Genome divergence and reproductive incompatibility among populations of Ganaspis near brasiliensis.

During the last decade, the spotted wing drosophila, Drosophila suzukii, has spread from eastern Asia to the Americas, Europe, and Africa. This fly attacks many species of cultivated and wild fruits with soft, thin skins, where its serrated ovipositor allows it to lay eggs in undamaged fruit. Parasitoids from the native range of D. suzukii may provide sustainable management of this polyphagous pest. Among these parasitoids, host-specificity testing has revealed a lineage of Ganaspis near brasiliensis, referred to in this paper as G1, that appears to be a cryptic species more host-specific to D. suzukii than other parasitoids. Differentiation among cryptic species is critical for introduction and subsequent evaluation of their impact on D. suzukii. Here, we present results on divergence in genomic sequences and architecture and reproductive isolation between lineages of Ganaspis near brasiliensis that appear to be cryptic species. We studied five populations, two from China, two from Japan, and one from Canada, identified as the G1 vs G3 lineages based on differences in cytochrome oxidase l sequences. We assembled and annotated the genomes of these populations and analyzed divergences in sequence and genome architecture between them. We also report results from crosses to test reproductive compatibility between the G3 lineage from China and the G1 lineage from Japan. The combined results on sequence divergence, differences in genome architectures, ortholog divergence, reproductive incompatibility, differences in host ranges and microhabitat preferences, and differences in morphology show that these lineages are different species. Thus, the decision to evaluate the lineages separately and only import and introduce the more host-specific lineage to North America and Europe was appropriate.

Coordination of SARS-CoV-2 wastewater and clinical testing of university students demonstrates the importance of sampling duration and collection time.

Wastewater surveillance has been a useful tool complementing clinical testing during the COVID-19 pandemic. However, transitioning surveillance approaches to small populations, such as dormitories and assisted living facilities poses challenges including difficulties with sample collection and processing. Recently, the need for reliable and timely data has coincided with the need for precise local forecasting of the trajectory of COVID-19. This study compared wastewater and clinical data from the University of Delaware (Fall 2020 and Spring 2021 semesters), and evaluated wastewater collection practices for enhanced virus detection sensitivity. Fecal shedding of SARS-CoV-2 is known to occur in infected individuals. However, shedding concentrations and duration has been shown to vary. Therefore, three shedding periods (14, 21, and 30 days) were presumed and included for analysis of wastewater data. SARS-CoV-2 levels detected in wastewater correlated with clinical virus detection when a positive clinical test result was preceded by fecal shedding of 21 days (p< 0.05) and 30 days (p < 0.05), but not with new cases (p = 0.09) or 14 days of shedding (p = 0.17). Discretely collected wastewater samples were compared with 24-hour composite samples collected at the same site. The discrete samples (n = 99) were composited examining the influence of sampling duration and time of day on SARS-CoV-2 detection. SARS-CoV-2 detection varied among dormitory complexes and sampling durations of 3-hour, 12-hour, and 24-hour (controls). Collection times frequently showing high detection values were between the hours of 03:00 to 05:00 and 23:00 to 08:00. In each of these times of day 33% of samples (3/9) were significantly higher (p < 0.05) than the control sample. The remainder (6/9) of the collection times (3-hour and 12-hour) were not different (p > 0.05) from the control. This study provides additional framework for continued methodology development for microbiological wastewater surveillance as the COVID-19 pandemic progresses and in preparation for future epidemiological efforts.

Long-read isoform sequencing reveals tissue-specific isoform expression between active and hibernating brown bears (Ursus arctos).

Understanding hibernation in brown bears (Ursus arctos) can provide insight into some human diseases. During hibernation, brown bears experience periods of insulin resistance, physical inactivity, extreme bradycardia, obesity, and the absence of urine production. These states closely mimic aspects of human diseases such as type 2 diabetes, muscle atrophy, as well as renal and heart failure. The reversibility of these states from hibernation to active season enables the identification of mediators with possible therapeutic value for humans. Recent studies have identified genes and pathways that are differentially expressed between active and hibernation seasons in bears. However, little is known about the role of differential expression of gene isoforms on hibernation physiology. To identify both distinct and novel mRNA isoforms, full-length RNA-sequencing (Iso-Seq) was performed on adipose, skeletal muscle, and liver from three individual bears sampled during both active and hibernation seasons. The existing reference genome annotation was improved by combining it with the Iso-Seq data. Short-read RNA-sequencing data from six individuals were mapped to the new reference annotation to quantify differential isoform usage (DIU) between tissues and seasons. We identified differentially expressed isoforms in all three tissues, to varying degrees. Adipose had a high level of DIU with isoform switching, regardless of whether the genes were differentially expressed. Our analyses revealed that DIU, even in the absence of differential gene expression, is an important mechanism for modulating genes during hibernation. These findings demonstrate the value of isoform expression studies and will serve as the basis for deeper exploration into hibernation biology.

Improved performance of the PacBio SMRT technology for 16S rDNA sequencing.

