05 using t-test;

two sample unequal variance; one tail distribution) (Fig. 1ii), as well as a reduction in faeces production (P < 0.05 using t-test; two sample unequal variance; one tail distribution) (Fig. 1iii). The reduction in body weight and faeces production of locusts was similar among check details all groups of locusts injected with different isolates of Acanthamoeba belonging to T1 and T4 genotypes. Of note, although locomotory behaviour was not quantified, after 5 days of infection locusts tended to be rather still and less excitable than non-infected locusts, often perching on a blade of wheat without attempting to eat. Acanthamoeba isolates of the T1 and T4 genotype each invade the locust brain Brains of locusts injected with Acanthamoeba were AZD5582 mouse dissected out and cultivated onto non-nutrient agar plates seeded with bacterial lawn. Amoebae were recovered from the brains of all groups of locusts injected with different Acanthamoeba isolates (data not shown). One hundred percent of amoebae-infected locusts showed the presence of amoebae in the brain lysates from day 5 onwards. As expected, lysates of non-infected control brains showed no growth of viable amoebae (data not shown). To further confirm the presence of amoebae within the CNS, brains from infected locusts were

fixed, PI3K Inhibitor Library sectioned and stained using Harris’ haematoxylin and eosin on days 3, 5 and 7 post-injection (three brains/isolate/day). Examination of the histological sections revealed that all amoebae

isolates tested were able to invade the locust brain (Fig. 2). Trophozoites were observed inside locust brains on days 5 and 7, post-injection, but not on day 3 (Fig. 2). In general, few amoebae were found in the brains on day 5 post-injection (sometimes as few as 1 or 2 amoebae in the whole brain, but sometimes quite numerous), whereas on day 7 amoebae were always BCKDHB very numerous (data not shown). Figure 2 Light micrographs of control-and Acanthamoeba- injected locust brains on different days post-infection. Locusts were injected with 106 amoebae/culture medium only and their brains were isolated, fixed and sectioned on days 3, 5 and 7 post infection. Trophozoites of amoebae were observed inside the locusts’ brains on days 5 (C) and 7 (D) post-infection, but not on day 3 (B) indicated by arrowheads. Disruption of the organisation within the brain tissue was also noticeable on days 5 and 7, but not on day 3. No amoeba or histopathological damage was observed in the control brains (A) and/or the capsule of the brain barrier. Note that the above images are representative micrographs of the genotype T4, but, similar results were observed with the T1 genotype. Magnification is × 400.