Filamentous proteophosphoglycan secreted by Leishmania promastigotes forms gel-like three-dimensional networks that obstruct the digestive tract of infected sandfly vectors

Development of Leishmania parasites in the digestive tract of their sandfly vectors involves several morphological transformations from the intracellular mammalian amastigote via a succession of free and gut wall-attached promastigote stages to the infective metacyclic promastigotes. At the foregut midgut transition of Leishmania-infected sandflies a gel-like plug of unknown origin and composition is formed, which contains high numbers of parasites, that occludes the gut lumen and which may be responsible for the often observed inability of infected sandflies to draw blood. This "blocked fly" phenotype has been linked to efficient transmission of infectious metacyclic promastigotes from the vector to the mammalian host. We show by immunofluorescence and immunoelectron microscopy on two Leishmania/sandfly vector combinations (Leishmania mexicana/Lutzomyia longipalpis and L. major/Phlebotomus papatasi) that the gel-like mass is formed mainly by a parasite-derived mucin-like filamentous proteophosphoglycan (fPPG) whereas the Leishmania polymeric secreted acid phosphatase (SAP) is not a major component of this plug. fPPG forms a dense three-dimensional network of filaments which engulf the promastigote cell bodies in a gel-like mass. We propose that the continuous secretion of fPPG by promastigotes in the sandfly gut, that causes plug formation, is an important factor for the efficient transmission to the mammalian host.     

New insights into the developmental biology and transmission mechanisms of Leishmania

Leishmania alternates between two main morphological forms in its life cycle: intracellular amastigotes in the mammalian host and motile promastigotes in the sandfly vector. Several different forms of promastigote can be recognised in sandfly infections. The first promastigote forms, which are found in the sandfly in the bloodmeal phase, are multiplicative procyclic promastigotes. These differentiate into nectomonad promastigotes, which are a non-dividing migratory stage moving from the posterior to the anterior midgut. When nectomonad promastigotes arrive at the anterior midgut they differentiate into leptomonad forms, a newly named life cycle stage, which resume replication. Leptomonad promastigotes, which are found in the anterior midgut, are the developmental precursors of the metacyclic promastigotes, the mammal-infective stages. Leptomonad forms also produce promastigote secretory gel, a substance that plays a key role in transmission by forming a physical obstruction in the gut, forcing the sandfly to regurgitate metacyclic promastigotes during bloodfeeding.     

Relationship between digestive enzymes and food habit of Lutzomyia longipalpis (Diptera: Psychodidae) larvae: Characterization of carbohydrases and digestion of microorganisms

The sandfly Lutzomyia longipalpis (Lutz and Neiva, 1912) is the main vector of American Visceral Leishmaniasis. In spite of its medical importance and several studies concerning adult digestive physiology, biochemistry and molecular biology, very few studies have been carried out to elucidate the digestion in sandfly larvae. Even the breeding sites and food sources of these animals in the field are largely uncharacterized. In this paper, we describe and characterize several carbohydrases from the gut of L. longipalpis larvae, and show that they are probably not acquired from food. The enzyme profile of this insect is consistent with the digestion of fungal and bacterial cells, which were proved to be ingested by larvae under laboratory conditions. In this respect, sandfly larvae might have a detritivore habit in nature, being able to exploit microorganisms usually encountered in the detritus as a food source.     

Transmission and scanning EM-immunogold labeling of Leishmania major lipophosphoglycan in the sandfly Phlebotomus papatasi

