Dependence of the higher termite, Nasutitermes exitiosus and the lower termite, Coptotermes lacteus on their gut flora

The importance of the gut microorganisms in the termites Nasutitermes exitiosus and Coptotermes lacteus was investigated by feeding them with antibiotics. With N. exitiosus, antibiotics which killed both the bacteria and the spirochaetes (ampicillin, kanamycin, chloramphenicol, erythromycin, cephaloridine, tetracycline) reduced the life span of the termite from 250 days to about 13 days, whereas antibiotics which had little effect on the flora (penicillin, methicillin) did not greatly reduce the life span of the termite. The essential role of the spirochaetes in N. exitiosus was shown by feeding metronidazole, or exposing the termites to pure oxygen. Both treatments killed the spirochaetes, but not the bacteria, resulting in a life span for the termite of 13–22 days. Acid fuchsin did not kill the spirochaetes. Fungi were not essential for N. exitiosus. In C. lacteus all treatments, except that with acid fuchsin, killed the protozoa, thereby reducing the life span of the termite from 69 days to 6–29 days.     

Protein-bound amino acid content of normally and abnormally faunated Formosan termites, Coptotermes formosanus

InCoptotermes formosanus workers containing all (normally faunated), none (completely defaunated), or all but one species (partially defaunated) of their symbiotic protozoa, protein-bound amino acid contents changed little in 1, 3, 5, or 8 weeks after defaunation. There were few differences in the amino acid contents of the three termite groups at any one time. Thus, the termites may be able to maintain their protein levels without protozoa, dead protozoa probably do not furnish needed nitrogen, and symbiotic protozoa gave no evidence of the ability to fix atmospheric nitrogen.     

Genetically Engineered Yeast Expressing a Lytic Peptide from Bee Venom (Melittin) Kills Symbiotic Protozoa in the Gut of Formosan Subterranean Termites

The Formosan subterranean termite, Coptotermes formosanus Shiraki, is a costly invasive urban pest in warm and humid regions around the world. Feeding workers of the Formosan subterranean termite genetically engineered yeast strains that express synthetic protozoacidal lytic peptides has been shown to kill the cellulose digesting termite gut protozoa, which results in death of the termite colony. In this study, we tested if Melittin, a natural lytic peptide from bee venom, could be delivered into the termite gut via genetically engineered yeast and if the expressed Melittin killed termites via lysis of symbiotic protozoa in the gut of termite workers and/or destruction of the gut tissue itself. Melittin expressing yeast did kill protozoa in the termite gut within 56 days of exposure. The expressed Melittin weakened the gut but did not add a synergistic effect to the protozoacidal action by gut necrosis. While Melittin could be applied for termite control via killing the cellulose-digesting protozoa in the termite gut, it is unlikely to be useful as a standalone product to control insects that do not rely on symbiotic protozoa for survival.     

Testing Protozoacidal Activity of Ligand-lytic Peptides Against Termite Gut Protozoa in vitro (Protozoa Culture) and in vivo (Microinjection into Termite Hindgut)

We are developing a novel approach to subterranean termite control that would lead to reduced reliance on the use of chemical pesticides. Subterranean termites are dependent on protozoa in the hindguts of workers to efficiently digest wood. Lytic peptides have been shown to kill a variety of protozoan parasites (Mutwiri et al. 2000) and also protozoa in the gut of the Formosan subterranean termite, Coptotermes formosanus (Husseneder and Collier 2009). Lytic peptides are part of the nonspecific immune system of eukaryotes, and destroy the membranes of microorganisms (Leuschner and Hansel 2004). Most lytic peptides are not likely to harm higher eukaryotes, because they do not affect the electrically neutral cholesterol-containing cell membranes of higher eukaryotes (Javadpour et al. 1996). Lytic peptide action can be targeted to specific cell types by the addition of a ligand. For example, Hansel et al. (2007) reported that lytic peptides conjugated with cancer cell membrane receptor ligands could be used to destroy breast cancer cells, while lytic peptides alone or conjugated with non-specific peptides were not effective. Lytic peptides also have been conjugated to human hormones that bind to receptors on tumor cells for targeted destruction of prostate and testicular cancer cells (Leuschner and Hansel 2004).In this article we present techniques used to demonstrate the protozoacidal activity of a lytic peptide (Hecate) coupled to a heptapeptide ligand that binds to the surface membrane of protozoa from the gut of the Formosan subterranean termite. These techniques include extirpation of the gut from termite workers, anaerobic culture of gut protozoa (Pseudotrichonympha grassii, Holomastigotoides hartmanni,Spirotrichonympha leidyi), microscopic confirmation that the ligand marked with a fluorescent dye binds to the termite gut protozoa and other free-living protozoa but not to bacteria or gut tissue. We also demonstrate that the same ligand coupled to a lytic peptide efficiently kills termite gut protozoa in vitro (protozoa culture) and in vivo (microinjection into hindgut of workers), but is less bacteriacidal than the lytic peptide alone. The loss of protozoa leads to the death of the termites in less than two weeks.In the future, we will genetically engineer microorganisms that can survive in the termite hindgut and spread through a termite colony as "Trojan Horses" to express ligand-lytic peptides that would kill the protozoa in the termite gut and subsequently kill the termites in the colony. Ligand-lytic peptides also could be useful for drug development against protozoan parasites.Download video file.^{(107M, mov)}     

