Impact of cysteine proteinase inhibition in midgut fluid and oral secretion on fecundity and pollen consumption of western corn rootworm (Diabrotica virgifera virgifera)

Abstract

Cysteine proteinases predominate in the midgut fluid (MF) and oral secretion (OS) of adult western corn rootworm (WCR) based on their mild acidic pH optima (pH 6.0), enhanced activities after treatment with thiol reducing agents, and inhibition by selective cysteine proteinase inhibitors (PIs). Four cysteine PIs including E-64, calpeptin, calpain inhibitor II, and leupeptin (also a serine PI) strongly inhibited azocaseinolytic activity in a dose-dependent manner in both the MF and OS. The most significant effect on adult female WCR of cysteine PI consumption with corn pollen was the reduction in fecundity, but female survival was not apparently affected. Mean fresh weights for all PI-fed females were also lower than control groups. All PI-fed groups [E-64, calpain inhibitor I (Cal I) and leupeptin] had a significantly lower daily egg production than respective corn pollen-fed controls. E-64 was more potent than leupeptin and Cal I on inhibiting fecundity, which correlates with their relative anti-proteinase potency in vitro. E-64, Cal I, and leupeptin at 1.5-2 nmol/beetle/day reduced fecundity down to 25-45% of control values. Reduced egg production by PI-fed beetles results from a combination of the direct inhibition of protein digestion and a post-ingestive negative feedback mechanism, which reduces food intake. The supplement of ten essential amino acids into the E-64-treated pollen enhanced up to 3.7-fold the number of eggs laid compared to the E-64-fed group without these amino acids, suggesting that egg production is dependent on the supply of essential amino acids from corn pollen proteolysis.

Anthropogenic increase in carbon dioxide compromises plant defense against invasive insects

“…E-64 in the diet of adult WCR and JB was a potent inhibitor of azocasein proteolysis, inhibiting both total and cysteine proteinases in the gut of WCR (cysteine, 76.3%; P ϭ 0.004; total, 84.3%; P ϭ 0.0008) and JB (cysteine, 64.8%; P Ͻ 0.0001; total 63.4%; P ϭ 0.0004) [supporting information (SI) Fig. S1], which is consistent with the role of cysteine proteinases as the primary digestion enzymes in these two species (10). Sequence analysis from WCR larval midguts revealed that, of 11 cysteine proteinases, 9 are cathepsin L-like enzymes and 2 are cathepsin B-like enzymes (27,28).…”

“…CystPIs can affect the behavior and performance of adult coleopterans. CystPI E-64 in the diet of adult female WCR reduced the number of eggs laid as a result of a combination of the direct inhibition of protein digestion and a postingestive negative feedback, which reduced food intake and insect mass (10). To determine the consequences of changes in soybean CystPI activity in naturally occurring herbivores, we estimated total and cysteine proteinase activity in the guts of JB and WCR that fed on plants grown under ambient or elevated CO 2 .…”

“…However, other compounds, such as proteinase inhibitors (PIs), can play a role in plant defense, affecting the digestibility of proteins and decreasing the availability of free amino acids required by insects for growth, development, and reproduction (8). Cysteine proteinases are common in the slightly acidic midgut (pH 5-7) of many coleopterans (9,10), and cysteine PIs (CystPIs) in plant tissues decrease growth and development by inhibiting these proteinases (10,11). Induction of the jasmonate signaling pathway in plants by herbivore damage leads to increased synthesis of CystPIs (12).…”

“…butane (E-64), inhibit gut cysteine (cathepsin L-like) activity, growth, and survival of larval and adult western corn rootworm (WCR; Diabrotica virgifera virgifera LeConte: Coleoptera) (10,13,15,16). Although WCR normally causes economic injury by damaging roots of corn (Zea mays), a variant of WCR feeds on soybean foliage and lays eggs in soybean fields (17).…”

Deleterious effects of plant cystatins against the banana weevil Cosmopolites sordidus

“…As suggested from studies reporting detrimental effects for the broad-spectrum inhibitor of papain-like proteases E-64 against Coleoptera, these negative effects of OsCYS1 and CpCYS1 were likely the result of a broad sensitivity of the insect digestive cathepsins to the ingested inhibitors. The negative effects of E-64 on growth and fecundity of several coleopteran species, including cystatin-resistant insects such as the Colorado potato beetle Leptinotarsa decemlineata (Bolter and Latoszek-Green, 1997) and the cowpea weevil Callosobruchus maculatus (Murdock et al, 1988), were systematically associated with a broadspectrum inhibition of cysteine proteases in midgut extracts (Michaud et al, 1993;Fabrick et al, 2002;Kim and Mullin, 2003). By contrast, the reported inefficiency of plant cystatins against a number of Coleoptera was typically associated with the presence of cystatin-insensitive cysteine proteases, thought to help the target insects compensating for the loss of sensitive protease activities following cystatin intake (Cloutier et al, , 2000Zhu-Salzman et al, 2003;Gruden et al, 2003Gruden et al, , 2004.…”