Ultrastructural Studies on the Trypanosomatid Phytomonas serpens in the Salivary Glands of a Phytophagous Hemipteran

Electron microscopy of salivary glands of the phytophagous hemipteran Phihia picta infected with Phytomonas serpens revealed the presence of flagellates in the gland periphery beneath the gland envelope, in the gland central lumen, between gland cells in the intercellular space and inside the gland cells. In the latter case, flagellates were found in the cytoplasm whether or not it was surrounded by a vacuolar membrane. Flagellates were always of the promastigote type, sometimes displaying a large twisted body. Morphological peculiarities of flagellates in different gland locations are recorded.     

Intranuclear Parasitism of the Ciliate Euplotes by a Trypanosomatid Flagellate

A trypanosomatid flagellate, Leptomonas sp., develops and multiplies in the macronucleus (only) of natural and laboratory-reared populations of the ciliate Euplotes. Up to 90% of the natural populations of Euplotes in our test pond had such nuclear infections. Laboratory infections were transmitted to this ciliate by feeding it liberated parasites. Paramecium resisted infections. All laboratory-induced infections were lethal to Euplotes, while control clones of the uninfected ciliates remained viable. This leptomonad, unlike Leptomonas karyophilus (found in Paramecium), shows no leishmanial forms in its several ciliate hosts and shows a varied pattern of locomotion.     

Axenic Cultivation of Phytomonas davidi Lafont (Trypanosomatidae), a Symbiote of Laticiferous Plants (Euphorbiaceae)*

SYNOPSIS. Phytomonas davidi (Trypanosomatidae) originally discovered by Lafont in 1909 on the island of Mauritius was rediscovered in Euphorbia cyathophora in Florida. Successful cultures were established in diphasic medium consisting of duck blood agar and modified Phillips’medium as overlay. Optimal growth was obtained when Mansour's medium was used as overlay and poorest growth when Cowperthwaite's medium buffered at pH 5.0 was utilized for this purpose. Marked changes tending toward choanomastigotes rather than the elongate twisted promastigotes were observed in cultures.     

Transovum Transmission of Trypanosomatid Cysts in the Milkweed Bug,Oncopeltus fasciatus

Leptomonas wallacei is a trypanosomatid that develops promastigotes and cystic forms in the gut of the hemipteran insect Oncopeltus fasciatus. Insect trypanosomatids are thought to be solely transmitted from one host to another through the ingestion of parasite-contaminated feces. However, here we show that L. wallacei cysts present on the eggshells of eggs laid by O. fasciatus can also act as infective forms that are transmitted to the insect offspring. Newly hatched O. faciatus nymphs are parasite-free, but some of them become contaminated with L. wallacei after feeding on eggshell remnants. The present study is the first report of transovum transmission of a trypanosomatid, a process that may have a relevant role in parasite’s within-host population dynamics.     

The Catabolism of Tryptophan to Indole-3-Ethanol by Crithidia fasciculata and Phytomonas davidi1

Crithidia fasciculata and Phytomonas davidi catabolize tryptophan (TRP) to indole-3-ethanol, which was identified by both thin layer and gas chromatography. The catabolic pathway involved in this metabolic conversion is suggested to be similar to that proposed for other members of the family Trypanosomatidae. Although this catabolism occurs at both 25° and 37°C, the catabolic rate is greater at 37°C, a non-permissive growth temperature. Conditions that inhibit protein synthesis would appear to favor the catabolism of tryptophan to indole-3-ethanol. The possible importance of this catabolic pathway to these organisms is discussed.     

Efficient Colonization of the Bean Bug Riptortus pedestris by an Environmentally Transmitted Burkholderia Symbiont

The bean bug Riptortus pedestris is specifically associated with the Burkholderia gut symbiont and acquires the symbiont from the environment every generation. Here, we investigated the infective dose of the symbiont by experimental administration. The 50% infective dose was remarkably low, only 80 cells, indicating efficient colonization of the symbiont.     

