Increased environmental microbial diversity reduces the disease risk of a mosquitocidal pathogen

ABSTRACT Increased diversity in host-associated microbial communities often benefits animal health by provisioning nutrients and protecting against pathogens. In contrast, whether the diversity of environmental microbial communities can also benefit animal health is much less studied. In aquatic environments, water enables environmental microbes to more directly interact with pathogens and animals than in terrestrial environments, which also suggests the potential for community diversity to benefit animal health. In this study, we addressed this question by identifying a strain of Chromobacterium haemolyticum named Rain 0013 (Ch_R13) that is a virulent pathogen of mosquito larvae, which live in aquatic habitats. Comparative genomic and functional data suggested many Chromobacterium are both mosquitocidal and opportunistic pathogens of other animals due to conservation of virulence factors that include a broad-spectrum toxin (cyanide). However, we also identified a strongly negative correlation between diversity of the environmental microbiota and disease severity due to Ch_R13 needing to achieve a threshold density to kill mosquito larvae. Experiments using defined (gnotobiotic) microbial communities further suggested certain environmental community members reduced Ch_R13 densities more than others. Altogether, our results indicate Ch_R13 and other Chromobacterium have the potential to broadly cause disease but the environmental communities of microbes in aquatic habitats attenuate disease risk. IMPORTANCE The host-specific microbiotas of animals can both reduce and increase disease risks from pathogens. In contrast, how environmental microbial communities affect pathogens is largely unexplored. Aquatic habitats are of interest because water enables environmental microbes to readily interact with animal pathogens. Here, we focused on mosquitoes, which are important disease vectors as terrestrial adults but are strictly aquatic as larvae. We identified a pathogen of mosquito larvae from the field as a strain of Chromobacterium haemolyticum. Comparative genomic analyses and functional assays indicate this strain and other Chromobacterium are mosquitocidal but are also opportunistic pathogens of other animals. We also identify a critical role for diversity of the environmental microbiota in disease risk. Our study characterizes both the virulence mechanisms of a pathogen and the role of the environmental microbiota in disease risk to an aquatic animal of significant importance to human health.


Adhesion and penetration protein app
Columns, solid dots, and asterisks are defined as in (A).

Mosquitoes
The University of Georgia (UGAL) strain of Aedes aegypti was established from wild-caught females in Athens, GA (2).The MRA-804 strain of Aedes albopictus was obtained from the Centers for Disease Control and Prevention (CDC) in Atlanta, GA, USA and has been in our insectary since 2012.The CDC MR4/BEI strain of Culex quinquefasciatus has been in our insectary since 2011.All mosquitoes were reared under a 12 h light:12 h dark photoperiod at 26°C and 70% relative humidity.Larvae of each species hatch from eggs and grow through four instars before pupating.Each was reared in pans at a density of ~150 larvae per liter of deionized water and fed daily rearing diet until pupation at ~6 days post-hatch.Rearing diet consisted of ground rat chow pellets (LabDiet 5001), lactalbumin (Sigma), and torula yeast extract (Bio-Serve) mixed in a ratio of 1:1:1 by volume, referred to as rat chow mix (RCM) (3).Adult mosquitoes were provided water and 10% sucrose in water ad libitum.For colony maintenance, adults from each generation were blood-fed 3-5 days post-eclosion to obtain eggs.Adults were blood-fed from an anesthetized male lab rat (Sprague Dawley), while An.gambiae was fed defibrinated rabbit blood (Hemostat Laboratories) using a membrane feeder.Anesthetization of rats and feeding were monitored by trained personnel following guidelines from the approved animal use protocol A2020 12-008-R1 approved by the University of Georgia Institutional Animal Care and Use Committee (IACUC), which is licensed by the US Department of Agriculture (USDA) and maintains an animal welfare Assurance, in compliance with Public Health Service policy, through the NIH Office of Laboratory Animal Welfare, and registration with the USDA APHIS Animal Care, in compliance with applicable federal regulations, guidance, and state laws governing animal use in research and teaching.Blood fed females were provided cups lined with wetted paper towel to oviposit upon 2-4 days post-blood feeding.

