Fig 1.
Anatomy of female Lutzomyia sand flies.
L. verrucarum (A) and L. longipalpis (B) 16h following ingestion of a B. bacilliformis-infected human blood meal. (C) External anatomy of L. verrucarum with red arrows illustrating route of blood meal from probosis through head, thorax, and abdomen. (D) Blood-fed L. verrucarum 24h following ingestion of an artificial blood meal * which is held within the abdominal midgut (AM) by a peritrophic membrane (PM) between the stomodeal valve (SV) and pyloric valve (PV). Flies digest blood meal by releasing enzymes into the PM-encased lumen of the AM. Esophagus (ES), Ileum (IL), Rectum (R), Malpighian tubules (M).
Fig 2.
B. bacilliformis colonization of sand flies 24h following ingestion of infected blood meal.
B. bacilliformis was highly concentrated in the abdominal midgut 24h following ingestion of an infected blood meal, colonization of L. verrucarum (A-B; magnified 1000x) and L. longipalpis (C-D; magnified 400x) was indistinguishable. At this time point, colonization was limited to abdominal midgut and bacteria were not observed in any other sand fly tissue. Image pairs were acquired with fluorescence (GFP) or phase contrast (PC) microscopy. B. bacilliformis exhibited a marked degree of motility in both species of sand fly (see S1 and S2 Videos).
Fig 3.
B. bacilliformis colonization of sand flies 48h post-blood meal.
At this time point, colonization of L. verrucarum (A-B) and L. longipalpis (C-D) was indistinguishable. Bacteria appeared to adhere to peritrophic membrane (PM) and although colonization was limited to abdominal midgut (shown here) on rare occasion, appeared outside the PM (arrow). The majority of bacteria changed morphology into non-motile small coccoid forms (see S3 Video), and appeared more numerous than at 24h. Images acquired (1000x) with fluorescence (GFP) or phase contrast (PC) microscopy.
Fig 4.
At this time point, colonization of the fly species appeared different, where B. bacilliformis were observed at relatively high density in locations outside the abdominal midgut of L. verrucarum, but not L. longipalpis. B. bacilliformis was found in the thoracic midgut (A-B) as well as the ileum (C-D) of L. verrucarum. B. bacilliformis colonization of L. longipalpis was limited to the abdominal midgut (E-H) and bacteria appeared to be digested along with the blood meal. Image pairs were acquired with fluorescence (GFP) or phase contrast (PC) microscopy.
Fig 5.
L. verrucarum at 5d post-blood meal.
After 5 d B. bacilliformis colonized the competent sand fly digestive tract. As observed at 72 h following the blood meal, bacteria were found in the abdominal midgut (A-B) as well as the ileum (C-F). Images were acquired with fluorescence (GFP) or phase contrast (PC) microscopy and the magnification used is indicated under each image pair.
Fig 6.
L. longipalpis at 7d post-blood meal.
A small number of bacteria were observed (arrowheads) in the cibarium (CI), which is immediately connected to the proboscis and pharynx (PH) (A-B). Observation of B. bacilliformis in L. longipalpis tissues other than the abdominal midgut was rare, and samples of the non-competent vector continued to demonstrate digestion/disappearance of bacteria over time (C-F). Images acquired with fluorescence (GFP) or phase contrast (PC) microscopy at 400x magnification.
Fig 7.
L. verrucarum at 14d post-blood meal.
Two weeks after competent sand flies imbibed infected blood, B. bacilliformis colonization persisted, but was limited to the digestive tract (A-D). We were unable to observe GFP+ bacteria on mouthparts (E-H) but relatively large brown-colored coccoid forms were present (arrowhead). Perhaps this is what Hertig referred to as organism x-prob (see S1 Fig).
Fig 8.
Viability of B. bacilliformis in L. longipalpis adults over time.
Four groups of 200 sand flies were offered B. bacilliformis-infected blood meals (6x106 bacteria/ml) and average colony forming units (CFUs) were determined over a 13-d time course and are plotted in log scale. Average numbers of bacteria per individual fly are provided, along with the age group where young and old are indicated (Y, O). Flies that have apparently cleared the infection are also shown, with the number of flies circled and age group indicated at each time point.