Cuscuta (dodders). Organelle genomes were generally used for studying plant phylogeny and genomic variations. Notably, plastomes in parasitic plants always show non-canonical features, such as reduced size and accelerated rates. However, few organelle genomes of this group have been sequenced, hindering our understanding of their evolution, and dodder mitogenome in particular.The morning glories (Convolvulaceae) are distributed worldwide and produce economically important crops, medicinal herbs, and ornamentals. Members of this family are diverse in morphological characteristics and trophic modes, including the leafless parasitic Cuscuta remained uncertain. The mitogenome was significantly inflated in Cuscuta japonica, which has exceeded over 800 kb and integrated massive DNAs from other species. In other dodders, mitogenomes were maintained in small size, revealing divergent evolutionary strategies. Mutations unique to plants were detected in the mitochondrial gene ccmFc, which has broken into three fragments through gene fission and splicing shift. The unusual changes likely initially happened to the common ancestor of the family and were caused by a foreign insertion from rosids followed by double-strand breaks and imprecise DNA repairs. The coding regions of ccmFc expanded at both sides after the fission, which may have altered the protein structure.We assembled 22 new mitogenomes and 12 new plastomes in Convolvulaceae. Alongside previously known ones, we totally analyzed organelle genomes of 23 species in the family. Our sampling includes 16 leafy autotrophic species and 7 leafless parasitic dodders, covering 8 of the 12 tribes. Both the plastid and mitochondrial genomes of these plants have encountered variations that were rarely observed in other angiosperms. All of the plastomes possessed atypical IR boundaries. Besides the gene and IR losses in dodders, some leafy species also showed gene and intron losses, duplications, structural variations, and insertions of foreign DNAs. The phylogeny reconstructed by plastid protein coding sequences confirmed the previous relationship of the tribes. However, the monophyly of ‘Merremieae’ and the sister group of Our family-scale analyses uncovered unusual scenarios for both organelle genomes in Convolvulaceae, especially in parasitic plants. The data provided valuable genetic resources for studying the evolution of Convolvulaceae and plant parasitism.The online version contains supplementary material available at 10.1186/s12915-022-01250-1. Convolvulaceae is cosmopolitan and exhibits a rich diversity of morphological characteristics comprising herbs, shrubs, trees, climbers, and leafless parasitic plants Cuscuta (dodders).Convolvulaceae Juss., also known as the morning glories and bindweeds, is a large family belonging to the order Solanales of the eudicots. It contains approximately 1900 species from 59 genera [Ipomoea [Evolvulus alsinoides [Ipomoea nil, which was obtained from the whole genome sequencing project [In the past decades, species in Convolvulaceae were classified into 12 tribes and six clades using several DNA markers . With grIpomoea –8 and doIpomoea –12. ManyIpomoea , 13. Addsinoides  and the  project .rpl2 in Convolvulaceae plastomes was a synapomorphy and a unique event in Asteridae [infA has been lost from the plastome, and inverted repeat (IR) boundaries have been altered in Ipomoea and dodders [I. nil is only 266 kb in length, which is much smaller than most known mitogenomes in angiosperms. Moreover, annotation of the mitogenome (GenBank ID: AP017303) reveals a very unusual ccmFc gene, with fragments located in three different positions. Further studies are required to reconcile these findings. Plastomes of dodders, like many other heterotrophic plants , ex, and two from the subgenus Grammica . These new plastomes showed similar features to the known ones in Cuscuta, including the losses of genes, introns, and IR regions . Rps16 and rpoC1 in Evolvulus, and rps16 and ycf3 in Dichondra have experienced intron losses. InfA was lost in Convolvulaceae, and BLASTP searches in Convolvulaceae nuclear peptides have confirmed the transfer to nucleus, like many other species [Our assemblies include at least 12 new plastomes, among which six genera were sequenced for the first time  region and the IR region, respectively , so their origins were mysterious. Foreign DNAs in plastomes were mainly known to be transferred from mitogenome, such as in Apiaceae [Dinetus and Dichondra plastomes could represent two additional examples of non-mitochondrial origin.Apiaceae –25, ApocApiaceae , AnacardApiaceae , OrobancApiaceae , and PoaApiaceae –32, whilCuscuta epilinum, Cu. europaea, and Cu. japonica, while only drafts were obtained for the four dodders from subgenus Grammica  due to large amounts of repeats. Mitogenomes in Convolvulaceae are mostly around 300 kb in length, which is shorter than most angiosperms, including their close relatives Solanales. The gene content of Convolvulaceae mitogenomes is similar to other angiosperms . Compared to Solanales, rpl2 and sdh3 were missing in all family members investigated. The rps7 was only found in I. biflora, and phylogenetic analysis indicated that it was closest to Rosales . Therefore, the rps7 most likely have been lost in the common ancestor of Convolvulaceae and I. biflora re-gained it by horizontal gene/DNA transfer (HGT). Mitogenomes of the Grammica were also the most degenerated in the family—they lost more genes, similar to their plastomes. Cu. epilinum, Cu. europaea, and Cu. japonica showed no significant differences in gene content  while its relative V. album outnumbers it 8.6-fold in size [V. scurruloideum evolved in an opposite direction, and how they maintained a small mitogenome are intriguing questions to be answered in the future.The parasitic plants have evolved at least 12 times independently in angiosperms , 34, whiCuscuta , 11, 18.oriaceae , 40;), Lhoraceae , 42;, Vitalaceae ;), and Anchaceae ;). There in size , 46. WhyOperculina macrocarpa (GenBank: KF242502) and Cressa cretica (NC_035516) were included. The plastid matrix contained 77,711 columns with 16,689 parsimony informative sites (PIS). The plastid tree has a similar topology to previous studies [Merremia and Operculina, were nested with Convolvuleae. Merremieae was suggested as polyphyletic [Sequences of plastid protein coding sequences (CDS), mitochondrial CDS, and nuclear 45S  were employed to build the maximum-likelihood (ML) trees of Convolvulaceae  system and is involved in the final stage of the maturation process [c-type cytochrome is an essential component of the mitochondrial electron transport chain and delivers electrons between complexes III and IV. Typically, the gene contains two exons and a group II cis-splicing intron, and the structure and sequence of the gene are remarkably conserved in almost all angiosperms. Differently, in all members of Convolvulaceae, it has been divided into three fragments. The first break occurred close to the 3′-end of the first exon, causing a ca. 43 aa loss (between positions 164 to 206 in tomato); the second break occurred within the intron without base losses . The second (FR2) and third (FR3) fragments were still connected in leafy species, with a ca. 3.5 kb insertion (2.8 kb of plastid origin) in between; in Cu. epilinum and Cu. europaea, the insertion was reduced to ca. 1.3 kb; while in other dodders, the connection was broken, and FR2 and FR3 had become separated.The genomic distribution of the three ies Fig. a. In leaccmFc fragments in Convolvulaceae probably implies they are still functional. We searched for DNA and amino acid sequences of ccmFc in known Convolvulaceae genomes to detect potential nuclear transfers, but no high-confidence matches were obtained. We then mapped the rRNA depletion transcriptome of sweet potato to the mitogenome to check for evidence of active expression. All three fragments yielded high expression levels , whereas FR2 and FR3 are two exons of another gene . To validate this, we synthesized cDNA of sweet potato, Dichondra, Cu. australis, Cu. japonica, tomato, and Arabidopsis from mRNA using reverse transcriptase (RT) and performed PCR analyses. Primer pair “F1 + R1” yielded a band in tomato and Arabidopsis but not Convolvulaceae species . Additionally, after the break, FR1 was separated from the stop codon. Thus, it required a new stop codon downstream; similarly, FR2 required a new initiation codon upstream. However, we determined that the closest stop and ATG initiation codons were far away, resulting in a ca. 185 and 118 aa expansion of ccmFc1 and ccmFc2, respectively. New RNA editing sites were absent in the expanded genic regions. We cloned cDNA to confirm the gene expansion  revealeFc2 Fig. d. The brcis- to trans-splicing remains unclear. What’s This Factor 9 (WTF9), a nuclear-encoded plant organelle RNA recognition protein, can bind directly to the 48 nucleotides of the ccmFc intron to regulate cis-splicing [trans-splicing shift, we used Arabidopsis WTF9 and HSP60 as references to search for homologs in genomes of Convolvulaceae and other angiosperms. WTF9 is retained in all species of Convolvulaceae and even duplicated in Ipomoea . Based on these, we speculated that the mechanism between cis- and trans-splicing might be similar, but further research is needed to test this