Effect of stress on heat shock protein levels, immune response and survival to fungal infection of Mamestra brassicae larvae.

Although the utilisation of fungal biological control agents to kill insect pests is desirable, it is known that the outcome of infection may be influenced by a number of criteria, including whether or not the target insect is stressed. In the current work, topical treatment of larvae of the lepidopteran pest, Mamestra brassicae, with conidia of Beauveria bassiana, followed by a heat stress (HS; 37°C for 1h) 48h later, resulted in a similar level of larval survival to that occurring for no heat stress (No-HS), fungus-treated larvae. By contrast, when the HS was applied 24h after fungal treatment, larval survival was significantly increased, indicating that the HS is protecting the larvae from B. bassiana. Similarly, exposure of larvae to a HS provided protection against Metarhizium brunneum (V275) at 48h (but not 24h) after fungal treatment. To elucidate the mechanism(s) that might contribute to HS-induced increases in larval survival against fungal infection, the effects of a HS on key cellular and humoral immune responses and on the level of selected heat shock proteins (HSP) were assessed. When larvae were kept under control (No HS) conditions, there was no significant difference in the haemocyte number per ml of haemolymph over a 24h period. However, exposure of larvae to a HS, significantly increased the haemocyte density immediately after (t=0h) and 4h after HS compared to the No HS controls, whilst it returned to control levels at t=24h. In addition, in vitro assays indicated that haemocytes harvested from larvae immediately after (0h) and 4h (but not 24h) after a HS exhibited higher rates of phagocytosis of FITC-labelled B. bassiana conidia compared to haemocytes harvested from non-HS larvae. Interestingly, the HS did not appear to increase anti-fungal activity in larval plasma. Western blot analysis using antibodies which cross react with Drosophila melanogaster HSP, resulted in a relatively strong signal for HSP 70 and HSP 90 from extracts of 50,000 and 100,000haemocytes, respectively, harvested from No-HS larvae. By contrast, for HSP 60, a lysate derived from 200,000haemocytes resulted in a relatively weak signal. When larvae were exposed to a HS, the level of all three HSP increased compared to the No HS control 4h and 16h after the HS. However, 24h after treatment, any heat stress-mediated increase in HSP levels was minimal and not consistently detected. Similar results were obtained when HSP 90, 70, and 60 levels were assessed in fat body harvested from heat stressed and non-heat stressed larvae. With regard to HSP 27, no signal was obtained even when a lysate from 200,000haemocytes or three times the amount of fat body were processed, suggesting that the anti-HSP 27 antibody utilised does not cross-react with the M. brassicae HSP. The results suggest that a HS-mediated increase in haemocyte density and phagocytic activity, together with an upregulation of HSP 90 and 70, may contribute to increasing the survival of M. brassicae larvae treated with B. bassiana and M. brunneum (V275).

Hydrolase activity in the venom of the pupal endoparasitic wasp, Pimpla hypochondriaca.

Venom from the pupal endoparasitoid, Pimpla hypochondriaca has previously been shown to contain a mixture of biologically active molecules. Currently, P. hypochondriaca venom was examined for the presence of hydrolase activity. Six hydrolases were consistently detected using the API ZYM semiquantitative colourimetric kit. The main hydrolases detected were; acid phosphatase, beta-glucosidase, esterase, beta-galactosidase, esterase lipase, and lipase. The most rapid and intense colour reaction was detected for acid phosphatase. The pH optimum and the specific activity of venom acid phosphatase was determined using p-nitrophenol phosphate as a substrate and were 4.8 and 0.47 nmol p-nitrophenol/min/microg of venom protein, respectively. The acid phosphatase activity was inhibited in a dose dependent manner by sodium fluoride (IC(50) 4.2 x 10(-4) M), and by cocktail inhibitor 2 (CI 2). P. hypochondriaca venom has previously been shown to display potent cytotoxic activity towards Lacanobia oleracea haemocytes maintained in vitro. The contribution of acid phosphatase in venom to this cytotoxic activity was investigated by titrating venom against CI 2 prior to the addition of L. oleracea haemocytes. The results suggest that, despite the relatively high levels of acid phosphatase activity in venom, venom acid phosphatase plays no role in the antihaemocytic activity of P. hypochondriaca venom in vitro.