Previous studies using immunostaining and light microscopy demonstrated expression of Leishmania major lipophosphoglycan (LPG) on parasites developing in the sandfly gut from 2 days post infection. By days 4 to 7 post infection, there appeared to be large amounts of parasite-free LPG deposited on/in the microvilli and epithelial cells lining the thoracic midgut, while forward migration of parasites and the morphological changes which accompany metacyclogenesis were associated with developmental modification of the LPG molecules. Studies presented here examine this process with much greater precision using electron microscopy and immunogold labeling techniques to study the different developmental forms (nectomonads, haptomonads, paramastigotes, and metacyclics) of promastigotes in the sandfly gut. Results obtained using LPG-specific monoclonal antibodies (WIC79.3, 45D3 and the metacyclic-specific 3F12) show (1) gold labeling over the cell surface, within the flagellar pocket, and extending along the entire length of the flagellum of electron-dense nectomonads observed in the abdominal and thoracic midgut regions on days 4 and 7 post infection, and of electron-lucid haptomonads in the foregut, (2) dense labeling around the flagellar tips, by which nectomonad forms bind to the midgut microvilli, but not on the microvilli themselves or within the epithelial cells lining the midgut, (3) significant metacyclic-specific (3F12) labeling on nectomonad forms in the lumen of the midgut and attached to the microvilli, and (4) dense labeling on the cell surface of electron-lucid paramastigotes in the esophagus and in the filamentous matrix surrounding paramastigote and metacyclic forms in the esophagus and pharynx. These results are discussed in the light of the proposed roles for LPG in parasite attachment to, and survival in, the sandfly gut.     

Post-engorgement dynamics of haemagglutination activity in the midgut of phlebotomine sandflies

Abstract. Haemagglutination activity (HA) was studied in gut extracts of both sexes of adults of six sandfly species. HA was sex dependent, with the activity in males more than 50 times lower man that of unfed females. In females, high HA was demonstrable in both the thoracic and abdominal midgut but not in the hindgut. In unfed flies the activity was similar in the midgut wall and the gut contents whereas, in fed females, a high increase was seen in the midgut contents. After blood-feeding, HA was elevated, reaching peak titres 2 days later and then falling to the base level or less immediately after defaecation. The magnitude of the HA response differed according to species, ranging from 2-fold in Lutzomyia carmelinoi up to 16-fold in Phlebotomus duboscqi. Quantitative differences between sandflies in their HA response may influence their ability to support the development of Leishmania spp.     

Regeneration of radiation-damaged digestive tissues in juvenile Pacific oysters (Crassostrea gigas)

Juvenile Pacific oysters, Crassostrea gigas, were irradiated with 16 and 40 krad and their tissues examined histologically. Degenerative syndromes and tissue regeneration processes were determined for the stomach, gut, collecting ducts, and digestive tubules. Following degeneration, tissue regeneration was observed in the digestive tissues of most oysters exposed to 16 krad and in a limited number exposed to 40 krad. Regeneration was first observed in the digestive tubules and subsequently in the stomach, gut, and collecting ducts. Cellular repopulation of the digestive tubules involved epithelialization with large, undifferentiated crypt cells which then differentiated into functional secretory and absorptive cells. Regeneration in the stomach, gut, and collecting ducts was initiated by proliferative islands of small basophilic cells. Mitotic division of those cells and their subsequent differentiation into functional epithelial cells resulted in the rapid restoration and apparent recovery of the affected tissues. The results of these studies indicate that radioresistance of juvenile C. gigas may in part be due to the remarkably efficient regenerative mechanisms involved in replacing injured or lost digestive tissues.     

Sugars in the alimentary canal of Lutzomyia youngi (Diptera: Phlebotominae)

Abstract. Gas chromatography (GC) analysis of 159 specimens (144 females and 15 males) of Lutzomyia youngi collected in Shannon traps in a coffee plantation in the Andean region of western Venezuela, where leishmaniasis is endemic, revealed the presence of fructose, sucrose, glucose and maltose in the gut and crop of the wild sandflies. The identification of the sugars was confirmed by comparing retention times with those observed for standard sugars and those obtained from sandflies experimentally fed on known sugar solutions. Although the sandflies in nature may ingest each of the four sugars, the results suggest that it is more probable there is an invertase enzyme (glycosidase?) in the gut or crop of the sandfly which hydrolyses ingested disaccharides (e.g. sucrose) to the constituent monosaccharides (i.e. fructose and glucose). Ecological and altitudinal distributions of sandfly species may be related to the availability of specific sugar sources, with epidemiological implications. Identification of the preferred sugar could make breeding easier and would facilitate further research on Leishmania–wector relationships.     

