Kuyo and his student police force, who beat up the Unwanted Harem and actually killed Tsukune; this was the first time he was injected with blood, and also caused the first instance where Inner Moka was in any real danger..
Ruby's master helped the transition along, in an arc that focused heavily on Fantastic Racism, which has since become one of the series' most prevalent themes. She was planning to wage war on humanity, and was willing to cut down her own adopted daughter to do so.
Midou and the outcast monsters, who beat up Inner Moka, killed Tsukune again, and caused this next blood injection to put the Evil in Superpowered Evil Side. After this incident, the main theme of Fantastic Racism between humans and monsters, and between monsters and other monsters, really began to take over.
Second season manga started out mostly silly and comedic, even the first serious villain, the doppleganger, had his own share of silliness. Then, in volume 3, we meet Miyabi, whose first act is to sexually harass Mizore, as well as steal her first kiss. This is NOT Played for Laughs, as the molestation destroys her emotionally, ultimately making her commit suicide. Fortunately, she was saved by Kurumu
Legato Bluesummer's introduction changes the tone in a single frame. It's a bright and sunny day, the kids are playing with Vash. Then out of nowhere, Legato. He killed and ate the friendly shopkeeper Vash was just speaking to, and feels it would be a downright shame if the little girl Vash just bought an ice cream for would have to be next. The entire scene is completely horrific, but what cements it as this is the opening shot of the usually lighthearted and goofy Vash looking legitimately terrified for the first time in the series that clearly indicates exactly how bad things are about to get.
Monev follows Legato's cue and begins shortly afterwards - while previously Vash was always able to stop the villains before they can do serious harm, the guy basically moves down everyone in his path (including women and children) to get his target - and he actually enjoys this collateral damage. This is also the first time that Vash really loses it, and comes close to killing Monev.
The first one is Udo Jin-e. The series had started rather lightly... and in came this loudBlood Knight with a permanent Slasher Smile, an until-then-unseen degree of cruelty, the capacity of either killing you bloodily or put you in an And I Must Scream position, and the dubious "honor" of showing us some glimpses of what would happen if Kenshin ever fully reverted to his Battousai side.
The second is Saitou Hajime. His introduction signaled the arrival of the Kyoto Arc (and the much more dangerous villain Shishio) whereas previously, the series had been a light-hearted action comedy where, a few serious villains like the aforementioned Jin'e and Aoshi aside, Kenshin's everyday life continued as a light-hearted and comical romp.
Kurata. Before he had appeared the only real "dark" parts of the anime resulted from Gotsumon's incredibly hostile attitude toward Ikuto/Keenan. When Kurata arrived, he brought genocide (including the deaths of both of Ikuto/Keenan's "parents", resulting in genuine Tear Jerker scenes) and singlehandedly made the season the darkest one yet. For once we had a genuinely evil human, and also BY FAR the most evil character ever to grace the franchise.
Devimon in the original Digimon Adventure. While the first few episodes of the show were simple Monster of the Week, his appearance in Episode 8 introduced a proper, intertwined story arc and highlighted that the show wasn't kidding around anymore. It got somewhat lighter - though with even more Myth Arc - in the short 'Etemon arc' - only for Myotismon to firmly seal the series in Cerebus Syndrome.
While Digimon Tamers was always more serious and thinky than the other series, Beelzemon's arrival causes a big shift. Darker and Edgier went from meaning "Digimon don't automatically respawn at the Primary Village anymore so fights actually count" to Impmon makes a Deal with the Devil, kills Leomon, and Jeri gets VERY close to the Despair Event Horizon. From there we go straight to an arc that gets very freaky and The Heartless uses Jeri's grief to power up its beasts by mind raping her for weeks on end. Things got bleak at that point. And after Beelzemon comes the True Enemy, the D-Reaper. It's a simple cleanup program that failed to take into account Technology Marches On and now seeks to delete the entire Digital World for being "too advanced." It's initial appearance already presents it as an Outside-Context ProblemOmnicidal Maniac. Then it escapes into the real world and starts getting to work at trying to delete it and the digital world, all while inflicting Mind Rape on an already broken Jeri so that it can feed off her despair.
Soul Eater started off as a zany wacky serious with a very eccentric cast. Then Medusa showed up with her abused and unstable child Chrona, and released Asura which caused a the series to take a darker shift where people started dying and going insane and the zany imagery became more nightmarish. Asura himself qualifies, any time he appears the series comic relief stops, with Medusa still having had some humorous scenes.
Things quickly got serious when KurumiTokisaki entered the picture. So much that there was a Breather Episode before her arc. Once she makes her appearance, civilians die en masse, and none of the protagonists are able to talk her out of it. It takes an even more powerful Spirit to take her down.