The Immune Response of Hemocytes of the Insect Oncopeltus fasciatus against the Flagellate Phytomonas serpens

The genus Phytomonas includes parasites that are etiological agents of important plant diseases, especially in Central and South America. These parasites are transmitted to plants via the bite of an infected phytophagous hemipteran. Despite the economic impact of these parasites, many basic questions regarding the genus Phytomonas remain unanswered, such as the mechanism by which the parasites cope with the immune response of the insect vector. In this report, using a model of systemic infection, we describe the function of Oncopeltus fasciatus hemocytes in the immune response towards the tomato parasite Phytomonas serpens. Hemocytes respond to infection by trapping parasites in nodular structures and phagocytizing the parasites. In electron microscopy of hemocytes, parasites were located inside vacuoles, which appear fused with lysosomes. The parasites reached the O. fasciatus salivary glands at least six hours post-infection. After 72 hours post-infection, many parasites were attached to the salivary gland outer surface. Thus, the cellular responses did not kill all the parasites.     

Biology and Structure of Phytomonas staheli sp. n., a Trypanosomatid Located in Sieve Tubes of Coconut and Oil Palms

SYNOPSIS. Phytomonas staheli sp. n. is described from the oil palm ( Elaeis guineensis ) and the coconut palm ( Cocos nucifera ) of Surinam, South America. The phytomonad is the probable cause of "Hartrot" in the coconut palm and "Marchitez sopresiva" in oil palms. Parasites are confined to the sieve tubes in palms. Some success was obtained at cultivation of the organism from Elaeis.     

Nutritional Requirements of Blastocrithidia culicis,a Trypanosomatid with an Endosymbiont1

ABSTRACT. The symbiont-containing Blastocrithidia culicis from Aedes vexans, unlike most lower trypanosomatids cultivated in defined media, require only three amino acids: methionine, histidine, and arginine; only three vitamins: thiamin, nicotinamide, and riboflavin; and neither heme nor purine. The bacterium-like endosymbiont presumably provides the trypanosomatid's other essential nutrients.     

Feeding of a Freshwater Flagellate,Bodo saltans,on Diverse Bacteria1

ABSTRACT Bodo saltans was isolated from a chalk stream and fed with pure cultures of seven bacteria obtained from the same river. The flagellates were allowed to migrate into suspensions of either of two bacterial species in a T-maze at 20–22°C. There was a significant difference (P < 0.01) between the numbers of flagellates which migrated into suspensions of different bacteria, which were subsequently arranged in an order of “attractiveness” to the flagellate. Bodo saltans grew successfully in monoxenic suspensions of all seven bacterial strains, but more rapid growth occurred with non-flagellated than with flagellated bacteria; this may be because while feeding, B. saltans tends to associate with surfaces where non-flagellated bacteria may also congregate. The efficiency with which B. saltans is able to utilize different bacteria may be influenced by the motility or secretory activities of the bacteria. There was no incontrovertible evidence that B. saltans responds to specific bacterial attractants.     

The Motility Symbiont of the Termite Gut Flagellate Caduceia versatilis Is a Member of the “Synergistes” Group

The flagellate Caduceia versatilis in the gut of the termite Cryptotermes cavifrons reportedly propels itself not by its own flagella but solely by the flagella of ectosymbiotic bacteria. Previous microscopic observations have revealed that the motility symbionts are flagellated rods partially embedded in the host cell surface and that, together with a fusiform type of ectosymbiotic bacteria without flagella, they cover almost the entire surface. To identify these ectosymbionts, we conducted 16S rRNA clone analyses of bacteria physically associated with the Caduceia cells. Two phylotypes were found to predominate in the clone library and were phylogenetically affiliated with the “Synergistes” phylum and the order Bacteroidales in the Bacteroidetes phylum. Probes specifically targeting 16S rRNAs of the respective phylotypes were designed, and fluorescence in situ hybridization (FISH) was performed. As a result, the “Synergistes” phylotype was identified as the motility symbiont; the Bacteroidales phylotype was the fusiform ectobiont. The “Synergistes” phylotype was a member of a cluster comprising exclusively uncultured clones from the guts of various termite species. Interestingly, four other phylotypes in this cluster, including the one sharing 95% sequence identity with the motility symbiont, were identified as nonectosymbiotic, or free-living, gut bacteria by FISH. We thus suggest that the motility ectosymbiont has evolved from a free-living gut bacterium within this termite-specific cluster. Based on these molecular and previous morphological data, we here propose a novel genus and species, “Candidatus Tammella caduceiae,” for this unique motility ectosymbiont of Caducaia versatilis.     

