Deficiency of Acetyl-CoA Carboxylase Impairs Digestion, Lipid Synthesis, and Reproduction in the Kissing Bug Rhodnius prolixus

Abstract

Rhodnius prolixus is a hematophagous insect, vector of Chagas disease. After feeding, as blood is slowly digested, amino acids are used as substrates to fuel lipid synthesis, and adult females accumulate lipids in the fat body and produce eggs. In order to evaluate the importance of de novo fatty acid synthesis for this insect metabolism, we generated acetyl-CoA carboxylase (ACC) deficient insects. The knockdown (AccKD) females had delayed blood digestion and a shorter lifespan. Their fat bodies showed reduced de novo lipogenesis activity, did not accumulate triacylglycerol during the days after blood meal, and had smaller lipid droplets. At 10 days after feeding, there was a general decrease in the amounts of neutral lipids and phospholipids in the fat body. In the hemolymph, no difference was observed in lipid composition at 5 days after blood meal, but at day ten, there was an increase in hydrocarbon content and a decrease in phospholipids. Total protein concentration and amino acid composition were not affected. The AccKD females laid 60% fewer eggs than the control ones, and only 7% hatched (89% for control), although their total protein and triacylglycerol contents were not different. Scanning electron microscopy of the egg surface showed that chorion (eggshell) from the eggs laid by the AccKD insects had an altered ultrastructural pattern when compared to control ones. These results show that ACC has a central role in R. prolixus nutrient homeostasis, and its appropriate activity is important to digestion, lipid synthesis and storage, and reproductive success.

Keywords: Rhodnius prolixus; acetyl-CoA carboxylase (ACC); de novo lipogenesis; fat body; oogenesis.

FIGURE 1

Acc knockdown in adult females compromised digestion and lifespan. Starving adult females were injected with 1 µg of dsRNA for Acc or Mal (control) and were fed 3 days later (A) Four and 10 days after blood meal, insects were dissected, and Acc RNA levels were determined by qPCR using Rhopr18S as a reference gene in ovaries (OV), fat bodies (FB), anterior midguts (AM), and posterior midguts (PM). Expression levels are relative to those from control insects (dashed line). Bars are means ± SEM for n = 3–5. (*), (**), (***), and (****): p < 0.05, 0.01, 0.001, and 0.0001, respectively, by Student’s t-test (B) Immunoblottings for ACC identification in fat bodies and ovaries’ homogenates (60–70 µg protein), obtained at day four after blood meal, representative images (n = 4) (C) At one, three, five, and 10 days after blood meal, insects were dissected and protein content was determined in total midguts. (****): p < 0.0001 by two-way ANOVA followed by Sidak’s post-test. Results are means ± SEM, n = 6. (D) Abdomens and midguts obtained from control and AccKD females, at 10 days after feeding. Arrows indicate the midguts. (E) The injected females were dissected at one and 10 days after blood meal, and the midgut protein content was analyzed by SDS-PAGE (12.5% polyacrylamide). Albumin and hemoglobin (Hb) bands are indicated. (F) Three days after dsRNA injection, insects were artificially fed on blood supplemented or not (control) with heparin. Females were weighed at days one and ten, and the results are expressed as the observed decrease in weight during this time. (****): p < 0.0001 by one-way ANOVA followed by Tukey’s test, n = 9–15. (G) After blood meal, the insect mortality was daily recorded. (****): p < 0.0001 by Log-Rank test, n = 27–28.

FIGURE 2

Acc knockdown affected perimicrovillar membranes. Starving adult females were injected with 1 µg of dsRNA for Acc or Mal (control) and were fed 3 days later. Insects were dissected at 10 days after blood meal, and the midguts were collected, washed, and processed for scanning electron microscopy. (A) dsMal; (B) dsAcc.

