A perspective on the impacts of microplastics on mosquito biology and their vectorial capacity.

Microplastics (plastic particles <5 mm) permeate aquatic and terrestrial ecosystems and constitute a hazard to animal life. Although much research has been conducted on the effects of microplastics on marine and benthic organisms, less consideration has been given to insects, especially those adapted to urban environments. Here, we provide a perspective on the potential consequences of exposure to microplastics within typical larval habitat on mosquito biology. Mosquitoes represent an ideal organism in which to explore the biological effects of microplastics on terrestrial insects, not least because of their importance as an infectious disease vector. Drawing on evidence from other organisms and knowledge of the mosquito life cycle, we summarise some of the more plausible impacts of microplastics including physiological, ecotoxicological and immunological responses. We conclude that although there remains little experimental evidence demonstrating any adverse effect on mosquito biology or pathogen transmission, significant knowledge gaps remain, and there is now a need to quantify the effects that microplastic pollution could have on such an important disease vector.

Interkingdom interactions shape the fungal microbiome of mosquitoes.

BACKGROUND: The mosquito microbiome is an important modulator of vector competence and vectoral capacity. Unlike the extensively studied bacterial microbiome, fungal communities in the mosquito microbiome (the mycobiome) remain largely unexplored. To work towards getting an improved understanding of the fungi associated with mosquitoes, we sequenced the mycobiome of three field-collected and laboratory-reared mosquito species (Aedes albopictus, Aedes aegypti, and Culex quinquefasciatus). RESULTS: Our analysis showed both environment and host species were contributing to the diversity of the fungal microbiome of mosquitoes. When comparing species, Ae. albopictus possessed a higher number of diverse fungal taxa than Cx. quinquefasciatus, while strikingly less than 1% of reads from Ae. aegypti samples were fungal. Fungal reads from Ae. aegypti were < 1% even after inhibiting host amplification using a PNA blocker, indicating that this species lacked a significant fungal microbiome that was amplified using this sequencing approach. Using a mono-association mosquito infection model, we confirmed that mosquito-derived fungal isolates colonize Aedes mosquitoes and support growth and development at comparable rates to their bacterial counterparts. Strikingly, native bacterial taxa isolated from mosquitoes impeded the colonization of symbiotic fungi in Ae. aegypti suggesting interkingdom interactions shape fungal microbiome communities. CONCLUSION: Collectively, this study adds to our understanding of the fungal microbiome of different mosquito species, that these fungal microbes support growth and development, and highlights that microbial interactions underpin fungal colonization of these medically relevent species.

Aedes aegypti gut transcriptomes respond differently to microbiome transplants from field-caught or laboratory-reared mosquitoes.

The mosquito microbiome is critical for host development and plays a major role in many aspects of mosquito biology. While the microbiome is commonly dominated by a small number of genera, there is considerable variation in composition among mosquito species, life stages, and geography. How the host controls and is affected by this variation is unclear. Using microbiome transplant experiments, we asked whether there were differences in transcriptional responses when mosquitoes of different species were used as microbiome donors. We used microbiomes from four different donor species spanning the phylogenetic breadth of the Culicidae, collected either from the laboratory or the field. We found that when recipients received a microbiome from a donor reared in the laboratory, the response was remarkably similar regardless of donor species. However, when the donor had been collected from the field, many more genes were differentially expressed. We also found that while the transplant procedure did have some effect on the host transcriptome, this is likely to have had a limited effect on mosquito fitness. Overall, our results highlight the possibility that variation in mosquito microbiome communities is associated with variability in host-microbiome interactions and further demonstrate the utility of the microbiome transplantation technique for investigating host-microbe interactions in mosquitoes.

Persistence of SARS-CoV-2 and its surrogate, bacteriophage Phi6, on surfaces and in water.

IMPORTANCE: The COVID-19 pandemic spurred research on the persistence of SARS-CoV-2 and its surrogates. Here we highlight the importance of evaluating viral surrogates and experimental methodologies when studying pathogen survival in the environment.

Variable microbiomes between mosquito lines are maintained across different environments.