Negative Fitness Consequences and Transmission Dynamics of a Heritable Fungal Symbiont of a Parasitic Wasp

Heritable bacterial symbionts are widespread in insects and can have many important effects on host ecology and fitness. Fungal symbionts are also important in shaping their hosts'' behavior, interactions, and evolution, but they have been largely overlooked. Experimental tests to determine the relevance of fungal symbionts to their insect hosts are currently extremely rare, and to our knowledge, there have been no such tests for strictly predacious insects. We investigated the fitness consequences for a parasitic wasp (Comperia merceti) of an inherited fungal symbiont in the Saccharomycotina (Ascomycota) that was long presumed to be a mutualist. In comparisons of wasp lines with and without this symbiont, we found no evidence of mutualism. Instead, there were significant fitness costs to the wasps in the presence of the yeast; infected wasps attacked fewer hosts and had longer development times. We also examined the relative competitive abilities of the larval progeny of infected and uninfected mothers, as well as horizontal transmission of the fungal symbiont among larval wasps that shared a single host cockroach egg case. We found no difference in larval competitive ability when larvae whose infection status differed shared a single host. We did find high rates of horizontal transmission of the fungus, and we suggest that this transmission is likely responsible for the maintenance of this infection in wasp populations.The majority of heritable bacterial symbionts associated with insects either provide nutritional benefits for hosts that feed on nutrient-poor diets, such as blood (e.g., Wigglesworthia sp. [1]) or sap (e.g., Buchnera spp. [33]), or manipulate the hosts'' reproduction to benefit their own transmission (e.g., Wolbachia spp. [38] or Cardinium sp. [40]). Thanks in part to these examples, research efforts have become more diverse, leading to the discovery of additional benefits, such as heat tolerance (29) and protection from parasitism (26).Despite growing interest in the cryptic roles of insect associates, fungal symbionts have largely been overlooked, and their prevalence, ecological importance, and evolutionary implications for hosts are still poorly understood. Yet we have reason to suspect that fungal symbionts may be as diverse and functionally important as bacteria in insects. Buchner''s (5) foundational work on arthropod-microbe symbioses included many fungi, and anecdotal reports of such symbioses are scattered throughout the literature (e.g., fire ants [3]; stingless bees [28]; earwigs, scale insects, flies, andrenid bees, and ants [39]; and leafhoppers [30]). Recent surveys of insects for fungi have resulted in an astonishing diversity, including fungi in beetles (35), a cockroach and five other neuropteran families (24), sap-feeding beetles, and flies and bees (15), and it has been suggested that the majority of unicellular fungal diversity may be in insects (35). It is often suggested that such associations are mutualistic, with the fungus presumably providing enzymes, essential amino acids, vitamins, or sterols (37) and the insect vectoring and providing a habitat for the fungus. Fitness consequences of these associations have been assessed in only a few cases, including associations in planthoppers (31), anobiid beetles (23, 32), and scolytid beetles (2). In most instances the significance of the relationship is not clear, especially in the many cases where the fungi are not obligate associates.In 1985, LeBeck (18) reported a unicellular fungal symbiont in Comperia merceti (Compere) (Hymenoptera: Encyrtidae), a gregarious endoparasitoid wasp that specializes on the egg cases of brown-banded cockroaches [Supella longipalpa (Serville) (Blattaria: Blattellidae)]. The fungus is found throughout the hemocoel in juvenile wasps, in adult males, and in the venom gland of adult females (18). In addition, the fungus is vertically transmitted from mother to offspring via the external surface of wasp eggs during oviposition into cockroach egg cases. Vertical transmission via the egg surface is a common method in other fungal symbiont systems (e.g., planthoppers [19]; lacewings [10]; and wood wasps, anobiid beetles, and cerambycid beetles [5]). LeBeck (18) characterized the fungus as a Candida sp. and suggested that it might alter the nutritional value of the host cockroach egg case for the benefit of the developing wasp larvae. However, this claim has never been tested. Further, the predacious diet of immature parasitic wasps would make them unusual candidates for nutritional symbionts; parasitic wasps consume other insects and do not ordinarily require the complementary nutrients that many fungal and bacterial symbionts provide to insects with unbalanced diets. To our knowledge, our study is the first to specifically test the role of an inherited fungus in an insect with a strictly predacious diet.C. merceti wasps house a single known fungal symbiont belonging to the Ascomycota (Saccharomycotina) and no detectable bacterial symbionts (9). Further, these wasps do not become infected with any of their host cockroaches'' symbionts (9). In in vitro trials of the C. merceti wasp fungus with other microbes there was no evidence of inhibition or any type of interaction (C. M. Gibson, unpublished). The current research tests the hypothesis that the wasps'' fungal symbiont is a mutualist and explores alternative means by which this fungus could be maintained in wasp populations.     