Once Sir Isaac Ray Peram Westcottstarts meddling with the protagonists, the story becomes more than just a race to seal the most Spirits and also bringing the story to a darker path.
Zophise from Zatch Bell!. Gash Bell tends to get a lot of these, because most of the enemies tend to be fairly goofy, it's jarring when you get someone who's playing for keeps.
Ur-Example in Dragon Ball is Tao. He was the first serious villain in the franchise with no humorous qualities to him and caused the first on-screen deaths in the franchise, which, back he appeared, was actually quite shocking due to how lighthearted the manga was.
Raditz from Dragon Ball Z, who jumped the series out of being comedy-focused altogether.
Frieza. Who has the honor of being one of the most evil villains in the entire franchise.
Majin Buu, in the Z anime's final arc. What started as a lighthearted tale of Gohan's days in high school while masquerading as a Henshin Hero took a turn for the dark with the appearance of this large, pink, apocalyptic blob. When he turns into Super Buu, he goes on to almost completely snuff out humanity. Then along comes Kid Buu, who destroys the earth.
General Rildo in Dragon Ball GT; until he showed up, GT was a throwback to the comedy of early DB.
In the Dragon Ball Z movies, Broly is this since unlike the previous villains in the movies, Broly has a truly horrific backstory where King Vegeta nearly had him killed due to his immense power and his father had to put a mind controlling device on him in order to control his powers and he would also stop at nothing to kill anyone in his path, especially Goku.
Mazinger Z has a weird example in Count Brocken. To put it simply, he is the first villain to harm(and actually killed) innocent people. And from that, he proceed to do some horrible stuff. He is so notable amongst the villains that in fact, right in this wiki, he is the most notable person that is mentioned when the Dark part of Mazinger Z is talked about, complete with creepy design than the already creepy Ashura and the show indeed goes into the darker parts after this. The weird part ? at the same chapter he appeared, he provided the single most hilarious moments in the entire series in a rather stupid way. Its kinda hard to think hes the same person who killed person on the screen for the first time.
Fate Averruncus from the manga. His first appearance saw him petrifying a bunch of people and essentially defeating the party (who had been putting up a good fight up to that point) all on his own. When he appeared a second time he averted Bloodless Carnage by impaling Negi with a hunk of stone and scattering everyone across the world. This was the trigger that eventually led Negi to begin using Black Magic. Oh, and Fate's ultimate goal is to erase the magic world from existence, along with everyone in it.
Evangeline serves as an earlier example. Once it's revealed that not only is she a vampire with a Power Limiter curse that also binds her to the school, but the one who cursed her was Negi's father, and she needs Negi's blood to break it, the series started its Genre Shift from harem comedy to shonen.
At least until Jail, in the third season, mentioned that he had all of the Numbers (functionally, and sometimes biologically, his daughters) impregnated with clones of himself.
The Huckebein from FORCE are taking the series to a new level of dark.
Chapter 48 of Magical Girl Lyrical Nanoha Vi Vid finally has Fabia Crozelg taking center stage by effortlessly taking out around half of the ViVid cast, including Einhart and Sieg.
Mukuro Rokudo from Katekyo Hitman Reborn! is an example of this trope; in the beginning of his story arc, a number of major characters are badly beaten. His appearance also signals a major Genre Shift in the series, which had been comedic until this point.
Although it started with a horrifically failed attempt at resurrecting their mother, the series begins kind of hopeful with Edward and Alphonse Elric traveling to find the Philosopher's Stone to set things right. Then they encounter the Sewing Life Alchemist Shou Tucker whose experiments that transformed his wife and daughter into pained chimeras gives a sign of the darker events that will follow.
The introduction of the homunculi lead the series into darker territory after Envy murders Hughes.
Naruto Always had relatively darker antagonists as the series progressed.
Takasugi Shinsuke is the first seriously evil character, and is featured in the series' first serious arc, benizakura. He is probably also the only main character in the show who doesn't have comedic side.
Later Nobunobu took that position since Takasugi and Kamui are out. And how? By decapitating the minor villain Ikeda Yaemon . And then by disbanding the Shinsengumi and sending Kondo and Matsudaira to execution and then by killing two hostesses and nearly kill Tae.
Right now another group of them are The Tendoshuu. They were the ones who send assasinate Shigeshige! Then they started ruling by using Nobunobu as a full puppet.
But now the prime example of the series is Utsuro, the human member of The Tendoshuu. His sole appearing is enough to send the whole series into a total serious status.
Though Tsutomu Nihei's Blame!! was never what you'd call light-hearted, the appearance of The Safeguard marked a dramatic shift in the storyline to something much more epic.
