A novel assay to isolate and quantify third-stage Dirofilaria immitis and Brugia malayi larvae emerging from individual Aedes aegypti

Abstract Background Mosquitoes transmit filarial nematodes to both human and animal hosts, with worldwide health and economic consequences. Transmission to a vertebrate host requires that ingested microfilariae develop into infective third-stage larvae capable of emerging from the mosquito proboscis onto the skin of the host during blood-feeding. Determining the number of microfilariae that successfully develop to infective third-stage larvae in the mosquito host is key to understanding parasite transmission potential and to developing new strategies to block these worms in their vector. Methods We developed a novel method to efficiently assess the number of infective third-stage filarial larvae that emerge from experimentally infected mosquitoes. Following infection, individual mosquitoes were placed in wells of a multi-well culture plate and warmed to 37 °C to stimulate parasite emergence. Aedes aegypti infected with Dirofilaria immitis were used to determine infection conditions and assay timing. The assay was also tested with Brugia malayi-infected Ae. aegypti. Results Approximately 30% of Ae. aegypti infected with D. immitis and 50% of those infected with B. malayi produced emerging third-stage larvae. Once D. immitis third-stage larvae emerged at 13 days post infection, the proportion of mosquitoes producing them and the number produced per mosquito remained stable until at least day 21. The prevalence and intensity of emerging third-stage B. malayi were similar on days 12–14 post infection. Increased uptake of D. immitis microfilariae increased the fitness cost to the mosquito but did not increase the number of emerging third-stage larvae. Conclusions We provide a new assay with an associated set of infection conditions that will facilitate assessment of the filarial transmission potential of mosquito vectors and promote preparation of uniformly infectious third-stage larvae for functional assays. The ability to quantify infection outcome will facilitate analyses of molecular interactions between vectors and filariae, ultimately allowing for the establishment of novel methods to block disease transmission.

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A novel assay to isolate and quantify third-stage filarial larvae emerging from individual mosquitoes

10.1101/2020.02.04.934653 ◽

2020 ◽

Author(s):

Abigail R. McCrea ◽

Elizabeth B. Edgerton ◽

Genevieve T. Oliver ◽

Fiona M. O’Neill ◽

Thomas J. Nolan ◽

...

Keyword(s):

Disease Transmission ◽

Post Infection ◽

Blood Feeding ◽

Economic Consequences ◽

Transmission Potential ◽

Filarial Nematodes ◽

Third Stage ◽

Novel Method ◽

Parasite Emergence ◽

New Strategies

AbstractBackgroundMosquitoes transmit filarial nematodes to both human and animal hosts, resulting in worldwide health and economic consequences. Transmission to a vertebrate host requires that ingested microfilariae develop into infective third-stage larvae capable of emerging from the mosquito proboscis onto the skin of the host during blood feeding. Determining the number of microfilariae that successfully develop to infective third-stage larvae in the mosquito host is key to understanding parasite transmission potential and to developing new strategies to block these worms in their vector.MethodsWe developed a novel method to efficiently assess the number of infective third-stage filarial larvae that emerge from experimentally infected mosquitoes. Following infection, individual mosquitoes were placed in wells of a multi-well culture plate and warmed to 37 °C to stimulate parasite emergence. Aedes aegypti infected with Dirofilaria immitis were used to determine infection conditions and assay timing. The assay was also tested with Brugia malayi infected Ae. aegypti.ResultsApproximately 30% of Ae. aegypti infected with D. immitis and 50% of those infected with B. malayi produce emerging third-stage larvae. Once D. immitis third-stage larvae emerge at 13 days post infection, the proportion of mosquitoes producing them, and the number produced per mosquito remain stable until at least day 21. The prevalence and intensity of emerging third-stage B. malayi were similar on days 12-14 days post infection. Increased uptake of D. immitis microfilariae increases the fitness cost to the mosquito but does not increase the number of emerging third-stage larvae.ConclusionsWe provide a new assay with an associated set of infection conditions that will facilitate assessment of the filarial transmission potential of mosquito vectors and promote preparation of uniformly infectious L3 for functional assays. The ability to quantify infection outcome will facilitate analyses of molecular interactions between vectors and filariae, ultimately allowing for the establishment of novel methods to block disease transmission.Graphical Abstract