Relationship between virulence and repellency of entomopathogenic isolates of Metarhizium anisopliae and Beauveria bassiana to the termite Macrotermes michaelseni

Termites encounter a diverse array of potentially useful and harmful fungi in their subterranean habitats. These vary from symbiotic to harmful species with varying levels of virulence. How these hemiedaphic insects survive in habitats with infective fungi is not well understood. Possible mediation of olfactory signals in avoiding contact with entomopathogenic fungi has been explored by a number of workers. In the present study, we initially found that Macrotermes michaelseni detected a virulent isolate of Metarhizium anisopliae from some distance and avoided direct physical contact. We hypothesized that there may be a relationship between virulence and repellency of different isolates of M. anisopliae and Beauveria bassiana to the termite. We compared these for selected isolates of the two fungi. Positive correlations between the two parameters for both sets of isolates of the fungi were obtained. The results show an interesting co-evolutionary phenomenon in which the termite's response to either M. anisopliae or B. bassiana is directly related to potential harm these fungi can inflict on the insect and that the virulent strains are more likely to be recognized from some distance and avoided.     

Role of bacteria in maintaining the redox potential in the hindgut of termites and preventing entry of foreign bacteria

The hindgut of the lower termites, Mastotermes darwiniensis and Coptotermes lacteus and the higher termite Nasutitermes exitiosus were made aerobic by exposure of the termites to pure oxygen, a procedure which killed their spirochaetes and their protozoa (lower termites only). The time taken for the hindgut to become anaerobic after the termites were restored to normal atmospheric conditions ranged from 2 to 4.5 hr. After oxygen treatment the number of gut bacteria increased some six- to ten-fold in all termite species, indicating that the bacteria are poised to use oxygen entering the gut. Removal of all the hindgut microbiota by feeding tetracycline caused the hindgut to become aerobic in M. darwiniensis and N. exitiosus. The transferring of M. darwiniensis to fresh wood, free of antibiotic, resulted in the return of the normal flora and the eventual establishment of anaerobic conditions in the hindgut. Thus the bacteria appear to be important in maintaining anaerobic conditions in the gut. Attempts to determine whether the protozoa (in the lower termites) played any part in maintaining the Eh of the hindgut were unsuccessful. Serratia marcescens failed to colonise the gut of normal C. lacteus and transiently colonized (for 5 days) the gut of normal N. exitiosus. Transient colonization by S. marcescens (from 6 to 10 days) occurred in N. exitiosus when its hindgut spirochaetes were killed and in C. lacteus when its spirochaetes and protozoa were killed, indicating a possible role for the spirochaetes and/or protozoa in influencing the bacteria allowed to reside in the hindgut. Exposure of normal termites to Serratia provoked an increase in the numbers of the normal gut bacteria.     

HMW1 Is Required for Cytadhesin P1 Trafficking to the Attachment Organelle in Mycoplasma pneumoniae