Gundam ZZ started out almost ridiculously lighthearted but after a bunch of Ineffectual Sympathetic Villains, Haman Karn gets into the plot and all of a sudden the heroes really need to worry. Another Gundam example is Mobile Fighter G Gundam though only the first few episodes are rather easy going until Devil Gundam and Kyouji are first mentioned.
Depending on if we're talking about the manga or the anime of Rozen Maiden, either Barasuishou or Kirakishou counts. It should be noted in Kirakishou's case that when the most cheerful and childlike character in the whole franchise gets eaten by her, you know there won't be much more comedy. Barasuishou on the other hand both directly and indirectly causes the deaths of all the other characters deliberately just to please her father, Enju, a jealous apprentice to Rozen, and with her dead serious attitude that everybody must participate in the Alice Game, it is not very surprising.
Butch and Cassidy be headed for this at their first appearance, being a Terrible Trio in their own right with a crooked Officer Jenny. It didn't last long, but at least other members of Team Rocket manage to stay properly intimidating — especially when they show up in force. Even Max admits that the cast is pretty lucky that they usually only have to deal with the Terrible Trio that stalks them around.
The Terrible Trio even had a bout as such come Best Wishes. While the heroes' dilemmas started to more often involve unruly Pokemon or Slice of Life dilemmas, Jessie, James and Meowth had gained more serious and fearsome personalities, appearing more sparsely with far more imposing schemes. This eventually fizzled out with their bumbling personalities returning later, though even after they make reminders they can turn deadly again at any time (a Kalos Quest two parter starts off with a war between Pokemon herds, before the trio play a sinister gambit that plays everything in their favour, forcing an Enemy Mine).
Pokémon Hunter J surpasses them in malevolence, being the darkest villain to ever appear in the series and to never suffer from Villain Decay, not to mention being one of the few characters that was actually killed off. While technically not a villain, Paul seems headed this way, being more abusive towards his Pokémon than anyone else.
Another example is Sabrina, who popped up earlier than Butch and Cassidy and before J did. Sabrina only appeared for two episodes, and after her arc everything returned for normal, but for years later, no villain surpassed Sabrina in terms of scarinessnote Even though Sabrina was closer to a Woobie, Destroyer of Worlds, as it is implied that she was incapable of controlling her psychic powers before Haunter came along. Let's face it: she transformed trainers she defeated into dolls, and was just plain creepy. J may be more evil and has no qualms committing murder, but at least she doesn't turn people into dolls. (Only she's a gym leader and not a villain)
Cyrus of Team Galactic intended to destroy the entire world and everyone with it (even the other members of Team Galactic) while possessing Azelf, Mesprit, Uxie, Dialga, and Palkia to create a new dimension for him to rule. And he apparently dies in the episode right after J.
N, Ghetsis, Colress, and Team Plasma in Best Wishes 2 Episode N.
Yami Marik. Contrary to the original Marik he was not content to hide in the shadows relying on minions and trickery, he would personally duel and make his opponents writhe in pain in the Shadow Games he inflicts on them. In both the original Japanese and the English dub, Marik was focused on Yami Yugi and only saw his friends as pawns to be used to get to him. Yami Marik on the other hand didn't care who he faced, he would make them suffer and eventually Mind Rape them when they lost. He also underwent an Evil Costume Switch, trading in Marik's Bare Your Midriff pink shirt for a sleeveless black one with a Badass Cape, the Eye of Ra was permanently displayed on his forehead, his hair was suddenly spiking into the air, and his voice is deeper with an echo effect.
The Three Tenors in Yu-Gi-Oh! 5D's. While the full extent of their plan isn't revealed for some time, when they first began to appear they rolled out the Meklord archetype and sent Yusei into a Heroic BSOD when suddenly the iconic Synchro monsters of the series that almost all the duelists rely on became useless, forcing them to re-evaluate their strategies in addition to the mystery of the new villains that had rolled into town.
Kiryu also counts for 5Ds. He's an old friend of Yusei's who was a gang leader in the past and is shown killing a police officer in flashback. He died in prison with his heart filled with hatred towards Yusei. When he first appears, his Earthbound Immortal makes its debut by eating the souls of the people on the battlefield, and he hands Yusei his only onscreen defeat.
Vector in Yu-Gi-Oh! ZEXAL. His first appearance marks the start of the Barians taking matter into their own hands. When he shows up again later, he reveals he's been pretending to be Yuma's friend for an entire arc, causing a Heroic BSOD and a brief rift between Yuma and Astral. After his defeat, his response is to wake up the evil god of the Barian World, solely so he can keep screwing with Yuma and Astral.