FIGURE 3

Decreased lipid synthesis and accumulation in the fat body upon Acc knockdown. Starving adult females were injected with 1 µg of dsRNA for Acc or Mal (control) and were fed 3 days later. (A) Three days after blood meal, insects were dissected and the isolated fat bodies were incubated in the presence of ³H-acetate for 1h for the determination of its incorporation into lipids. Bars are means ± SEM (n = 7). At 1, 3, 5, and 10 days after blood meal, insects were dissected and fat bodies were collected for (B) triacylglycerol and (C) protein content determination. The results are means ± SEM for five determinations. Alternatively, insects were dissected at day five after blood meal, the fat bodies were collected, and (D) the neutral lipid and (E) phospholipid composition was examined by TLC, followed by densitometry. Bars are means ± SEM for 7 (neutral lipids) or 5-6 (phospholipids) determinations. (*), (**), and (***): p < 0.05, 0.01, and 0.001, respectively, by Student’s t test (A,D,E) or two-way ANOVA followed by Sidak’s post-test (B,C). HC: hydrocarbon; TG: triacylglycerol; FA: fatty acid; DG: diacylglycerol; CH: cholesterol; PL: phospholipids; PA, phosphatidic acid; PE, phosphatidylethanolamine; PC, phosphatidylcholine; SM, sphingomyelin; PI, phosphatidylinositol; LPC: lysophosphatidylcholine.

FIGURE 4

Smaller lipid droplets in the fat body of Acc knockdown females. Starving adult females were injected with 1 µg of dsRNA for Acc or Mal (control) and were fed 3 days later. (A) At the fifth day after blood meal, freshly dissected fat bodies were stained with Nile Red and DAPI and observed under a laser scanning confocal microscope. Bars: 40 μm. (B) Maximum diameters of lipid droplets (LDs) were determined from four to five images obtained from at least four insects, in three experiments, and the results are means ± SD for at least 1037 LDs per group. (****): p < 0.0001 by Student’s t-test. (C) Diameter distribution histograms of the LDs from panel (B)

FIGURE 5

Hemolymph protein, amino acid, and lipid composition after Acc knockdown. Starving adult females were injected with 1 µg of dsRNA for Acc or Mal (control) and were fed 3 days later. At days five and ten after blood meal, hemolymph was collected for the determination of (A) the total protein content (means ± SEM, n = 8) and (B) the amino acid composition (data shown for three individual samples in each group). (C) The hemolymph lipid composition was also evaluated at the same days, and the results are expressed relative to control values for each lipid class (dashed line). Results are means ± SEM for six determinations. (*) and (**): p < 0.05 and 0.01, respectively, by Student’s t-test when compared to the corresponding control.

FIGURE 6

Acc knockdown impaired reproduction. Starving adult females were injected with 1 µg of dsRNA for Acc or Mal (control) and were fed 3 days later. At days one, three, five, and ten after blood meal, the insects were dissected, the ovaries were collected, and total contents of (A) proteins and (B) triacylglycerol were determined (n = 6). (C) After feeding, the cumulative oviposition was recorded (n = 35). (*) and (****): p < 0.05 and 0.0001, respectively, by two-way ANOVA followed by Sidak’s post-test. (D) Ovarioles dissected at day 12 after blood meal, from dsMal and dsAcc injected females, examined under a stereomicroscope. The arrowheads indicate the chorionated oocytes. Bar: 1.0 mm. (E) Total protein content in individual chorionated oocytes, dissected 11–13 days after feeding (means ± SEM; n = 4). (F) Analysis of proteins present in chorionated oocytes by SDS-PAGE (7.5% polyacrylamide). Vt: vitellin. (G) Triacylglycerol content in individual chorionated oocytes (means ± SEM; n = 9).

FIGURE 7

Acc knockdown females deposited eggs with defective chorion structure. The starving adult females were injected with 1 µg of dsRNA for Acc or Mal (control) and were fed 3 days later. (A) Chorionated eggs, deposited by dsMal and dsAcc injected females. Eggs were collected on different days of the oviposition cycle, and (B) the total number of hatched eggs in each group was determined. (****): p < 0.0001, by χ2 test, n = 1687. (C) Frequency of morphological alterations among eggs laid by females injected with dsAcc (n = 167). (D) Hydrocarbons extracted from the eggshells of eggs laid by AccKD and control females were evaluated by TLC, followed by densitometry (n = 3). (E) The laid eggs were processed for scanning electron microscopy, and their surface ultrastructure was examined.

FIGURE 8

Summary of effects of Acc knockdown in adult females of R. prolixus. After the inhibition of Acc gene expression by the injection of dsRNA, the fat bodies of the AccKD females synthesized and stored lower amounts of lipids, and had smaller lipid droplets. These females laid fewer eggs, which did not hatch and had chorion defects. Although digestion was disturbed, concentrations of amino acids and proteins in the hemolymph were not affected. LD: lipid droplet; TG: triacylglycerol.