Figure 1.
Visualization of binding of fluorescent Ligand-1 to gut protozoa of the Formosan subterranean termite, Coptotermes formosanus.
Blue fluorescence (excitation = 341 nm, emission = 471 nm) confirms that Ligand-1 binds to all the three species of the termite protozoa. Phagocytosed wood particles within the protozoa cytoplasm show some patchy autofluorescence. (A, B) Fluorescent and differential interference contrast (DIC) exposures of Pseudotrichonympha grassii, respectively. Binding of ligands was concentrated in the anterior region of P. grassii clearly showing the axostyle (a sheet of microtubules). (C, D) Fluorescent and DIC exposures of Holomastigotoides hartmanni, respectively. (E, F) Fluorescent and DIC exposures of Spirotrichonympha leidyi, respectively. Binding of Ligand-2 also showed a similar fluorescence pattern.
Figure 2.
Visualization of binding of fluorescent Ligand-1 to other groups of protozoa: (A–H) twelve species of gut protozoa from the eastern subterranean termite, Reticulitermes flavipes, and (I–P) four species of free-living aerobic protozoa.
(I, J) Fluorescent and differential interference contrast (DIC) exposures of Amoeba sp., respectively. (K, L) Fluorescent and DIC exposures of Euglena sp., respectively. (M,N) Fluorescent and DIC exposures of Paramecium sp., respectively. (O, P) Fluorescent and DIC exposures of Tetrahymena pyriformis, respectively. Blue fluorescence confirms that Ligand-1 binds to all the protozoa tested. Binding of Ligand-2 also showed a similar fluorescence pattern.
Figure 3.
Enhanced toxicity of Ligand-Hecate fusion peptide compared to Hecate alone.
Membranes of the termite protozoa lose their integrity five-fold faster when incubated with Ligand-Hecate fusion peptide as compared to incubation with Hecate alone at 1 µM concentration. (A, B) Differential interference contrast (DIC) images of P. grassii after 5 min of incubation with Ligand-Hecate. (C) DIC image of H. hartmanni after 5 min of incubation with Ligand-Hecate. (D) DIC image of S. leidyi after 5 min of incubation with Ligand-Hecate. (E) DIC images of all the three species of termite protozoa after 10 min of incubation with Ligand-Hecate. (F) DIC image of all the three species of termite protozoa after 10 min of incubation with Hecate alone. (G) DIC image of all the three species of termite protozoa after 10 min of incubation with the buffer without any peptide (control).
Figure 4.
Assays using Ligand-Hecate and Hecate peptides, and genetically engineered yeast strains.
(A) Mean lethal doses (LD50) of Ligand-Hecate and Hecate peptides against non-target microorganisms Escherichia coli, Pilibacter termitis, and Kluyveromyces lactis. The linking of Ligand-1 with Hecate significantly enhanced the mean lethal dose for each non-target microorganism. * indicates significant difference between treatments. (B) Toxicity of culture supernatants of different yeast strains against aerobic protozoa T. pyriformis. (C) Termite workers feeding on α-cellulose bait disk containing genetically engineered yeast cells in a bioassay setup. (D, E) Bait consumed by termite workers after five weeks. Addition of yeast into α-cellulose matrix did not deter termites from feeding and no significant difference was found in the diet consumption among different treatments. (F) Increasing number of yeast cells in the termite gut at two and three weeks of ingesting α-cellulose bait containing genetically engineered yeast strains. Control bait containing only α-cellulose did not show any CFU of K. lactis. (G) Bioassay setup to test transfer of the genetically engineered yeast cells to other nestmates. Termites fed on α-cellulose bait containing mPlum expressing yeast strain for two weeks (donors) were mixed with an equal number of workers from the same colony that were not fed on yeast bait (recipients, stained red with 1% Sudan Red 7B) and the mixed termites were fed on plain α-cellulose bait without any yeast in a Petri dish. (H) Number of mPlum expressing yeast cells (CFU) recovered from the donor and recipient termite guts two and four weeks after combining donors and recipients.
Figure 5.
Genetically engineered Kluyveromyces lactis yeast expressing the far red fluorescent protein mPlum (excitation – 590 nm and emission – 649 nm).
(A) Fluorescent and (B) Differential interference contrast (DIC) images of yeast cells expressing mPlum after 48 h of culture, respectively. (C, E) Fluorescent and (D, F) DIC images of yeast cells expressing mPlum inside the termite gut after two weeks of their continuous ingestion, respectively.
Figure 6.
α-cellulose bait containing Ligand-Hecate expressing yeast strain kills all the three species of protozoa found in C. formosanus workers within three weeks and the workers die within five weeks of bait ingestion.
(A) The gut of a worker with live protozoa at three weeks of ingesting plain α-cellulose bait. (B) Healthy protozoa exude out of worker gut when the gut is cut open. (C, D) The empty paunch of a worker possessing no protozoa at three weeks of ingesting the bait containing Ligand-Hecate yeast. (E) Healthy and (F) cellular debris of protozoa in the rectum of a worker at three weeks of ingesting the bait containing no yeast and Ligand-Hecate yeast, respectively. (G) Workers at five weeks of ingesting α-cellulose bait containing no yeast (left) and Ligand-Hecate expressing yeast (right). The worker fed on Ligand-Hecate bait is dead.