Antibacterial and proteolytic activity in venom from the endoparasitic wasp Pimpla hypochondriaca (Hymenoptera: Ichneumonidae).

Venom from the endoparasitic wasp, Pimpla hypochondriaca, is composed of a mixture of high and low molecular weight proteins, possesses phenoloxidase activity, has immunosuppressive properties, and induces paralysis in several insect species. In the present study we demonstrate that P. hypochondriaca venom also contains antibacterial and proteolytic activity. Antibacterial activity was detected against the Gram-negative bacteria Escherichia coli and Xanthamonas campestris but not against Pseudomonas syringae nor against two Gram-positive bacteria, Bacillus cereus and Bacillus subtilis. Endopeptidase and aminopeptidase activity in venom was detected using the synthetic fluorogenic substrates N-t-BOC-Phe-Ser-Arg-AMC, Arg-AMC and Leu-Arg. The aminopeptidase activity towards Arg-AMC was sensitive to amastatin (70% inhibition), an aminopeptidase inhibitor. Angiotensin-converting enzyme (ACE)-like enzyme activity was detected, by reverse-phase HPLC using the synthetic tripeptide Hip-His-Leu as a substrate. This activity was sensitive to captopril, an ACE inhibitor (IC(50) 3.8 x 10(-8) M). Using an antiserum raised against recombinant Drosophila melanogaster ACE-like enzyme, (rAnce), Western blot analysis revealed an immunoreactive protein, with a molecular weight estimate of 74 kDa, in P. hypochondriaca venom. The possibility that the endopeptidase, aminopeptidase and ACE are involved in the processing of peptide precursors in the venom sac is discussed.

Immunosuppressive properties of a protein (rVPr1) from the venom of the endoparasitic wasp, Pimpla hypochondriaca: Mechanism of action and potential use for improving biological control strategies.

Previously, it was determined that the presence of rVPr1 (a recombinant Pimpla hypochondriaca venom protein), in the haemocoel of two lepidopteran larvae, significantly increases their susceptibility to the biological control agents (BCAs), Bacillus thuringiensis (Bt) and Beauveria bassiana (Richards and Dani, 2010; Richards et al., 2011). The current work examines the mechanism of action of rVPr1 and demonstrates that it binds to the surface of some haemocytes and disrupts the organization of the haemocyte cytoskeleton. This binding is associated with a reduction in the ability of haemocytes to extend pseudopods, and to move and form aggregates in vitro over an 18 h period. Moreover, rVPr1 exerts these effects after a relatively short incubation period (1.5 h) and the haemocytes do not recover their ability to form aggregates after rVPr1 has been removed. In addition, rVPr1 significantly reduces haemocyte-mediated phagocytosis of Bt and B. bassiana in vitro (p < 0.05) and, following injection into the insect haemocoel, rVPr1 reduces the number of circulating haemocytes per ml of haemolymph (this being significantly different to the controls 3 h after injection [p = 0.05]). The finding that rVPr1 has an adverse effect on haemocyte function and number in vivo, supports the hypothesis that this wasp protein significantly increases the susceptibility of lepidopteran larvae to Bt and B. bassiana, by suppressing haemocyte-mediated immune responses in the insects which otherwise would be directed against these BCAs.

Venom from the pupal endoparasitoid, Pimpla hypochondriaca, increases the susceptibility of larval Lacanobia oleracea to the entomopathogens Bacillus cereus and Beauveria bassiana.

Cellular immune responses in insects protect them against parasites and pathogens that enter their hemocoel. Venom from the solitary pupal endoparasitoid, Pimpla hypochondriaca, has previously been shown to suppress certain key, cell-mediated immune responses of Lacanobia oleracea. Experiments were performed to determine if L. oleracea larvae injected with P. hypochondriaca venom would be more susceptible to Bacillus cereus, or Beauveria bassiana, when these microorganisms were subsequently injected. Mortality due to B. cereus (approximately 15 colony-forming units [CFU]/larva) and B. bassiana (approximately 2.4 x 10(3) conidia/larva) was enhanced by prior injection of 4 microg of venom. In addition, injection of venom/Dulbecco's phosphate-buffered saline (DPBS) or DPBS/B. bassiana reduced the rate at which larvae gained weight compared to control larvae. However, the greatest reduction in weight was recorded for larvae that had been injected with venom/B. bassiana conidia.