Mycoplasma pneumoniae proteins HMW1-HMW3 collectively are essential for cytadherence, but the function or requirement for each has not been defined. Cytadherence mutant M6 lacks HMW1 because of a frameshift in hmw1 and produces a truncated adherence-associated protein P30 because of a deletion at the 3′ end of p30. Genetic manipulation of this mutant was used to evaluate the role of HMW1 in cytadherence. Mutant M6 was transformed with a recombinant transposon containing a wild-type p30 allele. Transformants synthesized both truncated and full-length P30, from the resident and recombinant alleles, respectively. However, these transformants remained hemadsorption negative, suggesting that HMW1 is required for cytadherence. Wild-type M. pneumoniae cells are generally elongated, tapering to form the attachment organelle at one end of the cell. The cytadhesin protein P1 is normally densely clustered on the mycoplasma surface at this differentiated terminal structure. However, both mutant M6 and M6 transformed with recombinant p30 had a striking ovoid morphology with no tapering at the tip structure, making the attachment organelle indistinguishable. Furthermore, protein P1 was randomly distributed on the mycoplasma surface rather than clustered at a polar location. In contrast, mutant M6 transformed with a recombinant transposon expressing the wild-type hmw1 allele exhibited a near-normal morphology and localized P1 to the attachment organelle. Significantly, M6 transformed with an hmw1 gene truncated slightly at the 3′ end failed to restore proper morphology or P1 localization to the attachment organelle, suggesting a functional importance to the C-terminal domain of HMW1.     

Axostyle structure in the termite protozoon Pyrsonympha vertens

The axostyle of Pyrsonympha vertens is a cellular organelle composed of interconnected microtubules. In living organisms the axostyle has waves which originate at the anterior end of the protozoon and traverse the length to the posterior end of the protozoon so that an average of 3–4 waves are present in the organelle at any given point in time. The part of the axostyle between the waves is straight. In sections through the middle of the straight part, the microtubules are hexagonally packed, with predominant connections between tubules in rows across the width of the axostyle, but the microtubules are rectilinearly packed through the wave. The wave appears to involve changes in orientation and arrangement of the microtubules. The general structure of the microtubules, cross-bridges and axostyle in the straight and bent portions are described and related to the wave propagation by this organelle.     

Isolation and assessment of gut bacteria from the Formosan subterranean termite,Coptotermes formosanus (Isoptera: Rhinotermitidae), for paratransgenesis research and application

Paratransgenesis targeting the gut protozoa is being developed as an alternative method for the control of the Formosan subterranean termite (FST). This method involves killing the cellulose‐digesting gut protozoa using a previously developed antiprotozoal peptide consisting of a target specific ligand coupled to an antimicrobial peptide (Hecate). In the future, we intend to genetically engineer termite gut bacteria as “Trojan Horses” to express and spread ligand‐Hecate in the termite colony. The aim of this study was to assess the usefulness of bacteria strains isolated from the gut of FST as “Trojan Horses.” We isolated 135 bacteria from the guts of workers from 3 termite colonies. Sequencing of the 16S rRNA gene identified 20 species. We tested 5 bacteria species that were previously described as part of the termite gut community for their tolerance against Hecate and ligand‐Hecate. Results showed that the minimum concentration required to inhibit bacteria growth was always higher than the concentration required to kill the gut protozoa. Out of the 5 bacteria tested, we engineered Trabulsiella odontotermitis, a termite specific bacterium, to express green fluorescent protein as a proof of concept that the bacteria can be engineered to express foreign proteins. Engineered T. odontotermitis was fed to FST to study if the bacteria are ingested. This feeding experiment confirmed that engineered T. odontotermitis is ingested by termites and can survive in the gut for at least 48 h. Here we report that T. odontotermitis is a suitable delivery and expression system for paratransgenesis in a termite species.     

Fipronil enhanced natural occurrence of Fusarium solani (Hypocreales: Nectriaceae) on building infesting termite Heterotermes indicola Wasmann (Blattodea: Rhinotermitidae)

The Southeast Asian subterranean termite, Heterotermes indicola Wasmann (Blattodea; Rhinotermitidae), is recognized as a building infesting lower termite species in urban environment. The extensive use of chemical termiticides against aerial mud tubes and underground nests of H. indicola beneath the buildings could not suppress its infestation; however, it enhanced the environmental contamination and insecticide resistance. In the present study, we tried to control termites using naturally occurring entomopathogenic fungi Fusarium solani (Mart.) Sacc., along with sublethal concentrations of termiticide fipronil in no-choice feeding pathogenecity bioassay for 20?days. Termite mortality after 20?days of continuous exposure to highest fungal treatment 1?×?10⁹?conidia/mL was 10% exclusively, whereas 100% mortality was calculated just after 16?days of concurrently exposure to 5?ppm of fipronil and highest rate of fungus 1?×?10⁹?conidia/mL. These results indicated that insecticidal stress declined the immune response of termites and reduced the repellency of termites against fungal conidia by breaching the primary defense mechanism (allogrooming). This co-application of F. solani at suitable sublethal concentrations of fipronil showed the promising potential against termites that may reduce the selection pressure of pesticides and resistance risk by targeted pests, but further investigations are necessary for developing field trials.     

