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The Italians, who were deployed on the ship to protect against the growing threat from pirates in the Indian Ocean, were escorted off the vessel in the southern port of Cochin and arrested on Sunday evening.
"Their arrest has been recorded and detailed investigations are going on," P Chandrashekharan, additional director general of police, said, adding the two had been charged with murder.
The Italian foreign ministry said the men had immunity from Indian law as the Enrica Lexie was under the Italian flag as it sailed past the coast of Kerala from Singapore to Egypt when the shooting occurred on Wednesday.
"The Italian government believes that it is the Italian judiciary that is competent, given that the acts that happened in international waters on board a ship flying the Italian flag," the ministry said on Sunday.
Italian officials have said the Indian fishing boat behaved aggressively and was repeatedly warned by officers on board the ship before guards opened fire.
But India has protested against the deaths of the unarmed men and said that the Italian soldiers must face justice in local courts.
Ships are increasingly employing armed guards for protection off the coast of Somalia and further afield as vessels of all sizes have been seized by pirates who often secure huge ransoms for the crews