There's a non-villain example, although it takes a while before the "non-villainous" part is proven. Kurosaki's arrival is marked by him turning several innocent people into cards (a metaphor for death), being all-around sneaky and violent, and having the strange ability to make duel-monsters real. The Cerberus Syndrome hits in full when he duels the Bitch in Sheep's Clothing, unmasking him and subsequently revealing that the real Knight of Cerebus villains have been Hiding In Plain Sight all along. Kurosaki himself is a Properly ParanoidAnti-Hero whose pervious actions were part of an extreme yet understandable plot that could have potentially saved countless lives if he hadn't been misinformed on the current situation.note Twice. He first thought his targets were part of the genocidal army that destroyed his home. After learning the truth continued to card people in order to lure out Reiji Akabar, hoping that he could be used as a hostage to force Leo Akbar to stop the war and return all prisoners. After learning that Leo Akabar doesn't give a shit about his son andthat Reiji is planning on waging war against Leo, Kurosaki joined forces with Reiji and became a full Anti-Hero.
The Obelisk Force and Yuri, who fully live up to this trope by carding no less than 15 people without hesitation in their first appearance.
Neon Genesis Evangelion was a dark series, but the events of the "Action Arc" (from Asuka's arrival up to Leliel/Twelfth Angel) were relatively light-hearted, with Misato and Kaji making amends, Shinji starting to gain some confidence, finding a family and friends and having a Belligerent Sexual Tension relationship with Asuka. Then came Bardiel/The Thirteenth Angel, and everything went to Hell.
Medaka Box once was a lighthearted, comedic series about school adventures of a God-Mode Sue and her harem. After 14 chapters of that, the first serious antagonist, Unzen Myouri appeared, heralding oncoming Genre Shift into a fighting series with a brutal bloodbath (no one supposedly died, but a ton of people probably were put in hospital for years). And just as the arc, started by Unzen's appearance was about to end, Kumagawa Misogi stepped on the scene, signaling a lesser shift to even more Darker and Edgier tone.
Ringing Bell is all cute and adorable until the Wolf appears and the mood becomes very dark.
The series looked like it was going to be another typical magical girl show, albeit one with unusually dark undertones. Then came Episode 3, which introduced Charlotte, the third Witch. The moment when she ate fan favorite and Cool Big Sis Mami alive was the moment when the series showed just how dark of a Deconstruction of the genre that it could be. And this was only a taste of what would come later.
Unusually for this trope, Charlotte is defeated shortly after she killed Mami.
The first episode introduces four out of the five main girls, three of which are shown to be genuinely nice and kind-hearted people, and the fourth of which is somewhat mysterious and jaded. All other humans are similarly nice, normal people. Then one episode after Charlotte, we're introduced to a fifth magical girl, a psychopathic Jerk Ass who threatens to hunt down and kill Sayaka. That's Kyoko Sakura for you, folks. There's a reason why she didn't appear back when the show was pretending to be a typical Magical Girl show. She gets better and gets a Freudian Excuse, too.
Happens again with a heroic character in Puella Magi Madoka Magica the Movie: Rebellion. As soon as Homura discovers that she and her friends are in a witch barrier and takes off her glasses and hair ribbons, the movie's tone shifts from a faux-Tastes Like Diabetes parody to a sinister mystery. And she makes the movie even darker again, near the end, when she performs a Face–Heel Turn, becomes a self-proclaimed demon, and then takes over the universe after her friends rescue her, instead of just ascending to magical girl heaven.
Claymore never made any pretenses to be anything other than a dark series, but whenever Priscilla turns up, things are going to go south very, VERY fast.
Mag Mel from Bakugan. Previous villains were dark, but Mag Mel is by far the most terrifying villain yet with an extremely dark storyline. He was imprisoned for genocide and that's before the series even started! Once he appeared, he Mind Raped Dan and Drago in a very horrific manner. Once he was finally free, he started trying to burn Gundelia to the ground. In scope of sheer evil and darkness, he's the darkest and most horrifying villain in the entire series!
Aizen has gallows humour. He's willing to let Tousen cut off Grimmjow's arm just for the fun of it. His reaction to seeing comic relief unfold is to wonder whether or not it's a bad strategy to take him off of his guard. Even before he's revealed as a villain, an extended flashback shows Shinji commenting on how uptight he is.
Shukuro Tsukishima was this during the Xcution arc, though he did have a sense of humor. It just happened to be incredibly vicious.