The human pathogen Leishmania donovani secretes a histidine acid phosphatase activity that is resistant to proteolytic degradation

Promastigotes of all pathogenic Leishmania species secrete acid phosphatase (SAcP) activity during their growth in vitro. It has been suggested that this enzyme may play a role in the survival of the parasite within its sandfly-vector host. To carry out such functions, SAcP would have to be relatively resistant to endogenous sandfly gut-proteases. Therefore, the current study was undertaken to ascertain whether L. donovani SAcP activity was affected by treatment with various proteases. Native L. donovani SAcP was treated with a variety of serine-, thiol-, metallo- and mixed-proteases and subsequently assayed for enzymatic activity. Of the eleven proteases tested, only bromelain and subtilisin treatments caused a pronounced reduction in SAcP activity. Treatment of SAcP with seven out of the remaining nine proteases, resulted in an overall enhancement in SAcP enzymatic activity ranging from approximately 10% (e.g. with trypsin) to > or = 90% (e.g. with ficin). The resistance of the Leishmania SAcP to various proteases may prolong its functional life within the sandfly gut and help to facilitate parasite infection in this host.     

Dual origin of gut proteases in Formosan subterranean termites (Coptotermes formosanus Shiraki) (Isoptera: Rhinotermitidae)

Cellulose digestion in lower termites, mediated by carbohydrases originating from both termite and endosymbionts, is well characterized. In contrast, limited information exists on gut proteases of lower termites, their origins and roles in termite nutrition. The objective of this study was to characterize gut proteases of the Formosan subterranean termite (Coptotermes formosanus Shiraki) (Isoptera: Rhinotermitidae). The protease activity of extracts from gut tissues (fore-, mid- and hindgut) and protozoa isolated from hindguts of termite workers was quantified using hide powder azure as a substrate and further characterized by zymography with gelatin SDS-PAGE. Midgut extracts showed the highest protease activity followed by the protozoa extracts. High level of protease activity was also detected in protozoa culture supernatants after 24 h incubation. Incubation of gut and protozoa extracts with class-specific protease inhibitors revealed that most of the proteases were serine proteases. All proteolytic bands identified after gelatin SDS-PAGE were also inhibited by serine protease inhibitors. Finally, incubation with chromogenic substrates indicated that extracts from fore- and hindgut tissues possessed proteases with almost exclusively trypsin-like activity while both midgut and protozoa extracts possessed proteases with trypsin-like and subtilisin/chymotrypsin-like activities. However, protozoa proteases were distinct from midgut proteases (with different molecular mass). Our results suggest that the Formosan subterranean termite not only produces endogenous proteases in its gut tissues, but also possesses proteases originating from its protozoan symbionts.     

Cellulase Activity of Leishmania major in the Sandfly Vector and in Culture

ABSTRACT. The secretion of cellulose-degrading enzymes by Leishmania promastigotes in culture and in the sandfly vector was demonstrated. Two types of activity of cellulase enzyme-complexes were measured: endoglucanases, which randomly cleave cellulose chains and cellobioydrolases, which remove cellobiose from the nonreducing end of the molecule. The assays demonstrated that enzymes with these activities were secreted into the culture medium by Leishmania major, L. donovani , and L. braziliensis . These activities were also found in cultures of Sauroleishmania agamae, Leptomonas seymouri, Herpetomonas muscarum, Crithidia fasciculata and Trypanosoma brucei brucei that had a relatively low endoglucanase activity. Both endoglucanase and cellobiohydrolase activities were found in the gut of L. major-infected Phlebotomus papatasi , while gut preparations of uninfected sandflies had only cellobiohydrolase activity. The similar growth of L. major parasites in medium supplemented with either cellulose or glucose suggests these parasites can utilize cellulose.     

