Differential effects of silencing crustacean hyperglycemic hormone gene expression on the metabolic profiles of the muscle and hepatopancreas in the crayfish Procambarus clarkii

Abstract

In order to functionally characterize the metabolic roles of crustacean hyperglycemic hormone (CHH), gene expression of CHH in the crayfish (Procambarus clarkii) was knocked down by in vivo injection of CHH double-stranded RNA (dsRNA), followed by metabolomic analysis of 2 CHH target tissues (the muscle and hepatopancreas) using nuclear magnetic resonance spectroscopy. Compared to the levels in untreated and saline-injected (SAI) animals, levels of CHH transcript, but not those of molt-inhibiting hormone (a CHH-family peptide), in the eyestalk ganglia of CHH dsRNA-injected (DSI) animals were significantly decreased at 24, 48, and 72 hour post injection (hpi), with concomitant changes in levels of CHH peptide in the sinus gland (a neurohemal organ) and hemolymph. Green fluorescence protein (GFP) dsRNA failed to affect levels of CHH transcript in the eyestalk ganglia of GFP DSI animals. Number of metabolites whose levels were significantly changed by CHH dsRNA was 149 and 181 in the muscle and 24 and 12 in the hepatopancreas, at 24 and 48 hpi, respectively. Principal component analysis of these metabolites show that metabolic effects of silencing CHH gene expression were more pronounced in the muscle (with the cluster of CHH DSI group clearly being separated from that of SAI group at 24 hpi) than in the hepatopancreas. Moreover, pathway analysis of the metabolites closely related to carbohydrate and energy metabolism indicate that, for CHH DSI animals at 24 hpi, metabolic profile of the muscle was characterized by reduced synthesis of NAD+ and adenine ribonucleotides, diminished levels of ATP, lower rate of utilization of carbohydrates through glycolysis, and a partially rescued TCA cycle, whereas that of the hepatopancreas by unaffected levels of ATP, lower rate of utilization of carbohydrates, and increased levels of ketone bodies. The combined results of metabolic changes in response to silenced CHH gene expression reveal that metabolic functions of CHH on the muscle and hepatopancreas are more diverse than previously thought and are differential between the two tissues.

Fig 1. Effects of dsRNA treatment on transcript levels in the eyestalk ganglia of the crayfish Procambarus clarkii.

Animals were separated into 3 groups: the untreated, saline-injected, and dsRNA-injected groups. Using a relative real-time qPCR, CHH (A, C) and MIH (B) transcript levels were quantified for the samples of the eyestalk ganglia harvested from the untreated animals (blue bar), and the saline-injected (crimson bar) and dsRNA-injected animals (green bar) at 24, 48, and 72 h post injection (hpi) of CHH dsRNA (A, B), GFP dsRNA (C), or saline (A, B, C). Transcript levels are normalized to a reference gene (18S rRNA) and expressed relative to the untreated levels. **, and *** represent significantly different from the untreated or the saline-injected values at the level of 0.01 and 0.005, respectively (n = 5 for each data).

Fig 2. Effects of CHH dsRNA treatment on CHH peptide levels in the crayfish Procambarus clarkii.

Animals were separated into 3 groups: the untreated, saline-injected, and dsRNA-injected groups. Using a CHH-specific sandwich ELISA, CHH peptide levels were quantified for the samples of the sinus gland (A) and hemolymph (B) harvested from the untreated animals (blue bar), and the saline-injected animals (crimson bar) and CHH dsRNA-injected animals (green bar) at 24, 48, and 72 h post injection (hpi)of dsRNA or saline. *,**, and *** represent significantly different from the untreated or saline-injected values at the level of 0.05, 0.01 and 0.005, respectively (n = 5 for each data).

Fig 3. Effects of CHH dsRNA treatment on hemolymph glucose levels in the crayfish Procambarus clarkii.

Animals were separated into 3 groups: the untreated, saline-injected, and dsRNA-injected groups. Using a glucose assay, glucose levels were quantified for the hemolymph samples harvested from the untreated animals (blue bar), and the saline-injected animals (crimson bar) and CHH dsRNA-injected animals (green bar) at 24, 48, and 72 h post injection (hpi) of dsRNA or saline (n = 5 for each data).

Fig 4. Principal component analysis plot of tissue samples of the crayfish Procambarus clarkii showing differential metabolic profiles between saline-injected and CHH dsRNA-injected groups.

A, B: the muscle at 24 and 48 hpi, respectively; C, D: the hepatopancreas at 24 and 48 hpi, respectively. Triangle: CHH dsRNA injected group; Square: saline-injected group. Each symbol represents the metabolome of a single sample along two principal components.

Fig 5. Metabolic profiles of the muscle and hepatopancreas of the crayfish Procambarus clarkii 24 h after CHH dsRNA treatment.

Red rectangle: significantly decreased from saline-injected levels; blue rectangle: significantly increased from saline-injected levels; dark gray rectangle: not significantly changed; light gray rectangle: not detected. Data obtained at 24 hpi (as listed in Tables 1 and 2) were used for constructing the metabolic networks relating to carbohydrate and energy metabolism. Dotted lines indicate multiple metabolic steps are involved that are not individually specified.