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Long intergenic non-coding RNAs (lincRNAs) are appearing as an important class of regulatory RNAs with a variety of biological functions. The aim of this study was to identify the lincRNA profile in the dengue vector Aedes aegypti and evaluate their potential role in host-pathogen interaction. The majority of previous RNA-Seq transcriptome studies in Ae. aegypti have focused on the expression pattern of annotated protein coding genes under different biological conditions. Here, we used 35 publically available RNA-Seq datasets with relatively high depth to screen the Ae. aegypti genome for lincRNA discovery. This led to the identification of 3,482 putative lincRNAs. These lincRNA genes displayed a slightly lower GC content and shorter transcript lengths compared to protein-encoding genes. Ae. aegypti lincRNAs also demonstrate low evolutionary sequence conservation even among closely related species such as Culex quinquefasciatus and Anopheles gambiae. We examined their expression in dengue virus serotype 2 (DENV-2) and Wolbachia infected and non-infected adult mosquitoes and Aa20 cells. The results revealed that DENV-2 infection increased the abundance of a number of host lincRNAs, from which some suppress viral replication in mosquito cells. RNAi-mediated silencing of lincRNA_1317 led to enhancement in viral replication, which possibly indicates its potential involvement in the host anti-viral defense. A number of lincRNAs were also differentially expressed in Wolbachia-infected mosquitoes. The results will facilitate future studies to unravel the function of lncRNAs in insects and may prove to be beneficial in developing new ways to control vectors or inhibit replication of viruses in them.

Viral infections in mosquitoes trigger the RNA interference (RNAi) pathway, a key antiviral defense mechanism that generates virus-derived small RNAs (vsRNAs). Given the natural enrichment of vsRNAs during infection and their stability, small RNA sequencing (sRNA-seq) has emerged as a powerful tool for virome characterization. Culex tarsalis is a widely distributed mosquito species in North America and is an important vector of West Nile virus (WNV). Previous studies have shown that co-infection with insect-specific viruses (ISVs) can modulate WNV replication in Cx. tarsalis , highlighting the importance of characterizing the virome of this species. Here, we investigated the virome of Cx. tarsalis populations across 5 states of the Western United States using sRNA-seq. We analyzed samples from 17 geographic locations which were collected under suboptimal field conditions during the COVID-19 pandemic, presenting challenges related to sample integrity. Despite these challenges, sRNA-seq proved to be a reliable method for virome analysis. We identified a total of seven ISVs, all of which have been previously associated with Cx. tarsalis , along with their respective sRNA (siRNA and piRNA) profiles. The ISVs found here did not show a clear distribution pattern, but two of them (Marma virus and Culex narnavirus 1) were found in all sampled states. These findings not only deepen our understanding of ISVs, but also demonstrate the utility of sRNA-seq in non-ideal situations, enabling the collection and analysis of samples under real-world surveillance scenarios.

The genus Flavivirus contains more than 70 single-stranded, positive-sense arthropod-borne RNA viruses. Some flaviviruses are particularly medically important to humans and other vertebrates including dengue virus (DENV), West Nile virus, and yellow fever virus. These viruses are transmitted to vertebrates by mosquitoes and other arthropod species. Mosquitoes are also infected by insect-specific flaviviruses (ISFs) that do not appear to be infective to vertebrates. Cell fusing agent virus (CFAV) was the first described ISF, which was discovered in an Aedes aegypti cell culture. We found that while CFAV infection could be significantly reduced by application of RNAi against the NS5 gene, removal of the treatment led to quick restoration of CFAV replication. Interestingly, we found that CFAV infection significantly enhanced replication of DENV, and vice versa, DENV infection significantly enhanced replication of CFAV in mosquito cells. We have shown that CFAV infection leads to increase in the expression of ribonuclease kappa (RNASEK), which is known to promote infection of viruses that rely on endocytosis and pH-dependent entry. Knockdown of RNASEK by dsRNA resulted in reduced DENV replication. Thus, increased expression of RNASEK induced by CFAV is likely to contribute to enhanced DENV replication in CFAV-infected cells.

