I accidentally found something that could be a great speed skip for the sub-shark fight.

You can camp at the very left of the screen and not get hit by the sub-shark. You can get the jelly launcher + ammo, then when the sub-shark goes to the left to do his dash attack, UNLOAD YOUR ENTIRE JAR OF JELLYFISH INTO HIS BUTT. His health will drain VERY quickly, allowing you to kill him VERY quickly. If he somehow survives after you run out of ammo, you can always try to use the tire to butt bounce him, but I think he'd be dead already by the time you run out.

If this WAS already known about, I'm sorry for wasting your time. If you DIDN'T know about it, before, then you're welcome.

Taking this into account, this is what the sub-shark strategy would probably be: Run to the other side and grab the jelly launcher AND the extra ammo. If the sub shark does the dash attack BEFORE you get to the left side, either shoot a single shot, and/or butt bounce them. You could possibly land multiple hits with the jelly launcher, though, but if they're still alive, (Which they probably are) continue with the rest of this strategy. After that happens, (or if the sub-shark DIDN'T use a dash attack while you were going to the left side of the arena.) Just camp in the leftmost corner, and turn right. As soon as the sub-shark gets ready to do it's dash attack again, SPAM THE JELLY LAUNCHER UNTIL IT DIES. If it's somehow still alive after using all your ammo, you can still try to bounce on the tire and butt bounce on him to finish him off. I hope this helps!

So I just tried it out and I have no idea what you mean by "UNLOAD YOUR ENTIRE JAR OF JELLYFISH INTO HIS BUTT". He has invincibility frames in which you can't keep attacking him, which we already take in account for and try to minimize the time we spend NOT attacking him, so we chain shots from a distance with a delay in between, he dies and we're already right new to the item. Have you watched the way the speedrun does it? Can you post a video of your method? Because it doesn't sound faster.

I found it on an emulator, so even if I knew how to use a video recorder, I still don't think it'll be that useful, considering WHY emulator runs are banned. However, what I meant by what I said there was "Spam shots, but wait long enough in between shots for his invincibility frames (Or mercy invincibility, as I call it) to wear off before he gets hit again."

I meant that you safespot in the left corner, and attack him while he's charging up his dash attack until he dies. If he lives, maybe you could try to do whatever that weird ground pound-damage boost thing the WR run does at the start of the fight? Either way, I think this just makes actually KILLING him faster, but actually GETTING THE ITEM might not be affected very much by this strat.

Such is the way of a speedrunner; you try to perfect a strat, only to find out it's been obsoleted by a faster one that you didn't know about at the time.