A Soluble Pyrophosphatase Is Essential to Oogenesis and Is Required for Polyphosphate Metabolism in the Red Flour Beetle (Tribolium castaneum)

Abstract

Polyphosphates have been found in all cell types examined to date and play diverse roles depending on the cell type. In eukaryotic organisms, polyphosphates have been mainly investigated in mammalian cells with few studies on insects. Some studies have demonstrated that a pyrophosphatase regulates polyphosphate metabolism, and most of them were performed on trypanosomatids. Here, we investigated the effects of sPPase gene knocked down in oogenesis and polyphosphate metabolism in the red flour beetle (Tribolium castaneum) A single sPPase gene was identified in insect genome and is maternally provided at the mRNA level and not restricted to any embryonic or extraembryonic region during embryogenesis. After injection of Tc-sPPase dsRNA, female survival was reduced to 15% of the control (dsNeo RNA), and egg laying was completely impaired. The morphological analysis by nuclear DAPI staining of the ovarioles in Tc-sPPase dsRNA-injected females showed that the ovariole number is diminished, degenerated oocytes can be observed, and germarium is reduced. The polyphosphate level was increased in cytoplasmic and nuclear fractions in Tc-sPPase RNAi; Concomitantly, the exopolyphosphatase activity decreased in both fractions. Altogether, these data suggest a role for sPPase in the regulation on polyphosphate metabolism in insects and provide evidence that Tc-sPPase is essential to oogenesis.

Inorganic pyrophosphatase from the red flour beetle (Tribolium castaneum) modulates mitochondrial polyphosphate metabolism

“…The template sequence for the in vitro transcription of the pyrophosphatase gene was amplified based on cDNA containing adaptors for primers with promoting sequences for T7 RNA polymerase. Sequence and phylogenetic analyses have been previously reported by our group (Carvalho et al, ) for TC004566 T. castaneum ( Tc‐sPPase ). The following primer pairs were used: forward ggccgcggTCACGATGTCCCTTTGGTGG and reverse cccggggcGACCTTTTGCGGTTGCGAAT; this primer pair amplified a fragment of 736 bp (Carvalho et al, ).…”

“…Sequence and phylogenetic analyses have been previously reported by our group (Carvalho et al, ) for TC004566 T. castaneum ( Tc‐sPPase ). The following primer pairs were used: forward ggccgcggTCACGATGTCCCTTTGGTGG and reverse cccggggcGACCTTTTGCGGTTGCGAAT; this primer pair amplified a fragment of 736 bp (Carvalho et al, ). An amplicon from the gene conferring resistance to neocymin was amplified from the pTEX vector to generate an unrelated dsRNA control sequence.…”

“…Mitochondria were isolated by differential centrifugation, following the procedure described by Campos et al () and Carvalho et al (). Adult beetles (500 mg) were macerated and homogenized in 3 ml of 50 mM Tris‐HCl isolation buffer, pH 7.2, containing 0.5 M sucrose, 20 mM MgCl 2, 10 μM leupeptin, 1 μM pepstatin, 10 mM EGTA, and 1% fatty acid‐free albumin.…”

“…The activity was measured following the procedure described by Campos et al () and Carvalho et al (). The reaction mixture contained 50 mM Tris‐HCl buffer (pH 7.5), 5 mM MgCl 2 , 5 mM (molecular weight of the polyp) polyP 3 or polyP 15 (only as indicated) as the substrate, and heparin as an inhibitor.…”

“…The PPX activity during mitochondrial respiration was measured following the procedure described by Campos et al () and Carvalho et al (). The reaction mixture consisted of 0.5 mg of mitochondrial protein, 50 mM Tris HCl buffer (pH 7.4), 120 mM KCl, 1 mM EGTA, 5 mM MgCl 2 , and 0.2 mM adenosine diphosphate (ADP) in the absence of any Pi source.…”