The composition of the microbiome is shaped by both environment and host in most organisms, but in the mosquito Aedes aegypti the role of the host in shaping the microbiome is poorly understood. Previously, we had shown that four lines of Ae. aegypti harbored different microbiomes when reared in the same insectary under identical conditions. To determine whether these lines differed from each other across time and in different environments, we characterized the microbiome of the same four lines of Ae. aegypti reared in the original insectary and at another institution. While it was clear that the environment influenced the microbiomes of these lines, we did still observe distinct differences in the microbiome between lines within each insectary. Clear differences were observed in alpha diversity, beta diversity, and abundance of specific bacterial taxa. To determine if the line specific differences in the microbiome were maintained across environments, pair-wise differential abundances of taxa was compared between insectaries. Lines were most similar to other lines from the same insectary than to the same line reared in a different insectary. Additionally, relatively few differentially abundant taxa identified between pairs of lines were shared across insectaries, indicating that line specific properties of the microbiome are not conserved across environments, or that there were distinct microbiota within each insectary. Overall, these results demonstrate that mosquito lines under the same environmental conditions have different microbiomes across microbially- diverse environments and host by microbe interactions affecting microbiome composition and abundance is dependent on environmentally available bacteria.

Identification and characterization of two CRISPR/Cas systems associated with the mosquito microbiome.

The microbiome profoundly influences many traits in medically relevant vectors such as mosquitoes, and a greater functional understanding of host-microbe interactions may be exploited for novel microbial-based approaches to control mosquito-borne disease. Here, we characterized two novel clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems in Serratia sp. Ag1, which was isolated from the gut of an Anopheles gambiae mosquito. Two distinct CRISPR/Cas systems were identified in Serratia Ag1, CRISPR1 and CRISPR2. Based on cas gene composition, CRISPR1 is classified as a type I-E CRISPR/Cas system and has a single array, CRISPR1. CRISPR2 is a type I-F system with two arrays, CRISPR2.1 and CRISPR2.2. RT-PCR analyses show that all cas genes from both systems are expressed during logarithmic growth in culture media. The direct repeat sequences of CRISPRs 2.1 and 2.2 are identical and found in the arrays of other Serratia spp., including S. marcescens and S. fonticola , whereas CRISPR1 is not. We searched for potential spacer targets and revealed an interesting difference between the two systems: only 9 % of CRISPR1 (type I-E) targets are in phage sequences and 91 % are in plasmid sequences. Conversely, ~66 % of CRISPR2 (type I-F) targets are found within phage genomes. Our results highlight the presence of CRISPR loci in gut-associated bacteria of mosquitoes and indicate interplay between symbionts and invasive mobile genetic elements over evolutionary time.

Recovery of metagenomic data from the Aedes aegypti microbiome using a reproducible snakemake pipeline: MINUUR.

Background: Ongoing research of the mosquito microbiome aims to uncover novel strategies to reduce pathogen transmission. Sequencing costs, especially for metagenomics, are however still significant. A resource that is increasingly used to gain insights into host-associated microbiomes is the large amount of publicly available genomic data based on whole organisms like mosquitoes, which includes sequencing reads of the host-associated microbes and provides the opportunity to gain additional value from these initially host-focused sequencing projects. Methods: To analyse non-host reads from existing genomic data, we developed a snakemake workflow called MINUUR (Microbial INsights Using Unmapped Reads). Within MINUUR, reads derived from the host-associated microbiome were extracted and characterised using taxonomic classifications and metagenome assembly followed by binning and quality assessment. We applied this pipeline to five publicly available Aedes aegypti genomic datasets, consisting of 62 samples with a broad range of sequencing depths. Results: We demonstrate that MINUUR recovers previously identified phyla and genera and is able to extract bacterial metagenome assembled genomes (MAGs) associated to the microbiome. Of these MAGS, 42 are high-quality representatives with >90% completeness and <5% contamination. These MAGs improve the genomic representation of the mosquito microbiome and can be used to facilitate genomic investigation of key genes of interest. Furthermore, we show that samples with a high number of KRAKEN2 assigned reads produce more MAGs. Conclusions: Our metagenomics workflow, MINUUR, was applied to a range of Aedes aegypti genomic samples to characterise microbiome-associated reads. We confirm the presence of key mosquito-associated symbionts that have previously been identified in other studies and recovered high-quality bacterial MAGs. In addition, MINUUR and its associated documentation are freely available on GitHub and provide researchers with a convenient workflow to investigate microbiome data included in the sequencing data for any applicable host genome of interest.

