Mechanical transmission of dengue virus by Aedes aegypti may influence disease transmission dynamics during outbreaks.

BACKGROUND: Dengue virus outbreaks are increasing in number and severity worldwide. Viral transmission is assumed to require a minimum time period of viral replication within the mosquito midgut. It is unknown if alternative transmission periods not requiring replication are possible. METHODS: We used a mouse model of dengue virus transmission to investigate the potential of mechanical transmission of dengue virus. We investigated minimal viral titres necessary for development of symptoms in bitten mice and used resulting parameters to inform a new model of dengue virus transmission within a susceptible population. FINDINGS: Naïve mice bitten by mosquitoes immediately after they took partial blood meals from dengue infected mice showed symptoms of dengue virus, followed by mortality. Incorporation of mechanical transmission into mathematical models of dengue virus transmission suggest that this supplemental transmission route could result in larger outbreaks which peak sooner. INTERPRETATION: The potential of dengue transmission routes independent of midgut viral replication has implications for vector control strategies that target mosquito lifespan and suggest the possibility of similar mechanical transmission routes in other disease-carrying mosquitoes. FUNDING: This study was funded by grants from the National Health Research Institutes, Taiwan (04D2-MMMOST02), the Human Frontier Science Program (RGP0033/2021), the National Institutes of Health (1R01AI143698-01A1, R01AI151004 and DP2AI152071) and the Ministry of Science and Technology, Taiwan (MOST104-2321-B-400-016).

Honey bee foraging ability suppressed by imidacloprid can be ameliorated by adding adenosine.

Honey bees are important pollinators in most ecosystem, but they are currently facing many threats, which have led to a reduction in their population. Previous studies have indicated that neonicotinoid pesticide can impair the memory and learning ability of honey bees, which can eventually lead to a decline in their foraging and homing abilities. In this study, we investigated the homing ability barrier from the perspective of energy supply. We believe that when worker bees experience stress, their energy supply may shift from pro-movement to pro-resistance; this will lead to inadequate energy provision to the flight muscles, causing a reduction in wingbeat frequency and impairing the flight ability of the worker bees. To test this, the worker bees were treated with imidacloprid, and wing beats between the treatment groups were compared. Their glucose, glycogen, trehalose, and ATP contents were also measured, and their genes for energy metabolism and resistance were analyzed. The addition of adenosine improved the ATP content and helped recover the wingbeat frequency of the worker bees. The preliminary results obtained showed that wingbeat frequency and glucose content in the worker bees treated with imidacloprid were significantly lower than those in the control group. This result is consistent with our hypothesis and demonstrates that energy supply imbalances can prevent worker bees from returning to their hives.

CLEC5A mediates Zika virus-induced testicular damage.

BACKGROUND: Zika virus (ZIKV) infection is clinically known to induce testicular swelling, termed orchitis, and potentially impact male sterility, but the underlying mechanisms remain unclear. Previous reports suggested that C-type lectins play important roles in mediating virus-induced inflammatory reactions and pathogenesis. We thus investigated whether C-type lectins modulate ZIKV-induced testicular damage. METHODS: C-type lectin domain family 5 member A (CLEC5A) knockout mice were generated in a STAT1-deficient immunocompromised background (denoted clec5a-/-stat1-/-) to enable testing of the role played by CLEC5A after ZIKV infection in a mosquito-to-mouse disease model. Following ZIKV infection, mice were subjected to an array of analyses to evaluate testicular damage, including ZIKV infectivity and neutrophil infiltration estimation via quantitative RT-PCR or histology and immunohistochemistry, inflammatory cytokine and testosterone detection, and spermatozoon counting. Furthermore, DNAX-activating proteins for 12 kDa (DAP12) knockout mice (dap12-/-stat1-/-) were generated and used to evaluate ZIKV infectivity, inflammation, and spermatozoa function in order to investigate the potential mechanisms engaged by CLEC5A. RESULTS: Compared to experiments conducted in ZIKV-infected stat1-/- mice, infected clec5a-/-stat1-/- mice showed reductions in testicular ZIKV titer, local inflammation and apoptosis in testis and epididymis, neutrophil invasion, and sperm count and motility. CLEC5A, a myeloid pattern recognition receptor, therefore appears involved in the pathogenesis of ZIKV-induced orchitis and oligospermia. Furthermore, DAP12 expression was found to be decreased in the testis and epididymis tissues of clec5a-/-stat1-/- mice. As for CLEC5A deficient mice, ZIKV-infected DAP12-deficient mice also showed reductions in testicular ZIKV titer and local inflammation, as well as improved spermatozoa function, as compared to controls. CLEC5A-associated DAP12 signaling appears to in part regulate ZIKV-induced testicular damage. CONCLUSIONS: Our analyses reveal a critical role for CLEC5A in ZIKV-induced proinflammatory responses, as CLEC5A enables leukocytes to infiltrate past the blood-testis barrier and induce testicular and epididymal tissue damage. CLEC5A is thus a potential therapeutic target for the prevention of injuries to male reproductive organs in ZIKV patients.

