Induction of actin gene expression in the mosquito midgut by blood ingestion correlates with striking changes of cell shape

Ingestion of a blood meal by the female mosquito Anopheles gambiae (L., Diptera: Culicidae), results in a dramatic distention of the midgut epithelium. Here, we report that these events correlate with a transient increase of actin mRNA and protein abundance. The newly synthesized actin may provide a pool of actin protein needed to remodel epithelial cell cytoarchitecture. We also document changes in midgut epithelial cell morphology. Upon blood ingestion, the columnar cells flatten accompanied by the loss of microvilli on the lumenal side and the unfolding of the labyrinth on the basal side. These changes correlate with the large increase of epithelial surface area needed to accommodate the blood meal. Actin gene expression, actin synthesis and cell morphology all return to the pre-feeding state by 24 h after blood intake.     

Infection and replication sites of Spiroplasma kunkelii (Class: Mollicutes) in midgut and Malpighian tubules of the leafhopper Dalbulus maidis

Spiroplasma kunkelii distribution and infection mechanisms in the intestines and Malpighian tubules of Dalbulus maidis were investigated by transmission electron microscopy. Spiroplasmas were found between microvilli and in endocytic vesicles of the midgut epithelium. At the basal part, cytoplasmic vesicles contained multiple spiroplasmas with tube-like extensions and spiroplasmas accumulated between the laminae rara and densa of the basal lamina. Tip structures of flask-shaped spiroplasmas pierced the lamina densa that was discontinuous in close proximity to spiroplasmas. Spiroplasmas were found in hemolymph, crossed the basal lamina of Malpighian tubule epithelium and accumulated at high numbers in muscle cells that had cytopathogenic changes. S. kunkelii had perithrochous approximately 8nm diameter structures determined to be fimbriae protruding from the cell surface, and similar structures were adhering to the basal lamina of midgut epithelium and to external lamina of muscle cells. Further, spiroplasmas had pili-like appendages at one or both cell poles and appeared to conjugate. This is the first time that fimbriae and pili have been observed in a mollicutes.     

Host age and pathogen dosage impact cyst morphogenesis in the invertebrate pathogenic alga Helicosporidium sp. (Chlorophyta: Trebouxiophyceae)

Helicosporidium sp. is a pathogenic alga that replicates in the hemolymph of various invertebrate hosts. Morphogenesis of the infectious life stage, the cyst, occurs in the infected host, but to date cannot be induced in vitro. Using larvae of the heterologous host Helicoverpa zea, we examined potential factors influencing pathogenicity and in vivo cyst production of the alga and the impact of infection on host survival. Factors tested were cyst dosage administered per os (ranging from 10² to 10⁵ cysts per larva) and host age at exposure (third, fourth, and fifth larval instar). Cyst production occurred between 7 and 13 days after treatment, regardless of host age at treatment. Increasing dosage increased both percent infection and mortality, but cyst production did not track the total infection response. Increasing host age at exposure mitigated dosage effects on infection and mortality and also elevated cyst production in later-treated larvae. Only the highest dosage produced a significant decrease in the overall time to death. Moderate cyst dosages and later host ages were most effective at regenerating Helicosporidium cysts.     

Interaction of Leptomonas wallacei with the intestinal tract of its natural host Oncopeltus fasciatus (Hemiptera: Lygaeidae)

While investigating the distribution of Leptomonas wallacei in the intestine of the insect host Oncopeltus fasciatus, promastigotes and cyst-like forms of L. wallacei were observed only in the midgut ventricles V(3) and V(4) and the hindgut. In video-microscopy, once contact had occurred, the parasites remained attached to the midgut epithelium. Scanning electron microscopy revealed the adhesion of flagellates and cyst-like forms to the midgut wall and to the rectal pads of the hindgut. Using transmission electron microscopy, we observed that adhesion occurred mainly between the flagellum and the perimicrovillar membranes secreted by the midgut epithelium. No modifications were observed either in the parasite or in the epithelial cells. In the hindgut, adhesion to the superficial wax layer of the epithelial cells of the rectal pads was via flagellum. Host cell morphology appeared unaffected by L. wallacei.     

