Variation in the structure and function of invertebrate-associated bacterial communities

Microorganisms are intricately involved in the ecology of many insects, often contributing to host fitness and forming evolutionarily stable associations. The interactions between hosts and microbes can significantly alter their evolutionary trajectories, enabling them to adapt to novel environmental conditions. In this thesis I have examined how host ecology can shape the interactions of bacteria with insects of agricultural and epidemiological importance. I have described the bacterial communities associated with Bactrocera oleae (the olive fruit fly) and generated draft genome sequences for several members of the gut microbiota, including the symbiotic bacterium “Candidatus Erwinia dacicola”. Comparative genomic analyses indicate that Ca. E. dacicola and a novel facultative bacterium Tatumella TA1 may perform key nutritional functions for the host, including the synthesis of essential amino acids and ammonia assimilation from host nitrogenous waste products. Tatumella TA1 is consistently associated with all life stages of populations collected in Israel and Crete at low relative abundance, and encodes large adhesion proteins that may assist in attachment to the host epithelium or other members of the microbiota in the B. oleae gut. I have also examined the variation in frequency and relative abundance of facultative microbes that infect several Glossina spp. (the tsetse fly): the sole vector of African trypanosomes in Sub-Saharan Africa. In addition to three vertically transmitted endosymbionts (Wigglesworthia, Sodalis, and Wolbachia), tsetse flies are infected with two additional potential reproductive manipulators: Spiroplasma and Rickettsia, and a novel strain of Klebsiella. The draft genomes generated for these taxa over the course of this thesis provide the opportunity for future studies in to their role in host biology and how community interactions can shape the transmission and evolutionary dynamics of host-associated microbes

Interactions between VEGFR inhibitor and sprouting in <i>sema3fb</i> mutants.

A) A model of signaling pathways that regulate angiogenic sprouting, highlighting key genes controlling tip and stalk cell identity. B) Quantification of Tg(kdrl:mCherry) transgene expression levels in wild type and sema3fbca305 ISAs at 30hpf. N = 3: wild type (n = 13 embryos, average of 6500 a.u.) and sema3fbca305 (n = 14 embryos, average of 8400 a.u.). T-test with Welches correction, *p = 0.0186, a.u. = arbitrary unit of intensity. C). B) Fluorescent HCR in situ for vegfr2 and sflt1 RNA transcripts in whole-mount wild type and embryos sema3fbca305 embryos fixed at 30 hpf. D) Whole-mount Immunostaining for phosphoERK (pERK) in WT and sema3fbca305 embryos fixed at 30 hpf. E) Lateral confocal images of the trunk vasculature Tg(kdrl:mCherry) (white) in embryos treated with 0.5 μM SU5416 from 20hpf-30hpf. DLAV gaps (blue asterisks) and ISA truncated sprouts (yellow dashed line at the level of horizontal myoseptum are indicated. Scale bar, 100 μm. F) Quantification of ISA sprout length in 30 hpf embryos treated with 0.5 μM SU5416, N = 1: WT + DMSO (25 ISAs, 5 embryos, mean of 107±8 μm), WT + 0.5μM SU5416 (25 ISAs, 5 embryos, mean of 50±14 μm), sema3fbca305 + DMSO (30 ISAs, 6 embryos, mean of 82±17 μm), and sema3fbca305 +0.5μM SU5416 (30 ISAs, 6 embryos, mean of 82±19 μm). G) Percentage of ISA sprouts connected at DLAV in 30 hpf embryos treated with 0.5 μM SU5416, N = 1: WT + DMSO (25 ISA-DLAV, 5 embryos, mean 78% of ISA-DLAV/embryo), WT + 0.5 μM SU5416 (25 ISA-DLAV, 5 embryos, mean of 78%), sema3fbca305 + DMSO (30 ISA-DLAV, 6 embryos, mean of 51%), and sema3fbca305 + 0.5 μM SU5416 (30 ISA-DLAV, 6 embryos, mean of 82±19%). Error bars = ±SD. H) Lateral confocal images of the trunk vasculature Tg(kdrl:mCherry) (white) in embryos treated with low doses of DMH4 μM SU5416 from 20hpf-30hpf. ISA truncated sprouts (yellow dashed line at the level of horizontal myoseptum are indicated. Scale bar, 50 μm. I) Quantification of length of ISA sprouts in 30 hpf embryos treated with 15 μM DMH4, J) Quantification of length of ISA sprouts in 30 hpf embryos treated with 25 μM DMH4. I-J) N = 1; WT+ DMSO n = 3, 15 ISAs, average (ave.) 102±9 μm; WT+ μM DMH4 n = 2, 10 ISA, ave. 17±17 μm; WT+ 25 μM DMH4 n = 3, 15 ISAs, ave. 7±8 μm. sema3fbca305 + DMSO = 3, 15 ISA, ave. 87±18 μm, sema3fbca305 + 15 μM DMH4 n = 2, 10 ISA, ave. 40±16 μm; sema3fbca305 + 25 μM DMH4 n = 3,15 ISA, 32±13 μm. One-Way ANOVA Tukey’s multiple comparisons test, * means ¬¬p = 0.012, *****p = (TIF)</p