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Laboratory colonization by Dirofilaria immitis alters the microbiome of female Aedes aegypti mosquitoes

10.21203/rs.2.23991/v6 ◽

2020 ◽

Author(s):

Abdulsalam Adegoke ◽

Erik Neff ◽

Amie Geary ◽

Montana Ciara Husser ◽

Kevin Wilson ◽

...

Keyword(s):

Aedes Aegypti ◽

Dirofilaria Immitis ◽

Mosquito Species ◽

Bacterial Species ◽

Klebsiella Oxytoca ◽

Variable Region ◽

Blood Feeding ◽

Mosquito Vector ◽

Filarial Nematodes ◽

The Impact

Abstract Background: The ability of blood feeding arthropods to successfully acquire and transmit pathogens of medical and veterinary importance has been shown to be interfered with, or enhanced by, the arthropod’s native microbiome. Mosquitoes transmit viruses, protozoan and filarial nematodes, the majority of which contribute to the 17% of infectious disease cases worldwide. Dirofilaria immitis , a mosquito transmitted by filarial nematodes of dogs and cats, is vectored by several mosquito species including Aedes aegypti . Methods: In this study, we investigated the impact of D. immitis colonization on the microbiome of laboratory reared female A. aegypti . Metagenomic analysis of the V3-V4 variable region of the microbial 16SRNA was used for identification of the microbial differences down to species level. Results We generated a total of 1068 OTUs representing 16 phyla, 181 genera and 271 bacterial species. Overall, in order of abundance, Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes were the most represented phylum with D. immitis infected mosquitoes having more of Proteobacteria (71%) than uninfected mosquitoes (56.9%). An interesting finding in this study is the detection of Klebsiella oxytoca in relatively similar abundance in infected and uninfected mosquitoes, suggesting a possible endosymbiotic relationship, and has been previously shown to indirectly compete for nutrients with fungi on domestic housefly eggs and larva. While D. immitis colonization has no effect on the overall species richness, we identified significant differences in the composition of selected bacteria genus and phylum between the two groups. We also reported distinct compositional and phylogenetic differences in the individual bacteria species when commonly identified bacteria were compared. Conclusions In conclusion, this is the first study to the best of our knowledge to understand the impact of a filarial infection on the microbiome of its mosquito vector. Further studies are required to identify bacteria species that could play an important role in the mosquito biology. While the microbiome composition of A. aegypti mosquito have been previously reported, our study shows that in an effort to establish itself, a filarial nematode modifies and alters the overall microbial diversity within its mosquito host.

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Laboratory colonization by Dirofilaria immitis alters the microbiome of female Aedes aegypti mosquitoes

10.21203/rs.2.23991/v7 ◽

2020 ◽

Author(s):

Abdulsalam Adegoke ◽

Erik Neff ◽

Amie Geary ◽

Montana Ciara Husser ◽

Kevin Wilson ◽

...

Keyword(s):