hypothesis.The mechanism of the shift from splicing , 52. TheTrans-splicing has evolved independently several times in land plants, such as in lycophytes and gymnosperms [trans-splicing was less frequent. Aside from nad1, nad2, and nad5, only cox2 in Allium was reported to undergo trans-splicing [ccmFc in Convolvulaceae represents a novel type of trans-splicing in angiosperms. Gene fission has also occurred several times in plant mitogenomes, such as ccmFc in Marchantia and ccmFn in Brassicaceae, Allium, and Trifolium, caused either by deletion-based frameshift mutation or rearrangements [ccmFc in Convolvulaceae might be the most complicated, integrating gene fission, splicing shift, and expansions. We also searched the downstream of FR1 and upstream of FR2 against the NCBI nt database, finding that both were most similar to the mitochondrial sequences of rosids after masked hits of Convolvulaceae  repair, plant mitochondrial genes evolve more slowly than other genome compartments , 54. Thenosperms –60. In asplicing –64. Our ngements –68. As feae Fig. . We hyponctional , 71. In nctional , 72. Thec maturation can optimize extracellular electron transfer for higher electron flux in engineered Escherichia coli [An immediate question is how these changes would modify the structure of the CCM complex or even the efficiency of oxidative phosphorylation. The elevated substitution rates and potential changes in transmembrane helices may have affected the protein structure. Recent studies have shown that modifying cytochrome hia coli . Our rese family Convolvulaceae is famous for its extensive uses, diverse morphologies, and different trophic modes. In this study, our family-scale analyses revealed that the same amazement also exists in their genetic materials. Plastids (including chloroplasts) and mitochondria play crucial roles in cellular energy supply and metabolism. Normally the structure and genes of plastome and coding sequences of mitogenome are well-conserved. However, it has some changes in Convolvulaceae. Even in green leafy members, the IR boundaries are all atypical, and uncommon features such as intron losses, nuclear transfer (infA), duplications, and foreign insertions also exist. The mitogenome of Cu. japonica was significantly inflated by HGT-like sequences, whereas other dodders preserved in a small size like their leafy relatives—the mitogenome evolves divergently in dodders. Besides, dodder mitochondrial genes have more losses and higher substitution rates, but no HGT events. The most notable variation is the mitochondrial ccmFc. It is highly conserved in other plants but likely encountered gene fission, splicing shift, and coding expansion in the common ancestor of Convolvulaceae, which is unique in angiosperms. Nuclear genes involved in the splicing process seems still functional, raising the question of what differs between cis- and trans-splicing on mechanism. These unusual changes of ccmFc were likely caused by HGT insertion followed by double-strand breaks and imprecise repairs. Our data provide valuable genetic resources for studying the evolution and phylogeny of Convolvulaceae and plant parasitism. The results presented in this study are also helpful to understand the diversification of mitochondrial complexes and gene innovation.ThDichondra micrantha was also sequenced using the Oxford Nanopore promethION platform. Raw sequencing data for the other 10 species were obtained from NCBI SRA (https://www.ncbi.nlm.nih.gov/sra). Details of the reads are provided in Additional file Accessions of 12 Convolvulaceae species were collected from different places in China  as the reference. The assembly of mitogenomes was as described in our previous work [I. nil mitogenome. These contigs were connected and/or extended manually in GENEIOUS R10  by mapping reads back and checking both ends. Long repeats and plastid insertions were the two major challenges of the assembly. Repeat regions were resolved using short-read sequencing coverage; plastid insertions were resolved based on the locations of their plastid counterparts. The mitogenome could get one or several circles after the repeats and plastid insertions were properly determined. Cu. japonica yielded one linear chromosome since repeats at both ends could not be connected. The four dodders from subgenus Grammica  only obtained draft mitogenomes because of the large amounts of repeats. Putative mitochondrial protein-coding and rRNA genes were annotated based on similarity to known mitochondrial genes. tRNAs were predicted using tRNAscan-SE v2.0 [e-value 1e-5), and then counted using custom scripts.Short reads were filtered using TRIMMOMATIC v0.36 . Plastomous work . Brieflyous work  and FLYEous work , respect-SE v2.0 . Plastid-SE v2.0  against Ipomoea and Cuscuta, GSS also included the species-specific sequences. GSS were identified by searching each mitogenome against other Convolvulaceae mitogenomes, with e-value of 1e-5 and word size of 16. The GSS of Cu. japonica, Dichondra, and Evolvulus were searched against NCBI nt database to find the most potential donors, with only hits longer than 100 bp considered. Subsequently, the best matches were 184 grouped into orders . To test whether other dodders have experienced expansion, high-confidence HGT-like sequences of Cu. japonica (best hits >500 bp) were searched against other dodders, with the percentage of the coverage calculated. Finally, the heatmaps were generated using PHEATMAP (https://github.com/raivokolde/pheatmap) in R.Plastome (IR removed) and mitogenome syntenies were plotted using Python version MCscan of JCVI 192 utility libraries v1.1.17 . The DNA-bb 1000 -m GTR+G4+F -me 0.0001 runs 5. The trees were plotted using FIGTREE v.1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/). GenBank accessions were listed behind the species on the trees , I. nil (NC_031159), I. trifida (NC_034670), I. triloba (NC_037913), Cressa cretica (NC_035516), Cuscuta australis (NC_045885), Cu. campestris (NC_052920), Convolvulus arvensis (MW054627), Operculina macrocarpa (KF242502), Nicotiana tabacum (Z00044), Solanum melongena (MN218080), and Solanum lycopersicum (NC_007898). Mitochondrial CDS were obtained from our assemblies and I. nil (AP017303), N. tabacum (NC_006581), S. lycopersicum (NC_035963), and S. melongena (NC_050334). RNA editing sites were predicted using the PREP website  and removed manually. The nuclear 45S were obtained from de novo contigs and Nicotiana benthamiana (KP824745). Aligned plastid and mitochondrial (rps7 was excluded from an HGT) CDS were concatenated into one matrix, respectively. Then the three datasets  were built the ML trees as mentioned above. Solanaceae were always used as outgroups.The plastid CDS were obtained from our assemblies and I. batatas) were downloaded from ProteomeXchange PXD012999 [Protein mass spectrometry data for sweet potato , HSP60-2 (AT2G33210), HSP60-3A (AT3G13860), and HSP60-3B (AT3G23990) were used as references to search for homologs in Convolvulaceae, including sweet potato [I. trifida and I. triloba [I. nil [Cuscuta australis [Cu. campestris [Nicotiana tabacum [Amborella trichopoda v1.0 [Daucus carota v2.0 [Populus trichocarpa v3.0 [Oryza sativa v7 [Solanum lycopersicum iTAG2.4 [Solanum tuberosum v4.03 [Zostera marina v2.2 [https://phytozome.jgi.doe.gov/). The amino acid sequences were then aligned through MAFFT, and trees were then reconstructed using IQTREE (parameter: -bb 1000 -m LG+F+G4 runs 10 -me 0.0001).Amino acid sequences of t potato , I. trif3A AT3G130, and HS [I. nil , Cuscutaustralis , and Cu.9 AT3G243, HSP60-2 tabacum , Amboreloda v1.0 , Daucus ota v2.0 , PopulusB AT3G239 were useativa v7 , tomato  iTAG2.4 , potato um v4.03 , and Zosina v2.2   by normalizing each gene with Z-score.The expression level data of Resource ; those oDatabase , and thoDatabase . HeatmapTranscriptomes enriched with oligo-dT would introduce bias since organelle transcripts do not generally contain a polyA tail . Based oDichondra, tomato, Arabidopsis, and fresh vines of Cuscuta australis and Cu. japonica were used for RNA extraction using an Omega RNA isolation kit and reverse-transcribed into cDNA using random primers. Primers used for RT-PCR were designed using GENEIOUS (Additional file Fresh leaves of sweet potato, Additional file 1. Plastome content. The black, grey and blank cells represent intact, pseudo and missing, respectively. Coding genes with disrupted reading frames, premature stop codons, or non-triplet frameshifts were annotated as pseudogenes.Additional file 2. Mitogenome content. The black, grey and blank cells represent intact, pseudo and missing, respectively. Coding genes with disrupted reading frames, premature stop codons, or non-triplet frameshifts were annotated as pseudogenes.Additional file 3. Most mitochondrial genes in dodders.Additional file 4. BLASTN results of Cu. japonica genus- and species-specific sequences.Additional file 5. Plastid CDS, Mitochondrial CDS, and Nuclear 45S.Additional file 6 Similar situation observed for HSP60.Additional file 7. BLASTN searches of the downstream of FR1 and upstream of FR2. Erycibe was used as the query. Parameter: word_size 16, e-value 1e-5.Additional file 8. Collections of materials.Additional file 9. Data used for organelle genome assemblies.Additional file 10. Primers used to confirm gene fission and expansion.