Yhwach, the leader of the Vandenreich, is single-handedly responsible for taking Bleach into a darker direction than it has ever been. Aizen gets freed from prison to fight him. You know how bad one Knight of Cerebus is when you have to bring in the previous one just to fight him. Yhwach decimates the Gotei 13, kills Yamamoto and any Quincies he has no further use for, defeats the Royal Guard, and tricks Ichigo into killing the Soul King. He then absorbs the Soul King and remakes the Royal Realm in his image.
Higurashi: When They Cry seems like a light-hearted harem comedy until Miyo pops up and starts putting things in Keiichi's head.
Ooishi can tend to be this for specific arcs as well. When he shows up, you know Mood Whiplash is about to kick in. It even becomes a plot point in Tatarigoroshi-hen, in which Keiichi sees him as the personification of the end of their happy days.
Tiger & Bunny has several, each one putting the series deeper into much darker territory:
Lunatic - Vigilante Man, first to kill somebody in the series. Whenever he shows up, things starts turning grim. He has a Day in the Limelight in episode 16, probably the most depressing episode in the entire series.
Jake Martinez - while Lunatic was treated seriously, he was at least a Well-Intentioned Extremist, while Jake held the entire city hostage, and sent the main character and four of his friends to the hospitalFor the Evulz. He was the first villain who was shown to take pleasure in evildoing.
Doctor Rotwang and Cis from episode 15, who have been indirectly responsible for starting a chain of heartbreaking moments that prevailed through the rest of the series.
And finally the Big Bad Albert Maverick mindraping Barnaby in a scene strongly reminding of date-rape scenario, being the first to murder non-villainous cast member and framing Kotetsu for it.
SpaceMonsters from Gunbuster - for the first two episodes series is lighthearted and funny, being basically sport story with mechas and serious elements were nothing more but backstory. Once the first battle has come, it turned into desperate fight for survival and stayed that way even after the shift to the Super Robot Genre.
Phi Brain: Puzzle of God played with this - new villain, Herbert Müller, didn't started the shift to more serious tone. In fact his appearance stopped one that was already taking place, by putting delay on plans of previously established villain Rook, who resumed them after Müller was out of picture and became Knight Of Cerebus himself.
It's interesting to watch the increase of the power of the villains in One Piece.
Alvida and Morgan possessed no real threat to Luffy, and Buggy wasn't really much of a challenge either. Afterwards, Captain Kuro and Don Krieg were much more menacing, but still relatively weak: They were just bad guys who happened to show up at the time. Everything changed when Arlong came along, and while the series retained its light-hearted tone for the most part, seeing what the stakes are when a real monster gets involved changed the world of the series forever. Follow this up with an enemy Luffy can't fight in the form of Smoker, and it's clear that the end of the East Blue saga was when the series hit its stride.
And then Crocodile, Nico Robin and the Alabasta arc up the ante, going from relatively minor conflicts to an all-out civil war. It also marks the point where the heroes start ending up on the verge of defeat (repeatedly) by an opponent far beyond their level, as well as expanding on the series' Myth Arc (becoming far more than Luffy searching for some buried treasure and becoming King of the Pirates.)
Admiral Akainu finally averts the "Nobody dies outside of flashbacks" rule by killing Ace. Blackbeard follows it up soon after by killing Whitebeard and bringing about a new age of pirates.
Donquixote Doflamingo. While the main villains of the previous two arcs, Hody Jones and Caesar Clown, may have been nothing short of despicable, neither of them presented any real threat to our protagonist and were easily taken out. Then came the Dressrosa arc, and you have Luffy struggling for the first time since the timeskip just to keep up with Doflamingo, and even Trafalgar Law getting his right arm cut and being beaten half to death by the Shichibukai. Not only that, but in this arc we discover that Doflamingo was once a World Noble and that those who carry the D are considered their "natural enemies", which makes his conflict with Luffy and Law all the more meaningful. Add to this his alliance with Kaido and his underworld persona, "Joker", and Doflamingo is easily, so far, the most dangerous enemy the Straw Hat pirates have come across since the timeskip (and, arguably, the entire series).
One Piece usually has a strange Sorting Algorithm of Evil with its Big Bad bosses, ranging from dangerously competent to incompetent with each antagonist, with the occasional appearance of a serious threat like a Warlord like Kuma or Mihawk and the Three Admirals show up in which the situation drastically becomes more threatening. Then Akainu, the last of those three Admirals makes his debut and completely breaks the Status Quo by killing Ace, and the story's gotten significantly more Darker and Edgier in both its story and its enemies, while still retaining its positive elements.
Any admiral introduction pre-timeskip serves as one - Aokiji first appearance not only it showed characters the power of Marine admiral, but also serves as the beginning of unusually dark Enies Lobby saga, and Kizaru was one of the reasons crew got separated and Luffy was forced to fight alone on next several arcs, which was rather unfortunate, since Marinford war was one of them.