Knockdown resistance allele L1014F introduced by CRISPR/Cas9 is not associated with altered vector competence of Anopheles gambiae for o'nyong nyong virus.

Knockdown resistance (kdr) alleles conferring resistance to pyrethroid insecticides are widespread amongst vector populations. Previous research has suggested that these alleles are associated with changes in the vector competence of mosquitoes for arboviruses and Plasmodium, however non-target genetic differences between mosquito strains may have had a confounding effect. Here, to minimise genetic differences, the laboratory Anopheles gambiae Kisumu strain was compared to a CRISPR/Cas9 homozygous kdr L1014F mutant Kisumu-kdr line in order to examine associations with vector competence for o'nyong nyong virus (ONNV). Mosquitoes were infected using either blood feeds or intrathoracic microinjections. There were no significant differences in the prevalence of virus in mosquito body parts between kdr mutant and wildtype lines from either oral or intrathoracic injection routes. The ONNV titre was significantly higher in the legs of the wildtype strain at 7dpi following intrathoracic microinjection, but no other significant differences in viral titre were detected. ONNV was not detected in the saliva of mosquitoes from either strain. Our findings from per os infections suggest that the kdr L1014F allele is not associated with altered infection prevalence for ONNV, a key component of vector competence.

Wolbachia-induced inhibition of O'nyong nyong virus in Anopheles mosquitoes is mediated by Toll signaling and modulated by cholesterol.

Enhanced host immunity and competition for metabolic resources are two main competing hypotheses for the mechanism of Wolbachia-mediated pathogen inhibition in arthropods. Using an Anopheles mosquito - somatic Wolbachia infection - O'nyong nyong virus (ONNV) model, we demonstrate that the mechanism underpinning Wolbachia-mediated virus inhibition is up-regulation of the Toll innate immune pathway. However, the viral inhibitory properties of Wolbachia were abolished by cholesterol supplementation. This result was due to Wolbachia-dependent cholesterol-mediated suppression of Toll signaling rather than competition for cholesterol between Wolbachia and virus. The inhibitory effect of cholesterol was specific to Wolbachia-infected Anopheles mosquitoes and cells. These data indicate that both Wolbachia and cholesterol influence Toll immune signaling in Anopheles mosquitoes in a complex manner and provide a functional link between the host immunity and metabolic competition hypotheses for explaining Wolbachia-mediated pathogen interference in mosquitoes. In addition, these results provide a mechanistic understanding of the mode of action of Wolbachia-induced pathogen blocking in Anophelines, which is critical to evaluate the long-term efficacy of control strategies for malaria and Anopheles-transmitted arboviruses.

A cryopreservation method to recover laboratory- and field-derived bacterial communities from mosquito larval habitats.

Mosquitoes develop in a wide range of aquatic habitats containing highly diverse and variable bacterial communities that shape both larval and adult traits, including the capacity of adult females of some mosquito species to transmit disease-causing organisms to humans. However, while most mosquito studies control for host genotype and environmental conditions, the impact of microbiota variation on phenotypic outcomes of mosquitoes is often unaccounted for. The inability to conduct reproducible intra- and inter-laboratory studies of mosquito-microbiota interactions has also greatly limited our ability to identify microbial targets for mosquito-borne disease control. Here, we developed an approach to isolate and cryopreserve bacterial communities derived from lab and field-based larval rearing environments of the yellow fever mosquito Aedes aegypti-a primary vector of dengue, Zika, and chikungunya viruses. We then validated the use of our approach to generate experimental microcosms colonized by standardized lab- and field-derived bacterial communities. Our results overall reveal minimal effects of cryopreservation on the recovery of both lab- and field-derived bacteria when directly compared with isolation from non-cryopreserved fresh material. Our results also reveal improved reproducibility of bacterial communities in replicate microcosms generated using cryopreserved stocks over fresh material. Communities in replicate microcosms further captured the majority of total bacterial diversity present in both lab- and field-based larval environments, although the relative richness of recovered taxa as compared to non-recovered taxa was substantially lower in microcosms containing field-derived bacteria. Altogether, these results provide a critical next step toward the standardization of mosquito studies to include larval rearing environments colonized by defined microbial communities. They also lay the foundation for long-term studies of mosquito-microbe interactions and the identification and manipulation of taxa with potential to reduce mosquito vectorial capacity.

