functional and structural characterization of antennal apos in … · 2019-01-17 · bsci 296,...
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S U S H M A B O P P A N A
B S C I 2 9 6 , S P R I N G 2 0 1 4
J U L I A N H I L L Y E R
Functional and Structural Characterization of Antennal APOs
in Anopheles gambiae
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Mosquito Antennae
The antennae’s Johnston’s organs influence courtship and mating.
The reception of odorant and thermal cues by antennal receptor neurons controls oviposition and host-seeking behaviors.
Source: Zwiebel Lab
Flagellum
Pedicel
Scape
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Antennal Accessory Pulsatile Organs (APOs)
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AIMS
AIM 1: Functionally characterize the antennal APOs
AIM 2: Understand hemolymph flow within the antennal space
AIM 3: Substantiate previous studies on antennal APO structure
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Location of the antennal APOs
Posterior but close to the base of the antenna (the scape)
Symmetrically located on each side of the mosquito’s dorsal midline
Found within the inside edge of the mosquito’s compound eyes
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Methods: Contraction Rate Experiments
• Mosquitoes were cold anesthetized and injected with 1μm green fluorescent beads.
• Legs and wings were cut off, and mosquitoes were positioned dorsal side up.
• A 1 minute video of the APOs was acquired followed by 1 min. video of the heart.
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Antennal APO Contraction Rates
The right APO contracts an average of 0.978 ± 0.23 Hz.
The left APO has an average rate of 0.983 ± 0.237 Hz.
The left and right APO contract in sync with each other.
APO Contractions Over 15 Seconds
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Antennal APO vs. Heart Rates
The heart contracts, on average, at a rate of 1.78 Hz or 106.7 times a minute.
The antennal APOs contract 45% slower than the heart (paired t-test, p<0.0001).
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Methods: Particle Tracking Experiments
Mosquitoes were cold anesthetized and injected with 0.5 um fluorescent beads.
Mosquitoes were restrained and positioned in the same manner as mentioned before.
Particle tracking software was used to track 6 particles/antenna: 3 up and 3 down.
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Hemolymph Flow within Antennae
Particles travel up the antenna at an average velocity of 197 µm/sec and down at 51 µm/sec.
Maximum acceleration of particles going up the antenna is significantly greater when compared to downward traveling particles (t-test, p<0.0001).
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Methods: Staining and Histology
Mosquito heads were fixed, dehydrated, and infiltrated by JB-4 plus.
These heads were then transferred into molding trays and allowed to polymerize
Sectioning was carried out on a JB4 Sorvall Porter-Blum microtome, and sections were loaded, 10 at a time, onto slides.
The slides were stained with Azure II Blue.
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Antennal Vessel Structural Data
Sections showed clear evidence of an antennal vessel.
Antennal vessel model:
Narrow vessel allows hemolymph to be pumped up the antenna
Emptied out into the wider antennal hemocoel at the distal end of the appendage
Hemolymph returns to the head of the mosquito in a less linear manner and more slowly.
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Antennal APO Structural Data
Ampulla (Am) = sac-like chamber
where hemolymph accumulates
Z-body (ZB) = dense cluster of cells attached to the contractile muscle
Passive Systole Action
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Methods: FlyNap
FlyNap is a common anesthetic mixture consisting mainly of triethylamine.
Has a cardioacceleratory effect on mosquito heart rates
Method similar to contraction rate experiments using cold anesthesia.
VS.
Cold anesthesia FlyNap anesthesia
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Cold vs. FlyNap Anesthesia
APO and heart contraction rates for cold anesthetized mosquitoes were significantly lower than for FlyNap anesthetized mosquitoes (t-test, p<0.0001).
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Conclusions
Antennal APOs contract synchronously at an average of 0.98 Hz.
The antennal APOs contract 45% slower than the heart.
Hemolymph travels through the antenna via an antennal vessel.
The mosquito antennal APO structure was found to be in line with previous data.
FlyNap has an acceleratory effect on both APOs and the heart.
Acknowledgement: Dr. Julián Hillyer
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QUESTIONS???