Retropinna retropinna and Gobiomorphus cotidianus. A total of 64 fish were used for gill tissue meta-transcriptomic sequencing, from populations with contrasting life histories—landlocked  and diadromous—on the South Island and Chatham Islands. We observed that both viral richness and taxonomic diversity were significantly associated with life history and host species, with lacustrine R. retropinna characterised by higher viral alpha diversity than diadromous R. retropinna. Additionally, we observed transcripts of fish viruses from 12 vertebrate host-associated virus families, and phylogenetically placed eight novel RNA viruses and three novel DNA viruses in the Astroviridae, Paramyxoviridae, Orthomyxoviridae, Rhabdoviridae, Totiviridae, Poxviridae, Alloherpesviridae, and Adintoviridae in their evolutionary contexts. These results represent an important survey of the viruses that infect two widespread native fish species in New Zealand, and provide insight useful for future fish virus surveys.Viruses that infect fish are understudied, yet they provide important evolutionary context to the viruses that infect terrestrial vertebrates. We surveyed gill tissue meta-transcriptomes collected from two species of native freshwater fish from Aotearoa New Zealand— Fish represent basal evolutionary lineages to all extant terrestrial vertebrate species . Like alwaka carried unique Gondwanan species into ecological isolation, and during this process, early mammalian lineages became extinct [Aotearoa New Zealand separated from the Gondwanan supercontinent ca. 85–82 million years ago as part of the subcontinent Zealandia—known as Te Riu-a-Māui in Te Reo Māori ,5. The Z extinct . This un extinct ,8,9,10. Retropinna retropinna (common smelt) and Gobiomorphus cotidianus (common bully) are two widespread species of native New Zealand fish. Both are facultatively diadromous, normally spawning in freshwater and completing at least part of their life cycle in the sea, but they are also capable of living entirely in freshwater when they become landlocked [R. retropinna is a pelagic fish species found throughout lowland rivers, estuaries, and coastal lakes of New Zealand, including Rēkohu (Chatham Island) [G. cotidianus is a benthic fish species found in rivers and estuaries across the North and South Islands of New Zealand, as well as in isolated freshwater lake populations [ndlocked ,12. R. r Island) . G. cotiulations . The nicR. retropinna and G. cotidianus individuals collected from lacustrine and diadromous populations from the South Island and Rēkohu, Aotearoa New Zealand. In doing so, we identified multiple novel RNA and DNA viruses, and determined whether viral ecology differed among lacustrine and diadromous fish hosts.The objective of this study was to better understand the determinants of virus ecology in diadromous and lacustrine populations of two New Zealand endemic fish species. Using meta-transcriptomics, we surveyed the viromes of The use of native species of Aotearoa New Zealand for research was approved through iwi/imi consultation with the Ngāi Tahu Research Consultation Committee (University of Otago) and the Hokotehi Moriori Trust (Chatham Island). Animal ethics approval was obtained from the University of Otago Animal Ethics Committee .R. retropinna were collected from the Taieri River  on the South Island and Te Awainanga River  on Rēkohu, and lacustrine R. retropinna were collected from Lake Rotorua  and Tennants Lake , both on Rēkohu  and Otokaia Creek , and lacustrine G. cotidianus were collected from Lake Hayes  and Lake Wanaka   a.In total, 64 fish were captured using fyke nets or seine nets and transported to an indoor laboratory space within one hour of collection. Samples were collected as part of a larger study to examine gene expression changes in response to fresh and salt water, rather than specifically for virome analysis. For this reason, four fish were individually placed in 4 L containers of freshwater from their site of collection. Another four fish from the same cohort were gradually transferred to 4 L containers of 30 parts per thousand salt water made by combining source water with artificial sea salt . Fish were housed in fresh or salt water with aeration for 12 h prior to sacrifice. Fish were sacrificed with AQUI-S 20 E , length and weight measurements were taken, and gill tissue was then immediately excised, placed in RNA stabilisation solution , and frozen at –80 °C within one week. Tissue was stored at −80 °C until RNA was extracted.RNA was extracted from frozen tissue samples using the RNeasy Mini Kit (Qiagen). Total RNA was shipped in Bio-Bottles  on dry ice to Novogene (Singapore) where RNA sequencing libraries were prepared with poly-A enrichment to increase the proportion of mRNA, and indexed and pooled for 150 bp paired-end sequencing on an Illumina NovaSeq 6000.−5 was used for filtering viral transcripts, and a palmscan -hiconf high confidence threshold was used for filtering the identified RdRps. Putative viral transcripts identified in this way were then assigned to vertebrate -associated viruses or non-vertebrate (non-fish) viruses based on the best BLASTn/Diamond matches, as well as the associated taxonomic information in the NCBI taxonomy database [Virome assembly and analysis used a previously described analysis pipeline . Brieflydatabase  in conjudatabase . In instViral transcript abundances were estimated using the Trinity implementation of RNA-Seq by Expectation Maximization (RSEM) . Viral tTranslated viral contigs protein sequences of RdRp or DNA-dependent DNA polymerase (DdDp) were aligned with homologous representative protein sequences from viruses within the same viral family using MAFFT v7.450  . Amino aData handling and plotting generally used R (4.0.3) and the tidyverse . SamplinStatistical comparison of observed richness and an inverse Simpson index between life-history types (lacustrine versus diadromous) were stratified by fish species and modelled using generalised linear models (glm). For observed richness, hurdled glms were used that first tested the frequency of observing viral transcripts using a binomial family distribution, and secondly modelled the effect on observed richness in samples with richness > 0 using a Poisson family distribution. To test for effects of life history and species on an inverse Simpson index, Tweedie family glms were used to deal with the zero-inflated nature of the data, and used a gamma family distribution for generalisation. In both cases, log link functions were used to relate the generalised distributions to the linear predictors.Compositions of vertebrate-associated viral communities were compared using multivariate ordination methods. Analysis of homoscedasticity between test groupings was done using a Bray–Curtis distance matrix modelled in the betadisper function in vegan and tested with permutational tests for grouping species and life history, and the Tukey honest significant differences test for the source. To test for effects of species, life-history type, and location on viral composition, we used permutational multivariate analysis of variance with the adonis2 function (implemented in vegan) using the factor species, life history, and source in an NMDS model of Bray–Curtis dissimilarity of standardised abundance. Ordination plotting of standardised abundance used constrained correspondence analysis, also with species, life history, and location. ANOVA-like permutation analysis of the CCA model was used to corroborate the significance of the species, life history, and source as constraints. Ordination plots were generated using the ggord package .We also determined whether the fresh and salt water treatment that the fish were exposed to post-capture had any effect on virome composition following the above methodology.R. retropinna samples collected from Rēkohu/Chatham Island were named using a ta rē Moriori name, porure. Novel viruses identified in the G. cotidianus samples collected from the South Island were named using a Te Reo Māori name for G. cotidianus, Toitoi.Following consultation, novel viruses identified in R. retropinna and 32 G. cotidianus were collected from two lacustrine and two diadromous locations each from across the South Island of New Zealand and Chatham Islands . Porure astrovirus 1 RdRp transcripts were identified in two R. retropinna gill meta-transcriptomes collected from Tennants Lake on the Chatham Islands ; however, the host for this virus is unknown. The most similar astrovirus with a host association was Wenling gobies astrovirus  [We identified a novel positive-sense single-stranded RNA (ssRNA) virus in the c region a, which  Islands . The twoectively . The mosG. cotidianus gill meta-transcriptomes and have named these Toitoi astrovirus 1 and Toitoi astrovirus 2  [Two novel viruses from the avirus 2 b. The Toavirus 2 . Relativectively . Phylogerom fish b. Toitoi37394.1) , sharingParamyxoviridae named Toitoi paramyxovirus 1 was identified in a diadromous G. cotidianus collected in Otokaia Creek. Two transcripts of the virus were recovered with a cumulative length of 13,179 bp and had the highest relative abundance of any vertebrate virus at 0.011% (Bat paramyxovirus (AYM47517.1), sharing 52.76% amino acid identity   . Phylogeposition c.Rhabdoviridae family, termed Porure dimarhabodovirus 1, was identified in a R. retropinna sampled from a diadromous population in the Te Awainanga River on Chatham Island  amino acid identity, indicating Porure totivirus 1 was a novel totivirus species (A novel double-stranded RNA (dsRNA) virus in the ga River e. Transcdividual . We idendividual e. The to species .G. cotidianus fish collected from Waikouaiti River (Poxviridae transcript in the sample containing Toitoi poxvirus 1 was 0.00000014% of reads (Carp edema virus (BCT22690.1) [A novel fish poxvirus, termed Toitoi poxvirus 1, was identified in the gill meta-transcriptome of a ti River a. The reof reads . A 256 bof reads . The bes22690.1)  at 69.0%Alloherpesviridae were identified in G. cotidianus fish from the lacustrine population in Lake Hayes and the diadromous population in Otokaia Creek, ranging in relative abundances from 0.00000008% to 0.00000048% of reads (Percid herpesvirus 2 (AWU46759.1) [Transcripts from viruses in the of reads . Three tof reads . The bes46759.1) , and ranAdintoviridae viruses were identified in R. retropinna fish captured from Lake Tennants, Lake Rotorua, and Te Awainanga River on Rēkohu (Larimichthys croaker adintovirus (DAC81317), ranging from 48.2% to 73.6% amino acid identity (Transcripts from two novel n Rēkohu a. These n Rēkohu . Five ofn Rēkohu . Phylogen Rēkohu c. The toidentity .p = 0.813). The mean vertebrate host-associated viral richness was 1.53 (±0.834 SD) in R. retropinna, and 1.35 (±0.489 SD) in G. cotidianus. Fish species did have a significant association with the frequency of identifying vertebrate host-associated viral transcripts  (G. cotidianus and 46.9% of R. retropinna samples (p = 0.035) in R. retropinna (2.04 ± 1.43 SD) and G. cotidianus (1.53 ± 0.915 SD) gill-tissue meta-transcriptomes  was also observed for fish species (p = 0.0897)  [A summary of normalised viral abundances for viral transcripts identified across the gill-tissue meta-transcriptomes of the 64 fish sampled in this study is presented in = 0.023) a, with v samples . Fish spriptomes b. Non-ve species c. No sig 0.0897) d. Spearm100,000)  indicateR. retropinna inverse Simpson index for vertebrate host-associated viruses  , with lacustrine R. retropinna having lower richness than diadromous  c, with l 0.0493) c. Converadromous b. No sigp = 0.203) or life history groups , but did identify a significant difference in the dispersion of samples, with the Tairie source group being significantly different in dispersion from all other sources at p < 0.05. The adonis2 test indicated that species , life history , and source   had significant associations with beta diversity. Ordination of vertebrate-associated viromes using constrained correspondence analysis (CCA) . The CCA ordination plots illustrated the significant effects of the factor species  , and a p species a, life h species b, and so species c, as R. R. retropinna samples from Te Awainanga River clustered in proximity to the Totiviridae and Rhabdoviridae centroids. The diadromous G. cotidianus sources, Otokaia Creek and Waikouaiti River, were distinct from the lacustrine G. cotidianus collected from Lake Hayes and Lake Wanaka. The Waikouaiti River individuals clustered near the Hantaviridae and Orthomyxoviridae centroids, while the Otokaia Creek samples clustered near the Paramyxoviridae centroid. The Astroviridae centroid was clustered with the Lake Hayes, Lake Wanaka, Otokaia Creek, and Lake Tennants samples, but not Waikouaiti Creek or Te Awainanga River, representative of the frequency of Astoroviridae viral transcripts identified within the fish gill-tissue meta-transcriptomes (Sampling location (source) had the strongest influence on virome composition according to the adonis2 analysis, which can be observed in the CCA ordination of sample betadiversity c. Diadroriptomes c. In genImportantly, we found no association between fresh and salt water treatment on vertebrate- or non-vertebrate-associated virus, inverse Simpson index, beta diversity, nor on viral transcript abundance.R. retropinna and G. cotidianus gill-tissue meta-transcriptomes, we phylogenetically placed eight novel fish RNA viruses  and therefore different viral ecology may be at play. In addition, our analysis of sample dispersion indicated that lacustrine and diadromous populations had significantly different taxonomic compositions of vertebrate host-associated viromes (Notably, the viromes of freshwater fish from lacustrine and diadromous populations differed in their composition . Specifi viromes b, in turG. cotidianus and R. retropinna vertebrate host-associated viromes varied significantly in their composition (G. cotidianus is a benthic fish that lives and feeds on the bottom of aquatic ecosystems, while R. retropinna is a pelagic fish living in the water column [Overall, position a. This dr column ,12. It iG. cotidianus (Finally, we observed Toitoi astrovirus 1 in the lacustrine Lake Hayes and Lake Wanaka populations as well as the diadromous Otokaia Creek population of tidianus . Lake Ha


KMT2A gene (NM_001197104.2). In this article, we discuss a 5-year-old boy who has mild intellectual disability (ID), hypotonia, HC, hypertrichosis on the back, dysmorphic facies, psychomotor retardation, and growth delay. Trio-based whole-exome sequencing (trio-WES) was carried out on this patient and his parents, confirming the variants with Sanger sequencing. Trio-WES showed a de novo mutation of the KMT2A gene . On the basis of the clinical features and the results of the WES, WSS was diagnosed. Therefore, medical professionals should consider a diagnosis of WSS if patients have growth retardation and development delay as well as hirsutism, particularly HC.Wiedemann–Steiner syndrome (WSS) is a rare genetic disorder. Patients with WSS have characteristics of growth retardation, facial dysmorphism, hypertrichosis cubiti (HC), and neurodevelopmental delays. WSS is in an autosomal dominant inherited pattern caused by a mutation of the  Wiedemann–Steiner syndrome (WSS) (OMIM: #605130) is an autosomal dominant genetic disease. It was first described by Wiedemann et al. in 1989 . PatientKMT2A gene  (NM_001197104.2), which was found in 2012 by Jones et al. [KMT2A gene (NM_001197104.2) is located on chr11q23.3 and encodes a histone methyltransferase (HMT) enzyme. This enzyme regulates the gene expression profile in early embryonic development and hematopoiesis [KMT2A have been reported in the Leiden Open Variation Database (https://databases.lovd.nl/shared/genes/KMT2A) accessed on 5 July 2021 [http://www.hgmd.cf.ac.uk/ac/gene.php?gene=KMT2A) accessed on 5 July 2021, 163 variants have been documented. More patients have been diagnosed with WSS using whole-exome sequencing (WES).WSS is caused by the mutation of the opoiesis . To date NM_00119104.2, whuly 2021 ,14. In tKMT2A gene.Until now, the complete WSS phenotype has not been fully understood. In this article, we discuss a 5-year-old Taiwanese boy with the clinical features of WSS using trio-based WES (trio-WES) to identify a de novo missense pathogenic variation of the Z score = −0.55), a body length of 48 cm , and a head circumference of 35 cm . The patient’s parents were non-consanguineous, and they were phenotypically normal. The boy was referred to our genetic clinic due to mild ID and growth delay. At our clinic, the patient’s weight, and height were 16.6 kg , 104.9 cm , respectively. Physical examination showed dysmorphic facies and thick hair  accessed on 5 July 2021, ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) accessed on 5 July 2021, the Genome Aggregation Database (http://gnomad.broadinstitute.org/) accessed on 5 July 2021, the Exome Aggregation Consortium (http://exac.broadinstitute.org/) accessed on 5 July 2021, the 1000 Genomes Project (http://browser.1000genomes.org) accessed on 5 July 2021, and HGMD (http://www.hgmd.cf.ac.uk/) accessed on 5 July 2021. We confirmed the identified variants via Sanger sequencing and interpreted the variants using the guidelines of the American College of Medical Genetics and Genomics (ACMG) [We collected peripheral blood from the patient and his parents for genomic DNA at the age of 5 years. Trio-WES was carried out by the National Health Research Institutes . They used the Illumina HiSeq Xten system. The trimmed reads were mapped to the reference human genome (hg19). We identified the variants using the following databases: the Single Nucleotide Polymorphism Database (s (ACMG) .KMT2A