Aedes aegypti gut transcriptomes respond differently to microbiome transplants from field-caught or laboratory-reared mosquitoes.

The mosquito microbiome is critical for host development and plays a major role in many aspects of mosquito biology. While the microbiome is commonly dominated by a small number of genera, there is considerable variation in composition among mosquito species, life stages, and geography. How the host controls and is affected by this variation is unclear. Using microbiome transplant experiments, we asked whether there were differences in transcriptional responses when mosquitoes of different species were used as microbiome donors. We used microbiomes from four different donor species spanning the phylogenetic breadth of the Culicidae, collected either from the laboratory or field. We found that when recipients received a microbiome from a donor reared in the laboratory, the response was remarkably similar regardless of donor species. However, when the donor had been collected from the field, far more genes were differentially expressed. We also found that while the transplant procedure did have some effect on the host transcriptome, this is likely to have had a limited effect on mosquito fitness. Overall, our results highlight the possibility that variation in mosquito microbiome communities are associated with variability in host-microbiome interactions and further demonstrate the utility of the microbiome transplantation technique.

Predictors of Thirty-Day Hospital Readmissions in Systemic Lupus Erythematosus in the United States: A Nationwide Study.

OBJECTIVE: To evaluate independent risk factors for readmission and to determine the major reasons for readmission in a nationally representative sample of patients with systemic lupus erythematosus (SLE). METHODS: We used the Nationwide Readmissions Database to identify adults with SLE who were discharged from hospital to home during January-November of 2016 and 2017. Thirty-day all-cause readmissions were identified. A multivariable adjusted survey-specific logistic regression model was used to identify factors associated with readmission. RESULTS: A total of 132,400 hospitalized adults with SLE were discharged home during the study period; 88.3% were female, with a median age of 51.0 years (interquartile range 38.7-61.9 years). Of these, 18,973 individuals (14.3%) were readmitted within 30 days of discharge from their index hospitalization. In multivariable analyses, the factors associated with the highest odds for readmission were autoimmune hemolytic anemia (odds ratio [OR] 1.86 [95% confidence interval (95% CI) 1.51-2.29]), glomerular disease (OR 1.27 [95% CI 1.19-1.36]), pericarditis (OR 1.35 [95% CI 1.14-1.60]), heart failure (OR 1.34 [95% CI 1.24-1.44]), age 18-30 years (OR 1.28 [95% CI 1.17-1.41] versus age ≥​65 years), and Medicare (OR 1.20 [95% CI 1.13-1.28]) and Medicaid insurance (OR 1.26 [95% CI 1.18-1.34]). Sepsis (7.6%), SLE (7.4%), heart failure (3.5%), and pneumonia (3.2%) were among the most common causes for readmission. CONCLUSION: In this nationally representative study of SLE readmissions, the strongest risk factors for 30-day readmission were younger age, SLE-related manifestations, and public insurance. These results identify patient groups with SLE that would benefit from postdischarge interventions designed to reduce hospitalizations and improve health outcomes.

Vertical and Horizontal Transmission of Cell Fusing Agent Virus in Aedes aegypti.