Aedes Aegypti ◽

Dirofilaria Immitis ◽

Mosquito Species ◽

Bacterial Species ◽

Klebsiella Oxytoca ◽

Variable Region ◽

Blood Feeding ◽

Mosquito Vector ◽

Filarial Nematodes ◽

The Impact

Abstract Background: The ability of blood-feeding arthropods to successfully acquire and transmit pathogens of medical and veterinary importance has been shown to be interfered with, or enhanced by, the arthropod’s native microbiome. Mosquitoes transmit viruses, protozoan and filarial nematodes, the majority of which contribute to the 17% of infectious disease cases worldwide. Dirofilaria immitis, a mosquito-transmitted filarial nematodes of dogs and cats, is vectored by several mosquito species including Aedes aegypti.Methods: In this study, we investigated the impact of D. immitis colonization on the microbiome of laboratory reared female Ae. aegypti. Metagenomic analysis of the V3-V4 variable region of the microbial 16S RNA gene was used for identification of the microbial differences down to species level.Results: We generated a total of 1068 OTUs representing 16 phyla, 181 genera and 271 bacterial species. Overall, in order of abundance, Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes were the most represented phylum with D. immitis-infected mosquitoes having more of Proteobacteria (71%) than uninfected mosquitoes (56.9%). An interesting finding in this study is the detection of Klebsiella oxytoca in relatively similar abundance in infected and uninfected mosquitoes, suggesting a possible endosymbiotic relationship, and has been previously shown to indirectly compete for nutrients with fungi on domestic housefly eggs and larvae. While D. immitis colonization has no effect on the overall species richness, we identified significant differences in the composition of selected bacterial genera and phyla between the two groups. We also reported distinct compositional and phylogenetic differences in the individual bacterial species when commonly identified bacteria were compared.Conclusions: In conclusion, this is the first study to the best of our knowledge, this is the first study to understand the impact of a filarial infection on the microbiome of its mosquito vector. Further studies are required to identify bacteria species that could play an important role in the mosquito biology. While the microbiome composition of Ae. aegypti mosquito have been previously reported, our study shows that in an effort to establish itself, a filarial nematode modifies and alters the overall microbial diversity within its mosquito host.

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Laboratory colonization by Dirofilaria immitis alters the microbiome of female Aedes aegypti mosquitoes

10.21203/rs.2.23991/v3 ◽

2020 ◽

Author(s):

Abdulsalam Adegoke ◽

Erik Neff ◽

Amie Geary ◽

Montana Ciara Husser ◽

Kevin Wilson ◽

...

Keyword(s):

Aedes Aegypti ◽

Dirofilaria Immitis ◽

Mosquito Species ◽

Bacterial Species ◽

Klebsiella Oxytoca ◽

Variable Region ◽

Blood Feeding ◽

Mosquito Vector ◽

Filarial Nematodes ◽

The Impact

Abstract Background: The ability of blood feeding arthropods to successfully acquire and transmit pathogens of medical and veterinary importance has been shown to be interfered or enhanced by the arthropod’s native microbiome. Mosquitoes transmit bacteria, viruses, protozoan and filarial nematodes, majority of which contributes to the 17% of infectious disease cases worldwide. Dirofilaria immitis, a mosquito transmitted filarial nematodes of dogs and cats, is vectored by several mosquito species including Aedes aegypti.Methods: In this study, we investigated the impact of D. immitis colonization on the microbiome of laboratory reared female A. aegypti. Metagenomic analysis of the V3-V4 variable region of the microbial 16SRNA was used for identification of the microbial differences down to species level.Results: We generated a total of 1068 OTUs representing 16 phyla, 181 genera and 271 bacterial species. Overall, in order of abundance, Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes were the most represented phylum with D. immitis infected mosquitoes having more of Proteobacteria (71%) than uninfected mosquitoes (56.9%). An interesting finding in this study is the detection of Klebsiella oxytoca in relatively similar abundance in infected and uninfected mosquitoes, suggesting a possible endosymbiotic relationship. It has been previously shown to indirectly compete for nutrients with fungi on the domestic housefly eggs and larva. While D. immitis colonization has no effect on the overall species richness, we identified significant differences in the composition of selected bacteria genus and phylum between the two groups. We also reported distinct compositional and phylogenetic differences in the individual bacteria species when commonly identified bacteria were compared.Conclusions: In conclusion, this is the first study to the best of our knowledge to understand the impact of a filarial infection on the microbiome of its mosquito vector. Further studies is required to identify bacteria species that could play an important role in the mosquito biology. While the microbiome composition of A. aegypti mosquito have been previously reported, our study shows that in an effort to establish itself, a filarial nematode modifies and alters the overall microbial diversity within its mosquito host.