gene , which we confirmed via Sanger sequencing . His symptoms, which are compatible with WSS, include mild ID; growth delay; dysmorphic facies and thick hair; and hypertrichosis on the back, arms, and lower limbs but not on the abdomen.According to a previous study with a cohort of 33 French WSS cases , the resKMT2A gene. It is an important gene expression regulator in early development. HMT regulates multiple Wnt-related and Hox-related genes in histone H3 lysine 4 methylation [HMT is encoded by the hylation . Due to hylation , distinchylation .Patients with language delay and ID require special education classes with regular speech and occupational therapies. If symptoms of hypotonia are noted, patients should have carnitine supplementation . For patients with cardiac anomalies, surgical treatment should be considered. Because of growth retardation, children with WSS should have their pituitary function investigated. If biochemical evidence of growth hormone deficiency is noted, a pituitary magnetic resonance imaging scan should be considered, and patients should receive recombinant human growth hormone treatment. All these treatments can help patients with WSS survive with a better quality of life. WES, a powerful tool used to identify disease-causing variants, allows the relationship between genotype and phenotype to become clearer. Shashi et al.  stated tIn summary, we discussed a 5-year-old Taiwanese boy who was diagnosed with WSS via clinical features and trio-WES. He is the first patient with WSS presented in Taiwan. WSS is a rare genetic disorder that combines with sporadic syndrome and phenotypic heterogeneity. Therefore, our findings can be valuable in genetic diagnosis and mutation-based screening.


Apis mellifera, A. cerana, A. dorsata, and A. florea) in Thailand. All the bee venom extracts and melittins showed more robust antibacterial activities against Gram-positive  than Gram-negative bacteria  or a fungus , while the synthetic melittins also have antimicrobial activity at higher concentrations than the bee venom extract. Furthermore, the A. cerana venom extract showed the highest activity against the tested bacteria, followed by A. mellifera, A. florea, and A. dorsata. Therefore, A. cerana venom may be further developed for use in medical applications as a potential alternative agent against Gram-positive bacteria and antibiotic-resistant bacteria.Bee venom (BV), or apitoxin, is a complex substance produced by a gland in the abdominal cavity of bees. The main component of BV is melittin, which is a largely studied substance due to its biological properties. To date, the most well-known bee venom and melittin are derived from domesticated honey bees, while venom and melittin derived from wild honey bees have been under-investigated. Hence, this study primarily reports the antimicrobial activities of bee venom and synthetic melittin derived from four different honey bee species ( Apis mellifera) venom has been used since the ancient Egyptian era (4000 BC) [The honey bee is an essential economic insect; it is widely used in agriculture as a pollinator and produces several natural products. These products, such as honey, pollen, royal jelly, propolis, and, especially, honey bee venom, or Apitoxin, can be applied in the fields of pharmacy and medicine. Bee venom is produced by the venom glands located in the posterior of the abdomen and is stored in the venom reservoir. The venom is released through the sting apparatus to protect the bee from harm . The Wes4000 BC) . Traditi4000 BC) . Extensi4000 BC) ,5,6,7, a4000 BC) .2) and hyaluronidase); biologically active amines ; and non-peptide components  [Honey bee venom contains a variety of active substances, including peptides ; enzymes  . Melittio acids) , and reso acids)  and is ao acids) .Apis mellifera has been widely studied in Hymenoptera, whereas there is limited information regarding the native honey bee’s venom. Few studies have reported on the antioxidant activity of the crude venom of A. dorsata [A. florea.To our knowledge, the antimicrobial activities of different bee venoms and melittins have not yet been investigated. The venom of  dorsata  and antiA. dorsata, A. mellifera, A. florea, and A. cerana were 95.8 ± 3.2%, 76.5 ± 1.9%, 66.3 ± 8.6%, and 56.8 ± 1.8%, respectively [A. dorsata possesses the largest venom gland and venom sac, followed by A. cerana, A. mellifera, and A. florea. Moreover, the amount and composition of the lipid, protein, carbohydrate, and alkaline phosphatase are usually the highest in A. cerana, followed by A. mellifera and A. florea. The antimicrobial activity of bee venom and melittin from different honey bee species is poorly understood. This study collected crude venom from four honey bee species, and the melittin genes were discovered and used as the template for melittin synthesis. The antimicrobial activity of crude venom and synthetic melittin were compared against bacteria and a fungus.Interestingly, differences between different honey bee species affect the compositions of each of their venoms. The melittin contents vary in different honey bees; for example, the levels in A. cerana are the most effective in antimicrobial activity against Gram-positive bacteria and MRSA as opposed to Gram-negative bacteria and the fungus, followed by A. mellifera, A. florea, and A. dorsata. The crude bee venom showed higher antimicrobial activity compared to synthetic melittin. Based on these results, we suggest that different honey bee species have different melittin amino acid compositions that affect the antimicrobial activity of their venom, and crude bee venom is more effective with regard to antimicrobial activity than melittin alone.Our findings showed that the crude venom and melittin taken from A. florea and A. dorsata). The melittin gene sequences were obtained after PCR amplification of their genomic DNA deposited in GenBank (A. mellifera and A. cerana) as a template for melittin peptides synthesis  was similar to A. cerana (ACM) (96.30%) but different from A. dorsata (ADM) (85.19%) and A. florea (AFM) (77.78%).The multiple sequence alignments of four melittin amino acid sequences were predicted using MUSCLE . The resThe amino acid composition of the melittin sequences was computed using ExPAsy ProtParam. The primary sequence analysis results are shown in Apis species was predicted to contain an a-helix, b-turn, random coil, and extended strand, which revealed that the main structure of melittin is an a-helix , and melittin from A. cerana was found to contain serine (Ser). The change in the amino acid composition in the 18th position from asparagine to serine also changed the secondary structure, particularly in the a-helix region  against nine microorganisms were investigated using a two-fold serial dilution assay. The results showed that the MIC and MBC values of all the crude venoms against the Gram-positive bacteria were lower than those against the Gram-negative bacteria and yeast . Moreover, the crude venoms of A. cerana and A. mellifera possessed higher MIC and MBC values than the crude venoms of A. florea and A. dorsata . The crude venom of A. dorsata displayed low activity compared with the crude venom of A. florea .The antimicrobial activity of crude venom from four honey bee species . While the MIC and MBC/MFC values of AMM showed statistically significant differences with ADM (p = 0.011), the MIC value of AFC was not statistically significant with ADM (p = 0.107).In this experiment, four melittin peptides were synthesized to be tested with nine microorganisms. The MIC and MBC/MFC values of all the melittin peptides against the Gram-positive bacteria were lower in concentration compared to the values with regard to the Gram-negative bacteria and yeast, which are shown in M. luteus and MRSA were inhibited by all the crude venoms or their melittin peptides at lower concentrations than gentamycin. Incredibly, the crude venom of A. mellifera and A. cerana, as well as the melittin peptide of A. florea, showed statistically significant differences in terms of the MIC and MBC/MFC when compared with gentamycin at p < 0.05 , A. mellifera, and A. cerana against C. albicans   . While tA. mellifera and A. cerana shared high similarities but displayed some differences with A. dorsata and A. florea. This investigation suggests that the genetic differences in melittin are due to the evolution of honey bee species. A. mellifera and A. cerana possess a close relationship via their genetics, and they are a multiple-comb cavity nesting species, while A. dorsata and A. florea are single-comb open-air nesting species [A. mellifera showed high antimicrobial activity against Gram-positive mastitis pathogens, especially S. aureus and MRSA, as it was shown to inhibit Gram-negative bacteria (E. coli). Moreover, we also found that extracted venom from A. cerana showed the highest antimicrobial activity, followed by A. mellifera, A. florea, and A. dorsata. This result was in agreement with the findings of Surendra et al. [Apis using a disk diffusion assay, and A. cerana venom showed a higher inhibitory zone. Honey bee venom contains several bioactive substances, and the principal component is melittin, which many studies have reported as having high antimicrobial activity [The sequences of the melittin of four honey bee species were revealed via the PCR technique, and it was found that the melittin of  species . Honey b species  suggeste species  reporteda et al. , who meaactivity ,17,18. Tactivity . Additioactivity . Therefoactivity . Most Gractivity ,23.Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, S. aureus MRSA, and S. epidermidis) and Gram-negative bacteria  or a fungus . Single-nucleotide polymorphisms (SNP) have been studied in the melittin gene of A. mellifera and A. cerana, and it was found that the novel SNP of the melittin gene exists in two honey bee species [In this experiment, the antimicrobial activities of crude venom and each melittin peptide  were determined against standard human pathogenic microorganisms, including Gram-positive  in accordance with the manufacturer’s protocols. The DNA was stored at −80 °C until required.® Gel Extraction Kit  and sequencing .The extracted genomic DNA was used as a template for amplified melittin sequencing. This study designed the primers using Geneious version 8.1.8 (forward primer: 5′ GGA ATT CCA TAT GGG AAT TGG AGC AGT TC3′ and reverse primer: 5′GGC GCG GAT CCT TAT TAC TGT TGC CT3′). The melittin gene was synthesized using the KOD Hot Start Master Mix . The thermal cycling conditions were as follows: one cycle of initial denaturation at 95 °C for 2 min, 35 cycles of denaturation at 95 °C for 20 s, annealing at 55 °C for 10 s, and extension at 70 °C for 10 s. The PCR products were purified using the PureLinkFour sequences of the melittin peptide were chemically synthesized by SynPeptide Co., Ltd.  based on the deduced amino acid sequences, as shown in Staphylococcus aureus TISTR 517, Staphylococcus epidermidis DMST 15505, methicillin-resistant Staphylococcus aureus (MRSA) DMST 20625, Bacillus subtilis DMST 15896, and Micrococcus luteus DMST 15503, as well as three Gram-negative bacteria, including Klebsiella pneumoniae DMST 8216, Salmonella typhimurium DMST 562, and Escherichia coli ATCC 25922, and Candida albicans TISTR 5554 (yeast). All microorganisms were purchased from the American Type Culture Collection (ATCC), the Department of Medical Sciences Thailand (DMST), and the Thailand Institute of Scientific and Technological Research (TISTR).The antimicrobial properties of the crude venom from four honey bee species and melittin peptides were tested against five Gram-positive bacteria, including All microorganisms that were used for the determination of the minimum inhibitory concentrations (MICs) were freshly prepared before testing. Each culture was transferred to a sterile PBS, and the turbidity was adjusted by the 0.5 McFarland Standard . Each microorganism was added to the crude venom and melittin peptides at different concentrations (3.12–400 µg/mL) in 96-well plates following the Clinical and Laboratory Standards Institute (CLSI) guidelines ,36. SterA. dorsata and A. florea were obtained through this study, while the A. mellifera and A. cerana melittin sequences were retrieved from the National Center for Biotechnology Information (NCBI: https://www.ncbi.nlm.nih.gov/) (accessed on 10 March 2016) (http://web.expasy.org/protparam/) (accessed on 26 April 2021). For the analysis of the net charge, hydrophobic moment (mH), hydrophobicity (H), and melittin helical wheel plot, we used the online program HeliQuest [http://heliquest.ipmc.cnrs.fr/cgi-bin/ComputParamsV2.py) (accessed on 26 April 2021).The melittin gene sequences from ch 2016) . The dedeliQuest   and checked for normality of distribution. The Freidman and Wilcoxon signed-rank tests with Bonferroni adjustment for multiple comparisons were used to test the MIC and MBC/MFC data of the crude venoms against pathogenic microbes and test the MIC and MBC/MFC data of melittin peptides against pathogenic microbes as well. Comparisons of the MIC and MBC data among the three treatments in each honey bee species  were made using the Kruskal–Wallis test followed by the Dunn–Bonferroni multiple comparison test. The level of significance was concluded to be A. cerana displayed the most antimicrobial activity against Gram-positive bacteria and MRSA compared to the other honey bees. Nevertheless, the antimicrobial activity of honey bee venoms and melittins against Gram-negative bacteria and fungi is limited. Slight differences in the amino acid sequence, size, structuring, charge, amphipathicity, and hydrophobicity may play an essential role in the melittin properties. The present investigation revealed that bee venoms and melittin peptides, the primary component of bee venom, also act as an antimicrobial peptide in bee venom that could be a promising candidate for use as an antimicrobial agent.To summarize, we successfully investigated melittin genes from four species of honey bees. Most of the melittins were similar in terms of size and the number of amino acids. Melittin and crude venom from


Understanding an agricultural pest’s biology and ecology is essential for the creation of integrated pest management strategies. To elucidate what wild fruit resources the invasive blueberry pest, spotted-wing drosophila, can use to successfully reproduce, we conducted choice and no-choice laboratory assays. In our experiments, the fly was able to lay eggs in and develop to adulthood in eight of the eighteen fruiting plant species common in woodlands adjacent to commercial blueberry fields. Though none of these eight viable hosts were found to be preferred when the fly was given the choice between it and a commercial blueberry, the identified hosts could still be used by the drosophilid to maintain populations during as well as outside the blueberry growing season. Listing viable hosts better informs future studies and growers on how to balance pest management strategies targeting these viable fruit hosts while maintaining these marginal landscapes.Drosophila suzukii, an economically important pest of small and thin-skinned fruits, has caused annual crop losses up to 20% in the state of Georgia’s multimillion-dollar blueberry industry. The known host range of D. suzukii is large, yet the breadth of uncultivated and wild plants that can serve as alternative hosts in the southeastern United States is still not fully understood. Establishing comprehensive lists of non-crop D. suzukii hosts in woodlands near blueberry production will assist in the creation of more sustainable integrated pest management (IPM) strategies. Objectives of this study were to determine viability of wild fruiting plant species to this pest based on survivorship to adulthood and assess D. suzukii short-range preference between cultivated blueberries and wild fruit. Laboratory choice and no-choice assays were performed to determine if D. suzukii could complete its development on wild fruits sampled from the field. Results from our no-choice assays indicated that multiple species of wild fruits surveyed in Georgia were viable D. suzukii hosts including blackberry species, deerberry, hillside blueberry, common pokeweed, beautyberry, elderberry, evergreen blueberry, and large gallberry. Yet, none of these hosts were preferred by adult female D. suzukii as ovipositional substrates when compared to cultivated blueberries. However, these uncultivated species have the potential to sustain D. suzukii populations pre- and post-harvest season. This information can help farmers do more targeted management of these viable alternative hosts from wooded areas surrounding blueberry fields in order to minimize D. suzukii populations. Drosophila suzukii  (Diptera: Drosophilidae), commonly referred to as spotted-wing drosophila (SWD), is now a global pest of soft-skinned fruits as it has recently expanded its range across Europe and the Americas [Drosophila, D. suzukii use fruits to complete its life cycle and also as adult food resources [Drosophila Fallén, 1,823 species of this fly can oviposit into fresh pre-harvest fruit [D. suzukii larvae, and secondary infections [D. suzukii have a heavily sclerotized ovipositor which allows them to lay eggs in ripening and ripe fruit [D. suzukii-infested fruit [The invasive Asian vinegar fly, Americas ,3,4,5. TAmericas  and it iAmericas . Like otesources ,9. Unlikfections ,11,12,13pe fruit ,15. Econed fruit ,17,18.D. suzukii-related damage is blueberry (Vaccinium spp. L.) growers in the southeastern United States. Blueberry production has surged over the past 15 years in this region; the state of Georgia being a prime example where approximately 30,166 acres of