Specific esters are shared with apple , certain lactones are shared with peach and various terpenes are shared with citrus . Syntenic regions and orthologous genes could be exploited for flavor improvement in those species. Additional insights were gained for the strawberry gene regulatory landscape, SV diversity, complex interplays among cis- and trans- regulatory elements, and subgenome dominance. Previously, Hardigan et al. and Pincot et al. showed a large genetic diversity existing in breeding populations of Fragaria × ananassa, challenging previous assumptions that cultivated strawberry lacked nucleotide variation owing to the nature of its interspecific origin and short history of domestication . Our work corroborated their findings and showed that even highly domesticated populations harbor substantial expression regulatory elements and structural variants. Over half of the expressed genes in fruit harbored at least one eQTL, and 22 731 eGenes had impactful cis-eQTL. The distribution of trans-eQTL is not random, but rather is concentrated at a few hotspots controlled by putative master regulators . The aggregation of trans-eQTL also was observed in plant species such as Lactuca sativa and Zea mays . Furthermore, we observed a substantial number of trans-eQTL among homoeologous chromosomes, similar to observations in other allopolyploid plant species . In cotton, physical interactions among chromatins from different subgenomes have been identified via Hi-C sequencing , supporting a potential regulatory mechanism among homoeologous chromosomes. However, owing to the high similarity among four subgenomes and limited length of Illumina reads, large pot with drainage false alignment to incorrect homoeologous chromosomes could arise, leading to ‘ghost’ trans-eQTL signals.
Future studies are needed to scrutinize the homoeologous trans-eQTL and investigate the mechanism behind this genome-wide phenomenon. Higher numbers of trans-eQTL in the Fragaria vesca-like subgenome are consistent with its dominance in octoploid strawberry . By contrast, the highly mixed Fragaria viridis- and Fragaria nipponica- like subgenomes contained much smaller numbers of trans-eQTL. The characterization of naturally-occurring allelic variants underlying volatile abundance has direct breeding applications. First, this will facilitate the selection of desirable alleles via DNA markers. Second, understanding the causal mutations in alleles can guide precision breeding approaches such as gene editing to modify the alleles themselves and/or their level of expression. From a broader perspective, multi-omics resources such as this one will have value for breeding a wide array of fruit traits. Enhancing consumer satisfaction in fruit ultimately will depend on the improvement of the many traits that together enhance the overall eating experience.As the human population continues to grow, so will its demand for protein. In the Western portion of the world, livestock farming covers the vast majority of protein production; however, the insect farming industry is quickly expanding, producing protein-rich products with a relatively low ecological footprint. Insects are poikilotherms, meaning they do not rely on metabolic energy to regulate their body temperature. This allows them to allocate more energy toward growth, resulting in a higher feed conversion efficiency. Yellow meal worms are one of the most economically important insects in large-scale production. They are used as pet food and livestock feed with increasing commonality due to their high protein, fat, and polyunsaturated fatty acid contents. YMWs have recently been considered ideal for human nutrition, offering a more cost- and energy-efficient alternative protein source to livestock and aquaculture.
