Todomatsu pretending to not know Osomatsu in "The Melancholy of Osomatsu" starts to make sense once he reveals that he doesn't mention any of his brothers to the girls he talks to for fear of embarrassing him, and that he sees them as a detriment to advancing in society.
Todomatsu's secondary defining character trait is his desire to distance himself from his brothers so they don't drag him down socially. If you look at the Matsu colour scheme, Todomatsu is the only brother whose signature colour (pink) isn't part of the standard six-colour rainbow:
Related to the colour scheme; Iyami has a tendency to confuse Osomatsu and Todomatsu with each other. While this is a Continuity Nod from the *-Kun* days, Oso and Todo also happen to wear red and pink, colours that are closely related and could possibly be mistaken for each other depending on the lighting.
Remember how Jyushimatsu was the only one out of the older brothers who didn't sabotage Todomatsu's reputation in episode 7? At the time this had no explanation, but then the second skit on episode 14 revealed that Jyushimatsu also had a secret life like Totty (revealing that his secret account had been depleted due to fraudulent case). The reason why he didn't join his older brothers in episode 7 was due to understanding of the latter's situation.
Someone pointed out that that extra spout in Jyushimatsu's head when he does the water trick probably came from when Todomatsu's Harsh Word Impact from Episode 7 hit him◊ on the head.
Aside from the show graduating from -Kun to -San because of the Matsunos growing up, it's also a handy pun since this is the third anime starring the family.
Dayoon turning to a beautiful tan woman thanks to the Cute Girl drug made for a decent joke, but why specifically tan? In "Dayoon Tribe", there's a whole community of folk in his stomach who have the same hair color and skin color of the woman Dayoon turns into, bar the fact that they all have Dayoon's face. The only reason she turns out as pretty as she does is, again, thanks to the drug.
Translating the last episode's title as "Osomatsu-san, As It Was" ends up revealing the nature of the episode before it was even out; one of the meanings of "As it was" is how things used to be. (For reference, the Japanese title is "Osomatsu-san deshita", or "This was Osomatsu-san".)
Another layer to this: The phrase "Osomatsu-san deshita" has been used exactly twice in the series- Episode 1 and Episode 25. Both uses came after the series took a completely different turn than the rest of its fare (ReTooling the series beyond recognition in "Returns" and seemingly splitting up the brothers in "Letter") and were used to signal a change back to something more normal. For "Returns", the phrase was used to introduce the brothers as they grew up to be, and "As it Was" used it to signal a return to format.
Totoko committing Seppuku in "As it Was". According to the page, Totoko should have done the female jigai variation, as it would have been more ladylike. Besides the version shown being the most well known depiction outside of Japan, it also shows Totoko as she's always been in this series: decidedly unladylike.
One to do with advertising: The brothers ended up as the spokesmen for a non-alcoholic beer. For someone like Karamatsu or Ichimatsu, this drink would be perfect for them.
In Season 2's Celebration! Employment!, Matsuyo and Matsuzo calls over their sons to confront them being NEET adults. The order was: Osomatsu, Ichimatsu, Jyushimatsu, Karamatsu, Todomatsu, and finally Choromatsu. This is the reverse order of them leaving the house in Letter.
In the first Cavematsu sketch, the brothers all try to create, er...gratification devices, ranging from simple hollowed-out sticks to far more advanced and anachronistic technology. Is it any wonder that the largest and most mechanically-complex device was created by the cave version of Fappymatsu?
In November 2017, the Osomatsu San announces their product collaboration with the Ultra Series and the six Matsuno brothers are compared to their Ultra Brothers counterpart respectively. In case of Jyushimatsu, he is dressed as Ultraman Ace. Not only they were the fifth brothers, Jyushimatsu is also the only Matsuno siblings to have his own girlfriend (the country girl), alluding to how Ultraman Ace used to have two human hosts.
Was Osomatsu just playing along in "The Ichimatsu Incident", or did he really not recognize that his brothers had swapped clothing?
Opinions go either way on the matter. For evidence on the former, he seemed to be egging Ichimatsu on to confess that it's really just him (the "dark aura" thing, asking about borrowing "Karamatsu's" clothes, eating Ichimatsu's cat food in front of his face). But he seems equally as oblivious as well.
Jyushimatsu regularly goes out and plays baseball, but it's not a job and he's well out of high school. So who is he playing with?
He might just be practicing alone or with some of his brothers, as episode 9 shows.
Somebody pointed out that there was no beer at Ichimatsu's seat in episode 2. So how did he get drunk enough to have to be carried home if he didn't have anything to drink and his brothers more than likely wouldn't share?
It's mentioned in an interview that Ichimatsu and Karamatsu are more lightweight than their brothers, cue them having to drink water to sober up in that scene in ep 2.
In "Osomatsu in Hell", the sextuplet was sent to hell by King Enma due to the lack of good deed in their past life. Although they are truly justified by this, why didn't Enma sent Jyushimatsu alone to heaven? Considering that Jyushimatsu is the only one who shows care for Hijirisawa in the real world.
The final battle has it that almost all one-shot casts from previous episode returns to help the sextuplets to escape hell. Isn't it weird on how all of them ended up on hell? Could this mean that King Enma is an example of God Is Evil? I mean look at the list below:
Both of Chibita and Karamatsu's flower girls
Shazar and his girl (which may or may not disproves the theory that he is Hatabou's true identity.)
My guess is that in the case of F6's deaths, this may be a case of Death of Personality, which would explain the Hatabou = Shazar theory.
After Choromatsu departs in "Letter", Todomatsu goes up to Osomatsu and hits him, he's the next one to leave after getting a black eye. Karamatsu comes after and earlier he got into a nasty fight with Osomatsu after Jyushimatsu gets kicked during Choromatsu's goodbye party. Then Jyushimatsu, who has the aforementioned happen to him, leaves to move in with Dekapan. Ichimatsu is then the last one to leave the house, as he has none of his brothers left other than a depressed Osomatsu. Did the brothers leave because they got inspired by each other, or did Osomatsu's frustration scare them away?
What kind of job did Jyushimatsu get that caused him to get a broken arm?
Several fans have pointed out how Ichimatsu is noticeably skinnier than how he usually is. How long has he been homeless and when was the last time he ate something?
In Osomatsu-kun, Ichimatsu is very strong willed. Now though, he just an emotionless shell. What the hell happened to him?
Same thing that made Jyushimatsu the incomprehensible mess he is today, and Osomatsu criminally dependent on his brothers. Which is to say, we won't be getting that answer anytime soon.
Could just be the simple "time changes people" answer.
Kara's brothers leave him to die in favor of eating pear slices their mother brings them...while he's listening over the phone. If it wasn't bad enough he's apparently less important than fruit in the eyes of his brothers, and if his reaction to the pears is the same as all of theirs, there is a good chance he's being forced to listen to his family enjoying his favorite snack without him.
Although the "Valentine's Day" skit in season 2 (which ended with the Matsuno brothers turning into Namahages and hunting chocolates) is considered funny, one needs to remember that the one who leads the sextuplets is Jyushimatsu, instead of Osomatsu. This is played further when you realized that he is the only Matsuno brother to have ever experienced a romantic life (and gets rejected in the end), thus making him leading the Namahage troop all the more sour.
There's a fan parody showing Jyushimatsu, as a Namahage, breaking into a country house, only to find the Country Girl inside, ready to hand him chocolate.