Acknowledgements, Background, and Further Information
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Acknowledgements
Land Acknowldgement
Click here to learn more about about the history of the land this work was performed on.
Nyholm Lab
Many folks in the lab provided guidance, insights and encouragement in this work.
Imaging Help
The UConn Advanced Light Microscopy Core provided expert imaging advice.
Background
The ANG contains a community of bacteria that are discretely partitioned into tubules, allowing us to localize these bacterial populations with different host factors.
A) Ventral dissection of female adult E. scolopes. The ANG lies directly anterior to the nidamental gland (NG) and posterior to the symbiotic light organ (LO). B) Cartoon depiction of ANG structure. The ANG is a composite structure of individual, non-intersecting tubules that converge underneath each of the two lobes of the NG, where they deposit their bacterial populations into an intergland space between the ANG and NG. C) Cartoon depiction of bacterial tubule populations. The bacterial population of the ANG is mostly taxonomically partitioned. D) Light micrograph image of an ANG tubule cross section. The epithelial cells of some tubules release vesicles-like structures (black arrowheads) into the lumen, where the bacteria reside. E) A majority of bacteria-containing tubules are dominated by exclusively Alphaproteobacteria or Verrucomicrobia, with few others containing Gammaproteobacteria and mixed populations of represented taxa
Methods
Sample Prep and Lectin Staining:
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Whole tissues were dissected
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Fixed in 4% PFA
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Embedded in paraffin wax
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Sectioned on a microtome
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Mounted on slides
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Labeled with lectins and FISH probes
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Imaged on Nikon AXR confocal
Lectin Controls
​To confirm lectin specificity, control slides were stained with the lectin alongside its haptenic (target) sugar. On these slides, the absence of the lectin signal confirmed that the lectin was targeting that sugar.
Imaging and Quantification
All images were acquired on a Nikon AXR with a 20X PLAN APOCHROMAT lens. The acquisition parameters for each lectin were identical between technical and biological replicates. Images of stains were identically thresholded and abundance values were quantified by hand in Fiji. It took a while. Believe me, I tried to make an analysis pipeline work, but these tubules are so weird and irregular. Sometimes you gotta roll up your sleeves, put on your favorite Joni Mitchell record, and count away.
Lectin Staining of ANG Sections
We used many lectins besides WGA and PNA to stain the ANG tissue. A table of lectins that did stain ANG tissues is listed here.
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Lectins heterogeneously stain ANG tubules at varying abundances. A) Abundance of tubules containing each lectin. Lines denote means. B-F) Representative images of lectin-stained ANG sections. Each panel depicts image of an entire ANG section (.1) and enlarged image of the boxed region in panel 1 (.2). White arrowheads indicate tubules stained by respective lectin. Black arrowheads indicate tubules not stained by respective lectin. *On other biological replicates (n=2), AAL was found to stain the lumina of some tubules. Tukey groups in A denote significance.
Glycomic Analysis of Glycoprotein Fraction
To quantify carbohydrate abundance and sequence glycans of the adult ANG, we performed MADLI-TOF on both N- and O- glycans of ANG tissues (n=5). We just got back some exciting data that we've started exploring.
Lectin Staining of Recruitment Tissues
The recruitment tissue pores appear to have a glycan-rich secretion that occurs in the presence of symbiotic bacteria in the squid's habitat. These secretions may be mucus that help attract and recruit bacteria, similar to how the light organ secretes mucus in the presence of symbiotic bacteria.