Transmission of the Diachasmimorpha longicaudata rhabdovirus (DlRhV) to wasp offspring: an ultrastructural analysis

During oviposition, the parasitic wasp Diachasmimorpha longicaudata introduces an entomopoxvirus (DlEPV) and a rhabdovirus (DlRhV) into larvae of its tephritid fruit fly host Anastrepha suspensa. DlEPV and DlRhV replicate, respectively, in host hemocytes and epidermal cells. Both viruses, like many beneficial viruses of parasitic wasps, are retained in all wasp generations but their avenue(s) of transmission are unknown. This study tests the hypothesis that DlRhV is transmitted transovarially or through larval feeding on infected host hemolymph. Transmission electron microscopy (TEM) revealed no virions in pre-vitellogenic or vitellogenic ova, or in the lateral oviduct of D. longicaudata females. However, numerous virions occurred in subchorionic regions of 33-36-h-old oviposited eggs. This suggests that DlRhV is introduced into the egg either as (a) intact virions after chorionogenesis but prior to oviposition and/or as (b) unencapsidated RNA molecules, undetectable by TEM in pre-vitellogenic ova, that subsequently replicate and assemble into mature virions. DlRhV particles also occurred in the midgut lumen of 20-24-h-old wasp first instars, suggesting that they were ingested. These virions may have been released from the egg into the hemolymph during hatching or may have come from virions introduced by the female wasp directly into the host, separate from the egg. DlRhV particles were also evident in the intracellular vesicles and intercellular spaces of the larval midgut. Taken together, these data support the hypothesis that DlRhV is transovarially transmitted as virions and/or as unencapsidated RNA. Further studies are needed to determine whether the DlRhV that ultimately resides within the female wasp's accessory gland filaments is the progeny of the virus from the egg and/or larval midgut cells.     

Brain-midgut short neuropeptide F mechanism that inhibits digestive activity of the American cockroach, Periplaneta americana upon starvation

Immunohistochemical reactivity against short neuropeptide F (sNPF) was observed in the brain-corpus cardiacum and midgut paraneurons of the American cockroach, Periplaneta americana. Four weeks of starvation increased the number of sNPF-ir cells in the midgut epithelium but the refeeding decreased the number in 3h. Dramatic rises in sNPF contents in the midgut epithelium and hemolymph of roaches starved for 4 weeks were confirmed by ELISA. Starvation for 4 weeks reduced α-amylase, protease and lipase activities in the midgut of P. americana but refeeding restored these to high levels. Co-incubation of dissected midgut with sNPF at physiological concentrations inhibited α-amylase, protease and lipase activities. sNPF injection into the hemocoel led to a decrease in α-amylase, protease and lipase activities, whereas PBS injection had no effects. The injection of d-(+)-trehalose and l-proline into the hemocoel of decapitated adult male cockroaches that had been starved for 4 weeks had no effect on these digestive enzymes. However, injection into the hemocoel of head-intact starved cockroaches stimulated digestive activity. Injection of d-(+)-trehalose and l-proline into the lumen of decapitated cockroaches that had been starved for 4 weeks increased enzymes activities and suppressed sNPF in the midgut. Our data indicate that sNPF from the midgut paraneurons suppresses α-amylase, protease and lipase activities during starvation. Injection of d-(+)-trehalose/l-proline into the hemocoel of head-intact starved cockroach decreased the hemolymph sNPF content, which suggests that sNPF could be one of the brain factors, demonstrating brain-midgut interplay in the regulation of digestive activities and possibly nutrition-associated behavioral modifications.     

Host hemolymph proteins and protein digestion in larval Habrobracon hebetor (Hymenoptera: braconidae)

