Genomic organization and reproductive regulation of a large lipid transfer protein in the varroa mite, Varroa destructor (Anderson & Trueman)

Abstract

The complete genomic region and corresponding transcript of the most abundant protein in phoretic varroa mites, Varroa destructor (Anderson & Trueman), were sequenced and have homology with acarine hemelipoglycoproteins and the large lipid transfer protein (LLTP) super family. The genomic sequence of VdLLTP included 14 introns and the mature transcript coded for a predicted polypeptide of 1575 amino acid residues. VdLLTP shared a minimum of 25% sequence identity with acarine LLTPs. Phylogenetic assessment showed VdLLTP was most closely related to Metaseiulus occidentalis vitellogenin and LLTP proteins of ticks; however, no heme binding by VdLLTP was detected. Analysis of lipids associated with VdLLTP showed that it was a carrier for free and esterified C12 -C22 fatty acids from triglycerides, diacylglycerides and monoacylglycerides. Additionally, cholesterol and β-sitosterol were found as cholesterol esters linked to common fatty acids. Transcript levels of VdLLTP were 42 and 310 times higher in phoretic female mites when compared with males and quiescent deutonymphs, respectively. Coincident with initiation of the reproductive phase, VdLLTP transcript levels declined to a third of those in phoretic female mites. VdLLTP functions as an important lipid transporter and should provide a significant RNA interference target for assessing the control of varroa mites.

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

“…Preparations of the organs were made by dissecting the synganglia (Panel A), guts (Panel B), ovaries/lyrate organs (Panel C) and developing eggs (Panel D) from the bodies and the remaining carcasses (Panel E). V d spo, V d dib and V d shd transcript levels were normalized to the geometric mean of actin and glyceraldehyde 3‐phosphate dehydrogenase ( GAPDH) transcript levels, then calibrated to the V d spo‐phoretic mite level and presented as fold differences as reported previously (Cabrera et al ., ). Bars represent standard error of the mean.…”

“…With the identification of the three Halloween genes the presence of an enzymatic foundation for production of ecdysteroids in the varroa mites is established. Along with the validation of the presence of the Vdspo gene and transcript, the identification of cholesterol and β-sitosterol (Cabrera et al, 2013) as well as diketol (data not shown) in extracts of phoretic and reproductive varroa mites demonstrates the capacity to produce the initial precursor for the ecdysteroid pathway. Interestingly, the transcript for Vdspo was only found in preparations that contained the gut tissues from the varroa mites and the levels were clearly associated with reproductive activities.…”

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).…”

“…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.…”

“…Initiation of oogenesis is observed within 6 h after cell capping [31, 32]. Vitellogenesis is initiated within 10 h after cell capping as the vitellogenin transcripts become elevated [33], the large lipid transfer protein (LLTP) transcripts are depressed [34] and yolk spheres can be observed accumulating within the oocyte [35]. The first egg is laid approximately 70 h after cell capping [32, 36] and is a haploid male [37].…”