Detection and quantification of natural Wolbachia in Aedes aegypti in Metropolitan Manila, Philippines using locally designed primers.

Background: The Philippines bears health and economic burden caused by high dengue cases annually. Presently, the Philippines still lack an effective and sustainable vector management. The use of Wolbachia, a maternally transmitted bacterium, that mitigate arbovirus transmission has been recommended. Cytoplasmic incompatibility and viral blocking, two characteristics that make Wolbachia suitable for vector control, depend on infection prevalence and density. There are no current Wolbachia release programs in the Philippines, and studies regarding the safety of this intervention. Here, we screened for Wolbachia in Aedes aegypti collected from Metropolitan Manila, Philippines. We designed location-specific primers for qPCR to test whether this improved Wolbachia detection in Ae. aegypti. We explored if host sex and Wolbachia strain could be potential factors affecting Wolbachia density. Methods: Ae. aegypti mosquitoes (n=429) were screened for natural Wolbachia by taqman qPCR using location-specific Wolbachia surface protein primers (wspAAML) and known 16S rRNA primers. Samples positive for wspAAML (n=267) were processed for Sanger sequencing. We constructed a phylogenetic tree using IQ-TREE 2 to further characterize Wolbachia present in the Philippine Ae. aegypti. We then compared Wolbachia densities between Wolbachia groups and host sex. Statistical analyses were done using GraphPad Prism 9.0. Results: Wolbachia prevalence for 16S rRNA (40%) and wspAAML (62%) markers were high. Wolbachia relative densities for 16S rRNA ranged from -3.84 to 2.71 and wspAAML from -4.02 to 1.81. Densities were higher in male than female mosquitoes. Wolbachia strains detected in Ae. aegypti clustered into supergroup B. Some 54% (123/226) of these sequences clustered under a group referred to here as "wAegML," that belongs to the supergroup B, which had a significantly lower density than wAegB/wAlbB, and wAlbA strains. Conclusion: Location-specific primers improved detection of natural Wolbachia in Ae. aegypti and allowed for relative quantification. Wolbachia density is relatively low, and differed between host sexes and Wolbachia strains. An economical way of confirming sporadic or transient Wolbachia in Ae. aegypti is necessary while considering host sex and bacterial strain.

In vitro characterization of cell-fusing agent virus DNA forms in Aedes aegypti mosquitoes.

How non-retroviral endogenous viral elements (EVEs) are established is a long-standing question. Viral DNA (vDNA) forms of RNA viruses are likely to be EVE precursors. Cell-fusing agent virus (CFAV) is a major insect-specific virus (ISV) in the Aedes aegypti mosquitoes and one of the few existing non-retroviral RNA viruses found as EVEs. We characterized CFAV-derived vDNA in the cell line to understand the mechanism of why current viruses are rarely endogenized. vDNA production was affected by cell culture media independent of CFAV replication. vDNAs that correspond to different regions covering the entire viral genome were detected, implying multiple initiation sites exist. A considerable proportion of vDNAs corresponded to ssDNA. Higher vDNA copies were detected in the cytoplasm than the nucleus. Our findings provide valuable insights into the intracellular characteristics of ISV-derived vDNAs, which will aid in understanding the underlying mechanisms of non-retroviral EVE formation.

No detectable fitness cost of infection by cell-fusing agent virus in Aedes aegypti mosquitoes.

Aedes mosquitoes are well-known vectors of arthropod-borne viruses (arboviruses). Mosquitoes are more frequently infected with insect-specific viruses (ISVs) that cannot infect vertebrates. Some ISVs interfere with arbovirus replication in mosquito vectors, which has gained attention for potential use against arbovirus transmission. Cell-fusing agent virus (CFAV), a widespread ISV, can reduce arbovirus dissemination in Ae. aegypti. However, vectorial capacity is largely governed by other parameters than pathogen load, including mosquito survival and biting behaviour. Understanding how ISVs impact these mosquito fitness-related traits is critical to assess the potential risk of using ISVs as biological agents. Here, we examined the effects of CFAV infection on Ae. aegypti mosquito fitness. We found no significant reduction in mosquito survival, blood-feeding behaviour and reproduction, suggesting that Ae. aegypti is tolerant to CFAV. The only detectable effect was a slight increase in human attraction of CFAV-infected females in one out of eight trials. Viral tolerance is beneficial for introducing CFAV into natural mosquito populations, whereas the potential increase in biting activity must be further investigated. Our results provide the first insight into the link between ISVs and Aedes mosquito fitness and highlight the importance of considering all aspects of vectorial capacity for arbovirus control using ISVs.

Genome-wide detection of Wolbachia in natural Aedes aegypti populations using ddRAD-Seq.