Host plasma proteins and protein digestion in larval parasitoids were studied during trophic interactions of the ectoparasitoid Habrobracon hebetor Say (Hymenoptera: Braconidae), with a host, larvae of the Indianmeal moth, Plodia interpunctella Hübner (Lepidoptera: Pyralidae). We could detect no apparent differences in host hemolymph protein patterns up to 72 h after paralysation and/or parasitization by H. hebetor. A 190 kDa putative apolipophorin I present in host hemolymph could not be detected in the midguts of feeding H. hebetor larvae indicating that it is rapidly digested. The major 60 kDa storage proteins (putative hexamerins) in host hemolymph were detected in the parasitoid midgut and were completely digested 24 h after cessation of feeding and the beginning of cocoon formation. Host hemolymph had a pH of about 6.4. The pH optima of the midgut proteinases in the larval parasitoid were in the alkaline region, but midgut fluid in feeding parasitoid larvae was about pH 6. 8. Based on enzyme activity against selected artificial proteinase substrates including azocasein, N-alpha-benzoyl-L-Arg p-nitroanilide (BApNA), succinyl-Ala-Ala-Pro-Phe p-nitroanilide (SAAPFpNA), succinyl-Ala-Ala-Pro-Leu p-nitroanilide (SAAPLpNA), and inhibition by selected proteinase inhibitors, serine proteinases appear to be the predominant class of enzymes involved in protein digestion in the midguts of H. hebetor. There is also an active aminopeptidase (LpNA) associated with the microsomal fraction of midgut preparations. There was no evidence for preoral digestion or ingestion of proteinases from host hemolymph by the parasitoid larva. There was a very active BApNAase in the soluble fraction of midgut extracts. This activity increased on a per midgut basis up to 24 h after the beginning of cocoon formation but decreased rapidly by 48 h. Two major (P1 and P3) and several minor proteinases were detected in midgut extracts of H. hebetor analysed with gelatin zymograms. The apparent molecular mass of P1 varied from 95 to 49 kDa depending on protein loading. P3 had an apparent molecular mass of 39 kDa that was independent of protein loading. In summary, electrophoretic evidence indicates that host hemolymph protein patterns do not change significantly for at least 72 h after paralysation by H. hebetor. The role, if any, of envenomization in preventing breakdown of hemolymph proteins during this time remains to be determined. Because the predominant host hemolymph proteins, a putative apolipophorin I and the putative hexamerins, are readily digested by the serine proteinases present in the midguts of this parasitoid larva, these or similar proteins would provide an easily digested source of dietary amino acids that could be used for development of artificial diets for this beneficial insect.     

Absorption of mulberry root urease to the hemolymph of the silkworm, Bombyx mori

Mulberry leaves are the sole diet of the silkworm, Bombyx mori. The host urease is incorporated into the larval hemolymph and involved in nitrogen metabolism in the insect. To investigate the selective absorption of the host urease to the larvae, crude urease was prepared from mulberry leaves and roots. Root urease was identical to leaf urease on the basis of electrophoretic analyses: (1) the urease activity appeared in the same migration position in a native gel; (2) There was no difference in molecular mass of the subunit. The root urease was orally injected to the fifth instar larvae of the silkworm. Just before spinning, the larvae absorbed intact urease from the midgut lumen to the hemolymph without the loss of activity. The capacity to absorb urease occurred only at the specific stage. Localization of host urease in midgut tissue was observed using confocal laser scanning microscopy and transmission electron microscopy. Based on spatial distribution of immunofluorescent signals and immunogold particles, host urease specifically attached to the surfaces of microvilli existing in the apical side of columnar cells and appeared in the cytoplasm of the cells for transport to the hemolymph. The incorporation efficiency of root urease into the hemolymph was significantly higher than for ureases from jack bean seeds and Bacillus pasteurii. The urease that was transported to the hemolymph was electrophoretically altered, compared with the host urease extracted.     

Sporogonic development of Leucocytozoon smithi.

The sporogonic development of Leucocytozoon smithi in its black fly vector was studied by light and electron microscopy and was compared with that of other haemosporidians. Within 18 to 24 h after ingestion of gametocytes by black flies, ookinetes passing through the midgut epithelium were observed. Intracellular migration of ookinetes resulted in the apparent disruption and degeneration of host cells. Intercellular migration also occurred as was evidenced by the presence of ookinetes between midgut cells. Transformation of ookinete to spherical oocyst occurred extracellularly in three different sites. Although most oocysts were found between the host cell basal membrane and the basal lamina, large numbers also were found attached to the external surface of the basal lamina, projecting into the hemocoel. Ectopic development of oocysts in the midgut epithelium between cells was observed much less frequently than development on the basal side of the midgut. The oocyst wall of dense granules, believed to be of parasite origin, was distinguishable from the basal lamina of the host's midgut epithelium. As in other Leucocytozoidae, the cytoplasm of the oocyst differentiated into a single sporoblastoid from which 30-50 sporozoites were formed. Beginning on the third day post infection, elongation of segregated dense sporoblastoid material associated with pellicle thickening led to the formation of the finger-like sporozoite buds which projected into the oocyst cavity. Sporozoites within mature oocysts and salivary glands were structurally similar to sporozoites as described for other haemosporidians.     

