Evidence for a sugar receptor (lectin) in the peritrophic membrane of the blowfly larva, Calliphora erythrocephala Mg. (Diptera)

For the first time a sugar receptor (lectin) has been localized by electron microscopy in an invertebrate. The peritrophic membrane of the blowfly larva, Calliphora erythrocephala, is shown here to express lectins with high specificity for mannose. The lectin is restricted to the lumen side of the peritrophic membrane. The surface of the midgut epithelium is devoid of mannose-specific lectins. It is suggested that the midgut epithelium has lost these lectins during the course of evolution in favour of the peritrophic membrane which is secreted by specialized cells only at the beginning of the midgut.Peritrophic membranes and the midgut epithelium lack lectins specific for galactose. The lumen side of the peritrophic membrane of the larvae has mannose and/or glucose residues, and it is densely packed with two species of bacteria, Proteus vulgaris and P. morganii. These also have mannose-specific lectins as well as mannose residues on their pili. The existence of mannose-specific receptors and mannose residues on both, peritrophic membranes and bacteria, leads to the assumption of mutual adherence between the two surfaces.     

Ultrastructure of American foulbrood disease pathogenesis in larvae of the worker honey bee,Apis mellifera

Using electron microscopy, the pathogenesis of American foulbrood disease was followed from ingestion of Bacillus larvae spores by young, susceptible honey bee larvae to death of the host and sporulation of the pathogen. Interaction between the host peritrophic membrane and B. larvae vegetatives is described. Phagocytosis was demonstrated to be a mechanism of entry of pathogen into host midgut cells. No evidence of enzymatic digestion of peritrophic membrane or host-cell microvilli was found during the initial interaction of pathogen and host midgut cell, although eventual lysis of host gut cells may have been the result of enzymatic activity. Following entry of bacteria into the hemocoel, host death resulted from systemic bacteremia.     

Activated host neutrophils in the larval midgut lumen of the human bot fly Dermatobia hominis

Light microscopy (LM) and transmission electron microscopy (TEM) were used to observe activated polymorphonuclear neutrophils from mammalian hosts as well as invading bacteria in the midgut lumen of larvae of the human bot fly Dermatobia hominis. Other resident or recruited cells associated with dermal myiasis were fed on by larvae and digested more rapidly than neutrophils. The latter were observed moving towards bacteria and particles of food, extending the filopodia and engulfing material to be digested within phagosomes. The larval midgut lumen, thus, appears to be a suitable environment to produce neutrophil activation at least for short periods, as seen in mammalian hosts. Although interactions between phagocytes and bacteria in the midgut lumen may be important in bot fly larval development, further studies are required to confirm this.     

Tissue Localization of the Endosymbiotic Bacterium “Candidatus Blochmannia floridanus” in Adults and Larvae of the Carpenter Ant Camponotus floridanus

The distribution of endosymbiotic bacteria in different tissues of queens, males, and workers of the carpenter ant Camponotus floridanus was investigated by light and electron microscopy and by in situ hybridization. A large number of bacteria could be detected in bacteriocytes within the midguts of workers, young virgin queens, and males. Large amounts of bacteria were also found in the oocytes of workers and queens. In contrast, bacteria were not present in oocyte-associated cells or in the spermathecae of mature queens, although occasionally a small number of bacteria could be detected in the testis follicles of males. Interestingly, the number of bacteriocytes in mature queens was strongly reduced and the bacteriocytes contained only very few or no bacteria at all, although the endosymbionts were present in huge amounts in the ovaries of the same animals. During embryogenesis of the deposited egg, the bacteria were concentrated in a ring of endodermal tissue destined to become the midgut in later developmental stages. However, during larval development, bacteria could also be detected in other tissues although to a lesser extent. Only in the last-instar larvae were bacteria found exclusively in the midgut tissue within typical bacteriocytes. Tetracycline and rifampin efficiently cleansed C. floridanus workers of their symbionts and the bacteriocytes of these animals still remained empty several months after treatment had ceased. Despite the lack of their endosymbionts, these adult animals were able to survive without any obvious negative effect under normal cultivation conditions.     

