Global Wolbachia prevalence, titer fluctuations and their potential of causing cytoplasmic incompatibilities in tsetse flies and hybrids of Glossina morsitans subgroup species

Abstract

We demonstrate the high applicability of a novel VNTR-based (Variable-Number-Tandem-Repeat) molecular screening tool for fingerprinting Wolbachia-infections in tsetse flies. The VNTR-141 locus provides reliable and concise differentiation between Wolbachia strains deriving from Glossina morsitans morsitans, Glossina morsitans centralis, and Glossina brevipalpis. Moreover, we show that certain Wolbachia-infections in Glossina spp. are capable of escaping standard PCR screening methods by 'hiding' as low-titer infections below the detection threshold. By applying a highly sensitive PCR-blot technique to our Glossina specimen, we were able to enhance the symbiont detection limit substantially and, consequently, trace unequivocally Wolbachia-infections at high prevalence in laboratory-reared G. swynnertoni individuals. To our knowledge, Wolbachia-persistence was reported exclusively for field-collected samples, and at low prevalence only. Finally, we highlight the substantially higher Wolbachia titer levels found in hybrid Glossina compared to non-hybrid hosts and the possible impact of these titers on hybrid host fitness that potentially trigger incipient speciation in tsetse flies.

Graphical abstract

Fig. 1

Glossina phylogeny. The phylogenetic tree of the genus Glossina is based on IST-2 sequence data and was adapted from Chen et al. (1999). The figure depicts four groups of Glossina spp.: palpalis, morsitans, austeni, and fusca. Tsetse flies from the morsitans (G. m. morsitans, G. m. centralis, G. swynnertoni) and the fusca group (G. brevipalpis) were analyzed in this study (indicated by asterisks).

Fig. 2

VNTR-fingerprinting of Glossina spp. (A) VNTR-141-PCR of four Glossina species. Samples are either laboratory-reared or field collected single individuals of (a) G. m. morsitan Gmm, (b) G. m. centralis Gmc, (c) G. swynnertoni Gsw, and (d) G. brevipalpis Gbr. Black arrowheads indicate a 347-bp fragment in Gmm of chromosomal origin; a 464-bp fragment of cytoplasmic origin in Gmm, Gmc, and Gsw; and a larger 483-bp fragment in Gbr. (B) VNTR-141-PCR of wild type (wt) and aposymbiotic (apo) Gmm. The cytoplasmic 464-bp band is more prominent in wt, whereas the nuclear 347-bp band is strong in the apo Gmm. DNA quality was assessed via 12S rRNA-specific PCR.

Fig. 3

Organization of the VNTR-141 locus in Glossina spp. The 464-bp fragments of wGmm, wGmc, wGsw, and wGbr share a small 8-bp insertion between the 15-bp (blue) and 13-bp (yellow) repeat box and a 104-bp insertion (grid) at the 3′-end of the locus; the 347-bp chromosomal fragment of Gmm and the 483-bp wGbr fragment lack both. Green and blue boxes represent 15-bp repeats, yellow and red ones 13-bp repeats. The gray arrow represents the complete core 108-bp repeat of wAu from D. simulans. Abbreviations: wGmm, wGmc, wGbr, and wGsw symbolize Wolbachia from G. m. morsitans, G. m. centralis, G. brevipalpis, and G. swynnertoni, respectively. Black arrow indicates the 141-bp master unit from wMel-like strains.

Fig. 4

Detection of low-titer Wolbachia in tsetse flies. (A) Wolbachia-specific wsp-PCR of Glossina spp. (B) same gel after PCR-blot technique probed with a digoxygenin-labeled internal wsp-fragment (Arthofer et al., 2009). According to wsp-PCR, Wolbachia-infections are high in G. m. centralis (Gmc), intermediate in G. m. morsitans (Gmm), low in G. brevipalpis (Gbr), and not detectable in G. swynnertoni (Gsw). PCR-blot technique significantly enhances detection sensitivity, especially in G. m. morsitans, G. brevipalpis and G. swynnertoni. The Wolbachia-uninfected D. simulans, STC strain was used as negative control in both experiments.

Fig. 5

Wolbachia-prevalence in Glossina hybrids. (A–C) PCR-blot technique applied to parental and hybrid tsetse flies. Upper lanes show wsp-PCR, lower lanes show blot hybridizations using a digoxygenin-labeled internal wsp-probe (Arthofer et al., 2009). (A) Gmm mothers, Gmc fathers plus Gmm × Gmc F1 hybrids. (B) Gsw mothers, Gmc fathers, plus Gsw × Gmc and reciprocal hybrids. (C) Gsw mothers, Gmm fathers plus Gsw × Gmm and reciprocal hybrids. ♀ Symbolizes mothers, ♂ symbolizes fathers; hybrids are marked as F1° and F1^*, respectively. F1° hybrids result from ♀ × ♂ cross, whereas F1^* hybrids result from the reciprocal cross. (D) VNTR-141-PCR on Gmm × Gmc F1 hybrids of both sexes showing 464-bp (cytoplasmic) and 347-bp chromosomal fragments. (E) ISNew-PCR on same sample set showing a 324-bp (cytoplasmic) and an in silico calculated 400-bp fragment (chromosomal). (F) Quality of DNA was assessed via 12S rRNA PCR. The Wolbachia-uninfected Drosophila simulans STC strain was used as negative control in all experiments.

Fig. 6

Quantitative 16S rRNA Realtime-PCR on Glossina parental females and respective hybrid offspring (females are named first). (A) Gmm × Gmc hybrid females (F1 ♀) and males (F1 ♂); ♀ symbolizes Gmm mothers; F1 indicates hybrid daughters and sons. (B) Offspring from Gsw × Gmc cross indicated by F1; ♀ symbolizes Gsw mothers. Gsw female one is the mother of F1 hybrid number one; Gsw female two is the mother of F1 hybrid two. Same applies to (C and E). In (D), only one hybrid with the corresponding mother is presented. (C) Gmc × Gsw hybrids symbolized by F1, Gmc mothers are marked by ♀, (D) F1 indicates a Gsw × Gmm hybrid and ♀ the Gsw mother, (E) Gmm × Gsw hybrids symbolized by F1; ♀ indicates the Gmm mother. Differences in symbiont titer levels were considered statistically significant when P < 0.05 (^*), very significant when P < 0.01 (^**), and extremely significant when P < 0.001 (^***).