Fate of GFP-expressing Escherichia coli in the midgut and response to ingestion in a tick, Ornithodoros moubata (Acari: Argasidae)

Ticks are well-known vectors of various pathogens but migration of the pathogens in the tick midgut is not fully understood. In the present study, the fate of microbes in the midgut of Ornithodoros moubata was observed using green fluorescent protein (GFP)-expressing Escherichia coli. Fluctuations in the percentage of hemocytes in the hemolymph (Hc) and expression of an antimicrobial peptide, defensin, in the midgut was also investigated. Most E. coli gradually disappeared in the midgut after ingestion fluctuations in Hc coincided with the changes. Expression of defensin was also confirmed and slightly up-regulated after E. coli ingestion. Moreover, it was demonstrated that E. coli can not pass through the tick midgut epithelium after ingestion by the hemolymph cultures. It is known that various pathogens and host immunoglobulins ingested with a blood meal can enter into the hemocoel, which suggests the presence of unique and complex passage mechanisms for each molecule and organism. The results obtained here help to clarify that digestion enzymes is an important function of the tick midgut to protect against invading molecules and organisms.     

The nature of Thelohania solenopsae (Microsporidia) cysts in abdomens of red imported fire ants, Solenopsis invicta

Sixty four percent of Solenopsis invicta workers infected with Thelohania solenopsis contained 1-6 "cysts" ranging from 70 to 260 microm in diameter. Light and electron microscope analyses showed that cysts are hypertrophied adipocytes transformed by the parasites, each cyst presumably forming from a single cell. In the first step of the pathogenesis, Nosema-like spores functioning in autoinfection are produced; a diplokaryotic sequence leading to their formation causes fat body hypertrophy. When meiosis occurs, it switches parasite development to production of octospores and/or megaspores. Adipocytes become 2-4xlarger than normal in conjunction with intensive parasite multiplication and octospore maturation. Infected cells eventually lose their cellular organization and are converted into reservoirs for spores. There were no manifestations of cellular immunity, such as encapsulation or nodule formation. Similarly, there were no signs of specialized host-parasite interaction that might be interpreted as xenoma-like complexes. The role of the cysts in the parasite's life cycle is unclear. They may represent a defensive reaction of the host sacrificing the infected cells to segregate the infection. Alternatively, the cyst may help protect spores from environmental hazards and provide a concentrated infectious dose to aid horizontal transmission of the microsporidium. We propose to refer to hypertrophied adipocytes filled with T. solenospsae spores as "sporocytosacs", not "cysts."     

Viability of dinoflagellate cysts after the passage through the copepod gut

Several dinoflagellate species form nonmotile, thick-walled resting cysts in their life cycle. Cysts can be ingested by planktonic and benthic organisms, but there is scarce information concerning their survival after the passage through the digestive apparatus of the grazers. We tested the germination capability of cysts produced by two neritic dinoflagellates, Scrippsiella trochoidea (F. Stein) A.R. Loeblich and Scrippsiella ramonii Montresor, after their ingestion by four copepod species. Experiments have been carried out with four species: Acartia clausi Giesbrecht, 1889; Centropages typicus Kröyer, 1849; Temora stylifera Dana, 1849; and Clausocalanus lividus Frost and Fleminger, 1968. Copepods were fed either with motile cells or cysts, and feeding and clearance rates were estimated for A. clausi, C. lividus and T. stylifera. Grazing rates on both dinoflagellates was much higher for vegetative cells than for cysts. Resting cysts were isolated from the faecal pellets and incubated to test their germination capability. S. trochoidea cysts eaten by C. typicus and T. stylifera showed a high germination rate, while cysts of the same species were not viable after the passage through the gut of A. clausi and C. lividus. In contrast, S. ramonii cysts were never able to germinate after being ingested by copepods. The observed variation in viability among the two cyst types and the different survival rates observed for S. trochoidea cysts might be related to differences in cyst morphology and to differences in the digestive process among the tested copepod species.     