Background: Wolbachia, an endosymbiotic bacterium, is globally used to control arboviruses because of its ability to block arboviral replication and manipulate the reproduction of Wolbachia host, Aedes aegypti. Polymerase chain reaction (PCR)-based Wolbachia detection has been recently reported from natural Ae. aegypti populations. However, due to the technical limitations of PCR, such as primer incompatibility, PCR-based assays are not sufficiently reliable or accurate. In this study, we examined double digestion restriction site-associated DNA sequencing (ddRAD-Seq) efficiency and limitations in Wolbachia detection and quantification in field-collected Ae. aegypti natural populations in Metro Manila, the Philippines, compared with PCR-based assays. Methods: A total of 217 individuals Ae. aegypti were collected from Metropolitan Manila, Philippines. We separated it into 14 populations consisting of 7 female and male populations. We constructed a library for pool ddRAD-Seq per population and also screened for Wolbachia by PCR assays using wsp and 16S rRNA. Wolbachia density per population were measured using RPS17 as the housekeeping gene. Results: From 146,239,637 sequence reads obtained, 26,299 and 43,778 reads were mapped across the entire Wolbachia genome (with the wAlbA and wAlbB strains, respectively), suggesting that ddRAD-Seq complements PCR assays and supports more reliable Wolbachia detection from a genome-wide perspective. The number of reads mapped to the Wolbachia genome per population positively correlated with the number of Wolbachia-infected individuals per population based on PCR assays and the relative density of Wolbachia in the Ae. aegypti populations based on qPCR, suggesting ddRAD-Seq-based semi-quantification of Wolbachia by ddRAD-Seq. Male Ae. aegypti exhibited more reads mapped to the Wolbachia genome than females, suggesting higher Wolbachia prevalence rates in their case. We detected 150 single nucleotide polymorphism loci across the Wolbachia genome, allowing for more accurate the detection of four strains: wPip, wRi, TRS of Brugia malayi, and wMel. Conclusions: Taken together, our results demonstrate the feasibility of ddRAD-Seq-based Wolbachia detection from field-collected Ae. aegypti mosquitoes.

Intracellular Interactions Between Arboviruses and Wolbachia in Aedes aegypti.

Aedes aegypti is inherently susceptible to arboviruses. The geographical expansion of this vector host species has led to the persistence of Dengue, Zika, and Chikungunya human infections. These viruses take advantage of the mosquito's cell to create an environment conducive for their growth. Arboviral infection triggers transcriptomic and protein dysregulation in Ae. aegypti and in effect, host antiviral mechanisms are compromised. Currently, there are no existing vaccines able to protect human hosts from these infections and thus, vector control strategies such as Wolbachia mass release program is regarded as a viable option. Considerable evidence demonstrates how the presence of Wolbachia interferes with arboviruses by decreasing host cytoskeletal proteins and lipids essential for arboviral infection. Also, Wolbachia strengthens host immunity, cellular regeneration and causes the expression of microRNAs which could potentially be involved in virus inhibition. However, variation in the magnitude of Wolbachia's pathogen blocking effect that is not due to the endosymbiont's density has been recently reported. Furthermore, the cellular mechanisms involved in this phenotype differs depending on Wolbachia strain and host species. This prompts the need to explore the cellular interactions between Ae. aegypti-arboviruses-Wolbachia and how different Wolbachia strains overall affect the mosquito's cell. Understanding what happens at the cellular and molecular level will provide evidence on the sustainability of Wolbachia vector control.

Acute oral toxicity assessment of ethanolic extracts of Antidesma bunius (L.) Spreng fruits in mice.

The aim of the study is to assess the acute oral toxicity of ethanolic extracts of Antidesma bunius (L.) Spreng [Bignay] in ICR mice in accordance to OECD guideline 423. Single doses of Bignay fruit extracts ranging from 500 mg/kg to 2000 mg/kg, as well as a vehicle control, were given orally and monitored for 14 days. We observed that there is no mortality or adverse effects after treatment of mice indicating that Bignay extracts are safe for use in laboratory animals. Behavioral, respiratory, and neurologic changes, as well as changes in body weight, food and water consumption, did not occur during the experimental period. Hematological- (total-red and -white blood cells) and biochemical-profile analysis (alanine transaminase, blood urea nitrogen, and creatinine) remained within normal concentrations in treated mice regardless of sex. The morphology of visceral organs in all treated mice was typical in appearance when stained with H&E. It is assumed that the LD50 is greater than 2000 mg/kg and there is no mortality at the maximum dose used (2000 mg/kg). We have also determined the active components of dried Bignay by headspace GC-MS and by phytochemical analysis of ethanolic Bignay extracts. We have identified 16 compounds by GC-MS and 10 secondary metabolites. In this study, the assessment of extracted Antidesma bunius (L.) Spreng fruits [Bignay] on the value of safety measures in mice is described.