Bacteria
Bacteria used in the study and their origins are listed in Table S1.In addition to Ch_R13, these included Chromobacterium haemolyticum DSM 19808; C. haemolyticum NRRL B-11053, and eight commensal species (Microbacterium sp., Sphingobacterium sp., Flectobacillus sp., Rahnella sp., Serratia sp., Acinetobacter sp., Chryseobacterium sp., Comamonas sp.) we earlier isolated from CN laboratory or field cultures (4,5).We also used Escherichia coli K-12 substrain MG1655.E. coli has been identified in other A. aegypti cultures (5), while we have previously used this K12 MG1655 to produce monoxenic, GN A. aegypti cultures that develop into adults (6).Each of the above bacteria were stored as glycerol stocks at -80° C. Together, the eight commensal species plus E. coli K12 comprised the ALL-9 community described below that was used to produce gnotobiotic (GN) mosquito cultures.

Isolation, whole genome sequencing, and phylogenetic analysis of Ch_R13
Water from an outdoor container in Athens, GA USA named "Rain" was collected in September 2017 and returned to the laboratory where particulate detritus was removed by low-speed centrifugation at 250 x g followed by centrifugation of the supernatant at 6000 x g for 15 min.The resulting pellet was suspended in a 1:1 mixture of sterile glycerol:1x PBS and cryopreserved at -80C.Bacteria in the glycerol stock were plated on 1/10 diluted 869 agar plates and cultured at 25° C. Unique colony morphologies were selected and passaged three times to new agar plates to ensure individual isolates.Isolates were then suspended in a 1:1 mixture of sterile glycerol:1x PBS and cryopreserved at -80C.The DNeasy Blood and Tissue kit (Qiagen, Valencia, CA, USA) was used to isolate DNA from colonies exhibiting larvicidal activity (see below).DNA templates from these colonies were used to amplify a portion of the 16S rRNA gene with the primer set 27F short (5'-AGAGAGTTTGATCCTGGCTCAG-3') 1507R (5'-TACCTTGTTACGACTTCACCCCAG-3'), HotMaster Taq DNA polymerase (Quantabio, Beverly, MA, USA), and previously described polymerase chain reaction (PCR) conditions (3,7,8).Amplicons were visualized on a 1% agarose gel and cleaned with the QIAquick PCR purification kit (Qiagen, Valencia, CA, USA) before submitting for Sanger sequencing at Eurofins Genomics (Louisville, KY, USA).Resulting sequences were then compared to the NCBI nr database using BLASTn.
bioassays.Visibly fluorescent colonies were passaged to new selective media, and the 16S rRNA gene was sequenced (Eurofins Genomics, Louisville, KY, United States) to ensure correct species identity.Gycerol stocks were prepared to cryopreserve at -80°C the strain the strain used in assays (Ch_R13 E2-SpR ).

Axenic (AX), gnotbiotic (GN) and conventional (CN) mosquito cultures
AX larvae were produced by surface sterilizing A. aegypti eggs as previously described with resulting first instars hatching in sterile water (6).Ten first instars were then transferred to 6-well plates containing 5 ml of sterile water plus RCM diet per well to produce AX cultures whose sterility was confirmed using previously described PCR and culture-based assays (3,6).AX secondfourth instars used in assays were produced in darkness under AX rearing conditions as previously described using LP:YE diet (34) followed by transfer of larvae to culture wells containing 5 ml of sterile water plus RCM diet as used above for first instars.GN cultures with a defined community of commensal microbes were produced by adding one or members of the ALL9 community to AX cultures, which were grown and added as previously detailed (3).CN cultures were produced by placing first-fourth instar larvae from our general culture into 6-well culture plates containing 5 ml of sterile water plus RCM diet per well.