Introduction of Pacifistas served as one - crew has to fight one of them together, using all of their strength to win, only for Sentomaru to arrive with another one. That was the first time, that Luffy give order to run, without trying to fight back. And it was before admiral Kizaru and Bartolomew Kuma arrived on the scene.
Transformers: Beast Wars II is an incredibly light-hearted show with hilarious characters, that hardly ever takes itself seriously. And its villains are no exceptions - Galvatron - the Big Bad is a narcoleptic who spends most of the show asleep, leaving his inept kid brother Megastorm to run the show with effeminate The Dragon (Starscream), a thug who can only repeat his own name (BB), and Beavis and Butt-Head (Thrust and Dirge) under his command. Cue the last 10 episodes of the show, where Galvatron wakes up, takes command, and we learn that he has a gigantic Doomsday Device en route to Gaia, which he intends to use to destroy the whole planet and siphon the Anglomois energy. And then things go grimdark... Gigastorm (Megastorm after his makeover) is fatally wounded and dies in Galvatron's arms, and the whole series ends with all the Maximals sacrificing themselves to destroy the Nemesis, in the end flying up to 'robot heaven'. Granted, Beast Wars Neo retcons this.
Much like its Spiritual SuccessorClaymore, Berserk is nothing short of a Dark Fantasy series. However, the TV series is considerably Lighter and Softer than its manga counterpart, so for the majority of the TV series' run, it was basically composed of Guts and Griffith talking about dreams and ambition, the Hawks battling the Tudor Empire for the Kingdom of Midland and a bunch of political stuff, and developing characterization that wasn't derailed by Adaptation Induced Plotholes or Adaptation Explanation Extrication. But when Nosferatu Zodd was introduced with his prophecy halfway through the series, followed by the introduction of the Godhand toward the end especially the transformation of Griffith, we knew that this series was going to end on a bad note. Within the manga itself, the Skull Knight, despite not being a villain, manages to be this since whenever he shows it, tends to mean things will get bad.
Tsubasa Reservoir Chronicle has a rather interesting example where this trope is actually a heroic character, (or at least a good guy). Confused? Halfway through the manga, it's revealed that the main character Syaoran is actually a clone of another "Syaoran" that The Big Bad had locked away. The Original "Syaoran" eventually manages to escape and gets warped to where the heroes are, but by that time, Clone Syaoran (known as "Cloney" by the fans) has reverted to a heartless automaton. Cloney becomes an antagonist while "Syaoran" becomes the new main character, and from then on, the manga is a hell of a lot darker than it was before.
Just like in the games, Chaos was this. An Eldritch Abomination whose appearance becomes more monstrous as he "evolves" and gets more powerful. Unlike Eggman, he has no humorous traits, and his debut episode marked the series' shift toward more serious, continuity-heavy storytelling, contrasting previous episodes' lighthearted adventures and Monster of the Week format.
The Metarex in the second series (first two seasons outside of Japan) fit this trope really well. So basically, the first two seasons of the show were pretty much light-hearted and funny in tone. Then cue the arrival of the Metarex, a group of dangerous robots led by cyborg male Seedrians/Cosmo's race that plan to harness the power of the Planet Eggs to exterminate all animal life in the galaxy so plants can rule. The Metarex come in many different types of their own, like the one-shot villain Red Pine, the Laughably Evilbrute Yellow Zelkova, emotionless monsters like Pale Bay Leaf as well as Black Narcissus, who captured and tortured Chris and Cosmo and used them as bait to lure Sonic to him, and the tragically villainousMagnificent Bastard Dark Oak himself.
Fairy Tail has two. Jellal is the first villain who poses a serious threat to the cast's lives, and is trying to return Zeref himself, as opposed to the various demon's Zeref created that have been sought out in earlier arcs, or power skirmishes that have otherwise been the focus. In addition, while previous tragic backstories have been about losing or fighting with family members, his and Erza's past is full of all kinds of slavery, torture, and betrayal. After Jellal, every arc is some serious threat that will either kill the cast or destroy the country. The second is Hades, who is also the second villain to seek Zeref. He turns the first lighthearted arc in over 100 chapters into a fight for survival that ends with the series no longer being a Nobody Can Die story, many characters' sufferings turn out to have been for no reason, Zeref potentially unleashed on the world, and he inadvertently summons an evil dragon that leaves the core cast Legally Dead for seven years. Zeref himself invokes this trope after the time skip, where he plans to wipe out humanity.
In the Monster Rancher anime, General Durahan's arrival signaled the end of lighthearted episodes; he even appears right after a lighthearted one. He's also a literal knight. Even though he isn't around for very long, he still allows Mu to get the magic stone, which allows him to overcome his Weakened by the LightAchilles' Heel, the only thing keeping him from killing the protagonist.