Microbial Ecology of the Gut in Laboratory Stocks of the Migratory Grasshopper, Melanoplus sanguinipes (Fab.) (Orthoptera: Acrididae)

Mean pH values in pooled samples of foregut, midgut, and hindgut from adult Melanoplus sanguinipes, which had been raised in the laboratory on barley shoots and wheat bran, were 5.15, 6.39, and 5.98, respectively. Homogenates of midgut/hindgut sections and frass (feces) yielded colony counts of bacteria by the spread plate method of 5.7 to 5.9 and 5.3 to 5.5 log₁₀ colonies per mg, respectively; there were no significant differences (P > 0.05) between counts obtained on several media or on media incubated aerobically or anaerobically. There was no evidence of significant populations of protozoa, fungi, or obligately anaerobic bacteria associated with the gut. A total of 168 pure strains of bacteria isolated from the gut sections were characterized and assigned to 11 taxonomic groups, including Enterococcus spp., Serratia liquefaciens, Pseudomonas spp., and Enterobacter spp. Numbers of Enterococcus spp. in the gut were 2 to 3 orders of magnitude higher than those of the other genera. Strains representing only four of the groups were recovered from bran fed to the grasshoppers; the barley shoots, which were raised in sterile soil, appeared virtually sterile. Examination of the gut wall by scanning electron microscopy revealed the presence of epimural bacteria in the foregut and hindgut but not in the midgut. The distribution of epimural cocci and bacilli differed with the gut section examined. Numerous spherical to ovoid structures up to 10 μm in diameter, which were not identified, were associated with the microvillous surface of the midgut epithelium. Acetate was present in gut, hemolymph, and frass, and it was shown that representative isolates of Enterococcus spp. and Enterobacter agglomerans produced acetate when incubated in an aqueous suspension of bran. The egestion time of solid digesta, as measured with methylene blue-stained barley shoots, was 3.0 to 5.7 h. The results show that M. sanguinipes supported extensive indigenous populations of luminal and epimural bacteria in the gut which were composed predominantly of facultatively anaerobic species; the relatively short egestion time, indicating rapid passage of digesta through the gut, was consistent with the microscopic appearance of digesta residues in frass and could account, at least in part, for the absence of a significant population of obligately anaerobic bacteria from the gut.     

The goblet cavity matrix in the larval midgut of Hyalophora cecropia

The larval midgut of the silkmoth Hyalophora cecropia was examined using scanning electron microscopy. Goblet cells were observed to contain within their cavities a matrix plug. This matrix material was extruded onto the lumen side of the epithelium when the tissue was stretched. The rôle of this matrix material in maintenance of the capacity of the midgut to transport ions in vivo and in vitro is discussed.     

Pole Cell Migration through the Gut Wall of the Drosophila Embryo: Analysis of Cell Interactions

Early in development the precursors of germ cells in Drosophila migrate at the posterior pole of the embryo and translocate to the bottom of the developing posterior midgut primordium. At the end of germ band elongation the pole cells cross the gut wall to enter in association with the gonadal mesoderm. We used laser scanning confocal microscopy on whole-mount Rh-phalloidin-stained embryos and transmission electron microscopy to investigate how pole cells cross the epithelial wall of the posterior midgut primordium. Our results suggest that pole cells leave the midgut sac by traveling through the intercellular spaces of the epithelium. During this process the epithelial cells at the bottom of the posterior midgut primordium are greatly deformed, but their junctional complexes do not completely release, avoiding breaks in the epithelial wall.     

Bacterial symbionts of the giant jewel stinkbug Eucorysses grandis (Hemiptera: Scutelleridae)

Microbiological characterization of gut symbiotic bacteria in a limited number of stinkbugs of the families Acanthosomatidae, Plataspidae, Pentatomidae, Scutelleridae, Parastrachiidae, Alydidae and Pyrrhocoridae has shown symbiotic association with midgut bacteria to be common in phytophagous taxa of these heteropteran insects. Here we investigated the midgut bacterial symbiont of Eucorysses grandis, a stinkbug of the family Scutelleridae. A specific gammaproteobacterium was consistently identified in insects from five different geographic origins. The bacterium was detected in 64 of 64 insects sampled from three host populations. Phylogenetic analyses revealed that the bacterium constitutes a distinct lineage in the Gammaproteobacteria, neither closely related to the gut symbiont of another scutellerid stinkbug, Cantao ocellatus, nor to gut symbionts of other stinkbugs. Diagnostic PCR, in situ hybridization and electron microscopy demonstrated that the bacterium is located extracelluarly, in the midgut fourth section, which possesses crypts. These results indicate that the primary gut symbionts have multiple evolutionary origins in the Scutelleridae. A Sodalis-allied facultative symbiont was also identified in some insects from natural populations. Biological aspects of the primary gut symbiont and the secondary Sodalis-allied symbiont are discussed.     

