Three Halloween genes from the Varroa mite,Varroa destructor(Anderson & Trueman) and their expression during reproduction

Abstract

The ecdysteroid biosynthetic pathway involves sequential enzymatic hydroxylations by a group of enzymes collectively known as Halloween gene proteins. Complete sequences for three Halloween genes, spook (Vdspo), disembodied (Vddib) and shade (Vdshd), were identified in varroa mites and sequenced. Phylogenetic analyses of predicted amino acid sequences for Halloween orthologues showed that the acarine orthologues were distantly associated with insect and crustacean clades indicating that acarine genes had more ancestral characters. The lack of orthologues or pseudogenes for remaining genes suggests these pathway elements had not 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. All three transcripts were present in eggs removed from gravid female mites. A brood cell invasion assay was developed for acquiring synchronously staged mites. Mites within 4 h 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 may be capable of modifying 7-dehydro-cholesterol precursor and hydroxylations of other steroid precursors, but whether the mites directly produce ecdysteroid precursors and products remains undetermined.

A feeding protocol for delivery of agents to assess development in Varroa mites

“…These genes included two vitellogenins ( Vd Vg1, Vd Vg2) [33], a large lipid transfer protein ( Vd LLTP) [34], foraging ( Vd For) and malvolio ( Vd Mvl) [60], spook ( Vd spo), disembodied ( Vd dib), shade ( Vd shd) [59] and a co-transcription factor subolesin ( Vd sub) (Cabrera and Shirk, unpublished data). Comparison of the transcript levels for each of the nine selected genes between untreated control phoretic mites and the phoretic mites that survived the feeding protocol showed that that there were no significant differences after completing the feeding protocol (VdLLTP: t = -1.13, p = 0.3228, n = 3; VdVg1: t = 1.97, p = 0.1195, n = 3; VdVg2: t = 1.12, p = 0.3260, n = 3; VdFor: t = -1.20, p = 0.2964, n = 3; VdMvl: t = -1.87, p = 0.1341, n = 3; Vdspo: t = -1.35, p = 0.2475, n = 3; Vddib: t = -1.70, p = 0.1640, n = 3; Vdshd: t = -1.12, p = 0.3274, n = 3; Vdshd: t = -1.12, p = 0.3247; Vdsub: t = -0.84, p = 0.4468, n = 3); Fig 4).…”

“…All transcript levels were normalized to the geometric mean of actin and GAPDH transcript levels and presented as fold differences as reported previously [59]. Black bars are phoretic mites and gray bars represent mites completing feeding protocol.…”

“…RT-qPCR was conducted on the total RNA extracts from the surviving mites. Transcript levels were determined for the (A) large lipid transport protein (LLTP), (B) vitellogenin 1 (Vg1), (C) vitellogenin 2 (Vg2), (D) disembodied (Dib), (E) shade (Shd) and (F) spook (Spo) genes and normalized with the geometric mean for actin and GAPDH as reported previously [59]. T bars represent standard error of the mean.…”

“…These nine genes were selected based on previous work assessing differential transcription during the mite reproductive phase [33, 34, 59, 60]. Total RNA was isolated from these samples using the RNeasy Mini Kit (Qiagen Inc.), following the manufacturer’s recommendation.…”

“…Each reaction contained 1 μl of cDNA (diluted 1:50 with water), 0.6 μl of 10 μM-forward primer, 0.6 μl of 10 μM-reverse primer, 7.8 μl of water and 10 μl of Sso Advanced ™ SYBR ® green Supermix (1725274; Bio-Rad). Table 1 presents a list of the primers used to conduct reactions to assess transcript levels of Vd dib, Vd For, Vd LLTP, Vd Mvl, Vd shd, Vd spo, Vd sub, Vd Vg1, Vd Vg2 and the normalizing genes Vd Act and Vd GAPDH [59]. The transcript levels from each biological sample were normalized to the geometric mean for actin and GAPDH which was derived from the transcript levels (Cq values) of Vd Act and Vd GAPDH (geometric mean = √(Cq actin ) x (Cq GAPDH )) [61].…”