Demographic Characteristics and Population Simulation of Newly Invasive Fall Armyworm on Arachis hypogaea (Fabales: Fabaceae) and Dominant Green Manure Plant in Taiwan.

Spodoptera frugiperda (J.E. Smith) is a severe and fast-spreading pest of numerous agro-economic crops, including miscellaneous, vegetables, and green manure crops. Understanding pest ecology represents a core component in integrated pest management decision-making. In Taiwan, peanut (Arachis hypogaea L.) is an important miscellaneous crop, whereas sesbania (Sesbania roxburghii Merr.) is the most frequently used green manure crop. To improve the S. frugiperda management in Taiwan, the demographic characteristics and population simulation of this pest reared on peanut and sesbania leaves were analyzed using the age-stage, two-sex life table theory. The intrinsic rate of increase, finite rate of increase, and net reproductive rate of S. frugiperda were higher when reared on peanut (0.1625 d-1, 1.1764 d-1, 264.9 offspring) than on sesbania (0.0951 d-1, 1.0997 d-1, and 30.3 offspring). Population projection of S. frugiperda on peanut demonstrated that this crop is a more suitable host plant than sesbania. Yet, this suboptimal host still assures an increasing trend of more than 357-fold individuals in 75 d, from the initial cohort of 10 eggs. Our data suggest that green manure plants in fallowing fields may support the pest's survival all year round, and may be responsible for a successful establishment and unexpected outbreaks of this invasive pest on the neighboring crops in Taiwan. Our study thus highlights the importance of assessing the population dynamics and areawide pest management of an invasive polyphagous pest on a noneconomic crop to mitigate the potential risk of reinfestation and thus outbreaks.

Carbohydrate metabolism is a determinant for the host specificity of baculovirus infections.

Baculoviruses Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV) have highly similar genome sequences but exhibit no overlap in their host range. After baculovirus infects nonpermissive larvae (e.g., AcMNPV infecting B. mori or BmNPV infecting Spodoptera litura), we found that stored carbohydrates, including hemolymph trehalose and fat body glycogen, are rapidly transformed into glucose; enzymes involved in glycolysis and the TCA cycle are upregulated and produce more ATP; adenosine signaling that regulates glycolytic activity is also increased. Subsequently, phagocytosis in cellular immunity and the expression of genes involved in humoral immunity increase significantly. Moreover, inhibiting glycolysis and the expression of gloverins in nonpermissive hosts increased baculovirus infectivity, indicating that the stimulated energy production is designed to support the immune response against infection. Our study highlights that alteration of the host's carbohydrate metabolism is an important factor determining the host specificity of baculoviruses, in addition to viral factors.

Transgenic refractory Aedes aegypti lines are resistant to multiple serotypes of dengue virus.

The areas where dengue virus (DENV) is endemic have expanded rapidly, driven in part by the global spread of Aedes species, which act as disease vectors. DENV replicates in the mosquito midgut and is disseminated to the mosquito's salivary glands for amplification. Thus, blocking virus infection or replication in the tissues of the mosquito may be a viable strategy for reducing the incidence of DENV transmission to humans. Here we used the mariner Mos1 transposase to create an Aedes aegypti line that expresses virus-specific miRNA hairpins capable of blocking DENV replication. These microRNA are driven by the blood-meal-inducible carboxypeptidase A promoter or by the polyubiquitin promoter. The transgenic mosquitoes exhibited significantly lower infection rates and viral titers for most DENV serotypes 7 days after receiving an infectious blood meal. The treatment was also effective at day 14 post infection after a second blood meal had been administered. In viral transmission assay, we found there was significantly reduced transmission in these lines. These transgenic mosquitoes were effective in silencing most of the DENV genome; such an approach may be employed to control a dengue fever epidemic.

