Multi-host viruses in Argentine ants and honey bees: Increased viral disease in honey bees is associated with Argentine ants

<p><b>Emerging infectious diseases threaten public health, livestock economies, and wildlife. Human-mediated species introductions can alter host and pathogen communities that shape the dynamics of infectious diseases. Several RNA viruses that have been linked to population declines in wild pollinators and losses of managed honey bees have been detected in multiple other species and are suspected to circulate within insect communities. Yet, we lack an understanding of how disease dynamics are affected by the introduction of novel species. These introduced species include invasive ants, which can disturb honey bees and become a pest in apiaries. The Argentine ant (Linepithema humile) is a globally successful invader that has been observed to attack bees and multiple bee-associated viruses have been detected in this ant species.</b></p> <p>Here, I studied interactions between Argentine ants and European honey bees (Apis mellifera) and how these interactions affect viral dynamics in beehives. I first tested a range of pollinators and associated insects for RNA viruses that are pathogenic to honey bees. Bee-associated viruses showed evidence for active viral replication in several pollinator species but also in species that cohabit in beehives such as ants, spiders, and cockroaches. Using phylogenetic analyses, I found that viral transmission within communities was shaped by geographic origin rather than being restricted by species barriers. Next, I used a longitudinal field study to test whether Argentine ant presence affected pathogen infections and survival in beehives. Argentine ants tested positive for three bee-associated viruses even before beehives were moved into ant-infested sites. Increased levels of deformed wing virus in beehives in autumn were associated with ant presence, although hive mortality was not affected by ants over the duration of this experiment. I used RNA sequencing on a subset of honey bee samples collected during autumn to study the RNA virome and identify transcriptomic responses associated with ant presence. Twelve RNA viruses were found in beehives, among those, three plant-associated viruses and an unclassified RNA virus that had not previously been observed in honey bees. Deformed wing virus showed the highest viral titres in most hives, but was only marginally affected by ant presence. Sacbrood virus and tomato ringspot virus levels were increased in hives with ants, however, both viruses are not known to infect Argentine ants and the plant-associated tomato ringspot virus seems unlikely to affect bee health.</p> <p>Lastly, I tested the feasibility of controlling Argentine ants in apiaries using a novel pest control strategy. RNA interference is a conserved cellular gene regulation mechanism that could be used to silence specific genes in ants. Using double-stranded RNA (dsRNA) to silence two immune-related genes in Argentine ants was expected to increase pathogen susceptibility, which could then lead to higher pathogen levels that reduce ant numbers. My results indicated that no consistent immune silencing could be achieved in the field. Immune gene expression changes were observed, but pathogen titres were not affected, and ant numbers stayed high. Argentine ant control using a conventional insecticide significantly increased bee survival, whereas many hives in the dsRNA and control group abandoned their hives due to ant attacks. Although population control was not successful using the two Argentine ant-specific dsRNAs, insights into ant immunity and ant-bee interactions could improve the development of novel control strategies.</p> <p>Bee-associated viruses have repeatedly been detected in ant species, yet, this is one of the first studies to investigate whether ants affect viral dynamics in honey bees. I showed that invasive Argentine ants are associated with increases in viral pathogens in honey bees. The mechanisms by which ants affect bee disease are unknown, although there is some evidence for ants transmitting viruses or causing stress responses in bees that affect immunity. The findings of this thesis highlight the risk of invasive ant species disrupting pollination services. New and environmentally-friendly methods to control invasive species are urgently needed to improve bee health and limit the spread of invasive ants, such as Argentine ants. The high prevalence of bee-associated viruses and viral diversity in ants suggests that pathogens that are suitable for population control might be present in ant populations, although risks of spillovers into other species need to be carefully considered.</p>

Multi-host viruses in Argentine ants and honey bees: Increased viral disease in honey bees is associated with Argentine ants

