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Bacterial Infection and Immune Responses in Lutzomyia longipalpis Sand Fly Larvae Midgut.

Heerman M, Weng JL, Hurwitz I, Durvasula R, Ramalho-Ortigao M - PLoS Negl Trop Dis (2015)

Bottom Line: Depending on the aspects of their development, insects can acquire microbes present in soil, water, and plants.Sand fly larval stages acquire microorganisms from the soil, and the abundance and distribution of these microorganisms may vary depending on the sand fly species or the breeding site.Moreover, bacterial distribution, and likely the ability to colonize the gut, is driven, at least in part, by a gradient of pH present in the gut.

View Article: PubMed Central - PubMed

Affiliation: Department of Entomology, Kansas State University, Manhattan, Kansas, United States of America.

ABSTRACT
The midgut microbial community in insect vectors of disease is crucial for an effective immune response against infection with various human and animal pathogens. Depending on the aspects of their development, insects can acquire microbes present in soil, water, and plants. Sand flies are major vectors of leishmaniasis, and shown to harbor a wide variety of Gram-negative and Gram-positive bacteria. Sand fly larval stages acquire microorganisms from the soil, and the abundance and distribution of these microorganisms may vary depending on the sand fly species or the breeding site. Here, we assess the distribution of two bacteria commonly found within the gut of sand flies, Pantoea agglomerans and Bacillus subtilis. We demonstrate that these bacteria are able to differentially infect the larval digestive tract, and regulate the immune response in sand fly larvae. Moreover, bacterial distribution, and likely the ability to colonize the gut, is driven, at least in part, by a gradient of pH present in the gut.

No MeSH data available.


Related in: MedlinePlus

Confocal images of B. subtilis and P. agglomerans infection of sand fly larvae midguts.Anterior midgut image12h post feeding, EGFP-expressing Bs is able to infect the entire length of the midgut. A) DAPI staining; B) Shows Bs distributed throughout the anterior larval gut; C) Immuno-staining for cleaved caspase3 along the lumen of the midgut (the bright red staining in the bottom corner was due to auto-fluorescence associated with remnants of the colony chow previously fed to the larva). D) Merge. 12h post infection with GFP-expressing Pa localized to the posterior region of the midgut epithelium and induces apoptotic activity. E) DAPI staining depicting the midgut epithelium. F) Shows Pa localized on the apical portion of the lumen of the midgut. G) Immuno-staining for cleaved caspase3 along the lumen of the midgut depicting high levels of caspase3 activity. H) Merge. Bars = 50 µm.
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pntd.0003923.g004: Confocal images of B. subtilis and P. agglomerans infection of sand fly larvae midguts.Anterior midgut image12h post feeding, EGFP-expressing Bs is able to infect the entire length of the midgut. A) DAPI staining; B) Shows Bs distributed throughout the anterior larval gut; C) Immuno-staining for cleaved caspase3 along the lumen of the midgut (the bright red staining in the bottom corner was due to auto-fluorescence associated with remnants of the colony chow previously fed to the larva). D) Merge. 12h post infection with GFP-expressing Pa localized to the posterior region of the midgut epithelium and induces apoptotic activity. E) DAPI staining depicting the midgut epithelium. F) Shows Pa localized on the apical portion of the lumen of the midgut. G) Immuno-staining for cleaved caspase3 along the lumen of the midgut depicting high levels of caspase3 activity. H) Merge. Bars = 50 µm.

Mentions: Bs and Pa were assessed for their ability to infect the sand fly L3 larvae midgut following feeding, and their effect on induction of apoptosis. A monoclonal antibody targeting the cleaved caspase3 was used as an immunocytochemical marker to identify epithelial cells undergoing caspase-dependent programmed cell death, and to assess the integrity of the midgut. This antibody was used previously in Drosophila to detect c-Jun N-terminal kinase (JNK) pathway activation in response to infection [19]. In order to test if this was a viable approach, we fed larvae with LB-agar supplemented with the apoptosis inducer Paraquat, and compared its effects to larvae fed on LB-agar alone. The LB-agar fed larvae displayed a well-defined midgut epithelium with little background staining for active caspase3 (S3A Fig). In contrast, larvae fed on LB-agar supplemented with Paraquat showed midgut epithelia with significant loss of integrity, that were also severely flattened after mounting on the slide with reduced luminal space detectable by looking at nuclei alone (S3B Fig). The apoptotic effect of the Paraquat was further confirmed by the presence of a large population of cells showing heavy cytoplasmic specific staining for caspase3 (S3C and S3D Fig). After 12h of infection with Bs an extensive amount of luminal nucleic material is observed using DAPI staining (Fig 4A), and a massive infection can be seen in Fig 4B. Very little background caspase staining is observed in Bs-fed compared to the Paraquat treated controls (Fig 4C and 4D, and S3 Fig). In contrast, when Pa was used for infection, the Gram-negative bacteria induced staining comparable to that of the Paraquat control (Fig 4G and 4H and S3 Fig). However, we did not observe a similar breakdown in midgut superstructure.