sema3fb mutants display aberrant and persistent filopodia in the dorsal ISA.

A) Lateral images of the trunk vasculature with mosaic endothelial expression of the transgene fli1ep: Lifeact-EGFP highlighting actin (green) and endothelial cytoplasm using Tg(kdrl:mCherry; white) in ISAs at 30 hpf. DLAV gaps (blue asterisks) and truncated ISA sprouts (yellow arrowheads) are marked. Inset shows an enlarged view of single ISAs with Lifeact-EGFP expression that have reached the level of DLAV at 30hpf. Scale bar, 100 μm. B) Representative still images from time-lapse imaging from 28–30 hpf. Enlarged still images of stage-matched embryos with mosaic Lifeact-EGFP (green) in endothelial cells spanning the ISA and reaching the level of the DLAV by 28 hpf in both wild type and sema3fbca305 embryo. Endothelial cytoplasm is shown in red Tg(kdrl:mCherry). White arrowheads indicate filopodia present in connecting ISA sprouts within the boxed regions below the DLAV. C) Quantification of number Lifeact-EGFP positive filopodia on ISAs from 28–30 hpf from embryos of the indicated genotypes. N = 3: WT (14 EGFP positive ISAs/ 30 ISAs total, 6 embryos, mean of 4 filopodia/ISA) and homozygous sema3fbca305 (18 EGFP positive ISAs/35s ISAs total, 7 embryos, mean of 7 filopodia/ISA). Unpaired t-test with Welch’s correction,*p = 0.03 and ***p = 0.0002. Error bars = ±SD.</p

<i>sema3fb</i> mutants display aberrant and persistent filopodia.

A) Representative still images of single-cell expression of fli1ep: Lifeact-EGFP (green) in ISA endothelial cells from 28-30hpf wildtype and sema3fbca305 embryo time-lapse imaging. A dashed white line represents the horizontal myoseptum and selected areas for filopodia counts are highlighted in white boxes. B) Quantification of number Lifeact-EGFP positive filopodia on ISA at 28hpf from embryos of the indicated genotypes. Unpaired t-test, p = 0.3566. C) Quantification of number Lifeact-EGFP positive filopodia on ISA at 29hpf from embryos of the indicated genotypes. Unpaired t-test, p = 0.0029. D) Quantification of number Lifeact-EGFP positive filopodia on ISA at 30hpf from embryos of the indicated genotypes. N = 3 for each quantification: WT (14 ISAs, 6 embryos, mean of 3 filopodia/ISA) and homozygous sema3fbca305 (18 ISAs, 7 embryos, mean of 8 filopodia/ISA). Unpaired t-test, p = 0.0002. (TIF)</p

Angioblast migration distance and speed.

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Endothelial expressed sema3fb promotes endothelial cell sprouting.

