Ecdysteroid biosynthesis in Varroa mites: Identification of Halloween genes from the biosynthetic pathway and their regulation during reproduction

Biosynthesis of ecdysteroids involves sequential enzymatic hydroxylations by microsomal enzymes and mitochondrial cytochrome P450’s. Enzymes of the pathway are collectively known as Halloween genes. Complete sequences for three Halloween genes, spook (Vdspo), disembodied (Vddib) and shade (Vdshd), were identified in varroa mites and sequenced. Genomic sequence comparisons showed Vdspo contained 5 introns as opposed to a maximum of 2 introns in other arthropods while Vddib and Vdshd were similar to their arthropod orthologs. Phylogenetic analyses of predicted amino acid sequences for Halloween orthologs showed that the acarine orthologs were distantly associated with insect and crustacean clades indicating the acarine genes were more ancestral. The lack of orthologs or pseudogenes for remaining genes suggests these pathway elements had not yet evolved in ancestral arthropods. Vdspo transcript levels were highest in gut tissues, while Vddib transcript levels were highest in ovary-lyrate organs. In contrast, Vdshd transcript levels were lower overall but present in both gut and ovary-lyrate organs. Vddib and Vdshd transcripts were both elevated in eggs removed from gravid female mites. Vdspo transcript levels were significantly higher in reproductive mites than in phoretic mites while Vddib and Vdshd were not significantly different. A brood cell invasion assay was developed for acquiring synchronously staged mites. Mites within 4 hrs of entering a brood cell had transcript levels of all three that were not significantly different from mites on adult bees. These analyses suggest that varroa mites are capable of modifying 7-dehydro-cholesterol precursor and can complete other hydroxylations but whether the mite needs to produce ecdysteroid products is undetermined.