Cell fusing agent virus (CFAV) is an insect-specific flavivirus (ISF) found in Aedes aegypti mosquitoes. ISFs have demonstrated the ability to modulate the infection or transmission of arboviruses such as dengue, West Nile, and Zika viruses. It is thought that vertical transmission is the main route for ISF maintenance in nature. This has been observed with CFAV, but there is evidence of horizontal and venereal transmission in other ISFs. Understanding the route of transmission can inform strategies to spread ISFs to vector populations as a method of controlling pathogenic arboviruses. We crossed individually reared male and female mosquitoes from both a naturally occurring CFAV-positive Ae. aegypti colony and its negative counterpart to provide information on maternal, paternal, and horizontal transmission. RT-PCR was used to detect CFAV in individual female pupal exuviae and was 89% sensitive, but only 42% in male pupal exuviae. This is a possible way to screen individuals for infection without destroying the adults. Female-to-male horizontal transmission was not observed during this study. However, there was a 31% transmission rate from mating pairs of CFAV-positive males to negative female mosquitoes. Maternal vertical transmission was observed with a filial infection rate of 93%. The rate of paternal transmission was 85% when the female remained negative, 61% when the female acquired CFAV horizontally, and 76% overall. Maternal and paternal transmission of CFAV could allow the introduction of this virus into wild Ae. aegypti populations through male or female mosquito releases, and thus provides a potential strategy for ISF-derived arbovirus control. IMPORTANCE Insect-specific flaviviruses (ISFs), are a group of nonpathogenic flaviviruses that only infect insects. ISFs can have a high prevalence in mosquito populations, but their transmission routes are not well understood. The results of this study confirm maternal transmission of cell fusing agent virus (CFAV) and demonstrate that paternal transmission is also highly efficient. Horizontal transmission of CFAV was also observed, aided by evaluation of the pupal infection status before mating with an infected individual. This technique of detecting infection in discarded pupae exuviae has not been evaluated previously and will be a useful tool for others in the field of studying viral transmission in mosquitoes. Identifying these routes of transmission provides information about how CFAV could be maintained in wild populations of mosquitoes and can aid future studies focusing on interactions of CFAV with their hosts and other viruses that infect mosquitoes.

Lack of robust evidence for a Wolbachia infection in Anopheles gambiae from Burkina Faso.

The endosymbiont Wolbachia can have major effects on the reproductive fitness, and vectorial capacity of host insects and may provide new avenues to control mosquito-borne pathogens. Anopheles gambiae s.l is the major vector of malaria in Africa but the use of Wolbachia in this species has been limited by challenges in establishing stable transinfected lines and uncertainty around native infections. High frequencies of infection of Wolbachia have been previously reported in An. gambiae collected from the Valle du Kou region of Burkina Faso in 2011 and 2014. Here, we re-evaluated the occurrence of Wolbachia in natural samples, collected from Valle du Kou over a 12-year time span, and in addition, expanded sampling to other sites in Burkina Faso. Our results showed that, in contrast to earlier reports, Wolbachia is present at an extremely low prevalence in natural population of An. gambiae. From 5341 samples analysed, only 29 were positive for Wolbachia by nested PCR representing 0.54% of prevalence. No positive samples were found with regular PCR. Phylogenetic analysis of 16S rRNA gene amplicons clustered across supergroup B, with some having similarity to sequences previously found in Anopheles from Burkina Faso. However, we cannot discount the possibility that the amplicon positive samples we detected were due to environmental contamination or were false positives. Regardless, the lack of a prominent native infection in An. gambiae s.l. is encouraging for applications utilizing Wolbachia transinfected mosquitoes for malaria control.

Multiple Plasmodium falciparum drug resistance polymorphisms identified in a pregnant woman with severe malaria and a concomitant spontaneous abortion in Cross River, Nigeria, West Africa.

