafted to Kober 5BB or MGT 101-14 and infected with GLRaV-1 , GLRaV-3 , GLRaV-4 , GLRaV-1 and GLRaV-2 , or GLRaV-1 and GLRaV-3 were sampled during ripening in 2017 and 2018. In both years, fruits were sampled at prevéraison, véraison, mid-ripening, and at commercial harvest. These stages correspond to modified Eichhorn–Lorenz stages 34 , 35 , 36/37 , and 38 . Fruits in 2018 were sampled at developmental stages comparable to 2017 as determined by TSS. In 2017, berries were sampled on 7 July, 31 July, 14 August, and 31 August. In 2018, berries were sampled on 28 June, 30 July, 13 August, and 30 August. On each sampling date, six biological replicates were taken, with berries from one plant constituting one biological replicate. Two biological replicates per condition were sampled from each of three blocks. There were two exceptions. For Kober 5BB-grafted GLRaV-1,2 , three biological replicates were drawn from each of two plants in one block. For Kober 5BB-grafted GLRaV-3 , two of the six biological replicates were drawn from one plant. Approximately 20 berries were sampled per plant, with equal numbers of berries sampled from each side of the vine. Samples were then temporarily cooled on ice. Next, growing blueberries were rinsed with deionized water, deseeded, and snap-frozen in liquid nitrogen. Berries were stored at −80 °C until being crushed into a fine powder while frozen using a mechanical mill.
TSS were measured in technical triplicate using a digital refractometer. Four of six biological replicates, a total of 192 samples per year, were used for RNA-Seq and liquid chromatography coupled with mass spectrometry .Total RNA was extracted from 2 g of finely ground berry pericarp tissue as previously described . RNA purity was evaluated with a NanoDrop 2000 spectrophotometer , quantity with a Qubit 2.0 fluorometer , and integrity by electrophoresis. RNA-Seq libraries were prepared using the Illumina TruSeq RNA sample preparation kit v. 2 and barcoded individually following the manufacturer’s protocol. Final libraries were evaluated for quantity and quality with the High Sensitivity chip in a Bioanalyzer 2100 . Libraries were sequenced as 100-bp, single-end reads, using an Illumina HiSeq 4000 sequencer , producing an average of 18.07 ± 4.56 million reads per sample in 2017 and 14.69 ± 2.11 million reads per sample in 2018.RNA-Seq reads were trimmed using Trimmomatic v. 0.36 and the following settings: leading: 2, trailing: 2, sliding window: 4:20, min length: 70. Reads were mapped onto the primary assembly of the Cabernet Franc genome using HISAT2 and counts were generated using htseq-count with default parameters . All subsequent analyses were done using R in the R Studio environment . Data normalization and differential expression analyses were performed using DESeq2 v. 1.24.0 . A variance-stabilizing transformation was applied to expression data using DESeq2. VST data were centred in each GLRaV condition relative to the mean expression per gene in GLRaV given the same time, rootstock, and year. Centred data were used for MFA with the FactoMineR R package .
Genes were included in the MFA if they were differentially expressed versus GLRaV , and/or the effects of an infection differed between rootstocks in at least 1 year , and the direction of the effect relative to GLRaV was consistent in both years, even if a significant effect was only observed in 1 year. An MFA was repeated for each developmental stage separately. All hormone and metabolite data were included. Statistical over-representation tests were done using the cluster Profiler R package and VitisNet functional categories . To make use of the VitisNet functional annotations, Cabernet Franc genes were used to query Pinot Noir PN40024 sequences with BLASTp . The best hits, with no less than 80% reciprocal identity and coverage, were retained. A curated list of ABA biosynthesis and signalling genes annotated in PN40024 was retrieved from Pilati et al., 2017 . As described above, Cabernet Franc genes were used to query Pinot Noir PN40024 sequences. All target–query pairs had no less than reciprocal 97% identity and 86% reciprocal coverage .Approximately 50 mg of berry powder was weighed for the extraction and quantitation of ABA, SA, and JA. Exact weights were recorded to later calculate the exact amount of each of these analytes per milligram of fresh tissue. Four biological replicates were used. Extractions and analyses were randomized and performed in technical duplicate. Hormones were extracted following a method described by Pan et al. with a few modifications. Samples were subjected to 500 ml 2-propanol:H2O:HCl and spiked with 50 ng of d6 ABA, d5 JA, and d4 SA . Samples were vortexed, placed in an ultrasonic ice bath for 30 min, washed with dichloromethane, and centrifuged at 16,100 × g for 5 min. Nine-hundred microlitres of the lower phase was taken and dried. Samples were reconstituted in 100 µl 15% methanol and stored at −20 °C until analysis. A detailed description of the chromatographic separation, mass spectrometry data acquisition methodology, and multiple reaction monitoring transitions is included in Table S2.