Improved sequencing accuracy was obtained with 16S amplicons from environmental samples and a known pure culture when upgraded Pacific Biosciences (PacBio) hardware and enzymes were used for the single molecule, real-time (SMRT) sequencing platform. The new PacBio RS II system with P4/C2 chemistry, when used with previously constructed libraries (Mosher et al., 2013) surpassed the accuracy of Roche/454 pyrosequencing platform. With accurate read lengths of >1400 base pairs, the PacBio system opens up the possibility of identifying microorganisms to the species level in environmental samples.

Efficacy of a 3rd generation high-throughput sequencing platform for analyses of 16S rRNA genes from environmental samples.

Longer sequences of the bacterial 16S rRNA gene could provide greater phylogenetic and taxonomic resolutions and advance knowledge of population dynamics within complex natural communities. We assessed the accuracy of a Pacific Biosciences (PacBio) single molecule, real time (SMRT) sequencing based on DNA polymerization, a promising 3rd generation high-throughput technique, and compared this to the 2nd generation Roche 454 pyrosequencing platform. Amplicons of the 16S rRNA gene from a known isolate, Shewanella oneidensis MR1, and environmental samples from two streambed habitats, rocks and sediments, and a riparian zone soil, were analyzed. On the PacBio we analyzed ~500 bp amplicons that covered the V1-V3 regions and the full 1500 bp amplicons of the V1-V9 regions. On the Roche 454 we analyzed the ~500 bp amplicons. Error rates associated with the isolate were lowest with the Roche 454 method (2%), increased by more than 2-fold for the 500 bp amplicons with the PacBio SMRT chip (4-5%), and by more than 8-fold for the full gene with the PacBio SMRT chip (17-18%). Higher error rates with the PacBio SMRT chip artificially inflated estimates of richness and lowered estimates of coverage for environmental samples. The 3rd generation sequencing technology we evaluated does not provide greater phylogenetic and taxonomic resolutions for studies of microbial ecology.

A microRNA of infectious laryngotracheitis virus can downregulate and direct cleavage of ICP4 mRNA.

Viral microRNAs regulate gene expression using either translational repression or mRNA cleavage and decay. Two microRNAs from infectious laryngotracheitis virus (ILTV), iltv-miR-I5 and iltv-miR-I6, map antisense to the ICP4 gene. Post-transcriptional repression by these microRNAs was tested against a portion of the ICP4 coding sequence cloned downstream of firefly luciferase. Luciferase activity was downregulated by approximately 60% with the iltv-miR-I5 mimic. Addition of an iltv-miR-I5 antagomiR or mutagenesis of the target seed sequence alleviated this effect. The iltv-miR-I5 mimic, when co-transfected with a plasmid expressing ICP4, reduced ICP4 transcript levels by approximately 50%, and inhibition was relieved by an iltv-miR-I5 antagomiR. In infected cells, iltv-miR-I5 mediated cleavage at the canonical site, as indicated by modified RACE analysis. Thus, in this system, iltv-miR-I5 decreased ILTV ICP4 mRNA levels via transcript cleavage and degradation. Downregulation of ICP4 could impact the balance between the lytic and latent states of the virus in vivo.

MicroRNAs of Gallid and Meleagrid herpesviruses show generally conserved genomic locations and are virus-specific.

Many herpesviruses, including Marek's disease viruses (MDV1 and MDV2), encode microRNAs. In this study, we report microRNAs of two related herpesviruses, infectious laryngotracheitis virus (ILTV) and herpesvirus of turkeys (HVT), as well as additional MDV2 microRNAs. The genome locations, but not microRNA sequences, are conserved among all four of these avian herpesviruses. Most are clustered in the repeats flanking the unique long region (I/TR(L)), except in ILTV which lacks these repeats. Two abundant ILTV microRNAs are antisense to the immediate early gene ICP4. A homologue of host microRNA, gga-miR-221, was found among the HVT microRNAs. Additionally, a cluster of HVT microRNAs was found in a region containing two locally duplicated segments, resulting in paralogous HVT microRNAs with 96-100% identity. The prevalence of microRNAs in the genomic repeat regions as well as in local repeats suggests the importance of genetic plasticity in herpesviruses for microRNA evolution and preservation of function.

Sequence conservation and differential expression of Marek's disease virus microRNAs.

Marek's disease virus (MDV), a herpesvirus that causes a lymphoproliferative disorder in chickens, encodes a number of microRNAs derived primarily from two locations in the MDV genome. One cluster of microRNA genes flanks the meq oncogene, and a second cluster is found within the latency-associated transcript (LAT) region. The sequences of MDV microRNAs from a collection of field and reference strains with various levels of virulence were compared and found to be highly conserved. However, microRNAs from the meq cluster were detected at higher levels in lymphomas caused by a form of the virus designated very virulent plus (vv+; strain 615K, also known as T. King) than in those caused by a less virulent (very virulent [vv]) form (RB1B). For example, levels of mdv1-miR-M4, which shares a seed sequence with miR-155, a microRNA implicated in B-cell lymphoma, were threefold higher and levels of mdv1-miR-M2*/3p were more than sixfold higher in vv+ MDV-induced tumors than in vv MDV-induced tumors. In contrast, levels of the microRNAs from the LAT cluster were equivalent in tumors produced by vv and vv+ strains. Additionally, mdv1-miR-M4 is the MDV microRNA most highly expressed in tumors, where it accounts for 72% of all MDV microRNAs, as determined by deep sequencing. These data suggest that the meq cluster microRNAs play an important role in the pathogenicity of MDV.

