Live imaging of symbiosis: spatiotemporal infection dynamics of a GFP-labelled Burkholderia symbiont in the bean bug Riptortus pedestris

Abstract

Many insects possess endosymbiotic bacteria inside their body, wherein intimate interactions occur between the partners. While recent technological advancements have deepened our understanding of metabolic and evolutionary features of the symbiont genomes, molecular mechanisms underpinning the intimate interactions remain difficult to approach because the insect symbionts are generally uncultivable. The bean bug Riptortus pedestris is associated with the betaproteobacterial Burkholderia symbiont in a posterior region of the midgut, which develops numerous crypts harbouring the symbiont extracellularly. Distinct from other insect symbiotic systems, R. pedestris acquires the Burkholderia symbiont not by vertical transmission but from the environment every generation. By making use of the cultivability and the genetic tractability of the symbiont, we constructed a transgenic Burkholderia strain labelled with green fluorescent protein (GFP), which enabled detailed observation of spatiotemporal dynamics and the colonization process of the symbiont in freshly prepared specimens. The symbiont live imaging revealed that, at the second instar, colonization of the symbiotic midgut M4 region started around 6 h after inoculation (hai). By 24 hai, the symbiont cells appeared in the main tract and also in several crypts of the M4. By 48 hai, most of the crypts were colonized by the symbiont cells. By 72 hai, all the crypts were filled up with the symbiont cells and the symbiont localization pattern continued during the subsequent nymphal development. Quantitative PCR of the symbiont confirmed the infection dynamics quantitatively. These results highlight the stinkbug-Burkholderia gut symbiosis as an unprecedented model for comprehensive understanding of molecular mechanisms underpinning insect symbiosis.

Keywords: green fluorescence protein; gut symbiotic bacteria; insect symbiosis; midgut crypt; population dynamics; stinkbug; symbiont colonization.

Figure 1

Gut symbiosis in the bean bug R. pedestris. (a) Adult male. (b) Dissected midgut. Inset is an enlarged image of the symbiotic organ. Asterisk and arrowheads indicate midgut main tract and crypts, respectively. Abbreviations: M1, midgut first region; M2, midgut second region; M3, midgut third region; M4B, bulbous midgut region prior to M4 region; M4, midgut fourth region with crypts (symbiotic organ); H, hindgut.

Figure 2

Comparison of fitness parameters between the Burkholderia-infected (Bu⁺) and uninfected (Bu⁻) adult insects of R. pedestris. (a) Survival rate (=adult emergence rate) (b) time to adulthood, (c) body length, (d) maximum thorax width, (e) time to reproduction, (f) number of eggs. Numbers inside bars indicate sample sizes. Asterisks indicate statistically significant differences (**, P < 0.001; ***, P < 0.0001).

Figure 3

Infection processes of the GFP-labelled Burkholderia symbiont visualized by epifluorescence microscopy. (a) The junction of the M3 and M4B regions of a second instar nymph, 6 h after inoculation. (b) The M4 region of a second instar nymph, 24 h after inoculation. (c, d) The M4 region of a second instar nymph, 48 h after inoculation. (e) The M4 region of a second instar nymph, 72 h after inoculation. (f) The M4 region of a third instar nymph. (g) The M4 region of a fourth instar nymph. (h) The M4 region of a fifth instar nymph. Asterisks and arrowheads indicate the main tract and crypts of the midgut, respectively. In (d), host nuclear DNA was counterstained by 4',6-diamidino-2-phenylindole.

Figure 4

Population dynamics of the Burkholderia symbiont during colonization to and proliferation in the midgut symbiotic organ. Dissected midguts were subjected to quantitative PCR of symbiont dnaA gene copies. Mean of 17 or 18 insects is indicated at each data point. The symbiont inoculation window is indicated by dotted lines.

Figure 5

Infection processes of the GFP-labellled Burkholderia symbiont in second instar nymphs of R. pedestris inoculated at different doses (37, 370 and 3700 CFU/μL). The insects were dissected 72 h after inoculation, and their midgut M4 regions were observed by epifluorescence microscopy. (a) 37 CFU/μL, (b) 370 CFU/μL and (c) 3700 CFU/μL. Asterisks and arrowheads indicate the main tract and crypts of the midgut M4 region, respectively.

Figure 6

Infection processes of the GFP-labelled Burkholderia symbiont in R. pedestris reared in soybean pots. Cultured symbiont cells were inoculated to the soybean pots, to which newly moulted second instar nymphs were introduced. The insects were dissected, and their midgut M4 regions were observed by epifluorescence microscopy. (a) 48 h after introduction and (b) 72 h after introduction. Asterisks and arrowheads indicate the main tract and crypts of the midgut M4 region, respectively.