The Aquarium and Shark Lab in Hendersonville, North Carolina shared the heartbreaking update ... Charlotte passed away Sunday amid treatment for an illness.

After thanking fans for their love and support during Charlotte's unconventional journey, her caretakers noted the aquarium will remain temporarily closed ... but the other fish will still be looked after during this time.

Charlotte became an Internet sensation in February, after the aquarium announced her shocking pregnancy ... she had not shared a tank with a male stingray in 8 years. There were plenty of theories on Charlotte's mysterious pregnancy ... the most popular being parthenogenesis -- a type of asexual reproduction. Some scoffed, arguing another sea creature impregnated her.

It didn't take long for Charlotte's situation to make its way on to "Jimmy Kimmel Live," as well as "Saturday Night Live" -- where Ego Nwodim portrayed the viral stingray on 'Weekend Update.'

However, things took a sad turn by the spring.

At the end of May, when Charlotte still hadn't given birth, aquarium owner Brenda Ramer issued an ominous update ... Charlotte was never pregnant, rather was dealing with a reproductive disease, called diapause.

Brenda also hit back at those accusing the aquarium of propagating a pregnancy hoax, insisting they never fabricated details in Charlotte's journey.

Charlotte was estimated to be somewhere between 12 and 14 years old ... with the average stingray living around a decade.