Deformed wing virus infection affects the neurological function of Apis mellifera by altering extracellular adenosine signaling.

Deformed wing virus (DWV) infection is believed to be closely associated with colony losses of honeybee (Apis mellifera) due to reduced learning and memory of infected bees. The adenosine (Ado) pathway is important for maintaining immunity and memory function in animals, and it enhances antivirus responses by regulating carbohydrate metabolism in insects. Nevertheless, its effect on the memory of invertebrates is not yet clear. This study investigated how the Ado pathway regulates energy metabolism and memory in honeybees following DWV infection. Decreased Ado receptor (Ado-R) expression in the brain of infected bees resulted in a carbohydrate imbalance as well as impairments of glutamate-glutamine (Glu-Gln) cycle and long-term memory. Dietary supplementation with Ado not only increased the brain energy metabolism but also rescued long-term memory loss by upregulating the expression of memory-related genes. The present study demonstrated the regulation of the Ado pathway upon DWV infection and provides insights into the mechanisms underlying energy regulation and the neurological function of honeybees.

Real-time monitoring of deformed wing virus-infected bee foraging behavior following histone deacetylase inhibitor treatment.

Impairment in the learning/memory behavior of bees is responsible for the massive disappearance of bee populations and its consequent agricultural economic losses. Such impairment might be because of o both pesticide exposure and pathogen infection, with a key contributor deformed wing virus (DWV). The present study found that sodium butyrate (NaB) significantly increased survival and reversed the learning/memory impairment of DWV-infected bees. A next-generation sequencing analysis showed that NaB affected the expression of genes involved in glycolytic processes and memory formation, which were suppressed by DWV infection. In addition, we performed a large-scale movement tracking experiment by using a wireless sensor network-based automatic real-time monitoring system and confirmed that NaB could improve the homing ability of DWV-infected bees. In short, we demonstrated the mechanism of how epigenetic regulation can resume the memory function of honeybees and suggest strategies for applying NaB to reduce the incidence of colony losses.

MicroRNAs from Snellenius manilae bracovirus regulate innate and cellular immune responses of its host Spodoptera litura.

To avoid inducing immune and physiological responses in insect hosts, parasitoid wasps have developed several mechanisms to inhibit them during parasitism, including the production of venom, specialized wasp cells, and symbioses with polydnaviruses (PDVs). These mechanisms alter the host physiology to give the wasp offspring a greater chance of survival. However, the molecular mechanisms for most of these alterations remain unclear. In the present study, we applied next-generation sequencing analysis and identified several miRNAs that were encoded in the genome of Snellenius manilae bracovirus (SmBV), and expressed in the host larvae, Spodoptera litura, during parasitism. Among these miRNAs, SmBV-miR-199b-5p and SmBV-miR-2989 were found to target domeless and toll-7 in the host, which are involved in the host innate immune responses. Microinjecting the inhibitors of these two miRNAs into parasitized S. litura larvae not only severely decreased the pupation rate of Snellenius manilae, but also restored the phagocytosis and encapsulation activity of the hemocytes. The results demonstrate that these two SmBV-encoded miRNAs play an important role in suppressing the immune responses of parasitized hosts. Overall, our study uncovers the functions of two SmBV-encoded miRNAs in regulating the host innate immune responses upon wasp parasitism.

Identification of Immune Regulatory Genes in Apis mellifera through Caffeine Treatment.

Plants and pollinators are mutually beneficial: plants provide nectar as a food source and in return their pollen is disseminated by pollinators such as honeybees. Some plants secrete chemicals to deter herbivores as a protective measure, among which is caffeine, a naturally occurring, bitter tasting, and pharmacologically active secondary compound. It can be found in low concentrations in the nectars of some plants and as such, when pollinators consume nectar, they also take in small amounts of caffeine. Whilst caffeine has been indicated as an antioxidant in both mammals and insects, the effect on insect immunity is unclear. In the present study, honeybees were treated with caffeine and the expression profiles of genes involved in immune responses were measured to evaluate the influence of caffeine on immunity. In addition, honeybees were infected with deformed wing virus (DWV) to study how caffeine affects their response against pathogens. Our results showed that caffeine can increase the expression of genes involved in immunity and reduce virus copy numbers, indicating that it has the potential to help honeybees fight against viral infection. The present study provides a valuable insight into the mechanism by which honeybees react to biotic stress and how caffeine can serve as a positive contributor, thus having a potential application in beekeeping.

