Confocal Microscopy: Thomas

Confocal microscopy of the light organ crypts in juvenile euprymna scolopes reveals their morphological complexity and dynamic function in symbiosis

Basic info
• Euprymna scolopes is conlonized by vireo ficheri
• Uses counterillumination as a defense against predators
• Figure 1:
o B: ciliated
o C: 3 crypts on this side, crypts not symmetric but order is the same
o A: inksac develops and grows toward light organ and becomes like iris so that it can focus how much light it gives off at one point
Materials and methods
• Squids were mated once a week
• When eggs hatched put in 23 C water and 12/12h light/dark sequence
• Removed from water and put in vibrio fischeri free water
• Some squid inoculated with vibrio fischeri containing plasmid pKV111
o Encodes for color and antibiotic resistance
o Doesn’t change results
• The vibrio fischeri was diluted to 10 3- 10 4 CFU
• Squid were placed in own scintillation vials for 3 or 12 hours
• Moved then to free water and after 24h luminescences was measured
• Confocal microscopy was used to see the difference
Crypt morphology
• All crypts contain same four morphological regions
• DT1 is shortest and has largest cross sectional area, DT3 is the smallest cross section and longest
• Antechamber is medial to the duct
• The bottleneck connects the antechamber and the deep crypt
• Deep crypt is farthest from the pore and is the largest portion of the crypt
• Deep crypt one is the largest and most medial
• DC 1 is the most complex and split into three primary sections
• DC 3 is least complex
Localization of vibrio fischeri
• Colonization has a high success rate but many aspects vary
• Bacteria entered as early as 2h and first entered the antechamber, bottleneck and the DC between 4-8h
• By 17h many light organs were colonized
• By 24 h the hosts diel expulsion stats taking over
o Expels part of vibrio fischeri every morning
• DC 1 will expel more bacteria
• DC 3 expels hardly any
• After 4-5h the growth of vibrio fischeri occurs again
Symbiont-induced changes
• Decided to further study the morphologic changed of the deep crypts
• Concluded that in early stages of symbiosis DC regions can respond differently due to maturity
Deep crypts 2 and 3
• DC2,3 have four morphologic stages
• DC2 is usually one stage ahead of DC3
• DC2 always readily colonized by symbiote, DC3 not colonized if in stage 1
Crypt function
• What is use of antechamber
• May possibly have more functions
• The bottleneck serves as a physical barrier
o Does it close
o Does it expand during expulsion???
• May also limit subsequent colonization by V. fischeri
• Are bacteria just pushed through or does it change width
Juvenile crypt development
• All four regions in each of the ix crypts vary in size
• DC2 and 3 follow increase in size and complexity like DC1
• Development of antechamber and bottleneck not significantly preprogrammed
Role of symbionts
• Unexpected results
• Diel venting believed to be for regrowth of fresh V. fischeri
o Failed to duplicate DC 1
o What are biomechanics of venting
o Is prematurity of DC2 and DC3 a factor why it doenst eject as much as DC 1
Questions:
• during initial colonization, what is immunological changes
o dunno
• counterillumination get rid of shadow so predator will leave it alone
• regulation of light through the inksac
• V. fischeri glow
• Confocal
o A way of taking pictures; something to give a 3-d look
• Expulsion daily
o So the bacteria have less competition

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