The effects of temperature on host-pathogen interactions in D. melanogaster: who benefits?

Abstract

Drosophila melanogaster is widely used to study immune system function in insects. However, little work has been done in D. melanogaster on the effect of temperature on the immune system. Here we describe experiments that demonstrate that cooler temperatures enhance survival after infection and alter expression of immune-related genes in flies. This effect appears to be due not only to the fact that colder temperatures slow down bacterial growth, but also to the beneficial effects of cooler temperature on immune function. We explore the possibility that heat shock proteins, and in particular, Hsp83, may improve immune function at cool temperatures. We have long known that temperature can alter immune responses against microbial pathogens in insects. The approach described here allows us to determine whether this effect is due primarily to temperature-specific effects on the host or on its pathogen. These results suggest that both may be important.

Fig. 1

Survival curves (±1 S.E.) for females infected with P. aeruginosa (a) or L. lactis (b) at 17, 25, and 29 °C. In both cases, there is a significant difference between the temperature treatments (Proportional Hazards model, a: ${\chi }_2^2$, P<0.0001; b: ${\chi }_2^2$, P<0.0001). Median time to death (days post-infection) for P. aeruginosa: 17 °C = 3 days, 25 °C = 2 days, 29 °C = 1 day; L. lactis: 17 °C = 4 days, 25 °C = 2 days, and 29 °C = 1 day.

Fig. 2

Median colony counts for P. aeruginosa (solid bars) and L. lactis (hashed bars) infections, shown with first and third quartiles on a log scale. Early on in infection (1.5 and 3 h) there is not a clear pattern with regards to the effect of temperature on bacterial growth. However, at 8 and 13 h after infection, flies held at warmer temperatures had higher colony counts than those at cooler temperatures for both bacteria. Lawn plates were given a value of 2200. In the 29 °C treatment at 13 h, for L. lactis the majority of plates were lawns, and the median and quartiles were also lawns, so there are no quartile bars for that data point.

Fig. 3

Bacterial growth of P. aeruginosa (a) and L. lactis (b) in vitro at 17, 25, 29, and 37 °C. We find that warmer temperatures lead to faster bacterial growth (higher optical density).

Fig. 4

Measures of gene expression from real time RT-PCR analysis of flies infected with LPS at (a) 3 h and (b) 8 h; and with flies infected with killed L. lactis at (c) 3 h and (d) 8 h. Relative quantification (RQ) is on the y-axis (with 95% confidence intervals) and the immune genes are on the x-axis. An asterisk indicates a significant difference (P<0.05) in gene expression between the infected and sham flies. An asterisk with a bracket indicates a significant difference in gene expression between the two temperatures. We see an up-regulation of Cact, Mtk, and Pgrp-LC genes at 17 °C an up-regulation of Pgrp-LC and Mtk at 25 °C and only an up-regulation of Mtk at 29 °C.

Fig. 5

Survival curves (±1 S.E.) for the 3-h switch experiment for flies infected with (a) P. aeruginosa or (b) L. lactis at 17, 25, and 29 °C. After just 3 h at the respective temperatures, we see a significant difference in subsequent survival rate (Proportional Hazards model: P. aeruginosa, ${\chi }_2^2$, P<0.0001; L. lactis, ${\chi }_2^2$, P<0.0001). Median time to death for P. aeruginosa: 17 °C = 30 h, 25 °C = 28 h, 29 °C = 26 h; L. lactis: 17 °C = 30 h, 25 °C = 28 h, and 29 °C = 27 h.

Fig. 6

Survival curves (±1 S.E.) for flies maintained at 17, 25, or 29 °C before infection with (a) P. aeruginosa or (b) L. lactis. (a) There is no effect of pre-infection temperature regime on mortality when flies are infected with the gram-negative bacteria (Proportional Hazards model ${\chi }_2^2$, P = 0.756). (b) However, in flies infected with the gram-positive bacteria, there is a significant difference in mortality rate (Proportional Hazards model, ${\chi }_2^2$, P = 0.0019), with 29 °C flies having a greater mortality rate than 17 °C flies (Proportional Hazards model ${\chi }_1^2$, P = 0.0021).

Fig. 7

Mean gene expression (RQ; with 95% confidence intervals) for flies kept at 17 or 29 °C for 24 h, with no infection, relative to gene expression in 25 °C flies. Hsp83 is significantly higher in flies held at 17 °C than in flies held at 25 °C (t₂ = 5.13, P 0.036). Though not statistically significant, Mtk also shows a trend of increased expression at 17 °C (t₂ = 2.77, P = 0.109).