Toriko was a light hearted food hunting series with the occasional creepy GT Robots, and the arc after them is another finding secret recipe mission much like prior to their appearances. Then Tommyrod shows up. Unlike Starjun and Grinpatch, who were Noble Demon and Affably Evil villains respectively, Tommyrod kept all kinds of hideous parasitic insects within his body, who seriously tries to kill Toriko and actually manages to win the fight, despite getting an awesome asskicking, and from there the story gets much darker.
The series features some Demon Beasts/monsters that take the otherwise lighthearted and comedic anime on a turn for the darker and more dramatic. First up, we have Powered Masher / Masher 2.0 in a Darker and Edgier episode of the same name (in English, of course). Sure, Masher was already powerful enough in his original appearance in the second Knuckle Joe episode before that, but Masher 2.0 is even more dangerous and powerful than he was before. He did a good amount of damage to Knuckle Joe and even now comes equipped with a powerful laser. After Joe defeats Masher 2.0, everything goes back to being comical and happy.
Mumbies in Episode 84. This dude is considered to be even more dangerous than most of the bad guys Kirby's already faced before. Throwing in a creature that Kirby almost can't fight due to its trickery and supply of surprises was enough to give Kirby himself a hard time. And this thing's not the only one; there's even more Mumbies scattered across the universe, hidden on each planet all with the same goal of causing chaos and killing Star Warriors. After Kirby defeated this particular Mumbies, everything went back to happiness and humor. But not for long...
Twelve episodes later came the even Darker and Edgier five-part series finale, which featured the truly competent Destrayers. These things were the main reason why Pupu Village / Cappy Town was getting burned to a crisp and suffering from a Sugar Apocalypse. In fact, a blast fired from one of the Destrayers even knocked off a piece of... you guessed it... Kirby's WARP STAR! Yikes!
City Hunter is a strange version. It started serious (albeith with some dose of silliness), then got comedic in the Sayaka Ryujin arc and stayed comedic for most of its run, only for Sonia Field who wants to kill Ryo because he killed her father, who also was his partner at the time to bring back the seriousness. There had been other serious story arcs (chiefly Rosemary Moon's), and it happens that Sonia's father forced Ryo to kill him to protect her, but this time the story remained serious as it was before Sayaka Ryujin, culminating in the major antagonist of the initial part of the manga returning to settle the score.
Tenchi Muyo! GXP has Tarant Shunk, a man so ruthless that even other pirates fear him. The episode that introduces him is the first and only one in the entire series to show any violent bloodshed.
While the original Queen's Blade series (both the anime and the original gamebook continuity) didn't have any of them (except Aldra in the Rurou no Senshi manga), in the QB Rebellion sequel we have three of them for continuity: Captain Liliana and Ymir in the anime, as Liliana stole Vingt from Huit, and later Ymir uses dark powers for controlling the undead, and Sigui in the gamebook continuity, as she's the one of the few people that gives Annelotte, the main heroine from Rebellion, a real challenge.
Kamisama Kiss is mostly a Romantic Comedy but when Akura-Ou shows up things get darker and people start dying violent, bloody deaths and in large numbers.
Bobobo-bo Bo-bobo is a Widget Series filled with Shōnen and Surreal Humor, then Czar Baldy-Bald the III, and this misanthropic megalomaniac causes the series to get much more violent, especially by Shinsetsu where he achieves his Big Bad status again to make the empire worse than it was before.
While the series maintains an episodic Monster of the Week format even after her appearance, the addition of Northa to the cast of Fresh Pretty Cure! marked the arrival of a far stronger villain who was also dead serious about her intentions. Even the monsters of the week got stronger (thanks to her new sorewatase seeds). After Northa came the Dark Precure of HeartCatch Pretty Cure!, a villain who makes her mark by defeating the veteran Cure Moonlight in her first appearance. Every other appearance pushes Cures Blossom, Marine and Sixth Ranger Sunshine into corners until Moonlight gets back into the saddle.
Happiness Charge Pretty Cure's Phantom, the "Pretty Cure Hunter" is this. Unlike Northa or the Dark Precure, Phantom actually defeats Pretty Cure, depowering them, trapping them in crystal mirrors and shoving them in what appears to be a graveyard.
The Sailor Moon manga's first arc Queen Beryl as this role, when she started to actually act, it meant Usagi' love interest was brainwashed and Queen Metalia is revived. The anime biggest case was Sailor Galaxia. Both arcs, when she's starting acting mark an end to the series comedic moments, and lots of characters dying.