Adenosine Receptor Modulates Permissiveness of Baculovirus (Budded Virus) Infection via Regulation of Energy Metabolism in Bombyx mori.

Although the modulation of host physiology has been interpreted as an essential process supporting baculovirus propagation, the requirement of energy supply for host antivirus reactions could not be ruled out. Our present study showed that metabolic induction upon AcMNPV (budded virus) infection of Bombyx mori stimulated virus clearance and production of the antivirus protein, gloverin. In addition, we demonstrated that adenosine receptor signaling (AdoR) played an important role in regulating such metabolic reprogramming upon baculovirus infection. By using a second lepidopteran model, Spodoptera frugiperda Sf-21 cells, we demonstrated that the glycolytic induction regulated by adenosine signaling was a conservative mechanism modulating the permissiveness of baculovirus infection. Another interesting finding in our present study is that both BmNPV and AcMNPV infection cause metabolic activation, but it appears that BmNPV infection moderates the level of ATP production, which is in contrast to a dramatic increase upon AcMNPV infection. We identified potential AdoR miRNAs induced by BmNPV infection and concluded that BmNPV may attempt to minimize metabolic activation by suppressing adenosine signaling and further decreasing the host's anti-baculovirus response. Our present study shows that activation of energy synthesis by adenosine signaling upon baculovirus infection is a host physiological response that is essential for supporting the innate immune response against infection.

Snellenius manilae bracovirus suppresses the host immune system by regulating extracellular adenosine levels in Spodoptera litura.

Sufficient energy supply to the host immune system is important for resisting pathogens. Therefore, during pathogen infection, the host metabolism is reassigned from storage, growth, and development to the immune system. Previous studies in Drosophila melanogaster have demonstrated that systemic metabolic switching upon an immune challenge is activated by extracellular adenosine signaling, modulating carbohydrate mobilization and redistributing energy to the hemocytes. In the present study, we discovered that symbiotic virus (SmBV) of the parasitoid wasp Snellenius manilae is able to down-regulate the extracellular adenosine of its host, Spodoptera litura, to inhibit metabolism switching. The decreased carbohydrate mobilization, glycogenolysis, and ATP synthesis upon infection results in the host being unable to supply energy to its immune system, thus benefitting the development of wasp larvae. When we added adenosine to the infected S. litura larvae, we observed enhanced host immune responses that decreased the pupation rate of S. manilae. Previous studies showed that after pathogen infection, the host activates its adenosine pathway to trigger immune responses. However, our results suggest a different model: we found that in S. manilae, SmBV modulates the host adenosine pathway such that wasp eggs and larvae can evade the host immune response.

Identification of Regulatory Host Genes Involved in Sigma Virus Replication Using RNAi Knockdown in Drosophila.

The Drosophila melanogaster sigma virus, a member of the Rhabdoviridae family, specifically propagates itself in D. melanogaster. It contains six genes in the order of 3'-N-P-X-M-G-L-5'. The sigma virus is the only arthropod-specific virus of the Rhabdoviridae family. Sigma-virus-infected Drosophila may suffer from irreversible paralysis when exposed to a high CO2 concentration, but generally, no other symptoms are reported. A recent study reported that host gene expression in immune pathways was not changed in sigma-virus-infected Drosophila, which does not necessarily suggest that they are not involved in virus-host interactions. The present study aimed to identify host genes associated with sigma virus replication. Immune pathways JAK-STAT and IMD were selected for detailed study. The results showed that the genome copy number of the sigma virus increased after knocking down the immune pathway genes domeless and PGRP-LC in Drosophila S2 cells. The knocking down of domeless and PGRP-LC significantly up-regulated the expression of the L gene compared to the other viral genes. We propose that the immune pathways respond to sigma virus infection by altering L expression, hence suppressing viral replication. This effect was further tested in vivo, when D. melanogaster individuals injected with dsdome and dsPGRP-LC showed not only an increase in sigma virus copy number, but also a reduced survival rate when treated with CO2. Our study proved that host immunity influences viral replication, even in persistent infection. Knocking down the key components of the immune process deactivates immune controls, thus facilitating viral expression and replication. We propose that the immunity system of D. melanogaster regulates the replication of the sigma virus by affecting the L gene expression. Studies have shown minimal host-virus interaction in persistent infection. However, our study demonstrated that the immunity continued to affect viral replication even in persistent infection because knocking down the key components of the immune process disabled the relevant immune controls and facilitated viral expression and replication.