Giardia spp.: Distribution of contractile proteins in the attachment organelle

Giardia spp. trophozoites isolated from rat small intestine were examined by light microscopy, electron microscopy, SDS-gel electrophoresis, and immunocytochemistry. In SDS-gels of protein extracts of isolated Giardia spp. trophozoites protein bands corresponding to myosin, α-actinin, and actin were identified by comigration with avian myofibril proteins and molecular weight standards. Actin was specifically identified in SDS-gels by immunoautoradiography. Immunostaining for actin, α-actinin, myosin, and tropomyosin in trophozoites was demonstrated in the periphery of the ventral disc in an area corresponding to the lateral crest. Electron-dense fibrillar was observed in the lateral crest of the ventral disc by electron microscopy. Immunostaining for actin and α-actinin was also observed in the area of the median body, a microtubular organelle, and in electron-dense fibrillar material associated with the intracellular axonemes of the posterior-lateral flagella. The localization of these contractile proteins in the ventral disc suggests that they may play an important role in the mechanism of trophozoite attachment.     

Structure of the (E)-4-hydroxy-3-methyl-but-2-enyl-diphosphate reductase from Plasmodium falciparum

Terpenoid precursor biosynthesis occurs in human and many pathogenic organisms via the mevalonate and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways, respectively. We determined the X-ray structure of the Fe/S containing (E)-4-hydroxy-3-methyl-but-2-enyl-diphosphate reductase (LytB) of the pathogenic protozoa Plasmodium falciparum which catalyzes the terminal step of the MEP pathway. The cloverleaf fold and the active site of P. falciparum LytB corresponds to those of the Aquifex aeolicus and Escherichia coli enzymes. Its distinct electron donor [2Fe–2S] ferredoxin was modeled to its binding site by docking calculations. The presented structural data provide a platform for a rational search of anti-malarian drugs.     

Lipidome and proteome of lipid droplets from the methylotrophic yeast Pichia pastoris

Lipid droplets (LD) are the main depot of non-polar lipids in all eukaryotic cells. In the present study we describe isolation and characterization of LD from the industrial yeast Pichia pastoris. We designed and adapted an isolation procedure which allowed us to obtain this subcellular fraction at high purity as judged by quality control using appropriate marker proteins. Components of P. pastoris LD were characterized by conventional biochemical methods of lipid and protein analysis, but also by a lipidome and proteome approach. Our results show several distinct features of LD from P. pastoris especially in comparison to Saccharomyces cerevisiae. P. pastoris LD are characterized by their high preponderance of triacylglycerols over steryl esters in the core of the organelle, the high degree of fatty acid (poly)unsaturation and the high amount of ergosterol precursors. The high phosphatidylinositol to phosphatidylserine of ~ 7.5 ratio on the surface membrane of LD is noteworthy. Proteome analysis revealed equipment of the organelle with a small but typical set of proteins which includes enzymes of sterol biosynthesis, fatty acid activation, phosphatidic acid synthesis and non-polar lipid hydrolysis. These results are the basis for a better understanding of P. pastoris lipid metabolism and lipid storage and may be helpful for manipulating cell biological and/or biotechnological processes in this yeast.     

Effect of attachment of Corynebacterium rathayi on movement of Anguina agrostis larvae

Bird A. F. and Riddle D. L. 1984. Effect of attachment of Corynebacterium rathayi on movement of Anguina agrostis larvae. International Journal for Parasitology 14: 503–511. The movement of freshly hatched larvae (FHL₂) and dauer larvae (DL₂) of Anguina agrostis was compared on ‘agarose’ plates. The DL₂S moved faster and over greater distances. They were not attracted to Corynebacterium rathayi on agar plates, but contact with this bacterium, in most instances, markedly reduced their speed of movement. This reduction was found to be approximately proportional to the concentration of the bacteria (from 8 × 10⁵ to 8 × 10⁸ per ml) to which the DL₂ were exposed prior to observation of their movement, as was the number of bacteria observed to be adhering when viewed under the light microscope. This type of bacterial attachment appeared to be largely stage specific as it was much more pronounced and characteristically different in the DL₂ from that in the FHL₂. This interaction between the DL₂ and the bacterium was similar in material from fresh and from rehydrated nematode galls so that it was apparently not dependent on any surface cuticular changes associated with anhydrobiosis. Furthermore, pretreatment of the nematodes with the surfactant SDS did not influence attachment. An electron microscope study of sections cut through DL₂ exposed to bacteria showed that this interaction was indeed not a surface phenomenon but that the bacteria exerted a pathological effect on the nematode. The bacterium's capsular material actually penetrated and broke down the nematode epicuticle and part of the cortical zone. These observations make it easier to understand the dramatic physiological responses of this nematode to these bacteria.     