Larvicidal assays
The first larvicidal assays we conducted used 20 ml of water from container "Rain" (see above), sterile RCM diet and 20 CN A. aegypti, A. albopictus or C. quinquefasciatus first instars were added to 25 cm culture flasks (Genesee).Larvae were then observed every 24 h to assess the number of alive versus dead individuals present.The same assays were also conducted using sterile water or "Rain" water that was autoclaved or filter sterilized through an 0.2 µm filter (Millipore), which was then held at 4° C for many days before being retested.Assays using different strains of C. haemolyticum were conducted by growing bacteria in Luria Broth (LB) or sterile water containing sterile larva RCM rearing diet with abundance monitored by optical density (OD) at 600 nm while density was determined by concurrently collecting samples that were serially diluted to determine colony forming units (CFUs) per ml as previously described (3,35).
Bacteria were added at a starting density of 1x10 2 -1x10 6 CFUs per ml to CN, GN or AX cultures in 6 well culture plates (5 ml per well) containing 10 A. aegypti larvae (first-fourth instars) which were then examined at specific times post-inoculation to determine the number of alive versus dead larvae present.The unit of replication for all of the larvicidal assays was thus a culture well which contained a starting density of 10 or 20 CN, GN or AX larvae.Each treatment for a given assay was replicated a minimum of 6 times using culture wells that were established from independently generated starting pools of larvae and microbes.The percentage of surviving larvae or larvae that developed into adults per replicate were then compared to a control treatment that also consisted of 6 independently generated replicates.Data analysis is described in the Methods reported in the main text.
Ch_R13 densities in AX cultures were determined using colony counts as described above, while densities in CN cultures were estimated using a quantitative PCR (qPCR) assay that used specific primers (forward 5'-ACAAGATCGTCGCCGAATAC-3', reverse 5'-TAACAGCGGACAACTCATCG-3") to measure abundance of the single copy gyrase A gene.
Specificity of this amplicon was confirmed by PCR using DNA from Ch_R13 or bacteria from CN cultures where no product was detected.Bacterial DNA in CN cultures was isolated using the Qiagen DNeasy Blood and Tissue kit.After extraction, qPCR assays were run on a Rotor Gene Q (Qiagen): 3 min initial denaturation step at 95 °C, followed by 40 cycles of 20 s, denaturation at 95 °C, 20 s annealing at 50 °C and 20 s, extension at 72 °C.Data were acquired during the extension step and analyzed using Rotor-Gene application software.All reactions were conducted in quadruplicate.The data were then fit to a standard curve constructed using known amounts of the plasmid pTOPO TA vector containing the gyrase A amplicon that was serially diluted (36) to estimate genome copy number per ml of water.As with the larvicidal assays, the unit of replication for measuring Ch_R13 densities was a culture well while CFUs/ml or genome copies/ml for each treatment in a given assay was replicated a minimum of 6 times using independently generated samples.Data analysis is described in the Methods reported in the main text.

Cyanide concentrations and associated assays
Ch_R13 at a starting density of 10 2 CFUs per mL was inoculated into culture wells containing 5 ml of sterile water and sterile mosquito RCM diet as used in larvicidal assays.Growth of bacteria was then monitored by measuring optical density at 600 nm using a plater reader (Biotek Synergy) and colony forming units per ml as described above using water samples collected at different times.Cyanide concentrations were measured by an established assay (37).Briefly, small aliquots of water were collected from the above cultures at the same times that bacterial abundances were measured.A 100 mM dinitrobenzene (Sigma) and a 200 mM pnitrobenzaldehyde (Sigma) solution were prepared in 2-methoxyethanol (Sigma).A fresh 1:1 mixture of these two solutions was mixed with the experimental sample (77:23) to 100 µl followed by addition of 1.8 µl of NaOH.After 30 min at room temperature, 900 µl of 2-methoxyethanol was added followed measuring aliquots of each sample at 578 nm using the above plate reader with cyanide concentration determined by fitting the data to a standard curve generated by serial dilution of a potassium cyanide (KCN) stock solution.These assays which generated growth curves for Ch_R13 and associated measures of cyanide concentration in the water were measured three times using independently prepared starting samples.Effects on A. aegypti larvae were assessed using water containing cyanide from Ch_R13 cultures that was sterilized through a 0.2 µm filter (Pall) and serially diluted.One ml volumes of this water or a serially diluted KCN solution was placed in open 24 well culture plates (Genesee) followed by addition of 10 first instar A. aegypti and a small amount of sterile RCM rearing diet followed by assessment of the number of alive and dead larvae at different times.A hydroxocobalamin (vitamin B12a) (TCI America) stock solution was prepared in sterile water with 10 mM added to water containing a starting cyanide concentration of 10 mM immediately before addition of larvae or larvae and 1x10 2 CFUs of Ch_R13.The number of living and dead larvae as were assessed 1 and 24 h later.
Larvae were scored as living if mobile while larvae were scored as dead if immobile.Dead larvae also rapidly become more opaque due discoloration of the hemocoel.For these assays, culture wells containing a starting density of 10 larvae were the unit of replication.Each treatment was replicated a minimum of 6 times using independently prepared samples as described above for the larvicidal assays.Data analysis is described in the Methods reported in the main text.