The use of fiber-reinforced polymer (FRP) jackets as external confinement is becoming popular, especially in seismic areas, because of its ability to enhance the strength and ductility of reinforced concrete to perform as a sustainable symmetric structural member. Therefore, various researchers have worked out for the prediction of strength and strain models of FRP-confined concrete. This study presents the improved strain models for the FRP confined cylindrical concrete members. Different previously proposed models of axial strain of FRP-confined concrete were evaluated based on a large database of 678 specimens from previous experiments and an improved model was proposed using the general regression analysis technique. Furthermore, the proposed model was validated using the previous experimental work of FRP-wrapped concrete cylinders and their finite elements analysis (FEA) using the ABAQUS software. The accuracy of the proposed strain model was quite satisfactory in comparison with the previous experimental and FEA results of the present study. Moreover, the proposed empirical strain model was used for the parametric study to investigate the effect of different geometric and material parameters such as the compressive strength of unconfined concrete, diameter of the cylinder, elastic modulus and thickness of the FRP layers, on the axial strain of FRP-wrapped cylinders. A close agreement among the proposed strain models and experimental outputs was observed. This study will help in understanding the behavior of sustainable FRP-confined symmetric concrete members.

This study aimed to evaluate the effects of an organic acid blend and essential oils individually and in combination on growth performance, blood biochemical profile, gut health, and nutrient digestibility of broilers fed a higher level of an animal protein concentrate. Five hundred day-old Ross-308 male broiler chicks (average body weight, 39 ± 1.2 g) were randomly assigned to five replicated (5 replicates/treatment; 20 birds/replica) dietary treatments (100 birds/treatment). Birds in these group were given five different experimental diets that were prepared and designated as (i) basal diet (negative control, NC); (ii) basal diet plus Enramycin (positive control, PC), 50 mg/kg feed; (iii) basal diet with the addition of organic acid (OA) at 200 mg/kg feed; (iv) basal diet plus essential oils (EO) at 150 mg/kg feed; (v) basal diet plus combination of OA and EOs 200 and150 mg/kg feed (OA + EO). Experimental diets were prepared and fed in two phases i.e. starter (1–21 d) and finisher (22–42 d) phases of rearing. The findings of the present study revealed that feed intake did not vary significantly among the treatment groups, however, better (p < .05) body weight gain (BWG) and feed conversion ratio (FCR) was observed in OA, EO and OA + EO supplemented groups, respectively. The growth of Clostridium perfringens, Escherichia coli and Salmonella were reduced (p < .05) in OA, EO, OA + EO groups, while, Lactobacillus growth was positively improved (p < .05). Notably, the intestinal lesion score was significantly reduced, and villus height was improved in the OA, EO, OA + EO groups compared to birds in the NC group. Moreover, the serum level of calprotectin and liver enzymes were significantly reduced in the OA + EO treated group. At the end of the trial five birds from each experimental replicate of all five treated groups were shifted to metabolic cages on day-36 till day-42 for daily excreta collection and euthanized for ileal digesta to asses apparent metabolisable energy AME and nutrients digestibility. All dietary treated groups compered to birds in the NC group enhanced nutrients digestibility and AME. It was deduced that supplementing organic acids and essential oils are beneficial in improving birds performance, health, nutrient digestibility and subsequently could potentially replace antibiotic as growth promoters and could enhance the utilisation of animal protein concentrate without compromising performance and gut health in the poultry feed industry.

The present study was designed to study the effects of conventional and free range systems on egg quality attributes, fatty acid profile, and cholesterol contents of laying hen. Two hundred and forty R1 cross of Rhode Island Red (RIR) × Fyoumi (F) layers during the peak production were divided into two groups and reared in two different housing systems (free range vs. conventional systems). The statistical analysis of the results revealed that the housing system (modification of diet) significantly (p < 0.05) affected egg quality, fatty acid profile, and cholesterol contents. The highest (p < 0.05) whole egg and albumen weights were recorded for eggs produced in a conventional system and higher yolk weight and shell thickness were recorded in free range hens. Significantly (p < 0.05) lower concentration of saturated and higher concentration of mono- and polyunsaturated fatty acids were observed in eggs produced under a free range system. In addition, higher level of total omega-3 fatty acids and higher ratio of omega-6 to omega-3 fatty acids were observed in free range eggs as compared to those in the conventional system. From the results of the present study, we concluded that eggs produced from Rhode Island Red × Fyoumi laying hens in a free range system are qualitatively superior compared to those in the conventional system.

