Malpighamoeba infection compromises fluid secretion and P-glycoprotein detoxification in Malpighian tubules

Abstract

Malpighian tubules, analogous to vertebrate nephrons, play a key role in insect osmoregulation and detoxification. Tubules can become infected with a protozoan, Malpighamoeba, which damages their epithelial cells, potentially compromising their function. Here we used a modified Ramsay assay to quantify the impact of Malpighamoeba infection on fluid secretion and P-glycoprotein-dependent detoxification by desert locust Malpighian tubules. Infected tubules have a greater surface area and a higher fluid secretion rate than uninfected tubules. Infection also impairs P-glycoprotein-dependent detoxification by reducing the net rhodamine extrusion per surface area. However, due to the increased surface area and fluid secretion rate, infected tubules have similar total net extrusion per tubule to uninfected tubules. Increased fluid secretion rate of infected tubules likely exposes locusts to greater water stress and increased energy costs. Coupled with reduced efficiency of P-glycoprotein detoxification per surface area, Malpighamoeba infection is likely to reduce insect survival in natural environments.

Figure 1

Rhodamine B extrusion assay of Malpighian tubule function. (A) Locust dissection. (B) Locust dissected with Malpighian tubules exposed. (C) Three tubules removed from each locust and placed in a saline bath. The proximal end of each tubule was punctured with a sharpened capillary tube to allow the luminal fluid to be secreted. (D) After 30 min of preincubation, the saline was removed from the bath wells and replaced with 60 μM rhodamine B in saline; we discarded the droplets secreted after 30 and 60 min, analysing only those secreted at 90 min. (E) An example of a tubule incubated in the treatment bath containing rhodamine. The proximal end of the tubule is punctured to allow fluid secretion. (F) Examples of droplets secreted after 90 min of incubation by uninfected (above) and infected (below) Malpighian tubules. These droplets were removed from the puncture site in the proximal end and then placed onto a Petri dish for photography.

Figure 2

The Malpighian tubules of Malpighamoeba-infected locusts differ from those of uninfected locusts in appearance. (A) Uninfected tubules are thinner and more transparent. (B) The lumen of uninfected tubules is free from cysts. The dark structure running along the length of the tubule in this image is a tracheal branch. (C) Infected tubules are swollen and cloudy. (D) The lumen of infected tubules is filled with cysts.

Figure 3

The Malpighian tubules of Malpighamoeba-infected locusts differ from those of uninfected locusts in length, diameter and surface area. Infected locusts had longer (A) and wider (B) tubules than uninfected locusts. (C) Consequently, the surface area of infected tubules was greater than that of uninfected tubules. Large filled circles with error bars represent mean estimates from GLMM fits with the 95% confidence interval. Small circles represent the raw data.

Figure 4

Malpighamoeba infection affects the fluid secretion rate of Malpighian tubules. Tubule surface area positively correlates with fluid secretion rate, therefore infected tubules with larger surface tend to secrete more fluid than the smaller uninfected tubules.

Figure 5

Malpighamoeba infection affects rhodamine B extrusion by Malpighian tubules. (A) The uninfected tubule shown in Fig. 2B transporting rhodamine B. (B) Uninfected tubules secreted more concentrated droplets than the infected tubules. (C) The secretion rate positively correlates with the net extrusion rate of rhodamine B, but the steepness is more accentuated in the uninfected tubules compared to the infected ones. Black stars represent the mean fluid secretion rate for uninfected and infected tubules. (D) The net rhodamine extrusion rate per unit surface area is higher in uninfected than infected tubules. Small circles represent the raw data, large filled circles represent means estimates, and lines represent the regression line fitted from GLMM. Shaded areas and error bars represent the 95% confidence interval. All the values are back transformed to the original scale.

Figure 6

Locusts Malpighian tubules infected with Malpighamoeba locustae contain globular melanised encapsulations. (A) Malpighian tubules fused together with melanised encapsulations indicated by the arrow. (B) Isolated encapsulation.