blueberries were harvested in 2018, worth over US$300.3 million [D. suzukii infestation to some degree [D. suzukii for longer durations [One group that is threatened by  million . Other ve degree ,20,21,22urations ,24,25. TD. suzukii are captured more readily in the woodlands surrounding blueberry fields than among the berry crop itself [D. suzukii, collectively called alternative hosts. The list of identified viable plant hosts has been growing as alternative host studies are conducted wherever this pest expands its range [D. suzukii wild hosts, some may also be beneficial as habitat for natural enemies and pollinators [D. suzukii and maintaining natural habitat for other organisms providing ecosystem services.Past trapping data from southeast Georgia has revealed that, on average, p itself . A similp itself  and vinep itself . The habts range ,34,35,36linators ,37. A baD. suzukii within an agricultural landscape in multiple ways, which is why first assessing which plants can serve as reservoirs will allow more efficient integrated pest management efforts around such source material [D. suzukii can move over 100 m, indicating that movement between field margins and berry crops is possible for this pest [D. suzukii presence near agricultural landscapes during and after the cultivated crop growing season [Alternative hosts can facilitate and/or maintain higher populations of material ,34. Recehis pest ,39. Thisg season ,37,40.D. suzukii and the potential role of numerous wild plant species as D. suzukii hosts, an assessment of common fruiting plants surrounding blueberry fields in Georgia is necessary. Thus, the objectives of this study were to determine viability of wild fruiting plant species occurring in woodland habitats surrounding blueberry fields as hosts of D. suzukii based on oviposition and survivorship to adulthood, and assess D. suzukii short-range preference between cultivated blueberries and wild fruit.Based on the economic damage caused by Callicarpa americana L.; blackberry spp., Rubus spp. L.; cat sawbrier, Smilax glauca Walt.; Chinese privet, Ligustrum sinense Lour.; common pokeweed, Phytolacca americana L.; deerberry, V. stamineum L.; elderberry, Sambucus canadensis L.; evergreen blueberry, V. myrsinites Lam.; hillside blueberry, V. pallidum Aiton.; lanceleaf greenbrier, Smilax smallii Morong.; large gallberry, I. coriacea (Purch) Chapm.; muscadine grape, Vitis rotundifolia Michx.; myrtleleaf holly, I. myrtifolia Walt.; red bay, Persea borbonia (L.) Spreng.; red chokeberry, Aronia arbutifolia (L.) Pers.; saw palmetto, Serenoa repens (W. Bartram) Small; gallberry, I. glabra (L.) A. Gray; and Virginia creeper, Parthenocissus quinquefolia (L.) Planch.Fruit sampling was performed at nine sites across southeast Georgia counties known for high blueberry production . SamplesD. suzukii flies of both sexes used in this study were 5–7 days old and were reared as part of a laboratory colony in Clarke County, GA. The colony was established using specimens captured from wild populations in the same county during the summer of 2013. Cultures were maintained in 177-mL plastic square bottomed bottles  where flies were provided ~50 mL of standard diet , as discussed in Jaramillo et al. (2015). The lab colony of D. suzukii were kept in incubators  at 24 ± 1 °C, 65 ± 5% relative humidity, and a photoperiod of 14:10 h (L:D).Adult D. suzukii infestation, ripe fruit collected from woodlands bordering blueberry production were introduced to laboratory reared flies in choice and no-choice assay chambers. All collected fruit used in choice and no-choice assays were washed with dH2O and found under a dissecting microscope to be free of D. suzukii oviposition holes and wounds prior to use. Chambers consisted of 8oz plastic deli cups  with a 1/2 inch sand substrate, vented cover, and a wetted cotton ball which provided moisture to the flies. Berries rested on the sand substrate, which minimized mold growth and prevented the berries from rolling within the chambers. Two 5–7-day-old male and two 5–7-day-old female D. suzukii adults were introduced into the chambers for 48 h during the summer of 2015 and for 24 h in 2016. Assays were set up in laboratory settings of 24 ± 1 °C with a 70% relative humidity, where lighting was diffuse and ran on an approximately 14:10 h (L:D) cycle. Ten replications per trial for each species and assay type were run per sample collection and control assays using ripe cultivated blueberries were run concurrently with other no-choice assays. The number of wild plant berries used per cup in both assay types varied depending on berry size to match the approximate volume of cultivated blueberries. All collected fruit was used in laboratory trials within two days of collection date.To assess the susceptibility of wild fruit to No-choice assays were run with one species of fruit per cup. Choice assays were run with one cultivated blueberry and a number of berries of one wild fruit species, as mentioned above. Both species were placed equidistant from the cotton ball in choice assay chambers. After each exposure time (48 or 24 h), flies were aspirated out of assay chambers and eggs oviposited were counted under a dissecting microscope. The number of eggs were recorded by observing the egg breathing filaments which protrude out from each oviposition hole on the surface of the infested fruit. Fruit were then transferred to vented 2 oz plastic portion cups  lined with two layers of paper towel to absorb any berry leakage. Berries were then left to rear for three weeks under the same laboratory settings as mentioned above, after which any eclosion to adulthood was also recorded. The remaining fruit not used in our choice and no-choice assays were reared in vented 8oz plastic cups to assess natural infestation levels per tested fruiting species by recording any fly eclosion to adulthood after three weeks.D. suzukii count/berry/time exposure. In this study, susceptibility meant that oviposition occurred in the fruit and that at least one larva completed development to adulthood. No-choice oviposition and adult eclosion data (count/berry/time exposure) were log10(x + 1) transformed to meet the assumptions of normality and run through one-way ANOVA . No-choice egg and adult count data were analyzed by year and pooled for each plant species tested. Choice oviposition data (eggs/berry) were log10(x + 1) transformed to meet the assumptions of normality and analyzed using one-way ANOVA  by year and for each plant species confirmed to be susceptible in no-choice assays. The proportion of eggs deposited in alternative hosts versus cultivated ripe blueberries  were analyzed using one-way ANOVA and Student’s t-test  and reported as proportion of eggs that successfully completed the development to adult eclosion in no-choice assays. Significance was determined for the fixed effects at α = 0.05. All means were separated using Tukey–Kramer honestly significant difference tests and were back transformed for data presentation [The assay chambers were the experimental unit in this study and the response variable was the entation .P. americana, Rubus spp., V. stamineum, and V. pallidum  to 13.53 ± 1.03 (P. americana), though the overall D. suzukii adult emergence was low in comparison  observed in cultivated blueberry controls was significantly higher than in wild fruits assayed in 2015 except mparison . For exaer berry . Similarer berry . 17, 900 = 73.94, p < 0.0001; 2016: 6.50 ± 1.38, F18, 810 = 53.04, p < 0.0001) (Rubus spp. had adult eclosion of more than one fly per berry (6.33 ± 0.87) in 2015, whereas in 2016 both Rubus spp. (3.22 ± 0.61) and V. stamineum (1.98 ± 0.23) had adult eclosion above one fly per berry  . Of all er berry . Other wectively .D. suzukii to adulthood was significantly higher in Rubus spp. than all of the other wild species assayed, but was statistically similar to the cultivated blueberries in both 2015  and 2016   . Based oD. suzukii preferred to oviposit in ripe cultivated blueberries when compared to the wild susceptible fruits assessed in this study. In both 2015 and 2016, significantly more eggs were laid in cultivated blueberries than C. americana, Rubus spp., P. americana, V. stamineum, S. canadensis, V. myrsinites, V. pallidum, I. coriacea, and V. rotundifolia (P. americana (4.97 ± 0.59) was statistically similar to all other tested wild plant species except V. rotundifolia  in 2015.These tests indicated that adult female ndifolia . When coD. suzukii eggs in P. americana berries (8.10 ± 3.06) was statistically similar to V. stamineum, I. coriacea, and V. myrsinites and significantly higher when compared to Rubus spp., S. canadensis, V. pallidum, and C. americana . The proportion of D. suzukii eggs deposited into cultivated blueberries was statistically higher than all wild fruit species except I. coriacea   . D. suzukii infestation based on adult