Bacterial Infection and Immune Responses in Lutzomyia longipalpis Sand Fly Larvae Midgut.

Heerman M, Weng JL, Hurwitz I, Durvasula R, Ramalho-Ortigao M - PLoS Negl Trop Dis (2015)

Confocal images of B. subtilis and P. agglomerans infection of sand fly larvae midguts.Anterior midgut image12h post feeding, EGFP-expressing Bs is able to infect the entire length of the midgut. A) DAPI staining; B) Shows Bs distributed throughout the anterior larval gut; C) Immuno-staining for cleaved caspase3 along the lumen of the midgut (the bright red staining in the bottom corner was due to auto-fluorescence associated with remnants of the colony chow previously fed to the larva). D) Merge. 12h post infection with GFP-expressing Pa localized to the posterior region of the midgut epithelium and induces apoptotic activity. E) DAPI staining depicting the midgut epithelium. F) Shows Pa localized on the apical portion of the lumen of the midgut. G) Immuno-staining for cleaved caspase3 along the lumen of the midgut depicting high levels of caspase3 activity. H) Merge. Bars = 50 µm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4495979&req=5

pntd.0003923.g004: Confocal images of B. subtilis and P. agglomerans infection of sand fly larvae midguts.Anterior midgut image12h post feeding, EGFP-expressing Bs is able to infect the entire length of the midgut. A) DAPI staining; B) Shows Bs distributed throughout the anterior larval gut; C) Immuno-staining for cleaved caspase3 along the lumen of the midgut (the bright red staining in the bottom corner was due to auto-fluorescence associated with remnants of the colony chow previously fed to the larva). D) Merge. 12h post infection with GFP-expressing Pa localized to the posterior region of the midgut epithelium and induces apoptotic activity. E) DAPI staining depicting the midgut epithelium. F) Shows Pa localized on the apical portion of the lumen of the midgut. G) Immuno-staining for cleaved caspase3 along the lumen of the midgut depicting high levels of caspase3 activity. H) Merge. Bars = 50 µm.
Mentions: Bs and Pa were assessed for their ability to infect the sand fly L3 larvae midgut following feeding, and their effect on induction of apoptosis. A monoclonal antibody targeting the cleaved caspase3 was used as an immunocytochemical marker to identify epithelial cells undergoing caspase-dependent programmed cell death, and to assess the integrity of the midgut. This antibody was used previously in Drosophila to detect c-Jun N-terminal kinase (JNK) pathway activation in response to infection [19]. In order to test if this was a viable approach, we fed larvae with LB-agar supplemented with the apoptosis inducer Paraquat, and compared its effects to larvae fed on LB-agar alone. The LB-agar fed larvae displayed a well-defined midgut epithelium with little background staining for active caspase3 (S3A Fig). In contrast, larvae fed on LB-agar supplemented with Paraquat showed midgut epithelia with significant loss of integrity, that were also severely flattened after mounting on the slide with reduced luminal space detectable by looking at nuclei alone (S3B Fig). The apoptotic effect of the Paraquat was further confirmed by the presence of a large population of cells showing heavy cytoplasmic specific staining for caspase3 (S3C and S3D Fig). After 12h of infection with Bs an extensive amount of luminal nucleic material is observed using DAPI staining (Fig 4A), and a massive infection can be seen in Fig 4B. Very little background caspase staining is observed in Bs-fed compared to the Paraquat treated controls (Fig 4C and 4D, and S3 Fig). In contrast, when Pa was used for infection, the Gram-negative bacteria induced staining comparable to that of the Paraquat control (Fig 4G and 4H and S3 Fig). However, we did not observe a similar breakdown in midgut superstructure.

Bottom Line: Depending on the aspects of their development, insects can acquire microbes present in soil, water, and plants.Sand fly larval stages acquire microorganisms from the soil, and the abundance and distribution of these microorganisms may vary depending on the sand fly species or the breeding site.Moreover, bacterial distribution, and likely the ability to colonize the gut, is driven, at least in part, by a gradient of pH present in the gut.

View Article: PubMed Central - PubMed

Affiliation: Department of Entomology, Kansas State University, Manhattan, Kansas, United States of America.

ABSTRACT
The midgut microbial community in insect vectors of disease is crucial for an effective immune response against infection with various human and animal pathogens. Depending on the aspects of their development, insects can acquire microbes present in soil, water, and plants. Sand flies are major vectors of leishmaniasis, and shown to harbor a wide variety of Gram-negative and Gram-positive bacteria. Sand fly larval stages acquire microorganisms from the soil, and the abundance and distribution of these microorganisms may vary depending on the sand fly species or the breeding site. Here, we assess the distribution of two bacteria commonly found within the gut of sand flies, Pantoea agglomerans and Bacillus subtilis. We demonstrate that these bacteria are able to differentially infect the larval digestive tract, and regulate the immune response in sand fly larvae. Moreover, bacterial distribution, and likely the ability to colonize the gut, is driven, at least in part, by a gradient of pH present in the gut.

No MeSH data available.


Related in: MedlinePlus