Not only have they been proven to thrive on diets of agricultural byproducts, YMWs have also been shown to degrade polystyrene, a polymer found in plastics that is highly resistant to environmental degradation. Increased use of insects as a protein source for either humans or livestock poses the challenge of novel infectious disease outbreaks via pathogenic infection by any number of microorganisms . The high population density of insect farming exacerbates the spread of disease in these populations and increases the likelihood of epidemics and zoonotic transmission. Densoviruses are autonomous, nonenveloped, paraspherical DNA viruses that are 23 to 28 nm in diameter with icosahedral symmetry and contain 5 kb, single-stranded, linear DNA genomes. They belong to the subfamily Densovirinae within the Parvoviridae family. Densovirinae contains 1 unassigned species and 5 genera, which are Ambidensovirus, Brevidensovirus, Hepandensovirus, Iteradensovirus, and Penstyldensovirus. Comprising more than 20 viral species, most members of the subfamily Densovirinae are highly pathogenic for invertebrates. Currently, densoviruses are known to infect insects from 6 orders , decapod crustaceans , and echinoderms . In addition, densoviruses have been devastating to commercially produced invertebrate populations, such as the cricket, silkworm, and shrimp. Densovirus infections were initially referred to as “densonucleosis,” a term used to indicate the large, dense, and homogeneous appearance of infected nuclei of greater wax moth larvae due to replication of a small virus . A significant number of densoviruses are polytropic with a variable host range, while other densovirus infections are host- and organ-specific. The high pathogenicity of densovirus to pests, especially mosquitos, favors its use as a biological control agent, although the long-term impact of viral spillover among non-target native insect populations remains unknown. Insects represent the most bio-diverse group of animals on Earth, with more than 2000 species being consumed by humans worldwide. Unlike viruses affecting vertebrates/ mammals, densoviruses are as diverse as their invertebrate hosts. This is reflected in the wide range of biological behaviors displayed among different densovirus species. Still, little is known about viruses affecting insects, including those used for food and feed. Characterization of novel densovirus infections among distinct invertebrate populations is of utmost importance to recognize outbreaks and accelerate responses. This is especially important in agricultural populations whose infections pose tremendous risk of interspecific spillover and economic devastation. Herein, we describe an outbreak of densovirus infection with high mortality in a commercial mealworm farm in 2020. This report focuses on the characterization of the molecular, bright-field, and electron microscopic features of this infection and its clinical significance.Problems began when a large commercial insect breeder was unable to raise larvae of YMW beetles.
Anecdotally, the disease was first observed in 5- to 6-week-old post-hatching larvae, 3 to 5 weeks before the average harvesting age. YMW larvae presented with lethargy, size variability, cessation of eating, and “spinning” prior to death. All YMW larvae were affected and died before reaching the harvesting size. These YMW larvae were fed a proprietary diet on which the farm had previously yielded good success. The feed was tested for various organophosphorus insecticides, which were not detected. Due to the progression of the disease, which affected various in stars and presumably multiple production cycles, the farmer halted YMW larvae production practices. After several months of farming interruption and total reconstruction of the facilities, drainage collection pot the breeder eventually resumed normal production of YMWs.For detection, identification, and quantification, viruses were extracted, cleaned, concentrated, and contrasted using a modified nebulization method originally described by Cruickshank et al. Briefly, YMWs were frozen at –80°C. To extract the virus from the tissue/cellular compartments, YMWs were subjected to 2 freeze/thaw cycles before bead-mill homogenization in a Bead Ruptor 12 at 5 m/s velocity for 45 seconds. Removal of macrodebris from this suspension was achieved by decantation via low-speed centrifugation at 2000g for 10 minutes at 30°C. The supernatant was filtered with a series of 5.0, 1.2, and 0.8 μm syringe filters to remove prokaryotes and microdebris. The supernatant was then subjected to ultra centrifugation at 285,000g for 110 minutes at 5°C . The resulting supernatant was discarded and the pellet containing concentrated virus was resuspended in 200 μL of boiled double-distilled water. Next, a 2:1:1 dilution of virus pellet-1% sodium phosphotungstate solution -bovine serum albumin contrast was established. For viral screening, 100 μL of this mixture was microaerosolized onto a 200-mesh formvar/carbon-coated electron microscope grid using a glass nebulizer. For viral quantification, 1 μL of the 2:1:1 mixture was applied as a drop to an Alcian blue–treated 200-mesh formvar/carbon-coated electron microscope grid . The detected virus was identified by finding repetitive profiles with consistent dimensions, morphological features, and structural characteristics that match the dimensions and features accepted and published by the Report of the International Committee on Taxonomy of Viruses.19To quantify the amount of viral particles per gram of tissue, the number of viruses in a known area was calculated.17 Enumerations of viral particles in a total of 20 individual 0.35 μm2 areas were performed and averaged. This enabled an estimation of the total number of viral particles on a single 3-mm diameter grid, based on its calculated surface area of 7.1 mm2 . Even distribution of viruses on the grid was ensured by the presence of bovine serum albumin in the suspension-stain mixture. Because this grid was covered by a known volume of viral pellet suspension , the concentration of viral particles in the entire suspension could be determined. From here, the number of viral particles present in the known weight of mealworm tissue processed was estimated and scaled to determine the approximate number of viral particles contained in 1 g of mealworm tissue. To analyze the tissue ultrastructure, whole bodies of 3 diseased YMWs and 3 unaffected individuals were fixed in Karnovsky’s fixative . Coronal sections of the body tissue were taken in 2 to 3 mm segments at 4 levels: head , thorax , abdomen , and podal appendages.
