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 degrees C for 1 h) 48 h 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 24 h 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 48 h (but not 24 h) 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 24 h period. However, exposure of larvae to a HS, significantly increased the haemocyte density immediately after (t = 0 h) and 4 h after HS compared to the No HS controls, whilst it returned to control levels at t = 24 h. In addition, in vitro assays indicated that haemocytes harvested from larvae immediately after (0 h) and 4 h (but not 24 h) 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 100,000 haemocytes, respectively, harvested from No-HS larvae. By contrast, for HSP 60, a lysate derived from 200,000 haemocytes 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 4 h and 16 h after the HS. However, 24 h 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 200,000 haemocytes 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). (C) 2016 Elsevier Ltd. All rights reserved.