Proliferation and distribution of cells that transiently express a catecholaminergic phenotype during development in mice and rats

Cells that transiently express a catecholaminergic phenotype have previously been shown to appear in the rat gut during development. In the present study the immunocytochemical demonstration of the enzymes, tyrosine hydroxylase (TH) and dopamine-β-hydroxylase (DBH), were used as markers to examine tissues of rats and mice for catecholaminergic cells. The simultaneous radioautographic demonstration of labeling of identified catecholaminergic cells by tritiated thymidine was used to assess their ability to proliferate. Transient catecholaminergic cells were not limited to rat gut. They were also found in the gut of the mouse where they were present by 10 days' gestation and disappeared before Day 13. Similar cells were found in the mouse kidney, the mantle layer of the sacral spinal cord, and the dorsal mesentery. In mice, transient catecholaminergic cells contained TH but did not react with antiserum to DBH. Transient catecholaminergic cells in the rat gut and other locations synthesized DNA. We conclude that transient catecholaminergic cells (1) occur in both rat and mouse embryos, although the cells of mice may not contain DBH; (2) appear in other organs as well as the gut; (3) are able to proliferate. The ultimate fate of these cells remains to be demonstrated.     

Rhesus monkey model for Leishmania major transmitted by Phlebotomus papatasi sandfly bites

Leishmaniasis research needs a near-human model for investigations of natural infection processes, immunological responses and evaluation of treatments. Therefore, we developed a reproducible system using Leishmania major Yakimoff & Schokhor (Trypanosomatidae: Kinetoplastida), the cause of Old World zoonotic cutaneous leishmaniasis (ZCL), transmitted to rhesus monkeys Macaca mulatta (Zimmerman) (Primates: Cercopithecidae) by sandfly bites of experimentally infected Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae). Eight monkeys of presumed Indian origin (Leishmania naive) were exposed to bites of female sandflies that had been infected with L. major by membrane-feeding on human blood seeded with amastigotes isolated from hamster footpad lesions. Infection rates of membrane-fed sandflies averaged > 85% seven days after the infective feed, with uniformly high numbers of promastigotes in the stomodaeal valve region of the sandfly gut. Nodules and ulcerating dermal lesions developed on 7/8 monkeys 2-4 weeks post-bite and persisted for 3-7 months. Monkeys also developed satellite lesions beyond the area of sandfly bites on the head, but not on the chest. Three re-challenged monkeys developed lesions that healed faster than lesions from their primary challenges. After infection, monkeys developed delayed type hypersensitivity (DTH) responses to a panel of Leishmania skin test antigens (LSTA) and, when tested by ELISA and IFA, showed significant post-infection antibody titres which typically rose for approximately 170 days and then gradually receded during the next 100 days following the first challenge. After the second challenge, antibody titres spiked higher within approximately 50 days and receded more rapidly. In contrast, four rhesus macaques of Chinese origin developed no lesions following infected sandfly bites, although they raised antibodies and LSTA reactions, indicating subclinical infection.     

Chitinase secreted by Leishmania functions in the sandfly vector.

Leishmania major parasites ingested with host blood by the sandfly Phlebotomus papatasi multiply confined within the peritrophic membrane. This membrane consists of a chitin framework and a protein carbohydrate matrix and it is secreted around the food by the insect midgut. Histological sections of infected flies show lysis of the chitin layer in the anterior region of the peritrophic membrane that permits the essential forward migration of a concentrated mass of parasites. Both the location and the nature of this disintegration are specific to infected flies. At a later stage the parasites concentrate in the cardiac valve region and subsequently this segment of the fore gut loses its cuticular lining. We have found that chitinase and N-acetylglucosaminidase are secreted by cultured L. major promastigotes, but not by sandfly guts. Hence lysis of the chitin layer of the peritrophic membrane could be catalysed by these enzymes of the parasites. Activity of both enzymes was also observed in other trypanosomatids, including L. donovani, L. infantum, L. braziliensis, Leptomonas seymouri, Crithidia fasciculata and Trypanosoma lewisi.     