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Laboratory colonization by Dirofilaria immitis alters the microbiome of female Aedes aegypti mosquitoes

10.21203/rs.2.23991/v2 ◽

2020 ◽

Author(s):

Abdulsalam Adegoke ◽

Erik Neff ◽

Amie Geary ◽

Montana Ciara Husser ◽

Kevin Wilson ◽

...

Keyword(s):

Aedes Aegypti ◽

Dirofilaria Immitis ◽

Mosquito Species ◽

Bacterial Species ◽

Klebsiella Oxytoca ◽

Variable Region ◽

Blood Feeding ◽

Mosquito Vector ◽

Filarial Nematodes ◽

The Impact

Abstract Background: The ability of blood feeding arthropods to successfully acquire and transmit pathogens of medical and veterinary importance has been shown to be interfered or enhanced by the arthropod’s native microbiome. Mosquitoes transmit bacteria, viruses, protozoan and filarial nematodes, majority of which contributes to the 17% of infectious disease cases worldwide. Dirofilaria immitis, a mosquito transmitted filarial nematodes of dogs and cats, is vectored by several mosquito species including Aedes aegypti.Methods: In this study, we investigated the impact of D. immitis colonization on the microbiome of laboratory reared female A. aegypti. Metagenomic analysis of the V3-V4 variable region of the microbial 16SRNA was used for identification of the microbial differences down to species level.Results: We generated a total of 1068 OTUs representing 16 phyla, 181 genera and 271 bacterial species. Overall, in order of abundance, Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes were the most represented phylum with D. immitis infected mosquitoes having more of Proteobacteria (71%) than uninfected mosquitoes (56.9%). An interesting finding in this study is the detection of Klebsiella oxytoca in relatively similar abundance in infected and uninfected mosquitoes. It has been previously shown to indirectly compete for nutrients with fungi on the domestic housefly eggs and larva. While not statistically significant, D. immitis infection alters bacterial diversity by reducing the bacterial species richness and abundance.Conclusions: In conclusion, this is the first study to the best of our knowledge to understand the impact of a filarial infection on the microbiome of its mosquito vector. While the microbiome composition of A. aegypti mosquito have been previously reported, our study shows that in an effort to establish itself, a filarial nematode modifies and alters the overall microbial diversity within its mosquito host.

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Laboratory colonization by Dirofilaria immitis alters the microbiome of female Aedes aegypti mosquitoes

10.21203/rs.2.23991/v4 ◽

2020 ◽

Author(s):

Abdulsalam Adegoke ◽

Erik Neff ◽

Amie Geary ◽

Montana Ciara Husser ◽

Kevin Wilson ◽

...

Keyword(s):

Aedes Aegypti ◽

Dirofilaria Immitis ◽

Mosquito Species ◽

Bacterial Species ◽

Klebsiella Oxytoca ◽

Variable Region ◽

Blood Feeding ◽

Mosquito Vector ◽

Filarial Nematodes ◽

The Impact

Abstract Background: The ability of blood feeding arthropods to successfully acquire and transmit pathogens of medical and veterinary importance has been shown to be interfered with, or enhanced by, the arthropod’s native microbiome. Mosquitoes transmit viruses, protozoan and filarial nematodes, the majority of which contribute to the 17% of infectious disease cases worldwide. Dirofilaria immitis, a mosquito transmitted by filarial nematodes of dogs and cats, is vectored by several mosquito species including Aedes aegypti.Methods: In this study, we investigated the impact of D. immitis colonization on the microbiome of laboratory reared female A. aegypti. Metagenomic analysis of the V3-V4 variable region of the microbial 16SRNA was used for identification of the microbial differences down to species level.Results We generated a total of 1068 OTUs representing 16 phyla, 181 genera and 271 bacterial species. Overall, in order of abundance, Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes were the most represented phylum with D. immitis infected mosquitoes having more of Proteobacteria (71%) than uninfected mosquitoes (56.9%). An interesting finding in this study is the detection of Klebsiella oxytoca in relatively similar abundance in infected and uninfected mosquitoes, suggesting a possible endosymbiotic relationship, and has been previously shown to indirectly compete for nutrients with fungi on domestic housefly eggs and larva. While D. immitis colonization has no effect on the overall species richness, we identified significant differences in the composition of selected bacteria genus and phylum between the two groups. We also reported distinct compositional and phylogenetic differences in the individual bacteria species when commonly identified bacteria were compared. Conclusions In conclusion, this is the first study to the best of our knowledge to understand the impact of a filarial infection on the microbiome of its mosquito vector. Further studies are required to identify bacteria species that could play an important role in the mosquito biology. While the microbiome composition of A. aegypti mosquito have been previously reported, our study shows that in an effort to establish itself, a filarial nematode modifies and alters the overall microbial diversity within its mosquito host.

