To the best of our understanding, this study is the first to utilize 16S metagenomic analysis to understand the significance of a filarial nematode on the microbiome of a mosquito vector under laboratory conditions. This technique offers the advantage of detecting both culturable and unculturable pathogenic and non-pathogenic microbes from a given DNA or RNA. As previously reported from previous metagenome studies on mosquitoes and similar arthropods or insects (Mancini et al., 2018; Coon et al., 2014; Coatsworth et al., 2018), Figure 1A shows that the phylum Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes was detected from the mosquito groups tested. Similar studies have also identified the phylum Proteobacteria as one of the dominant phylum in the microbiome of A. aegypti mosquitoes (Ramirez et al., 2012; Audsley et al., 2017). We observed an inverse relationship in the abundances of the phylum Proteobacteria and Bacteroidetes in our study with D. immitis infected mosquitoes having a higher abundance of the phylum Proteobacteria when compared to the uninfected mosquitoes and vice-versa. The Proteobacteria group are the largest phylum found in different environments, plants and animals (http://www.bacterio.net/; Degli and Martinez, 2017) with members ranging from free living commensals to pathogenic microbes. We found that the genus Klebsiella was present in relatively similar abundances in both infected and uninfected mosquitoes. Although no known function has been associated to this group in insects or arthropods, Klebsiella belongs to a class of gram Proteobacteria known as Gammaproteobacteria with previous detection in field and laboratory raised Culex quinquefasciatus, A. albopictus, and A. aegypti (Gonçalves et al., 2019; Yadav et al., 2015; Wu et al., 2019).
The presence of the genus Enterobacter in infected mosquito at approximately 20 folds of uninfected mosquitoes was also observed. This bacteria genus has been detected in similar microbiome studies of A. aegypti where they have been associated with their role in blood meal digestion due to their hemolytic activities (Coon et al., 2016). This most likely explains why the genus Enterobacter have been commonly associated with females of other mosquito species (Minard et al., 2013). A likely explanation for the increase in the abundance of this genus in the presence of D. immitis colonization could be a response to deplete the abundance of the filarial nematode through specific mechanisms by the bacteria. An elegant study by Cirimotich and colleagues showed the inhibition of Plasmodium infection in Anopheles gambiae mosquitoes mediated by a bacterium designated as Enterobacter Esp_Z. The inhibition was due to the production of reactive oxygen species by the bacteria. The presence of Enterobacter could also facilitate or enhance mosquito infection by D. immitis. Bacteria have also been shown to facilitate pathogen infectivity within arthropod vectors. One of such ways they achieve this is by producing enzyme-like substances that degrades compartment barriers within the arthropods which then aids in efficient colonization by the pathogen. Enhancins or enhancing-like proteins are produced by baculovirus such as Tricoplusia ni granuloviris and bacteria such as Bacillus and have been shown to degrade the peritrophic matrix of insect hence facilitating gut colonization by pathogens (Toprak et al., 2012; Fang et al., 2009; Parkhill et al., 2001). A recent study on the enhancing producing commensal bacteria from A. aegypti identified Enterobacter as an enhancin expressing bacteria (Wu t al., 2019).
Another bacteria genus that also increased with the presence of D. immitis infection is Elizabethkingia. Previous report has identified this genus from the midgut of laboratory reared (Gonçalves et al., 2019) and field collected (Audsley at al., 2017) A. aegypti. These studies did not associate any known role to this bacterium.
Bacteria in the genus Chryseobacterium, Ochrobactrum, Sphingobacterium, and Pseudomonas were all present in higher abundance in uninfected mosquito group. The detection of Chryseobacterium from our study agrees with similar detection from previous report in laboratory reared mosquitoes (Chen et al., 2016; Wang et al., 2011). Pseudomonas, a Gram-negative, Gammaproteobacteria was previously shown to have reduced abundance in Wolbachia positive A. aegypti mosquitoes. A similar observation was also made from our study where the abundance of Pseudomonas was inversely correlated with the presence of D. immitis infection in the mosquito as shown in Figure 2.
Bahia et al. reported blocking of the Plasmodium parasite by Pseudomonas putida isolated from A. gambiae.