Immunofluorescence microscopy
AX cultures containing A. aegypti first instars were inoculated with 1 x 10 2 CFUs per ml of Ch_R13 E2-SpR followed by collection of larvae at different times and dissection of the digestive tract in in phosphate buffer saline (PBS, pH 7.4).Samples were fixed in 4% paraformaldehyde in PBS for 20 min at room temperature.After rinsing three times in PBS, guts were permeabilized for 20 min in PBS plus 0.2% Triton X-100 (PBT) for 20 min and incubated with F432 Fluorescein Phalloidin (Thermo Fisher) at room temperature for 1 h.After rinsing, samples were slide mounted in glycerol containing HOECHST 33342 (1 µg per ml).For vital dye staining, AX and CN cultures containing first instars were inoculated with Ch_R13 followed by collection of digestive tracts that were incubated with propidium iodide (Thermo Fisher) at room temperature for 20 min followed by addition of HOECHST 33342 (1 µg per ml).All samples were then slidemounted with cover slips and examined using a Zeiss LSM 710 inverted confocal microscope with acquired images processed using Adobe Photoshop.

Protease and chitinase assays
Milk agar plates were used to assess protease activity and chitin agar plates were used to measure chitinase activity (38).Milk agar plates were made by adding 2.5 g yeast extract and 7.5 g agar to 350 ml of water; 15 g of milk powder was dissolved in 150 ml of water; both solutions were then autoclaved, cooled, mixed, and poured into plates.Blood agar plates were made using LB and sterile rabbit blood.Chitin powder (20 g) was dissolved in 500 ml of concentrated hydrochloric acid and continuously stirred at 4°C for 1 h.The hydrolyzed chitin was washed several times with distilled water to remove the acid and to bring the pH to the range of 6 to 7.
The colloidal chitin was then filtered and stored at 4°C.Chitin plates were made as follows: 7 g of K2HPO4, 0.5 g of MgSO4 heptahydrate, 2 g of yeast extract, and 15 g of agar were dissolved in 850 ml of water; 20 g of colloidal chitin was dissolved in 150 ml of water; both solutions were then autoclaved, cooled, mixed, and poured into plates.Each plate was quartered followed by addition of a 7 mm circular piece of sterile filter paper to which 10

Fig
Fig S1 Percentage of CN A. aegypti, A. albopictus, or C. quinquefasciatus first instars that were

Fig S4
Fig S4 Genomic synteny among members of the C. haemolyticum group.(A) Dendrogram of

Fig S6 Ch_R13 ,
Fig S6 Ch_R13, DSM 19808 and NRRL B-11053 have similar mortality effects in CN than AX

Fig S7
Fig S7 Ch_R13 grows to higher densities in AX than CN cultures.(A) Growth of Ch_R13 in AX cultures from a starting density of 10 2 CFUs (two replicates) as measured by colony counts.(B)

Fig S8
Fig S8 Percentage of first instar A. aegypti that develop into adults in GN cultures with the ALL9

Fig S9
Fig S9 Starting density affects the abundance of of Ch_R13 in GN cultures containing the ALL9

Table S2 .
Bacterial strains used in the study.

Table S3 .
Multivariate binomial regression analysis of the percentage of larvae that pupated in the data set presented in Fig.4.Replicate was included as a random effect for the model that examined individual species effects and the model that examined 2species interactions.In both cases, the estimated among-replicate variance was very small (0.000217 and 0.001592, respectively) when compared to the magnitude of any treatment effects.

Table S4 .
Multivariate binomial regression analysis of the percentage of larvae that survived to adulthood in the data set presented in Fig.4.Replicate was removed as a random effect for both models due to the estimated among-replicate variance approximating zero.