To be honest, you probably could shoot most of these stills, along with the deer since it was just grazing, at a shutter of 1/60-1/120th and had the ISO much much lower. A lower ISO means less noise, and the noise can cause an image to not look sharp. Also, I don't know about the lens you were using, but most have image stability which will give you at least 2 more stops of hand holding ability which translates to a longer shutter duration than you'd normally be able to hand hold at "x" focal length.

AI-Servo is the correct setting to use, NOT one shot. When working with such a long lens as close as you need to be, depth of field is very shallow. Any movement of either the bee or the camera that changes the lens to subject distance will result in an out of focus bee. AI-Servo will allow the lens to adjust focus as the distance changes. I might consider using not spot focus but expanded point focus. That uses one main focus point with 4 surrounding points to assist. As the bee moves around a bit in the center of the frame, the camera will track the bee. Another setting that may help is to use AF case 2 that delays refocusing when the focus points briefly leave the subject.

There are a couple of responses that mention confusion about spot AF with the Canon 5d Mk IV. The square with the tiny dot in the middle is called "spot focus" and it uses a very small area of the focus sensor. It can be very useful when you need to be precise with the location you wish to focus on. BUT, it is not very good at tracking moving objects. The single box with no dot is called single point focus and uses a larger area to focus on. It is still rather small but extends just a bit beyond the edges of the square. It is much better at tracking movement and is the better choice to match with AI-Servo.

This is the newer version of the Canon 100-400 I just got and I’m not sure if it is my technique or what but photos just seem soft too many times. All are at 1/1000 and ISO 1000 which the Canon 5D M IV should be able to handle. So the first bee, it is very cropped the whole thing is just soft or not in focus. Second bee, I really had the little box right on the bee and it seemed to focus to the right. Flowers, most my flowers just seem soft to me all the time. Wasp, at at f10 shouldn't the whole thing be in focus? The back end seems to be... Deer noise and just not that sharp. Last hummingbird, I do get photos like this that makes me think ok I do like this lens. So I was in shutter priority, this camera always seems to overexpose so I had it set to underexpose a little. I was also in Al Servo for most, occasionally I did switch out, but most except the deer were most likely in Al Servo. You can't get really close to things like the bee so cropping is necessary.
What technique should be used to get sharp photos? Am I too picky, not good at focusing (use auto focus and hand held). Anyway, what ever anyone does that has this lens to get good results on a more regular basis I would really like to know!
Thanks for your time. Will check tomorrow to see if anyone has suggestions.
Beth