Gut content fossilization and evidence for detritus feeding habits in an enrolled trilobite from the Cambrian of China

Enrolment was a major defensive strategy for trilobites that significantly contributed to their evolutionary success. The ability to enrol also helped to constrain the morphological evolution of trilobites, which in part was driven by the need to improve this capability to encapsulate the soft parts of the body within the mineralized dorsal exoskeleton. Here, we describe a unique example of gut content fossilization in an enrolled trilobite from the Cambrian of China, and we propose a taphonomic scenario that considers the possible implication of enrolment in this exceptional preservation. A micro‐facies analysis indicates that the specimen was entombed during an obrution event and remained intact due to limited infaunal activity. The encapsulation of the body did not prevent the decay of soft tissues, but it permitted the delicate gut content to be protected during diagenesis. In isolating the decaying soft tissues from the external environment, enrolment might also have favoured the establishment of microenvironmental conditions conducive to the precipitation of pyrite framboids. Within the gut, the formation of such crystals may have led to the consolidation of the ingested material. These results suggest that fossilized gut contents might be quite common in enrolled trilobites. Textural and compositional analyses reveal that the gut content is similar to the sediment surrounding the fossil except for the presence of pyrite framboids that indicate higher initial organic matter content. The complete enrolment of the body argues against an accidental ingestion of this material or a diagenetic origin for it. Accordingly, detritus feeding habits are inferred for this ptychopariid trilobite.     

Why do larval helminths avoid the gut of intermediate hosts?

In complex life cycles, larval helminths typically migrate from the gut to exploit the tissues of their intermediate hosts. Yet the definitive host's gut is overwhelmingly the most favoured site for adult helminths to release eggs. Vertebrate nematodes with one-host cycles commonly migrate to a site in the host away from the gut before returning to the gut for reproduction; those with complex cycles occupy sites exclusively in the intermediate host's tissues or body spaces, and may or may not show tissue migration before (typically) returning to the gut in the definitive host. We develop models to explain the patterns of exploitation of different host sites, and in particular why larval helminths avoid the intermediate host's gut, and adult helminths favour it. Our models include the survival costs of migration between sites, and maximise fitness (=expected lifetime number of eggs produced by a given helminth propagule) in seeking the optimal strategy (host gut versus host tissue exploitation) under different growth, mortality, transmission and reproductive rates in the gut and tissues (i.e. sites away from the gut). We consider the relative merits of the gut and tissues, and conclude that (i) growth rates are likely to be higher in the tissues, (ii) mortality rates possibly higher in the gut (despite the immunological inertness of the gut lumen), and (iii) that there are very high benefits to egg release in the gut. The models show that these growth and mortality relativities would account for the common life history pattern of avoidance of the intermediate host's gut because the tissues offer a higher growth rate/mortality rate ratio (discounted by the costs of migration), and make a number of testable predictions. Though nematode larvae in paratenic hosts usually migrate to the tissues, unlike larvae in intermediates, they sometimes remain in the gut, which is predicted since in paratenics mortality rate and migration costs alone determine the site to be exploited.     

Sandfly maxadilan exacerbates infection with Leishmania major and vaccinating against it protects against L. major infection.

Bloodfeeding arthropods transmit many of the world's most serious infectious diseases. Leishmania are transmitted to their mammalian hosts when an infected sandfly probes in the skin for a bloodmeal and injects the parasite mixed with its saliva. Arthropod saliva contains molecules that affect blood flow and modulate the immune response of the host. Indeed, sandfly saliva markedly enhances the infectivity of L. major for its host. If the salivary molecule(s) responsible for this phenomenon was identified, it might be possible to vaccinate the host against this molecule and thereby protect the host against infection with Leishmania. Such an approach represents a novel means of controlling arthropod-borne disease transmission. Here, we report that a single molecule, maxadilan, in sandfly saliva can exacerbate infection with L. major to the same degree as whole saliva, and that vaccinating against maxadilan protects mice against infection with L. major.     