Dengue is the most burdensome vector-borne viral disease in the world. Dengue virus (DENV), the etiological cause of dengue, is transmitted primarily by the Aedes aegypti mosquito. Like any arbovirus, the transmission cycle of dengue involves the complex interactions of a multitude of human and mosquito factors. One point during this transmission cycle that is rich in these interactions is the biting event by the mosquito, upon which its saliva is injected into the host. A number of components in mosquito saliva have been shown to play a pivotal role in the transmission of dengue, however one such component that is not as well characterized is extracellular vesicles. Here, using high-performance liquid chromatography in tandem with mass spectrometry, we show that dengue infection altered the protein cargo of Aedes aegypti extracellular vesicles, resulting in the packaging of proteins with infection-enhancing ability. Our results support the presence of an infection-dependent pro-viral protein packaging strategy that uses the differential packaging of pro-viral proteins in extracellular vesicles of Ae. aegypti saliva to promote transmission. These studies represent the first investigation into the function of Ae. aegypti extracellular vesicle cargo during dengue infection.

MicroRNAs (miRNAs) are a group of small noncoding RNAs that regulate gene expression during important biological processes including development and pathogen defense in most living organisms. Presently, no miRNAs have been identified in the mosquito Culex tarsalis (Diptera: Culicidae), one of the most important vectors of West Nile virus (WNV) in North America. We used small RNA sequencing data and in vitro and in vivo experiments to identify and validate a repertoire of miRNAs in Cx. tarsalis mosquitoes. Using bioinformatic approaches we analyzed small RNA sequences from the Cx. tarsalis CT embryonic cell line to discover orthologs for 86 miRNAs. Consistent with other mosquitoes such as Aedes albopictus and Culex quinquefasciatus, miR-184 was found to be the most abundant miRNA in Cx. tarsalis. We also identified 20 novel miRNAs from the recently sequenced Cx. tarsalis genome, for a total of 106 miRNAs identified in this study. The presence of selected miRNAs was biologically validated in both the CT cell line and in adult Cx. tarsalis mosquitoes using RT–qPCR and sequencing. These results will open new avenues of research into the role of miRNAs in Cx. tarsalis biology, including development, metabolism, immunity, and pathogen infection.

Animal industry seeks cost-effective solutions to enhance performance and health of domestic animals. This study investigated the effects of supplementing Bacillus spp. probiotics and xylanase on 2000 one-day-old Japanese quails, randomly assigned to four treatment groups (10 replicates). The control group received no supplementation, while the others were supplemented with a Bacillus-based probiotic at 7.5 × 10 7 cfu/kg of feed, xylanase enzyme (2,000 U/kg) alone or in combination. Quails receiving both probiotic and enzyme exhibited significantly (p < 0.01) higher weekly and overall weight gain, and lower feed conversion ratios compared to the control group. Dressing percentage was higher (p < 0.01), and mortality lower in birds supplemented with a combination of enzyme and probiotic. Antibody titres against infectious bronchitis and infectious bursal disease were significantly (p < 0.01) higher in quails receiving combined probiotic and enzyme supplementation, while titres against Newcastle disease virus were higher (p < 0.01) in groups supplemented with probiotic and enzyme individually or in combination. Additionally, digestibility was significantly (p < 0.01) higher in groups receiving combined enzyme and probiotic supplementation, with higher apparent metabolizable energy compared to the control. The populations of beneficial Lactobacillus increased, while harmful E. coli and Salmonella decreased significantly in quails supplemented with both probiotic and enzyme. In conclusion, supplementing xylanase enzyme and probiotic together in Japanese quails positively influenced growth, nutrient digestibility, immune response, and cecal microbiota.

The endosymbiont Wolbachia is known to block replication of several important arboviruses, including dengue virus (DENV), in the mosquito vector Aedes aegypti. So far, the exact mechanism of this viral inhibition is not fully understood. A recent study in Drosophila melanogaster has demonstrated an interaction between the pelo gene and Drosophila C virus. In this study, we explored the possible involvement of the pelo protein, that is involved in protein translation, in Wolbachia-mediated antiviral response and mosquito-DENV interaction. We found that pelo is upregulated during DENV replication and its silencing leads to reduced DENV virion production suggesting that it facilities DENV replication. However, in the presence of Wolbachia, specifically in female mosquitoes, the pelo protein is downregulated and its subcellular localization is altered, which could contribute to reduction in DENV replication in Ae. aegypti. In addition, we show that the microRNA aae-miR-2940-5p, whose abundance is highly enriched in Wolbachia-infected mosquitoes, might mediate regulation of pelo. Our data reveals identification of pelo as a host factor that is positively involved in DENV replication, and its suppression in the presence of Wolbachia may contribute to virus blocking exhibited by the endosymbiont.