The same samples used for RNA-Seq and hormone analyses were used to measure water-soluble metabolites by LC-MS. Approximately 200 mg of frozen berry powder was weighed. Extractions and analyses were randomized and performed in technical duplicate. Hormones were extracted in 1 ml 1% HCl in HPLC-grade water and spiked with salicin as an internal standard . The samples were vortexed, placed in an ultrasonic ice bath for 30 min, and centrifuged. The supernatants were collected and frozen at −80 °C until analysis. A detailed description of the chromatographic separation, mass spectrometry data acquisition methodology, and MRM transitions is included in Table S2.Habitat complexity is critical for the functioning of ecological communities in both terrestrial and aquatic systems. Processes such as resource foraging, colonization, and species interactions often depend on the level of heterogeneity in the configuration of physical elements in a habitat . Vegetation connectivity and structure are important components of habitat complexity and can influence species interactions and community patterns at local scales. In aquatic systems, more complex habitats made up of macrophytes support communities that are more diverse and abundant, and allow for greater food capture than systems without vegetation . In terrestrial systems, vegetation structure—such as the biomass of foliage and the variety of plant architectures—generally influences species composition, and increases species richness and abundance of numerous taxa . Additionally, vegetation structure can influence mobility and foraging success of vertebrates and invertebrates . In tropical ecosystems, ants are among the most abundant and bio-diverse of taxonomic groups and are considered important predators, herbivores, and seed dispersers . Ants are cursorial central-place foragers—organisms that forage from a central place to which they return with food to feed with the colony . Therefore, foraging and discovery of food resources is strongly constrained by the need to construct and follow trails along vegetation . This is particularly relevant for ants using the arboreal stratum as their primary foraging space . For instance, the availability of vegetation connections can maximize ants’ foraging efficiency, locomotion, and velocity , square plant pots as well as contribute to changes in community composition and species richness . The availability of such resources can ultimately lead to differences in resource utilization by ant communities . In tropical agricultural systems, especially agroforests, ants play important ecological roles , and management practices can strongly influence ant behavior and their potential for providing biological pest control services . Indeed, one of the oldest known records of the use of ants for pest control dates to 304 A.D in citrus plantations in China. In these systems, artificial connections made of bamboo were used by farmers to facilitate foraging by the Weaver Ant to suppress damaging phytophagous insects. In that same study, Huang and Yang report anecdotal evidence that suggests equal yields in orchards that use chemicals vs. orchards that use ant bridges to control for pests. Similarly, Peng and Christian , report lower levels of fruit damage in cashew with the presence of weaver ants. However, as vegetation complexity declines in agroecosystems, tree density and diversity may also decrease , as well as the possibility to generate connections between the arboreal vegetation, which might impact arthropod populations . The lack of connectivity between trees in managed systems can have a significant impact on the mobility of worker ants and their ability to control resources. This impact may be particularly marked at greater distances from the nest, where ant dominance may be lower . This in turn may influence the ecosystem services provided by ants, particularly the suppression of pest outbreaks . Shaded coffee plantations, which maintain high levels of shade and structural complexity , can sustain complex networks of organisms, which can result in biological pest control . In coffee systems, ants are a functionally diverse and abundant group of ground and arboreal-nesting arthropods and are considered important biological control agents .
Ants are predators of the most devastating coffee pest, the coffee berry borer , a beetle that drills cavities in coffee berries and severely damages the seed . Several species of arboreal ants, with nests attached to or inside tree trunks, branches, or twigs, control adult and immature stages of this pest either through direct predation or deterrence . Ants of the genus Azteca are numerically dominant in shaded coffee plantations. These ants forage intensively on coffee plants , and deter CBB adults by removing them from the coffee plant, therefore lowering fruit damage . In shaded coffee plantations, Azteca sericeasur ants nest on shade trees and access adjacent coffee plants through the leaf litter or available pathways, such as fallen branches, vines, and other vegetation , matching the description by Longino for this species in forest habitats. In more intensively managed coffee systems, with fewer and more distant nesting trees, connectivity may be sparse or absent and artificial connections might buffer against this loss. Vegetation structure and arboreal characteristics in coffee plantations are likely to be important factors influencing ant foraging behavior and nesting in arboreal ants . However, the influence of vegetation connectivity on the foraging of this dominant arboreal ant and its effect on pest removal in coffee plantations has not yet been studied. Previous work has documented the importance of arboreal connections for ants and biological control in agricultural systems. For example, various studies and farmers’ manuals suggest that connecting nests to adjacent trees using bamboo strips enables weaver ants to colonize new trees, which increases ants’ efficiency in removing pests, including the pentatomid insect Tesserarotoma papillosa . However, there is little evidence about the effect of increasing arboreal connectivity on biological control using experimental data. We report an experiment testing the influence of adding connections between shade trees and coffee plants and its effects on CBB removal on coffee plants. To our knowledge, this is the first study providing experimental data on the effect of adding connectivity on ant activity and pest removal in coffee agroecosystems. Specifically, we tested one hypothesis: Connectivity affects CBB removal in this system by increasing recruitment rates of A. sericeasur ants to prey items; we predicted that A. sericeasur ants use artificial connections between nesting trees and coffee plants; plants with connectivity have higher ant activity than isolated plants; plants with connections have grater removal rates of CBB by A. sericeasur ants; and A. sericeasur activity and CBB removal rates by A. sericeasur ants decrease with increased distance from A. sericeasur nests.We conducted the study in a 300 ha shaded coffee plantation in the Soconusco region of Chiapas, Mexico. The coffee plantation is located at 1,100 m a.s.l. in the Sierra Madre de Chiapas Mountains. The natural vegetation types are high and mid-elevation perennial forest and the climate is semitropical with rainfall typically occurring between May and October . The coffee plantation can be characterized as a commercial polyculture, where coffee plants grow under the canopy of shade of trees, mostly in the genus Inga , providing an average canopy cover of 75% .Within the farm, we haphazardly selected 20 non-overlapping sites located at least 10 m away from each other with one Inga micheliana tree containing an A. sericeasur carton nest on the tree trunk . Azteca sericeasur is a polydomous, arboreal ant species , which occurs in ~13% of trees at our study site , and forages on coffee plants .