The use of kairomones by Megaponera foetens (Fab.) (Hymenoptera: Formicidae) in the detection of its termite prey

Scout ants of the obligate termite predator, Megaponera foetens, respond to cues in fresh soil sheeting laid down by foraging termites by returning to their nests laying a recruitment trail. Effective cues are found in the sheeting of Macrotermes and Odontotermes, but not Microtermes and Ancistrotermes. They can be extracted using organic solvent, but have disappeared from the sheeting after about. 24 h. Solvent extracts of minor worker termites have the same effect on scout ants as fresh sheeting. The ability of M. foetens to detect fresh soil sheeting may increase the efficiency of predation on Macrotermes and Odontotermes species.     

Developmental changes in morphology and toxin content of the salivary gland of the australian paralysis tick Ixodes holocyclus

Binnington K.C. and Stone B.F. 1981. Developmental changes in morphology and toxin content of the salivary gland of the Australian paralysis tick Ixodes holocyclus.International Journal for Parasitology11: 343–351. Histological study of the salivary gland of female I. holocyclus has shown that 2 of the 4 cell types present are richest in granules in the unfed stage but have discharged these granules after an attachment period of 24 h. The presence of a toxin in homogenates of salivary glands from unfed females and its absence after 24 h of attachment may be associated with the loss of granules from these 2 cell types shortly after attachment. Another cell type shows a gradual increase in granule content throughout feeding and a fourth, a peak in granule content after an attachment period of 120 h. The latter cell type may produce the paralysing toxin since ticks do not paralyse the host until they have fed for about this time and homogenised glands are most toxic at 120–144 h.     

Crystal structure of serine acetyl transferase from Brucella abortus and its complex with coenzyme A

Brucella abortus is the major cause of premature foetal abortion in cattle, can be transmitted from cattle to humans, and is considered a powerful biological weapon. De novo cysteine biosynthesis is one of the essential pathways reported in bacteria, protozoa, and plants. Serine acetyltransferase (SAT) initiates this reaction by catalyzing the formation of O-acetylserine (OAS) using l-serine and acetyl coenzyme A as substrates. Here we report kinetic and crystallographic studies of this enzyme from B. abortus. The kinetic studies indicate that cysteine competitively inhibits the binding of serine to B. abortus SAT (BaSAT) and noncompetitively inhibits the binding of acetyl coenzyme A. The crystal structures of BaSAT in its apo state and in complex with coenzyme A (CoA) were determined to 1.96 Å and 1.87 Å resolution, respectively. BaSAT was observed as a trimer in a size exclusion column; however, it was seen as a hexamer in dynamic light scattering (DLS) studies and in the crystal structure, indicating it may exist in both states. The complex structure shows coenzyme A bound to the C-terminal region, making mostly hydrophobic contacts from the center of the active site extending up to the surface of the protein. There is no conformational difference in the enzyme between the apo and the complexed states, indicating lock and key binding and the absence of an induced fit mechanism.     

Characterization of the Theileria parva sporozoite proteome

East Coast fever is a lymphoproliferative disease caused by the tick-borne protozoan parasite Theileria parva. The sporozoite stage of this parasite, harboured and released from the salivary glands of the tick Rhipicephalus appendiculatus during feeding, invades and establishes infection in bovine lymphocytes. Blocking this initial stage of invasion presents a promising vaccine strategy for control of East Coast fever and can in part be achieved by targeting the major sporozoite surface protein p67. To support research on the biology of T. parva and the identification of additional candidate vaccine antigens, we report on the sporozoite proteome as defined by LC–MS/MS analysis. In total, 4780 proteins were identified in an enriched preparation of sporozoites. Of these, 2007 were identified as T. parva proteins, representing close to 50% of the total predicted parasite proteome. The remaining 2773 proteins were derived from the tick vector. The identified sporozoite proteins include a set of known T. parva antigens targeted by antibodies and cytotoxic T cells from cattle that are immune to East Coast fever. We also identified proteins predicted to be orthologs of Plasmodium falciparum sporozoite surface molecules and invasion organelle proteins, and proteins that may contribute to the phenomenon of bovine lymphocyte transformation. Overall, these data establish a protein expression profile of T. parva sporozoites as an important starting point for further study of a parasitic species which has considerable agricultural impact.