eclosion through this study. In 2015, a total of 5 D. suzukii flies were reared from S. canadensis fruit, 15 from P. Americana, and 22 flies from Rubus spp. These three species, P. americana, S. canadensis, and Rubus spp., also had natural infestation in 2016 with a total of 4, 8, and 35 flies reared from them, respectively. The remaining assessed species, though many were found to be susceptible in laboratory assays, were not infested in the field.Berries taken from the field and reared in the laboratory setting had minimal D. suzukii could complete its life cycle to adulthood: Rubus spp., V. stamineum, V. pallidum, P. americana, C. americana, S. canadensis, V. myrsinites, and I. coriacea. Of these plants, Rubus spp. and P. americana had been recently described as susceptible species in multiple other studies [I. coriacea, V. stamineum, V. pallidum, and S. canadensis to be susceptible to D. suzukii infestation [C. americana can act as a viable host for D. suzukii. The plant species mentioned above are listed as viable hosts because at least one instance of oviposition occurred  and at least one instance of an adult eclosing out of fruit of each species occurred during this study in 2015–2016. However, there is a disconnect between these no-choice data and the natural infestation levels observed in the field-collected fruit [Rubus spp., S. canadensis, and P. americana had natural infestation, and all infestation levels were low in the single digits. Through our no-choice assays, we identified eight fruiting plant species of the eighteen tested in which  studies ,42. Furtestation ,37,43. Ted fruit ,20. Of tV. stamineum, V. pallidum, C. americana, V. myrsinites, and I. coriacea could be due to many factors. The size, density, and dispersal potential of D. suzukii populations in the field setting can be drastically affected by fragmentation in landscape vegetation and diversity of host plant choices. Woodlands surrounding blueberry production in the state of Georgia rarely exceed 100 m in any direction but the spacing between these fragmented woodlands is commonly only 10–50 m, which is well within the dispersal distance known for this pest [D. suzukii could be spread thin throughout this landscape, leading to the small amounts of natural infestation observed in our field-collected wild fruit samples [This discrepancy between susceptibility in lab yet zero infestation in the field for his pest ,38,39. D samples .D. suzukii during the summer and winter months. Average ambient temperatures experienced during Georgia summers exceed the maximum limits for D. suzukii activity, suggesting that food and ovipositional resources need to be within such favorable microclimates [D. suzukii, but so can common forest resources, such as mushrooms and bird manure [D. suzukii adults [D. suzukii resources must be conducted to better understand how the marginal landscape surrounding agricultural fields affect the population and distribution of this invasive insect pest. Plotting viable wild host ripening periods against year-round D. suzukii activity data could aid in creating infestation models as relevant factors [In the state of Georgia, planted pine habitat is common surrounding blueberry production, which can become overgrown with herbaceous fruiting plants if left unmanaged. These spaces might provide microclimates with favorable conditions for climates ,47,48,49climates  found thd manure , as welli adults . Further factors .D. suzukii oviposition in the field based on characteristics of fruit quality might cause differing results in susceptibility. The skin resistance of grapevine berries was shown to be the determining factor in the number of eggs oviposited when compared to other fruit parameters [D. suzukii oviposition levels and that fruit with higher pH and brix are more attractive to D. suzukii for oviposition and development [In addition, possible physical limitations preventing rameters . Skin perameters ,54,55,56elopment ,58,59. IP. americana, I. coriacea, and V. pallidum, yet minimal development to adulthood was observed. Many challenges in these tests could account for such a trend. The fruit of plants producing smaller berries, such as elderberry, were occasionally seen drying out within the assay chambers before completion of the tests. Furthermore, fungal hyphae growth on Rubus spp., even in the dry sand substrate of our choice and no-choice assay chambers, was seen throughout the study, leading to lower survivorship ratios than expected [Oviposition in no-choice assays was consistently high for species such as expected . Additioexpected  assessedexpected .D. suzukii survivorship did increase in plant groups such as Rubus spp., P. americana, and V. stamineum. However, the wild plant species that are attractive as ovipositional substrates and that do not yet support D. suzukii adult development will act as egg sinks for this pest and will not contribute to their populations [D. suzukii numbers if occurring in riparian zones around cultivated fields, functioning like a dead-end trap plants, especially if they reduce the infestation levels in the more attractive cultivated crop as Ulmer et al. [C. americana, Lamiacae (mint family), produce essential oils that have insecticidal properties when administered as fumigants and are toxic on contact to D. suzukii [In 2016, due to the small ratios of eggs to adult eclosion, the exposure time for all laboratory assays was halved from the previous year to 24 h because of the possibility of intraspecific competition between the larvae. Though oviposition rates were mostly consistent from 2015 to 2016, ulations . Plant sr et al.  found inr et al. . Interes suzukii . All of D. suzukii compared to ripe cultivated blueberries, when choosing where to oviposit their eggs. These findings align with Rodriguez-Saona et al. [D. suzukii populations before and after the blueberry growing season, when the most attractive fruits are in the bordering woodlands and not the agricultural fields. In Georgia, this blueberry fruit availability spans from late April through mid-July with the majority of ripe and/or fallen overripe fruit present from mid-to-late-summer. The inverse situation, where alternative hosts are preferred over cultivated fruit, might increase populations of D. suzukii around berry crops but could also lure the populations away from the farm and into the woodlands [D. suzukii activity within the growing season is critical [D. suzukii to lay eggs in one berry versus another may also be influenced by natal host species [Rubus spp. and P. americana, they showed that flies prefer to oviposit their eggs in the same species from which they emerged and that this has implications on their fitness [None of the alternative hosts assessed were preferred by adult female a et al.  results a et al. . This shoodlands . These voodlands ,64,65,66critical . Fitnesscritical ,42 indic species ,42. By u fitness ,42. SuchD. suzukii in the state of Georgia. Taken together, choice, no-choice, and field infestation numbers, this study has revealed eight species of wild fruiting plants ubiquitous around Georgia blueberry production to be susceptible to D. suzukii. This is the first report of C. americana being identified as a viable host of D. suzukii while the remaining species belong to genera with known hosts. It is not surprising that three of the susceptible species were in the same genus, Vaccinium, as cultivated blueberries grown in this region. These data also indicate that these alternative hosts are most likely sustaining D. suzukii populations during and after the blueberry season, but are likely not increasing the fly numbers in season due to cultivated blueberries being a preferred host. Future studies should further address the susceptibility of wild fruiting plants around blueberry fields and other agricultural settings by using field and laboratory studies in tandem. Understanding the role of not only the physical properties of the wild berries but also chemical cues from those wild berries in ovipositional site selection by D. suzukii will contribute more to the body of work surrounding alternative hosts [D. suzukii could be implemented in the blueberry cropping systems during harvest season and pre- and post-harvest to track the D. suzukii movement and correlate it to berry presence, not just trap captures [D. suzukii viable hosts and reducing the loss of beneficial plants by creating refuge for natural enemies and pollinators. Plants found to be susceptible in this study can be minimized around blueberry production through physical removal, controlled burns and/or herbicides; however, further research is needed to assess effectiveness of these methods in controlling D. suzukii populations and their impact on forest and pollinator health.This was the first comprehensive study to address alternative hosts of ve hosts . Researccaptures . These d