Killing of Trypanosomatid Parasites by a Modified Bovine Host Defense Peptide,BMAP-18

Background

Tropical diseases caused by parasites continue to cause socioeconomic devastation that reverberates worldwide. There is a growing need for new control measures for many of these diseases due to increasing drug resistance exhibited by the parasites and problems with drug toxicity. One new approach is to apply host defense peptides (HDP; formerly called antimicrobial peptides) to disease control, either to treat infected hosts, or to prevent disease transmission by interfering with parasites in their insect vectors. A potent anti-parasite effector is bovine myeloid antimicrobial peptide-27 (BMAP-27), a member of the cathelicidin family. Although BMAP-27 is a potent inhibitor of microbial growth, at higher concentrations it also exhibits cytotoxicity to mammalian cells. We tested the anti-parasite activity of BMAP-18, a truncated peptide that lacks the hydrophobic C-terminal sequence of the BMAP-27 parent molecule, an alteration that confers reduced toxicity to mammalian cells.

Methodology/Principal Findings

BMAP-18 showed strong growth inhibitory activity against several species and life cycle stages of African trypanosomes, fish trypanosomes and Leishmania parasites in vitro. When compared to native BMAP-27, the truncated BMAP-18 peptide showed reduced cytotoxicity on a wide variety of mammalian and insect cells and on Sodalis glossindius, a bacterial symbiont of the tsetse vector. The fluorescent stain rhodamine 123 was used in immunofluorescence microscopy and flow cytometry experiments to show that BMAP-18 at low concentrations rapidly disrupted mitochondrial potential without obvious alteration of parasite plasma membranes, thus inducing death by apoptosis. Scanning electron microscopy revealed that higher concentrations of BMAP-18 induced membrane lesions in the parasites as early as 15 minutes after exposure, thus killing them by necrosis. In addition to direct killing of parasites, BMAP-18 was shown to inhibit LPS-induced secretion of tumour necrosis factor alpha (TNF-α), a cytokine that is associated with inflammation and cachexia (wasting) in sleeping sickness patients. As a prelude to in vivo applications, high affinity antibodies to BMAP-18 were produced in rabbits and used in immuno-mass spectrometry assays to detect the intact peptide in human blood and plasma.

Conclusions/Significance

BMAP-18, a truncated form of the potent antimicrobial BMAP-27, showed low toxicity to mammalian cells, insect cells and the tsetse bacterial symbiont Sodalis glossinidius while retaining an ability to kill a variety of species and life cycle stages of pathogenic kinetoplastid parasites in vitro. BMAP-18 also inhibited secretion of TNF-α, an inflammatory cytokine that plays a role in the cachexia associated with African sleeping sickness. These findings support the idea that BMAP-18 should be explored as a candidate for therapy of economically important trypanosome-infected hosts, such as cattle, fish and humans, and for paratransgenic expression in Sodalis glossinidius, a bacterial symbiont in the tsetse vector, as a strategy for interference with trypanosome transmission.     

Primary Gut Symbiont and Secondary,Sodalis-Allied Symbiont of the Scutellerid Stinkbug Cantao ocellatus