BACKGROUND: The development of resistance by Plasmodium falciparum to anti-malarial drugs impedes any benefits of the drug. In addition, absence or delayed availability of current anti-malarial drugs in remote areas has the potential to results to parasite escape and continuous transmission. CASE PRESENTATION: The case of a 29-year old pregnant woman from Biase Local Government Area in Cross River State Nigeria presenting with febrile illness and high body temperature of 38.7 °C was reported. She looked pale and vomited twice on arrival at the health facility. Her blood smear on the first day of hospitalization was positive for P. falciparum by RDT, microscopy (21,960 parasite/µl) and real-time PCR, with a PCV of 18%. She was treated with 600 mg intravenous quinine in 500 ml of 5% Dextrose/0.9% Saline 8-hourly for 24 h. On the second day of hospitalization, she complained of weakness, persistent high-grade fever and vaginal bleeding. A bulging amnion from an extended cervix was observed. Following venous blood collection for laboratory investigations, 600 µg of misoprostol was inserted into the posterior fornix of her vagina as part of her obstetric care. Parenteral quinine was discontinued, and she was given full therapeutic regimen of artemether-lumefantrine 80/480 mg tablets to be taken for 3 days beginning from the second day. Her blood samples on the second and third day of hospitalization remained positive for P. falciparum by all three diagnostic methods. Single nucleotide polymorphism (SNP) assay on all three P. falciparum isolates revealed the presence of variants associated with multiple drug resistant markers. DISCUSSION: Infecting P. falciparum isolates may have been resistant to initial quinine treatment resulting from parasite cross-resistance with other quinoline associated resistant markers such as 86Y and 184 F. CONCLUSIONS: Therefore, the likely transmission of similarly resistant parasites in the study area calls for reinforcement of interventions and adherence to current World Health Organization guidelines in administering only approved drugs to individuals in order to mitigate parasite escape and eventual transmission to other susceptible individuals.

Transcriptomic and small RNA response to Mayaro virus infection in Anopheles stephensi mosquitoes.

Mayaro virus (MAYV) is an arboviral pathogen in the genus Alphavirus that is circulating in South America with potential to spread to naïve regions. MAYV is also one of the few viruses with the ability to be transmitted by mosquitoes in the genus Anopheles, as well as the typical arboviral transmitting mosquitoes in the genus Aedes. Few studies have investigated the infection response of Anopheles mosquitoes. In this study we detail the transcriptomic and small RNA responses of An. stephensi to infection with MAYV via infectious bloodmeal at 2, 7, and 14 days post infection (dpi). 487 unique transcripts were significantly regulated, 78 putative novel miRNAs were identified, and an siRNA response is observed targeting the MAYV genome. Gene ontology analysis of transcripts regulated at each timepoint shows a number of proteases regulated at 2 and 7 dpi, potentially representative of Toll or melanization pathway activation, and repression of pathways related to autophagy and apoptosis at 14 dpi. These findings provide a basic understanding of the infection response of An. stephensi to MAYV and help to identify host factors which might be useful to target to inhibit viral replication in Anopheles mosquitoes.

Methods of SARS-CoV-2 Inactivation.

Inactivation methods allow for hazard group 3 (HG3) pathogens to be disposed of and used safely in downstream experiments and assays to be carried out at lower containment levels. Commonly used viral inactivation methods include heat inactivation, fixation methods, ultraviolet (UV) light and detergent inactivation. Here we describe known methods used to inactivate SARS-CoV-2 for safe downstream biological assays.

Neuroinvasion and Neurotropism by SARS-CoV-2 Variants in the K18-hACE2 Mouse.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) not only affects the respiratory tract but also causes neurological symptoms such as loss of smell and taste, headache, fatigue or severe cerebrovascular complications. Using transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2), we investigated the spatiotemporal distribution and pathomorphological features in the CNS following intranasal infection with SARS-CoV-2 variants, as well as after prior influenza A virus infection. Apart from Omicron, we found all variants to frequently spread to and within the CNS. Infection was restricted to neurons and appeared to spread from the olfactory bulb mainly in basally oriented regions in the brain and into the spinal cord, independent of ACE2 expression and without evidence of neuronal cell death, axonal damage or demyelination. However, microglial activation, microgliosis and a mild macrophage and T cell dominated inflammatory response was consistently observed, accompanied by apoptotic death of endothelial, microglial and immune cells, without their apparent infection. Microgliosis and immune cell apoptosis indicate a potential role of microglia for pathogenesis and viral effect in COVID-19 and the possible impairment of neurological functions, especially in long COVID. These data may also be informative for the selection of therapeutic candidates and broadly support the investigation of agents with adequate penetration into relevant regions of the CNS.