5. Lionfish Venom

Question 1

lionfish in the atlantic ocean

Answer 1

lionfish are native to the pacific ocean –> ended up in the atlantic as an invasive species bc atlantic fish not used to eating it with its venom

Question 2

describe the lionfish stinging mechanism

Answer 2

• 13 spines on the back
• venom glands within spines produce venom but the rest of the fish is not poisonous
• even just 1 spine or the skin brushing against the spines can inject venom

Question 3

what is nociception?

Answer 3

pain sensation –> activation of the pain pathway

Question 4

how can we identify which part of venom is responsible for activating pain pathway?

Answer 4

identify behaviour effects to find cellular targets and pathways

isolate + identify the toxin to understand toxin-receptor interactions and learn how predators are resistant

Question 5

what is the pain measurement in mice?

Answer 5

inject some venom into feet of mice and record how long the mice lick their feet

Question 6

why do we know that a protein component is responsible for the pain response from venom?

Answer 6

add trypsin or heat to denature proteins before injection –> lose pain response

Question 7

why do we know that the protein component must have cysteine bridges?

Answer 7

add reducing agent before injection –> lose pain response

Question 8

describe the transgenic mice used

Answer 8

pain sensing neurons express fluorescent Ca2+ sensor when Ca2+ is bound/channel is activated

Question 9

does venom target peptidergic or non-peptidergic nociceptors?

Answer 9

non-peptidergic

Question 10

describe the use of capsaicin

Answer 10

induces Ca2+ response and activates peptidergic pain receptors to cause fluorescence

Question 11

2 results of fluorescent Ca2+ from applying venom

Answer 11

1. Ca2+ fluorescence rapidly rises and slowly decreases
2. Ca2+ fluorescence has a delayed increased and oscillates

Question 12

why does venom cause oscillation of Ca2+ fluorescence?

Answer 12

When the channel is activated, ATP is activated to induce oscillatory response in secondary responders

Question 13

what receptor does venom act on? data to support using pharmacological blockers

Answer 13

venom acts on P2X3

• Inject venom + saline = high pain response
• Inject venom + P2X3 blockers = reduced pain response

Question 14

what is the second aim?

Answer 14

to identify the algogenic/pain-causing component of the venom –> i.e. the toxin

Question 15

is the toxin EDTA-sensitive?

Answer 15

no, but other parts of the venom are

Question 16

workflow for identifying the toxin

Answer 16

A. grind tissue and collect venom gland
- make cDNA library
- sequence to get transcriptome and predict proteome
B. collect whole venom
- fraction the venom
- MS
- predict proteome
C. compare the proteomes from the gland and whole venom
- find parts that are the same in both
D. identified 4 toxins that could activate P2X3 in high content screening –> observe pain response in animals

Question 17

what is the 3rd aim?

Answer 17

characterize receptor target and evolved resistance mechanisms

Question 18

why do venom-resistant mechanisms exist in animals?

Answer 18

animals are able to eat other venomous animals so they have mechanisms of resistance against the toxins

Question 19

4 mechanisms of resistance

Answer 19

1. target-site insensitivity
2. toxin scavenging molecules
3. off-target repurposing
4. auto-resistance

Question 20

what is target-site insensitivity?

Answer 20

mutation in receptor so it cannot bind toxin anymore

Question 21

target-site insensitivity with P2X3

Answer 21

P2X3 binds ATP to give response but toxin can also induce this response –> mutate P2X3 so it can still bind ATP but not toxin

Question 22

what are toxin scavenging molecules?

Answer 22

molecules that sequester the toxin so it cannot act on its target

Question 23

what is off-target repurposing?

Answer 23

use a decoy receptor that is not associated with the toxin function but can bind the toxin

Question 24

what is autoresistance?

Answer 24

resistance to toxin from their own species bc animals often cannibalize themselves

Question 25

what animal can eat the lionfish? why?

Answer 25

moray eel
- their wall epithelium is so thick that the spikes cannot enter

Question 26

describing cloning of venom receptors

Answer 26

cloned venom receptors from:
- Zig zag eel –> not in same environment as lionfish so no evolutionary pressure to modify receptor
- Honeycomb moray eel –> in same environment as lionfish so there is evolutionary pressure to modify receptor for resistance
- Lionfish

then tested each receptor’s sensitivity to lionfish venom

Question 27

what is the goal of looking at various venom receptors?

Answer 27

find differences between human and eel receptors then swap domains to see if we can confer sensitivity in eel receptors or remove sensitivity in human receptors