Unexpectedly, only few bacteria species were differentially altered in D. immitis infected and uninfected mosquitoes. Klebsiella (K.) oxytoca, a Gammaproteobacteria belonging to the genus Klebsiella, was found at relatively similar abundance in infected and uninfected mosquitoes (Fig 3). A possible explanation for the presence of K. oxytoca in similar abundances in infected and uninfected mosquitoes could be an indication of the bacteria been a likely endosymbiont that could be vertically and horizontally acquired as previous studies have reported the detection of K. oxytoca from laboratory reared and field collected C. quinquefasciatus, A. aegypti (Gonçalves et al., 2019). A symbiotic role will explain the reason why it was detected in such abundances in both mosquito groups. A probiotic role has also been associated with the bacteria in male medfly (Ceratitis capitate), where it was shown to reverse radiation induced loss of copulatory performance (Ami et al., 2009). A study on the fungicidal effects of bacterial colonies found on the domestic housefly eggs revealed that fungal growth is inhibited by the presence of K. oxytoca by producing antifungal metabolites and nutrient depletion (Lam et al., 2009). Determining the microbial communities in mosquito eggs and their antimicrobial activities will give a broader insight into members that inhibit microbial growth. The expression of Enhancin proteins by certain bacteria species have been documented and shown to breakdown the peritrophic matrix of insect vectors (Fang et al., 2009; Parkhill et al., 2001). Wu et al. (2019) recently showed that Serratia (S.) marcescens facilitates dengue virus invasion of A. aegypti by producing the protein Enhancin which breaks down the mucin component of the peritrophic matrix barrier. Interestingly, the same study also reported that K. oxytoca expresses an Enhancin protein that degrades another type of mucin in A. aegypti but with no effect on dengue infection. Taken together, K. oxytoca could be proposed as having symbiotic relationship with the mosquito as well as a likely synergist by facilitating invasion and replication of specific pathogens, an observation that has been previously reported proposed in the Amblyomma maculatum tick (Budachetri et al., 2018).
Elizabethkingia (E.) meningoseptica and Enterobacter hormaechei were two of the notable bacteria species whose relative abundance increased with D. immitis infection. E. meningoseptica is a bacteria species with notable detection in several species of laboratory reared mosquitoes (Gonçalves et al., 2019; Boissie` re et al. 2012; Rani et al. 2009). It has also been detected in the eggs of Anopheles stephensi indicating a vertical means of transmission (Ngwa et al., 2013). Ngwa et al. (2013) also reported antiplasmodial and antimicrobial activity associated with E. meningoseptica in A. stephensi. While evaluation of antimicrobial or antiparasitic characteristics of the microbial communities detected was beyond the scope of our study, we propose that the increased abundance of E. meningoseptica in infected mosquitoes could 1) indirectly stem from its ability to prime the mosquito’s innate immune system, hence making it possible to inhibit the filarial infection and/or 2) directly produce metabolites that inhibit D. immitis.
hormaechei is another bacterium that demonstrated increased abundance in D. immitis infection as shown in Figure 3. The genus Enterobacter have been proposed to aid in blood digestion in hematophagous insects due to their hemolytic activities (Gaio et al., 2011). Several reports have also reported different mosquito refractoriness or susceptibility to pathogen infection in the presence of different Enterobacter species. Cirimotich et al. (2011) reported refractoriness of Anopheles gambiae to Plasmodium infection in the presence of an Enterobacter species. Another species of Enterobacter (E. cloacae) was also reported to express a mucin degrading Enhancin protein that breaks down the mucin component of the A. aegypti peritrophic matrix (Wu et al., 2019) although this wasn’t shown to facilitate pathogen colonization, in respect to Dengue virus infection.
Another interesting data from this study was the inverse correlation between D. immitis infection and microbial diversity and richness. In mosquitoes, the innate immune response is activated in the presence of invading pathogens as it was previously shown that activation of the toll pathway and production of reactive oxygen species have been reported in mosquitoes challenged with Plasmodium (Molina-Cruz et al., 2008) and filarial nematodes (Edgerton et al., 2020). These immune effectors while countering pathogen, could in extension disrupt the normal mosquito microbiome community which could explain the overall reduction in the microbial richness observed from this study as seen in Figure 4B and C. Our observation of reduced microbial richness in infected mosquitoes was in contrast to what was reported in a previous study which shows a more diverse microbiome in Plasmodium infected Anopheles mosquitoes (Bassene et al., 2018).
This study fills a knowledge gap on the interaction between a mosquito vector and a filarial pathogen of veterinary significance. We were able to show that while same bacterial species were found to be present in both mosquito groups, the relative abundances of individual species changes with the infection status, with infected mosquitoes presenting a reduced microbial richness. This indicates a likely consequence of the nematode in altering favoring or inhibiting growth of members of the bacterial community.