Symbiotic associations with midgut bacteria have been commonly found in diverse phytophagous heteropteran groups, where microbiological characterization of the symbiotic bacteria has been restricted to the stinkbug families Acanthosomatidae, Plataspidae, Pentatomidae, Alydidae, and Pyrrhocoridae. Here we investigated the midgut bacterial symbiont of Cantao ocellatus, a stinkbug of the family Scutelleridae. A specific gammaproteobacterium was consistently identified from the insects of different geographic origins. The bacterium was detected in all 116 insects collected from 9 natural host populations. Phylogenetic analyses revealed that the bacterium constitutes a distinct lineage in the Gammaproteobacteria, not closely related to gut symbionts of other stinkbugs. Diagnostic PCR and in situ hybridization demonstrated that the bacterium is extracellularly located in the midgut 4th section with crypts. Electron microscopy of the crypts revealed a peculiar histological configuration at the host-symbiont interface. Egg sterilization experiments confirmed that the bacterium is vertically transmitted to stinkbug nymphs via egg surface contamination. In addition to the gut symbiont, some individuals of C. ocellatus harbored another bacterial symbiont in their gonads, which was closely related to Sodalis glossinidius, the secondary endosymbiont of tsetse flies. Biological aspects of the primary gut symbiont and the secondary Sodalis-allied symbiont are discussed.Insects are among the largest animal groups on the earth, embracing 750,000 to several millions of species (37, 52). Diverse insects are symbiotically associated with microorganisms, especially bacteria (5-7). In some insects, symbiotic bacteria are harbored in specialized host cells called bacteriocytes (or mycetocytes), constituting obligate mutualistic associations. For example, Buchnera aphidicola is harbored within bacteriocytes in the abdominal body cavity of almost all aphids and provides essential amino acids that are lacking in the phloem sap diet of the insects (9, 47). Wigglesworthia glossinidia is localized in a midgut-associated bacteriome of tsetse flies and plays pivotal roles in biosynthesis of B vitamins that are deficient in the vertebrate blood diet of the insects (2, 34). These obligate endocellular symbionts are often collectively referred to as “primary symbionts.”In contrast, there are facultative endosymbiotic microorganisms not essential for their host insects, often collectively called “secondary symbionts.” For example, many aphids are known to harbor various facultative symbionts, which belong to distinct lineages in the Gamma- and Alphaproteobacteria (33, 43) and the Mollicutes (10). While the majority of those facultative bacteria are either parasitic or commensalistic for their hosts, some of them affect the host fitness beneficially in particular ecological contexts (29, 32, 36, 44, 51). In addition to the obligate primary symbiont Wigglesworthia, tsetse flies harbor the facultative secondary symbiont Sodalis glossinidius, whose biological function for the hosts is currently elusive (3, 8).Members of the suborder Heteroptera, known as true bugs and consisting of over 38,000 described species, are characterized by their sucking mouthparts, half-membranous forewings, and incomplete metamorphosis (46). In the Heteroptera, symbiotic associations with bacteria are mainly found in phytophagous groups, especially in stinkbugs of the infraorder Pentatomomorpha. These stinkbugs generally possess many sacs or tubular outgrowths, called crypts or ceca, in a posterior region of the midgut, whose lumen is densely populated by a specific bacterial symbiont (7, 16). In some cases, experimental elimination of the symbiotic bacteria resulted in retarded growth and high mortality of the host insects (1, 13, 21, 26, 27, 39), indicating that these gut symbionts play important biological roles. Most of the gut symbionts are vertically transmitted through host generations by such mechanisms as egg surface contamination in the families Pentatomidae and Acanthosomatidae (1, 27, 39, 40, 42), coprophagy in the Cydnidae and Coreidae (22, 45), and capsule transmission in the Plataspidae (20), whereas a case of environmental acquisition has been reported from the Alydidae (26). Thus far, gut symbiotic bacteria of some members of the Acanthosomatidae, Plataspidae, Pentatomidae, Alydidae, and Pyrrhocoridae have been characterized using molecular techniques (21, 23, 25, 27, 38), while phylogenetic and biological aspects of gut symbiotic bacteria have been untouched in many other stinkbug groups.These gut symbiotic bacteria are, despite their extracellular localization, regarded as “primary symbionts” of the stinkbugs. On the other hand, some stinkbugs may, in addition to the gut symbiotic bacteria, also be associated with facultative “secondary symbionts.” For example, Wolbachia infections have been detected from diverse stinkbugs, most of which are probably of parasitic or commensalistic nature (24). Besides Wolbachia, there has been no report on facultative, secondary symbionts from stinkbugs.Members of the family Scutelleridae, often referred to as jewel bugs or shield-backed bugs, are stinkbugs characterized by their greatly enlarged convex scutellum that usually covers the entire abdomen. Some tropical species are also known for their vivid and beautiful body coloration (46). The family contains approximately 80 genera and 450 species, and in Japan, at least 7 genera and 9 species have been recorded (50). In the early 20th century, the presence of symbiotic bacteria was histologically described in midgut crypts of several scutellerid species (16, 31, 42). Since these pioneer works, however, no studies have been conducted on the symbiotic bacteria of scutellerid stinkbugs.Here we investigated the midgut symbiont of Cantao ocellatus, a scutellerid stinkbug widely distributed in Asian countries, including Japan, and known to guard their eggs and newborn nymphs (Fig. ​(Fig.1A)1A) (50). In addition to the gut symbiont, we also identified a Sodalis-allied facultative secondary symbiont from gonads of the insect.Open in a separate windowFIG. 1.(A) Adult female of Cantao ocellatus, guarding hatchlings under her body. (B) Dissected midgut from an adult female of C. ocellatus. 1st, midgut 1st section; 2nd, midgut 2nd section; 3rd, midgut 3rd section; 4th, midgut 4th section with crypts; hg, hindgut. (C) Enlarged image of the midgut 4th section with crypts. Arrowheads indicate three rows of crypts, while a fourth row is hidden behind. Glandular crypts (gc) are developed in adult females specifically, which may be involved in egg surface contamination with the symbiont. (D) An in situ hybridization image of the midgut 4th section, in which red and green signals indicate the gut symbiont and the host nucleus, respectively. Each arrow shows a crypt. (E) An enlarged image of the symbiotic bacteria in the crypts.     