A) Lateral view of sema3fb expression at 30hpf by ISH. Inset shows expression in the dorsal aorta (DA) and intersegmental arteries (ISAs). B) Schematic representation of the zebrafish vasculature at 30 hpf. Inset: The ISAs sprout from the DA and connect to form the Dorsal Longitudinal Anastomotic Vessel (DLAV) by 30 hpf. C-E) Lateral confocal images of the trunk vasculature (black) of 30 hpf (C) wild type sibling (sib), (D) heterozygous (het) sema3fbca305/+ and (E) homozygous (hom) sema3fbca305 mutants. Gaps in the DLAV (blue asterisks) and truncated ISA sprouts (yellow arrowhead) are noted. Abbreviations: DA (Dorsal Aorta), and PCV (Posterior Cardinal Vein). Anterior, left; Dorsal, up. Scale bar, 100 μm. F) ISA Sprout length at 30 hpf in wild type (WT) sibs (mean length of 106±10 μm), het sema3fbca305/+ (92±19μm), and hom sema3fbca305 (91±18μm), ****pca305/+ (50% connected) and hom sema3fbca305 (42% connected), ****pca305/+ (8.5±2.7 μm), and hom sema3fbca305 (9.2±2.9 μm)., **pca305/+ = 22 embryos (n = 163 ISAs), and hom sema3fbca305 = 9 embryos (n = 75 ISAs), 2-Way ANOVA Tukey’s multiple comparisons test. Error bars = ±SD.</p

Loss of <i>sema3fb</i> disrupts ISA migration.

A) Lateral confocal time-lapse images from 25–29 hpf double transgenic Tg(kdrl:mCherry;fli1a:nEGFP) endothelial cells (magenta) and nuclei (white). The location of the horizontal myoseptum (green dashed line) and DLAV (blue dashed line) are noted to highlight ISA growth over time. Scale bar, 50 μm. B) Average ISA Sprout Length at 30-minute intervals from 25–29 hpf: WT vs sema3fbca305 at 25.0 hpf, p = 0.474; at 25.5 hpf p = 0.262; at 26.0 hpf p = 0.081; at 26.5 hpf *p = 0.023; at 27.0 hpf *p = 0.020; at 27.5 hpf **p = 0.030; at 28.0 hpf **p = 0.008; at 28.5 hpf **p = 0.007; at 29.0 hpf *p = 0.024. C-E) Quantification of ISA migration speeds (μm/min). C) At 26–27 hpf WT = 0.15 μm/min and sema3fbca305 = 0.12 μm/min, p = 0.157. D) At 27–28 hpf WT = 0.19 μm/min and sema3fbca305 = 0.13 μm/min, *p = 0.020. E) At 28–29 hpf: WT = 0.16 μm/min and sema3fbca305 = 0.19 μm/min, p = 0.461. B-E) N = 2; WT = 7 embryos (n = 33 ISAs) and sema3fbca305 = 7 embryos (n = 35 ISAs), Unpaired t-test with Welch’s correction. F-H) Lead angioblast distance from DA at 1hr intervals. F) At 27 hpf mean distance from DA: WT = 55.12±14.06 μm and sema3fbca305 = 47.18±5.75 μm, *p = 0.030. G) At 28 hpf mean distance from DA: WT = 71.57±15.47 μm and sema3fbca305 = 55.47±9.65 μm, ***p = 0.0008. H) At 29 hpf mean distance from DA: WT = 85.19±18.03 μm and sema3fbca305 = 68.36±16.64 μm, **p = 0.008. F-H) N = 1: WT = 4 embryos (20 ISAs) and sema3fbca305 = 3 embryos (15 ISAs), Unpaired t-test with Welch’s correction. I) Lateral confocal images of 30hpf double transgenic Tg(kdrl:mCherry;fli1a:nEGFP) endothelial cells (ECs, magenta) and nuclei (white). EC nuclei clumps (blue arrows/arrowheads) are noted. Scale bar, 100 μm. Inset: Schematics show method for measuring distance between EC nuclei and highlight EC nuclei clumps in ISAs. J) Number of EC nuclei (angioblasts) per ISAs at 30 hpf. WT (mean of 3 nuclei/ISA), heterozygous (het) sema3fbca305/+ (3 nuclei/ISA), and homozygous (hom) sema3fbca305 (3 nuclei/ISA). K) Quantification of inter-endothelial nuclei spacing per ISA at 30 hpf. WT (mean 28±13 μm), het sema3fbca305/+ (23±13μm), and hom sema3fbca305 (22±14 μm), ***p = 0.0002 and ****p2), het sema3fbca305/+ (mean 60±24 μm2), and hom sema3fbca305 (mean 56±23 μm2),**p = 0.0069 and ****psema3fbca305/+ = 19 embryos (n = 190 ISAs), and homsema3fbca305 = 11 embryos (n = 110 ISAs),. 2-Way ANOVA Tukey’s multiple comparisons test Error bars = ±SD.</p