<p><b>Emerging infectious diseases threaten public health, livestock economies, and wildlife. Human-mediated species introductions can alter host and pathogen communities that shape the dynamics of infectious diseases. Several RNA viruses that have been linked to population declines in wild pollinators and losses of managed honey bees have been detected in multiple other species and are suspected to circulate within insect communities. Yet, we lack an understanding of how disease dynamics are affected by the introduction of novel species. These introduced species include invasive ants, which can disturb honey bees and become a pest in apiaries. The Argentine ant (Linepithema humile) is a globally successful invader that has been observed to attack bees and multiple bee-associated viruses have been detected in this ant species.</b></p> <p>Here, I studied interactions between Argentine ants and European honey bees (Apis mellifera) and how these interactions affect viral dynamics in beehives. I first tested a range of pollinators and associated insects for RNA viruses that are pathogenic to honey bees. Bee-associated viruses showed evidence for active viral replication in several pollinator species but also in species that cohabit in beehives such as ants, spiders, and cockroaches. Using phylogenetic analyses, I found that viral transmission within communities was shaped by geographic origin rather than being restricted by species barriers. Next, I used a longitudinal field study to test whether Argentine ant presence affected pathogen infections and survival in beehives. Argentine ants tested positive for three bee-associated viruses even before beehives were moved into ant-infested sites. Increased levels of deformed wing virus in beehives in autumn were associated with ant presence, although hive mortality was not affected by ants over the duration of this experiment. I used RNA sequencing on a subset of honey bee samples collected during autumn to study the RNA virome and identify transcriptomic responses associated with ant presence. Twelve RNA viruses were found in beehives, among those, three plant-associated viruses and an unclassified RNA virus that had not previously been observed in honey bees. Deformed wing virus showed the highest viral titres in most hives, but was only marginally affected by ant presence. Sacbrood virus and tomato ringspot virus levels were increased in hives with ants, however, both viruses are not known to infect Argentine ants and the plant-associated tomato ringspot virus seems unlikely to affect bee health.</p> <p>Lastly, I tested the feasibility of controlling Argentine ants in apiaries using a novel pest control strategy. RNA interference is a conserved cellular gene regulation mechanism that could be used to silence specific genes in ants. Using double-stranded RNA (dsRNA) to silence two immune-related genes in Argentine ants was expected to increase pathogen susceptibility, which could then lead to higher pathogen levels that reduce ant numbers. My results indicated that no consistent immune silencing could be achieved in the field. Immune gene expression changes were observed, but pathogen titres were not affected, and ant numbers stayed high. Argentine ant control using a conventional insecticide significantly increased bee survival, whereas many hives in the dsRNA and control group abandoned their hives due to ant attacks. Although population control was not successful using the two Argentine ant-specific dsRNAs, insights into ant immunity and ant-bee interactions could improve the development of novel control strategies.</p> <p>Bee-associated viruses have repeatedly been detected in ant species, yet, this is one of the first studies to investigate whether ants affect viral dynamics in honey bees. I showed that invasive Argentine ants are associated with increases in viral pathogens in honey bees. The mechanisms by which ants affect bee disease are unknown, although there is some evidence for ants transmitting viruses or causing stress responses in bees that affect immunity. The findings of this thesis highlight the risk of invasive ant species disrupting pollination services. New and environmentally-friendly methods to control invasive species are urgently needed to improve bee health and limit the spread of invasive ants, such as Argentine ants. The high prevalence of bee-associated viruses and viral diversity in ants suggests that pathogens that are suitable for population control might be present in ant populations, although risks of spillovers into other species need to be carefully considered.</p>

Mapping of sequences in the 5’ region and 3’ UTR of tomato ringspot virus RNA2 that facilitate cap-independent translation of reporter transcripts in vitro

Tomato ringspot virus (ToRSV, genus Nepovirus, family Secoviridae, order Picornavirales) is a bipartite positive-strand RNA virus, with each RNA encoding one large polyprotein. ToRSV RNAs are linked to a 5’-viral genome-linked protein (VPg) and have a 3’ polyA tail, suggesting a non-canonical cap-independent translation initiation mechanism. The 3’ untranslated regions (UTRs) of RNA1 and RNA2 are unusually long (~1.5 kb) and share several large stretches of sequence identities. Several putative in-frame start codons are present in the 5’ regions of the viral RNAs, which are also highly conserved between the two RNAs. Using reporter transcripts containing the 5’ region and 3’ UTR of the RNA2 of ToRSV Rasp1 isolate (ToRSV-Rasp1) and in vitro wheat germ extract translation assays, we provide evidence that translation initiates exclusively at the first AUG, in spite of a poor codon context. We also show that both the 5’ region and 3’ UTR of RNA2 are required for efficient cap-independent translation of these transcripts. We identify translation-enhancing elements in the 5’ proximal coding region of the RNA2 polyprotein and in the RNA2 3’ UTR. Cap-dependent translation of control reporter transcripts was inhibited when RNAs consisting of the RNA2 3’ UTR were supplied in trans. Taken together, our results suggest the presence of a CITE in the ToRSV-Rasp1 RNA2 3’ UTR that recruits one or several translation factors and facilitates efficient cap-independent translation together with the 5’ region of the RNA. Non-overlapping deletion mutagenesis delineated the putative CITE to a 200 nts segment (nts 773–972) of the 1547 nt long 3’ UTR. We conclude that the general mechanism of ToRSV RNA2 translation initiation is similar to that previously reported for the RNAs of blackcurrant reversion virus, another nepovirus. However, the position, sequence and predicted structures of the translation-enhancing elements differed between the two viruses.