Morphological and enzymatic analysis of the midgut of Anopheles darlingi during blood digestion

The midgut of adult female Anopheles darlingi is comprised of narrow anterior and dilated posterior regions, with a single layered epithelium composed by cuboidal digestive cells. Densely packed apical microvilli and an intricate basal labyrinth characterize each cell pole. Before blood feeding, apical cytoplasm contains numerous round granules and whorled profiles of rough endoplasmic reticulum. Engorgement causes a great distension of midgut. This provokes the flattening of digestive cells and their nuclei. Simultaneously, apical granules disappear, the whorls of endoplasmic reticulum disassemble and 3h post bloodmeal (PBM), nucleoli enlarge manyfold. An intense absorptive process takes place during the first 24 h PBM, with the formation of large glycogen inclusions, which persist after the end of the digestive process. Endoproteases activities are induced after bloodmeal and attain their maximum values between 10 and 36 h PBM. At least two different aminopeptidases seem to participate in the digestive process, with their maximum activity values at 36 and 48 h PBM, respectively. Coarse electrondense aggregates, possibly debris from digested erythrocytes, begin to appear on the luminal face of the peritrophic membrane from 18 h PBM and persist during all the digestive process, and are excreted at its end. We suggest that these aggregates could contain some kind of insoluble form of haem, in order of neutralize its toxicity.     

The invasion of the midgut of the mosquito Culex (Culex) quinquefasciatus Say, 1823 by the helminth Litomosoides chagasfilhoi Moraes Neto, Lanfredi and De Souza, 1997

The Litomosoides chagasfilhoi helminth was studied as a model for microfilaria invasion of the midgut of Culex quinquefasciatus mosquito, vector of Wuchereria bancrofti helminth, causative agent of the human filariasis. Histology and transmission and scanning electron microscopy were utilized to show the topography of mosquito midgut invasion by the helminth. An analysis of midguts dissected at different time points after a blood meal demonstrated that the microfilariae interacted and crossed the peritrophic matrix and the midgut epithelium of C. quinquefasciatus. The microfilariae invaded preferentially the mosquito abdominal midgut and the invasion process occurred between 2 and 3h after the blood feeding. In some cases, microfilariae caused an opening in the midgut that separated the epithelial cells, while in others cases, the worms caused the detachment of cells from the epithelium. Ultimately, L. chagasfilhoi crossing activity appeared to damage the midgut. It was also observed that the microfilariae lost their sheaths during their passage through the fibrous material of the peritrophic matrix, before they reached the midgut epithelium. Since the exsheathment process is necessary for the continuity of larvae development, it seems that the passage through the peritrophic matrix is an important step for the parasite's life cycle. This experimental model revealed details of the interaction process of helminthes within the vector midgut, contributing to the knowledge of factors involved in the vector competence of C. quinquefasciatus as a vector of filariasis.     

Dual control of midgut trehalase activity by 20-hydroxyecdysone and an inhibitory factor in the bamboo borer Omphisa fuscidentalis Hampson

During larval diapause lasting 9 months from September to May, trehalase activity in the midgut of the bamboo borer Omphisa fuscidentalis Hampson (Lepidoptera: Crambidae) was low from December to April, followed by a fourfold increase in May that remained high during the pupal stage in July. An application of juvenile hormone analog (JHA) produced increases in the ecdysteroid titer, while trehalase activity was increased by both JHA and 20-hydroxyecdysone (20E) injection. The trehalase activity in the midgut of diapausing larvae was doubled by incubating the midgut with 20E for 48h. During diapause as well as after JHA application, expression of two ecdysone receptor isoform genes (EcR-A and EcR-B1) in the midgut increased simultaneously with the increase in hemolymph ecdysteroid titer, followed by an increase in trehalase activity. The hemolymph of diapausing larvae contained a trehalase inhibitor and inhibitory activity was high during diapause. After 20E injection, trehalase inhibition decreased as midgut trehalase activity increased. Taken together, at least two factors may participate in the change in midgut trehalase activity: increase in trehalase activity and decrease in trehalase inhibitor activity, both of which may be induced by 20E.     