Quantitative PCR mean fold change data.

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<i>sema3fb</i> mutants have increased VEGF receptor expression and activity.

A) RT-qPCR analysis of key endothelial markers in wild type and sema3fbca305 FACS isolated Tg(kdrl:mCherry) positive endothelial cells at 26hpf (inset). N = 2, 2-Way ANOVA Tukey’s multiple comparisons test, *p = 0.0184, **p = 0.0021, and ****pS3 Table for fold-change details). B) Fluorescent HCR in situ of 30 hpf whole-mount wild type and embryos sema3fbca305 embryos. Representative images show punctate overlapping expression of vegfr2 (white) and sflt1 (red) mRNA transcripts within the DA and ISAs (dashed white outline). C) Quantification of HCR in situ pixel density in ISAs and DA, wild type (WT, n = 3 embryos, 15 ISAs) and sema3fbca305 (n = 3 embryos, 15 ISAs), Unpaired Student’s t-test with Welch’s correction WT vs. sema3fbca305: vegfr2 *p = 0.047 and sflt1 *p = 0.036. D) Whole-mount Immunostaining for phosphoERK (pERK) in WT and sema3fbca305 embryos fixed at 30 hpf. Representative images show Tg(kdrl:mCherry) positive ISAs (purple) and pERK positive ECs (green). Inset: pERK positive ISAs are traced using kdrl:mCherry expression (dashed white line) and dashed oval outlines highlight individual ECs with pERK staining within each ISA. E) Number of pERK positive ISAs at 30hpf. F) Quantification of average pERK fluorescence intensity in embryos at 30 hpf. D-E) N = 3, WT (n = 21 embryos, mean of 5 pERK positive ISAs), and homzygous sema3fbca305 (n = 19 embryos, mean of 5 pERK positive ISAs). 2-Way ANOVA Tukey’s multiple comparisons test, *p = 0.012. G) Schematic of Vegfr2 inhibition time course, embryos are treated at 20 hpf with either 0.1%DMSO or Vegfr2 inhibitors and removed from treatment for live imaging at 30hpf. H) Representative confocal images of trunk vasculature (black) of 30 hpf embryos treated with DMSO control or 0.2 μM SU5416. DLAV gaps (blue asterisks) and truncated ISA (yellow arrowheads) are marked. Scale bar, 100 μm. I) Length of ISA sprouts in treated embryos at 30 hpf: WT + DMSO (n = 25 ISAs, mean of 104±9 μm), WT + 0.2 μM SU5416 (n = 25 ISAs, mean of 92±17 μm), sema3fbca305 + DMSO (n = 30 ISAs, mean of 85±17 μm), and sema3fbca305 + 0.2μm SU5416 (n = 30 ISAs, mean of 98±11 μm) **p = 0.0039 and ****psema3fbca305 + DMSO (n = 30 ISA-DLAV, mean 46±16%), and sema3fbca305 +0.2 μm SU5416 (n = 30 ISA-DLAV, mean 73±10%), **p = 0.0084, ***p = 0.0002, and ****psema3fbca305 + DMSO = 6 embryos, sema3fbca305 + 0.2 μm SU5416 = 6 embryos,. 2-Way ANOVA Tukey’s multiple comparisons test. Error Bars = ±SD.</p

ISA length at different time intervals.

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