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Laboratory colonization by Dirofilaria immitis alters the microbiome of female Aedes aegypti mosquitoes

10.21203/rs.2.23991/v8 ◽

2020 ◽

Author(s):

Abdulsalam Adegoke ◽

Erik Neff ◽

Amie Geary ◽

Montana Ciara Husser ◽

Kevin Wilson ◽

...

Keyword(s):

Aedes Aegypti ◽

Dirofilaria Immitis ◽

Mosquito Species ◽

Bacterial Species ◽

Klebsiella Oxytoca ◽

Variable Region ◽

Blood Feeding ◽

Mosquito Vector ◽

Filarial Nematodes ◽

The Impact

Abstract Background: The ability of blood-feeding arthropods to successfully acquire and transmit pathogens of medical and veterinary importance has been shown to be interfered with, or enhanced by, the arthropod’s native microbiome. Mosquitoes transmit viruses, protozoan and filarial nematodes, the majority of which contribute to the 17% of infectious disease cases worldwide. Dirofilaria immitis, a mosquito-transmitted filarial nematodes of dogs and cats, is vectored by several mosquito species including Aedes aegypti.Methods: In this study, we investigated the impact of D. immitis colonization on the microbiome of laboratory reared female Ae. aegypti. Metagenomic analysis of the V3-V4 variable region of the microbial 16S RNA gene was used for identification of the microbial differences down to species level.Results: We generated a total of 1068 OTUs representing 16 phyla, 181 genera and 271 bacterial species. Overall, in order of abundance, Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes were the most represented phylum with D. immitis-infected mosquitoes having more of Proteobacteria (71%) than uninfected mosquitoes (56.9%). An interesting finding in this study is the detection of Klebsiella oxytoca in relatively similar abundance in infected and uninfected mosquitoes, suggesting a possible endosymbiotic relationship, and has been previously shown to indirectly compete for nutrients with fungi on domestic housefly eggs and larvae. While D. immitis colonization has no effect on the overall species richness, we identified significant differences in the composition of selected bacterial genera and phyla between the two groups. We also reported distinct compositional and phylogenetic differences in the individual bacterial species when commonly identified bacteria were compared.Conclusions: To the best of our knowledge, this is the first study to understand the impact of a filarial infection on the microbiome of its mosquito vector. Further studies are required to identify bacteria species that could play an important role in the mosquito biology. While the microbiome composition of Ae. aegypti mosquito have been previously reported, our study shows that in an effort to establish itself, a filarial nematode modifies and alters the overall microbial diversity within its mosquito host.

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Laboratory colonization by Dirofilaria immitis alters the microbiome of female Aedes aegypti mosquitoes

10.21203/rs.2.23991/v5 ◽

2020 ◽

Author(s):

Abdulsalam Adegoke ◽

Erik Neff ◽

Amie Geary ◽

Montana Ciara Husser ◽

Kevin Wilson ◽

...