Sporogonic Development of Leucocytozoon smithi

The sporogonic development of Leucocytozoon smithi in its black fly vector was studied by light and electron microscopy and was compared with that of other haemosporidians. Within 18 to 24 h after ingestion of gametocytes by black flies, ookinetes passing through the midgut epithelium were observed. Intracellular migration of ookinetes resulted in the apparent disruption and degeneration of host cells. Intercellular migration also occurred as was evidenced by the presence of ookinetes between midgut cells. Transformation of ookinete to spherical oocyst occurred extracellularly in three different sites. Although most oocysts were found between the host cell basal membrane and the basal lamina, large numbers also were found attached to the external surface of the basal lamina, projecting into the hemocoel. Ectopic development of oocysts in the midgut epithelium between cells was observed much less frequently than development on the basal side of the midgut. The oocyst wall of dense granules, believed to be of parasite origin, was distinguishable from the basal lamina of the host's midgut epithelium. As in other Leucocytozoidae, the cytoplasm of the oocyst differentiated into a single sporoblastoid from which 30–50 sporozoites were formed. Beginning on the third day post infection, elongation of segregated dense sporoblastoid material associated with pellicle thickening led to the formation of the finger-like sporozoite buds which projected into the oocyst cavity. Sporozoites within mature oocysts and salivary glands were structurally similar to sporozoites as described for other haemosporidians.     

Cystoisospora belli: in vitro multiplication in mammalian cells

Intracellular development of Cystoisospora belli was demonstrated in 4 different mammalian cell lines. Human ileocecal adenocarcinoma (HCT-8), epithelial carcinoma of lung (A549), Madin-Darby bovine kidney (MDBK), and African green monkey kidney (VERO) were exposed in vitro to C. belli sporozoites, which had been isolated from the feces of HIV-AIDS patients. Parasites invaded all the cellular types between 4 and 12h after exposure and multiplication was demonstrated after 24 h. Grater number of merozoites formed in VERO cells, followed by HCT-8. In the MDBK and HCT-8 cells, the parasitophorous vacuole was less evident and immobile merozoites were observed in the cytoplasm. In VERO cells, one or several parasitophorous vacuoles contained up to 16 mobile sporozoites. No oocysts were found in any of the cell types used. VERO cells may be suitable for studies of the interaction between parasite and host cells.     

Precocious development of hydatid cysts in a macropodid host

This study describes the pathological changes associated with an experimental infection of captive wallabies with Echinococcus granulosus. Adult and juvenile tammar wallabies (Macropus eugenii) were infected orally with 0, 1,000, 2,500 or 8,000 E. granulosus eggs. Lung cyst progression was monitored by chest radiography every 4 months until 16 months p.i. Animals were necropsied from 9 to 16 months after infection. Cysts were detected radiographically from 4 months onwards. The number of cysts per animal varied from one to 10 and the majority (36/40) of cysts established in the lungs. Infection rate was low (35.5%), but cyst development was more rapid and onset of fertility much earlier than has been recorded in sheep. Cyst growth resulted in loss of functional lung capacity, up to an estimated 28% within 14 months of infection. Degenerative changes in cysts were less common in tammars than has been reported in sheep, with gross degeneration of cysts identified in only two animals. Complications associated with lung cyst development included fatal anaphylaxis, pneumothorax and atelectasis. Seven of the 11 infected tammars died or were euthanased as a result of infection during the experiment. From the parasite's perspective, infection of this host allows a shortened life cycle and correspondingly greater biotic potential. We believe this is the first published study that demonstrates the susceptibility of tammar wallabies to hydatid disease and confirms their suitability as a laboratory model for studying the disease in macropodids.     

A study of regional gut endoderm potency by analysis of Cdx2 null mutant chimaeric mice

Inactivation of Cdx2 by homologous recombination results in the development of forestomach epithelium at ectopic sites in pericaecal areas of the midgut of heterozygote mice. Local factors subsequently result in the secondary induction of tissues exhibiting an orderly sequence of tissue types between the ectopic forestomach tissue and the surrounding colon. Clonal analysis of this secondarily generated tissue using Y chromosome painting in chimaeric mice indicates that once differentiated to express Cdx2, host colonic epithelium can only form small intestinal-type epithelium, while Cdx2 mutant cells give rise to a succession of gastric-type tissue but never to a small intestine morphology. Our results indicate a difference in potency between forestomach and midgut precursor endodermal cells.