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The Liberal Democrats overturned a significant Conservative majority to win the Tiverton and Honiton seat in Devon, while Labour retook Wakefield in West Yorkshire.
Andrew Bowie, the MP for West Aberdeenshire and Kincardine, acknowledged his party had a “very difficult night”.
His comments echo those of Oliver Dowden, the party’s co-chair, who resigned on Friday following the by-election defeats.
It follows months of criticism of Mr Johnson after he was issued with a fixed-penalty notice for his involvement in Downing Street parties during coronavirus lockdowns.
Mr Johnson, speaking in Rwanda on Friday, has said he will “keep going” as leader despite the results.
Speaking to BBC Radio Scotland’s Good Morning Scotland programme, Mr Bowie said he would vote against the Prime Minister again if presented with a choice – but he stressed the country needs to focus on the “big issues”.
He said: “I made my feelings quite clear only two weeks ago in the vote of confidence when I voted that I had no confidence in the current leadership of the Conservative Party.
“I do believe that we shouldn’t be naval gazing and worrying about who is the current leader and we do need to be focusing on the big issues facing the country, such as the cost-of-living situation and supporting the government of Ukraine.
“But no, I don’t think we can carry on as if it’s business as usual.”
When asked what MPs should do about their lack of faith in Mr Johnson, he replied: “I think that is for others to be discussing this morning.”
However, he said voters had made their opinions clear in the by-elections and many MPs and party members will now be “discussing amongst ourselves how we move forward”.
He added: “I’ve been in this game long enough to know that predicting anything is a fool’s game.
“I wouldn’t want to hazard a guess what is going to happen in the next hour, let alone the next day or the next week or month.”
Asked about his position if presented with another vote of no confidence in the PM, Mr Bowie said: “If the question was put again I wouldn’t be changing my position, no.”
Mr Johnson survived the no confidence vote on June 6 with the backing of 211 Tory MPs, while 148 voted against him.