Keyword(s):

Aedes Aegypti ◽

Dirofilaria Immitis ◽

Mosquito Species ◽

Bacterial Species ◽

Klebsiella Oxytoca ◽

Variable Region ◽

Blood Feeding ◽

Mosquito Vector ◽

Filarial Nematodes ◽

The Impact

Abstract Background: The ability of blood feeding arthropods to successfully acquire and transmit pathogens of medical and veterinary importance has been shown to be interfered with, or enhanced by, the arthropod’s native microbiome. Mosquitoes transmit viruses, protozoan and filarial nematodes, the majority of which contribute to the 17% of infectious disease cases worldwide. Dirofilaria immitis , a mosquito transmitted by filarial nematodes of dogs and cats, is vectored by several mosquito species including Aedes aegypti . Methods: In this study, we investigated the impact of D. immitis colonization on the microbiome of laboratory reared female A. aegypti . Metagenomic analysis of the V3-V4 variable region of the microbial 16SRNA was used for identification of the microbial differences down to species level. Results: We generated a total of 1068 OTUs representing 16 phyla, 181 genera and 271 bacterial species. Overall, in order of abundance, Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes were the most represented phylum with D. immitis infected mosquitoes having more of Proteobacteria (71%) than uninfected mosquitoes (56.9%). An interesting finding in this study is the detection of Klebsiella oxytoca in relatively similar abundance in infected and uninfected mosquitoes, suggesting a possible endosymbiotic relationship, and has been previously shown to indirectly compete for nutrients with fungi on domestic housefly eggs and larva. While D. immitis colonization has no effect on the overall species richness, we identified significant differences in the composition of selected bacteria genus and phylum between the two groups. We also reported distinct compositional and phylogenetic differences in the individual bacteria species when commonly identified bacteria were compared. Conclusions: In conclusion, this is the first study to the best of our knowledge to understand the impact of a filarial infection on the microbiome of its mosquito vector. Further studies are required to identify bacteria species that could play an important role in the mosquito biology. While the microbiome composition of A. aegypti mosquito have been previously reported, our study shows that in an effort to establish itself, a filarial nematode modifies and alters the overall microbial diversity within its mosquito host.

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Laboratory colonization by Dirofilaria immitis alters the microbiome of female Aedes aegypti mosquitoes

10.21203/rs.2.23991/v1 ◽

2020 ◽

Author(s):

Abdulsalam Adegoke ◽

Erik Neff ◽

Amie Geary ◽

Montana Ciara Husser ◽

Kevin Wilson ◽

...

Keyword(s):

Aedes Aegypti ◽

Dirofilaria Immitis ◽

Mosquito Species ◽

Bacterial Species ◽

Klebsiella Oxytoca ◽

Variable Region ◽

Blood Feeding ◽

Mosquito Vector ◽

Filarial Nematodes ◽

The Impact

Abstract Background The ability of blood feeding arthropods to successfully acquire and transmit pathogens of medical and veterinary importance has been shown to be interfered or enhanced by the arthropod’s native microbiome. Mosquitoes transmit bacteria, viruses, protozoan and filarial nematodes, majority of which contributes to the 17% of infectious disease cases worldwide. Dirofilaria immitis , a mosquito transmitted filarial nematodes of dogs and cats, is vectored by several mosquito species including Aedes aegypti . Methods In this study, we investigated the impact of D. immitis colonization on the microbiome of laboratory reared female A. aegypti . Metagenomic analysis of the V3-V4 variable region of the microbial 16SRNA was used for identification of the microbial differences down to species level. Results We generated a total of 1068 OTUs representing 16 phyla, 181 genera and 271 bacterial species. Overall, in order of abundance, Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes were the most represented phylum with D. immitis infected mosquitoes having more of Proteobacteria (71%) than uninfected mosquitoes (56.9%). An interesting finding in this study is the detection of Klebsiella oxytoca in relatively similar abundance in infected and uninfected mosquitoes. It has been previously shown to indirectly compete for nutrients with fungi on the domestic housefly eggs and larva. While not statistically significant, D. immitis infection alters bacterial diversity by reducing the bacterial species richness and abundance. Conclusions In conclusion, this is the first study to the best of our knowledge to understand the impact of a filarial infection on the microbiome of its mosquito vector. While the microbiome composition of A. aegypti mosquito have been previously reported, our study shows that in an effort to establish itself, a filarial nematode modifies and alters the overall microbial diversity within its mosquito host.

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