Sensui from YuYu Hakusho who killed the main character of the damn series and succeeded in opening the tunnel to demon world, which potentially could have wiped out human life. He's also a manipulative corrupter, ruthlessly sacrificing children as pawns and introduced really dark nihilistic themes into the series.
Animal Land starts off with Tarouza, a human child, dealing with relatively simple challenges from trying to get the various animals to get along in harmony, to defending against gigantic carnivores. Then comes Jyu, another human child who is a Social Darwinist, who sees all that Tarouza is trying to do be an upfront against nature and thus burns down all of the hard work Tarouza did in seven years. Jyu is then upstaged in more ways than one by Giller. He is a mysterious Mad Scientist type character who causes all sorts of harm towards the animals in the world. Also he uses Animalistic Abominations known as Chimeras.
After Satsuki's betrayal and attempted decapitation failed, Ragyo Kiryuin showed just what a living nightmare she was. For all her campiness, her villainy is played brutally straight, shown when she thrashes Satsuki and imprisons her, and then brainwashes Ryuko and sews Junketsu into her skin before sending her to kill Nudist Beach, and then putting into action a plan to blow up Earth.
Tenchi Muyo! is typically a lighthearted harem comedy, in both the original OVA and in Tenchi Universe. The exceptions in both occur when Kagato shows up. While more powerful villains than him appeared in the OVA, they never had as much of an impact as him.
The movie Tenchi In Love is mostly like the rest of the lighthearted "Universe" series with barely a mentioned of the heavily built up Eldritch Abomination Kain brought up at the start of the movie. But then towards the end of the film, Kain shows up, and unlike villains in the franchise before him, he actually kills somebody, in an outright horrific manner to boot.
Tenchi in Tokyo is probably the lightest and most comical series in the Tenchi Universe. However, when Yugi shows up, the show starts to get much darker as she tries to split up Tenchi from the girls and dark plot twists start appearing in the show.
The series had a strong mixture of comedy and heartwarming moments in addition some tearjerkers where numerous likable characters were killed off. Then the manga introduces the Wild Hunt, a secret police organization who happen to be way worse than the Jaegars.
InuYasha started out with the Monster of the Week formula with Inuyasha and Kagome spending each episode confronting various demons in order to retrieve the Shikon Jewels. However, when Naraku made his appearance, the series started revolving around the team's efforts to defeat Naraku while also exploring the Myth Arc of Naraku and how he tortured each character in the past, such as being the one who nearly destroys Inuyasha and Kikyo's relationship with each other.
Once Aion appears in the anime of Chrono Crusade the series grows incredibly dark as the first several episodes are fairly light-hearted. His first appearance marks the first major tonal shift.
My Monster Secret is a romantic comedy about dumb and cute monster girls trying to hide their secrets. Then the demon Shirogane Karen appears, being the first character to appear genuinely threatening to find out whether Youko's secret have been revealed or not. This lasts for about half a chapter, after which it's obvious she's just another idiot among the cast, and the series went back to its silly nature.
The God of Death. Secondary characters get killed off once he appears, though they get better, and he comes very close to killing Class E.
Akari Yukimura, AKA the true identity of Kaede Kayano. Her first appearance alone makes everyone, in-universe and out, rethink everything they knew about Kayano. And from there, it leads directly to some very major reveals about Koro-sensei's past.
The series started out pretty lighthearted with Tohru having comedic adventures with the Sohma family. All that changes when Akito made her appearance and the manga started focusing more on the tragic side effects that the Sohma Family's curse had on its members.
Ren, Akito's mother, is revealed to be even worse since she was the one who was responsible for mistreating Akito and causing Akito to become the villain that she has become.
The Gunsmith Cats manga starts lightly, until Gray shows up and almost kills Rally by the time his arc ends. The villains afterwards get worse, ending with the infamous Goldie.
Valkyrie Drive: Mermaid takes a more darker tone with the introduction of Momoka and her two unnamed partners. Momoka's defeated Akira, who was largely responsible for keeping the peace on the island and helps Charlotte become governor. Charlotte, being more of a tyrant tries to forcible round up all the girls not in Feste.
Black Cat is normally about Train and his friends going on adventures to capture criminals as sweepers, but whenever Creed is introduced, things get dark really fast for Train and his friends.
Death Note is by no means a dark series and while L was a Worthy Opponent for Light, the series gets even darker with the introduction of Mello and Near. Not only were Mello and Near much more cunning than L was, their tactics eventually led to Light's death. Although, this is a rather odd example as Near is an Anti-Hero like L; it's just that Light's character becomes much darker at this point as he becomes a full blown villain and Mello and Near happened to show up once this progression takes place.