Insecticidal Toxic Proteins Produced by Photorhabdus luminescens akhurstii,a Symbiont of Heterorhabditis indica

We describe the isolation and characterization of an insect pathogenic bacterium from the entomopathogenic nematode Heterorhabditis indica (Karnataka strain), an isolate from the southern regions of India. The strain has been identified and characterized by phenotypic, biochemical tests and PCR-RFLP analysis of the 16S rRNA gene as Photorhabdus luminescens subsp. akhurstii. The insecticidal toxin complex produced by this bacterium has been purified through a series of steps including ultrafiltration, anion exchange chromatography, and gel filtration chromatography. The toxin consists of two protein complexes of approximately 1,000 kD and was active against the larvae of Spodoptera litura and Galleria mellonella.     

The Ejectisomes of the Flagellate Chilomonas paramecium: Visualization by Freeze-Fracture and Isolation Techniques1

Freeze-fracture procedures were used to visualize ejectisomes and adjacent plasma membrane specializations in the flagellate protozoan Chilomonas paramecium. The ejectisomes are membrane-bounded, cylindrically rolled, extrusive organelles. Small ones occur in large number beneath the plasma membrane of the body and considerably larger ones are located around the gullet membrane. The intra-membrane particle distribution is different in each type. In small ejectisomes, the portion of the membrane in contact with the plasma membrane of the body has a P-face rosette of five particles while the plasma membrane has not been observed with a rosette. Small ejectisomes and plasma membrane both contain aggregations of particles a short distance from the contact or docking site. Slightly beneath the plasma membrane is the periplastic sheet with which we speculate the small ejectisomes interact during the docking phenomenon. No obvious rosettes have been observed in large ejectisomes. Some other ejectisomal structures are presented and discussed.     

Balantidium prionurium n. sp., Symbiont in the Intestine of the Surgeonfish,Prionurus punctatus1

During the spring of 1984, the ciliate Balantidium prionurium n. sp. was collected from the intestinal lumen of the herbivorous surgeonfish, Prionurus punctatus, from the Gulf of California. The symbionts were found in five fish from two well separated collection sites. Morphostatic specimens average 51 μm x 42 μm, and thus fall within the size range of several other fish balantidia. But the presence of Balantidium in a saltwater fish has not been reported, and such a host difference alone supports at least provisional recognition of this organism as a new species.     

Axenic Cultivation and Ultrastructural Study of a Phytomonas sp. Isolated from the Milkweed Plant Euphorbia hyssopifolia1

ABSTRACT. A large number of trypanosomatids was seen in the latex of the milkweed plant Euphorbia hyssopifolia found in Rio de Janeiro, Brazil. The parasites, which belong to the genus Phytomonas, were isolated and axenically cultivated in a biphasic culture medium. The form and the structural organization of the parasites as found in the intact plant, in the isolated latex, and in the culture medium was studied by scanning and transmission electron microscopy.