Deep sequencing of chicken microRNAs.

BACKGROUND: The use of new, deep sequencing technologies has greatly accelerated microRNA discovery. We have applied this approach to the identification of chicken microRNAs and to the comparison of microRNAs in chicken embryo fibroblasts (CEF) infected with Marek's disease virus (MDV) to those present in uninfected CEF. RESULTS: We obtained 125,463 high quality reads that showed an exact match to the chicken genome. The majority of the reads corresponded to previously annotated chicken microRNAs; however, the sequences of many potential novel microsRNAs were obtained. A comparison of the reads obtained in MDV-infected and uninfected CEF indicates that infection does not significantly perturb the expression profile of microRNAs. Frequently sequenced microRNAs include miR-221/222, which are thought to play a role in growth and proliferation. A number of microRNAs (e.g., let-7, miR-199a-1, 26a) are expressed at lower levels in MDV-induced tumors, highlighting the potential importance of this class of molecules in tumorigenesis. CONCLUSION: Deep sequencing technology is highly suited for small RNA discovery. This approach is independent of comparative sequence analysis, which has been the primary method used to identify chicken microRNAs. Our results have confirmed the expression of many microRNAs identified by sequence similarity and identified a pool of candidate novel microRNAs.

Marek's disease virus encodes MicroRNAs that map to meq and the latency-associated transcript.

MicroRNAs (miRNAs) are a class of small (approximately 22-nucleotide) regulatory molecules that block translation or induce degradation of target mRNAs. These have been identified in a wide range of organisms, including viruses. In particular, the oncogenic gammaherpesviruses Kaposi's sarcoma herpesvirus and Epstein-Barr virus encode miRNAs that could potentially regulate either viral or host genes. To determine if Marek's disease virus (MDV), an oncogenic alphaherpesvirus of chickens, encodes miRNAs, we isolated small RNAs from MDV-infected chicken embryo fibroblasts (CEF) and used the 454 Life Sciences sequencing technology to obtain the sequences of 13,679 candidate host and viral small RNAs. Eight miRNAs were found, five of which flank the meq oncogene and three that map to the latency-associated transcript (LAT) region of the genome. The meq gene is unique to pathogenic serotypes of MDV and is transcriptionally active during latency and transformation, and the LAT region of the MDV genome is antisense to the immediate-early gene ICP4. Secondary structure analysis predicted that the regions flanking the miRNAs could form hairpin precursors. Northern blot analysis confirmed expression of all miRNAs in MDV-infected CEF, MDV-induced tumors, and MDV lymphoblastoid cell lines. We propose that the MDV miRNAs function to enable MDV pathogenesis and contribute to MDV-induced transformation of chicken T cells.

Chicken genomics resource: sequencing and annotation of 35,407 ESTs from single and multiple tissue cDNA libraries and CAP3 assembly of a chicken gene index.

Its accessibility, unique evolutionary position, and recently assembled genome sequence have advanced the chicken to the forefront of comparative genomics and developmental biology research as a model organism. Several chicken expressed sequence tag (EST) projects have placed the chicken in 10th place for accrued ESTs among all organisms in GenBank. We have completed the single-pass 5'-end sequencing of 37,557 chicken cDNA clones from several single and multiple tissue cDNA libraries and have entered 35,407 EST sequences into GenBank. Our chicken EST sequences and those found in public databases (on July 1, 2004) provided a total of 517,727 public chicken ESTs and mRNAs. These sequences were used in the CAP3 assembly of a chicken gene index composed of 40,850 contigs and 79,192 unassembled singlets. The CAP3 contigs show a 96.7% match to the chicken genome sequence. The University of Delaware (UD) EST collection (43,928 clones) was assembled into 19,237 nonredundant sequences (13,495 contigs and 5,742 unassembled singlets). The UD collection contains 6,223 unique sequences that are not found in other public EST collections but show a 76% match to the chicken genome sequence. Our chicken contig and singlet sequences were annotated according to the highest BlastX and/or BlastN hits. The UD CAP3 contig assemblies and singlets are searchable by nucleotide sequence or key word (http://cogburn.dbi.udel.edu), and the cDNA clones are readily available for distribution from the chick EST website and clone repository (http://www.chickest.udel.edu). The present paper describes the construction and normalization of single and multiple tissue chicken cDNA libraries, high-throughput EST sequencing from these libraries, the CAP3 assembly of a chicken gene index from all public ESTs, and the identification of several nonredundant chicken gene sets for production of custom DNA microarrays.