Lipid metabolism dysfunction following symbiont elimination is linked to altered Kennedy pathway homeostasis

Abstract

Lipid metabolism is critical for insect reproduction, especially for species that invest heavily in the early developmental stages of their offspring. The role of symbiotic bacteria during this process is understudied but likely essential. We examined the role of lipid metabolism during the interaction between the viviparous tsetse fly (Glossina morsitans morsitans) and its obligate endosymbiotic bacteria (Wigglesworthia glossinidia) during tsetse pregnancy. We observed increased CTP:phosphocholine cytidylyltransferase (cct1) expression during pregnancy, which is critical for phosphatidylcholine biosynthesis in the Kennedy pathway. Experimental removal of Wigglesworthia impaired lipid metabolism via disruption of the Kennedy pathway, yielding obese mothers whose developing progeny starve. Functional validation via experimental cct1 suppression revealed a phenotype similar to females lacking obligate Wigglesworthia symbionts. These results indicate that, in Glossina, symbiont-derived factors, likely B vitamins, are critical for the proper function of both lipid biosynthesis and lipolysis to maintain tsetse fly fecundity.

Keywords: Bacteriology; Physiology.

Graphical abstract

Figure 1

Reproduction and lipid metabolism is dysfunctional in aposymbiotic flies Proportion of females giving birth (A) N = 4, and lipid content in the surviving larvae (B) are significantly reduced in aposymbiotic flies (t test, p value 0.007 and 0.0003, respectively). N = 8–10. (C–H) Nile blue staining reveals significant differences in the neutral (pink) and charged lipid (blue) composition in aposymbiotic and control flies. (I) Volume of the fat body is increased significantly in aposymbiotic flies (t test, p value 0.008). N = 12–14. (J) Image quantification reveals significantly higher charged lipids in the control compared to the aposymbiotic flies (t test, P value 0.002). N = 12–14.

Figure 2

Lipidomics reveals a significant shift in lipid moetities following symbiont removal Compounds showing a significantly different fold change in their abundance in control (yellow) and aposymbiotic (green) flies are shown. Red denotes increase in compounds of the Kennedy pathway in aposymbiotic flies, while blue indicates lipids showing higher fold change in control individuals. Specifically, there is a major dysfunction in the Kennedy pathway for phosphatidylcholine synthesis. N = 5, ∗ indicates significance based on a t test. Details of specific results are shown in Figures S1–S6.

Figure 3

Increased expression of choline-phosphate cytidylyltransferase 1 (ctt1) is associated with tsetse fly pregnancy (A) Previous RNA-seq studies^, reveal that cct1 is expressed in pregnant/lactating flies. (B) qPCR indicated that the expression pattern for ctt1 is correlated with the pregnancy cycle, similar to a milk protein critical to feed the developing larvae (milk gland protein 1, mgp1). N = 4. (C) Expression of cct1 is localized in both the milk glands and fat body, suggesting critical roles in lipid breakdown and milk production. N = 4, ∗, denotes significance based on a Kal’s proportion test or t test. Inset - cct1 in situ hybridization, red, along with milk gland protein (MGP) immunohistochemistry, green, and DAPI staining of nuclei, blue, of a cross section of milk gland tubules and fat body. Negative controls not treated with Digoxigenin-labeled sense RNA probes displayed no signal.

Figure 4

Suppression of cct1 produces aposymbiotic phenotypes of altered lipid metabolism during pregnancy (A) RNA interference reduces the expression of cct1. N = 5, ∗, denotes significance based on a t test. mgp1, milk gland protein 1. GFP, green flourescent protein. Primers listed in Table S1. (B and C) Total progeny production and progeny generated per day are reduced following suppression of cct1. N = 3 groups of 10. Different letters denote significance based on an ANOVA. acid smase1, asmase1. (D and E) Milk phospholipids and phosphatidylcholine are reduced when cct1 is reduced. N = 5. ∗, denotes significance based on a t test. (F) Aposymbiotic flies show increased expression of cct1 as a potential compensatory mechanism to allow for lipolysis during pregnancy. N = 6. ∗, denotes significance based on a t test. (G) Glyphosate treatment increased lipid levels within flies. N = 10. ∗, denotes significance based on a t test. (H) Supplementation of the bloodmeals with yeast extract or Wigglesworthia-containing bacteriome extract prevented the obese phenotype in aposymbiotic flies. N = 10, ∗, denotes significance based on a t test.

Figure 5

Summary of the dynamics between symbiont loss and reproduction during tsetse fly pregnancy in relation to altered lipolysis The percentage annotations in the panel depicting the Kennedy Pathway represent the relative amount of the specific lipid metabolite in the aposymbiotic tsetse fly relative to the untreated, wild-type control which would be equivalent to 100%.