Cultivated and wild grapevines in Tennessee possess overlapping but distinct virus populations

Viruses and viroids prevalent in a population of 42 wild grapevines (i.e., free-living, uncultivated grapevines; Vitis spp.) were compared to those in a population of 85 cultivated grapevines collected in Tennessee, USA by RNA-seq analysis of pools of ribosomal RNA-depleted total RNA. The sequences of 10 viruses (grapevine fleck virus, grapevine leafroll-associated virus 2, grapevine rupestris stem pitting-associated virus, grapevine Syrah virus 1, grapevine vein-clearing virus, grapevine virus B, grapevine virus E, tobacco ringspot virus, tomato ringspot virus and a novel nano-like virus) and two viroids (hop stunt viroid and grapevine yellow speckle viroid 1) were detected in both grapevine populations. Sequences of four viruses (grapevine associated tymo-like virus, grapevine leafroll-associated virus 3, grapevine red blotch virus and grapevine virus H) were identified only from cultivated grapevines. High, moderate and low numbers of sequence reads were identified only from wild grapevines for a novel caulimovirus, an enamovirus, and alfalfa mosaic virus, respectively. The presence of most virus sequences and both viroids was verified independently in the original samples by reverse transcription-polymerase chain reaction followed by Sanger sequencing. Comparison of viral sequences shared by both populations showed that cultivated and wild grapevines harbored distinct sequence variants, which suggests that there was limited virus movement between the two populations. Collectively, this study represents the first unbiased survey of viruses and viroids in both cultivated and wild grapevines within a defined geographic region.

A threat to stone fruit and grape production: tomato ringspot virus (ToRSV) transmission by X. americanum s.l. (sensu lato).

Prunus stem pitting (PSP) is a lethal disease caused by the Tomato ringspot virus (ToRSV) and transmitted by the dagger nematode. All peach varieties and most other stone fruit are susceptible to ToRSV. The same virus also causes a decline in certain wine grape varieties. In order to develop strategies to prevent the occurrence of these diseases it is important to understand the interaction between Xiphinema americanum and ToRSV. This chapter discusses the economic importance, geographical distribution, host range, damage symptoms, biology and life cycle, interactions with other nematodes and pathogens, recommended integrated management, and management optimization of X. americanum. Future research requirements and future developments are also mentioned.

Weeds as Reservoirs of Viruses in Agrobiocenoses of Legumes in Ukraine

This paper is the review of literature data on the prevalence of weeds as possible reservoirs of plant viruses in agroecosystems of Ukraine. The information presented here about the most distributed weeds as reservoirs of harmful plant viruses will be useful for understanding the pathogens ecology, analyzing the virus epidemiology and for disease management. Since legumes are the main crops grown in Ukraine, the paper focuses on weeds spreading in the agrosystems of cultivated plant. The paper provides information about the primary sources of soybean viruses (Soybean mosaic virus, Cucumber mosaic virus, Alfalfa mosaic virus, Tomato ringspot virus) and bean viruses (Bean yellow mosaic virus, Bean common mosaic virus) as well as the main factors contributing the virus transmission in agrocenosis.

A Survey for Nine Major Viruses of Grapevines in Tennessee Vineyards

Despite the significance of grape production to the fruit industry in Tennessee (TN), no published information has been available on viruses affecting grapevines in the state. Hence, a survey was conducted during the 2016 and 2017 growing seasons to determine the status of nine major viruses of grapevines in TN vineyards by taking advantage of classical serological assays and confirmatory nucleic acid-based diagnostic approaches. A total of 349 samples from 23 grapevine cultivars mostly displaying viral-like symptoms were collected from 23 commercial vineyards. All samples were assayed by DAS-ELISA for arabis mosaic virus (ArMV), grapevine leafroll-associated virus (GLRaV)-1, GLRaV-2, GLRaV-3, GLRaV-4, grapevine fanleaf virus (GFLV), tobacco ringspot virus (TRSV), and tomato ringspot virus (ToRSV). Selected serologically positive samples were also tested by RT-PCR, followed by Sanger sequencing of the generated amplicons. Additionally, 19 grapevines displaying symptoms characteristic of grapevine red blotch virus (GRBV) were also assayed by PCR followed by confirmatory sequencing-based methods. Collectively, these assays verifiably detected GLRaV-1, GLRaV-2, GLRaV-3, ToRSV, and GRBV in TN vineyards. This is the first record of the presence of these viruses in TN vineyards. ArMV, GLRaV-4, GFLV, and TRSV were not detected. The majority of samples tested positive for a single virus, whereas mixed infections with more than one virus were detected in 37% of samples.

Influence of chemotherapy on development and production of virus free in vitro strawberry plants

The objective of the study was to determine effects of ribavirin on development and health status of in vitro grown strawberry cultivars ‘Honeoye’ and ‘Elkat’ infected with viruses Strawberry mild yellow-edge virus (SMYEV), Tomato ringspot virus (ToRSV) and Arabis mosaic virus (ArMV). Antiviral compound ribavirin was added in concentrations 20, 40, 80 and 160 mg/l to the same MS medium as for multiplication. Growth reduction was noted on medium with 160 mg/l ribavirin and to a lesser degree in the 40 and 80 mg/l treatments. At the end of chemotherapy, in vitro clones free of viruses detected previously in the initial plants were obtained for both selected cultivars across all ribavirin concentrations. The highest number of plants (94) with negative results of ELISA testing was noted on medium with the highest ribavirin concentration 160 mg/l and the lowest (73) on medium with the lowest concentration 20 mg/l of ribavirin. The treated plants look symptomless and appear morphologically equal to the untreated control plants. Results indicate that ribavirin treatment of in vitro plants is a suitable method for eliminating SMYEV, ToRSV and ArMV from strawberry.