Digestive chitinolytic activity in marine fishes of Monterey Bay, California

Chitinolytic activities, both chitinase (EC 3.2.1.14) and minimum chitobiase (beta-N-acetyl-D-glucosaminidase; EC 3.2.1.30), were measured in stomach and intestinal tissues and their contents, from 13 fish species. Higher activities were found in the tissues than in the gut contents, and higher activities were seen in the stomachs than in the intestines. Demersal species exhibited chitobiase activities very close to their chitinase activities, suggesting that these fishes can degrade chitin completely to its soluble, absorbable monomer, N-acetyl-glucosamine. This suggests that these species may catabolize chitin not just to penetrate prey exoskeletons but also to derive nutrients from the chitin itself. In contrast, three mesopelagic species exhibited low chitobiase but high chitinase activities. This chitobiase limitation correlated strongly with gastrointestinal tract morphology, with the myctophids having the greatest chitobiase limitation and the shortest alimentary tracts. The high chitinase activities measured in the myctophids reflect their ability to rapidly disrupt prey exoskeletons ingested during their nightly feeding in surface waters. Their chitobiase activities are greatly reduced because with rapid meal evacuation through a short gut there is little time for processing and limited energetic advantage in the complete degradation of chitin. These results suggest multiple roles for chitinolytic enzymes in marine fishes and that feeding habits and frequency may have a bearing on the evolution of their digestive enzymes systems.     

Uric Acid-Degrading Bacteria in Guts of Termites [Reticulitermes flavipes (Kollar)

Uricolytic bacteria were present in guts of Reticulitermes flavipes in populations up to 6 x 10 cells per gut. Of 82 strains isolated under strict anaerobic conditions, most were group N Streptococcus sp., Bacteroides termitidis, and Citrobacter sp. All isolates used uric acid (UA) as an energy source anaerobically, but not aerobically, and NH(3) was the major nitrogenous product of uricolysis. However, none of the isolates had an absolute requirement for UA. Utilization of heterocyclic compounds other than UA was limited. Fresh termite gut contents also degraded UA anaerobically, as measured by CO(2) evolution from [2-C]UA. The magnitude of anaerobic uricolysis [0.67 pmol of UA catabolized/(gut x h)] was entirely consistent with the population density of uricolytic bacteria in situ. Uricolytic gut bacteria may convert UA in situ to products usable by termites for carbon, nitrogen, energy, or all three. This possibility is consistent with the fact that R. flavipes termites from UA, but they do not void the purine in excreta despite the lack of uricase in their tissues.     

Sandfly pheromones. Their biology and potential for use in control programs

Lutzomyia longipalpis (Diptera: Psychodidae) is the vector of Leishmania chagasi the causative agent of visceral leishmaniasis (VL) in South and Central America, particularly Brazil, where the greatest incidence occurs. The disease is fatal if untreated. Although huge efforts have been made to control VL the incidence is increasing. Vector control remains an important element of disease control but residual spraying and other strategies have failed to make any lasting impact. Manipulation of sandfly chemical communication offers the opportunity to add new techniques and tools to reduce sandfly populations and thereby reduce Leishmania transmission. This paper reports the current understanding of several areas of sandfly chemical ecology and their prospects for application.     

Identification of plasmalogen in the gut of silkworm (Bombyx mori)

Herbivorous insect species are constantly challenged with endogenous and exogenous oxidative stress. Consequently, they possess an array of antioxidant enzymes and small molecular weight antioxidants. Lipid-soluble small molecular antioxidants, such as tocopherols, have not been well studied in insects but may play important antioxidant roles. In this study, we identified plasmalogen phosphatidylethanolamines (pPEs) as well as α-, β/γ-, δ-tocopherol in the larvae of the silkworm Bombyx mori by LCMS analyses and examined their distribution. Plasmalogen are reported to inhibit the metal ion induced oxidation. The composition of tocopherols was the same among gut contents, gut tissues, and the other tissues. However, plasmalogens, a unique class of glycerophospholipids rich in polyunsaturated fatty acids and containing a vinyl ether bond at the sn-1 position, were mainly distributed in gut tissues. Plasmalogens might protect gut tissues from oxidation stress.