These sections were postfixed with 1% osmium tetroxide in 0.1 M sodium cacodylate buffer. Samples were processed as described elsewhere.1 Briefly, samples were dehydrated in a 25% to 100% ethyl alcohol gradient series for 2 hours using an automated tissue processor . The tissue was then infiltrated with 1:1 and subsequently 3:1 EMbed 812 resin:propylene oxide for 1 and 2 hours, respectively. The tissue was then infiltrated with 100% resin for 3 hours before embedding and incubation at 58°C for 24 hours to polymerize the resin . Embedded samples were trimmed and sectioned on a Leica UC7 ultramicrotome . One-micrometer thick sections were mounted on glass slides and stained with toluidine blue to select areas of interest. From the selected areas, thin sections were obtained and collected on a 100-mesh nickel grid . Grids were contrasted with 5% uranyl acetate for 20 minutes and Satos’ lead citrate for 6 minutes. All samples were visualized using a JEOL 1400Plus transmission electron microscope . Images were obtained and analyzed using a OneView Camera Model 1095 with the Gatan Microscope Suite 3.0 .Total nucleic acid was extracted from 200 µL of the resuspended EM pellet using the MagMax Pathogen RNA/DNA kit following the recommendations of the manufacturer. The extracted DNA was purified using AMPure XP beads and used as input to a library generated with the Ligation Sequencing Kit SQK-LSK109 . First, DNA was end-repaired using the NEBNext Ultra II DNA End Prep and Repair kit , purified using AMPure XP beads in a ratio of 1:1 volume of beads per sample, and eluted in 30 µL of nuclease-free water. Sequencing adapters were ligated to the DNA using NEBNext Quick T4 DNA ligase by incubation at room temperature for 10 minutes. The adapter-ligated DNA library was purified with AMPure XP beads in a ratio of 1:2.5 volume of beads per sample, followed by 2 washes with S Fragment buffer and elution in 7 µL of elution buffer . The library was loaded onto an FLO-MIN106 9.4 Flowcell on a GridION sequencer . Prior to starting the run, the sequencing kit SQK-LSK109 with barcoding expansion pack EXP-NBD196 and the “super accuracy” basecaller were selected in the MinKNOW data analysis software. After sequencing for 24 hours, FASTQ files containing “pass” reads were loaded into Geneious Prime and mapped to a reference densovirus genome using the MiniMap2 plugin. Using the mealworm densovirus genome sequence, as well as all densovirus whole genomes available in GenBank, a multiple sequence alignment was produced using the MUSCLE algorithm in Geneious. Phylogenetic analysis using a maximum likelihood method was performed with the program MEGA . The Hasegawa-Kishino-Yano model of nucleotide substitution with gamma distributed rates was used, and bootstrap values were calculated using 1000 pseudo-replicates.Upon gross examination, YMW larvae infected with densovirus presented with variably underdevelopment curved bodies and multifocal-to-generalized, dark cuticular discoloration . Organs and body tissue were soft, discolored, and reduced in volume when compared with those unaffected. Histologically, the lesions in infected YMWs were characterized by cytoplasmic and nuclear enlargement with InI bodies and death of epithelial cells of the cuticular epidermis, pharynx and esophagus , rectum , and respiratory tubules—tracheae and tracheoles .