Ultrastructure and immunolocalization of digestive enzymes in the midgut of Podisus nigrispinus (Heteroptera: Pentatomidae)

The predatory stinkbug Podisus nigrispinus has been utilized in biological control programs. Its midgut is anatomically divided into anterior, middle and posterior regions, which play different roles in the digestive process. We describe the midgut ultrastructure and the secretion of digestive enzymes in the midgut of P. nigrispinus. Midguts were analyzed with transmission electron microscopy and the digestive enzymes amylase, cathepsin L, aminopeptidase and α-glucosidase were immunolocalized. The ultrastructural features of the digestive cells in the anterior, middle and posterior midgut regions suggest that they play a role in digestive enzyme synthesis, ion and nutrient absorption, storage and excretion. The digestive enzymes have different distribution along the midgut regions of the predator P. nigrispinus. Amylase, aminopeptidase and α-glucosidase occur in three midgut regions, whereas cathepsin L occurs in the middle and posterior midgut regions. The anterior midgut region of P. nigrispinus seems to play a role in water absorption, the middle midgut may be involved in nutrient absorption and the posterior midgut region is responsible for water transport to the midgut lumen.     

Gut symbiotic bacteria of the genus Burkholderia in the broad-headed bugs Riptortus clavatus and Leptocorisa chinensis (Heteroptera: Alydidae)

The Japanese common broad-headed bugs Riptortus clavatus and Leptocorisa chinensis possess a number of crypts in the posterior region of the midgut, whose lumen contains a copious amount of bacterial cells. We characterized the gut symbiotic bacteria by using molecular phylogenetic analysis, light and electron microscopy, in situ hybridization, and PCR-based detection techniques. Restriction fragment length polymorphism analysis of 16S rRNA gene clones suggested that a single bacterium dominated the microbiota in the crypts of the both bug species. The predominant 16S rRNA gene sequences obtained from different individuals and species of the bugs were not identical but were very similar to each other. Homology searches in the DNA databases revealed that the sequences showed the highest levels of similarity (96% to 99%) to the sequences of Burkholderia spp. belonging to the beta subdivision of the class Proteobacteria. In situ hybridization with specific oligonucleotide probes confirmed the localization of the Burkholderia symbiont in the lumen of the midgut crypts. Electron microscopy showed that the lumen of the crypts was filled with rod-shaped bacteria of a single morphotype. Molecular phylogenetic analysis demonstrated that the Burkholderia symbionts of the bugs formed a well-defined monophyletic group, although the group also contained several environmental Burkholderia strains. The phylogenetic relationship of the Burkholderia symbionts did not reflect the relationship of the host bug species at all. The sequences from R. clavatus and the sequences from L. chinensis did not form clades but were intermingled in the phylogeny, suggesting that horizontal transmission of the symbiont might have occasionally occurred between populations and species of the bugs.     

Structural and ultrastructural studies of Anticarsia gemmatalis midgut cells infected with the baculovirus A. gemmatalis nucleopolyhedrovirus

Anticarsia gemmatalis is a lepidopteran insect susceptible to A. gemmatalis nucleopolyhedrovirus (AgNPV), which is being used in a large scale, in Brazil, as a biological control agent against this serious soybean pest. Baculovirus usually infects its insect host through the midgut epithelium. In the midgut, it replicates in the nuclei of epithelial cells, producing progeny virus and establishing systemic infection. The AgNPV infection of A. gemmatalis midgut was studied using light and electron microscopy. It was observed that AgNPV enters the midgut mainly through columnar cells. Although the virus was not found in the nuclei of columnar cells until late on infection, it is believed that these cells are the primary sites of infection and replication. This fact can be explained by the continuous regeneration of the midgut epithelium. Besides, the infection may be occurring in isolated cells, making it more difficult to be visualized by electron microscopy. At 48 h post infection, hemocytes and tracheoblasts are infected and polyhedra are formed later in these cells, which are the secondary sites of infection.     

An electron microscopic study of Borrelia in the body of the female ixodid tick Ixodes persulcatus

Borrelia burgdorferi s. lato in naturally infected females of tick Ixodes persulcatus were examined by transmission electron microscopy. The Borreliae were found in midgut and ovary. Location and ultrastructure of bacteria indicate extracellular migration through the midgut epithelium as a preferential way. In gonad, the borreliae intracellular situate in ovarian epithelium and oocytes before and at the beginning of vitellogenesis. The demonstration of numerous spirochetes in the oocytes provides the support for transovarial transmission of the agent. Two morphological types of borreliae were observed.     

Stages of Infection during the Tripartite Interaction between Xenorhabdus nematophila, Its Nematode Vector, and Insect Hosts

Bacteria of the genus Xenorhabdus are mutually associated with entomopathogenic nematodes of the genus Steinernema and are pathogenic to a broad spectrum of insects. The nematodes act as vectors, transmitting the bacteria to insect larvae, which die within a few days of infection. We characterized the early stages of bacterial infection in the insects by constructing a constitutive green fluorescent protein (GFP)-labeled Xenorhabdus nematophila strain. We injected the GFP-labeled bacteria into insects and monitored infection. We found that the bacteria had an extracellular life cycle in the hemolymph and rapidly colonized the anterior midgut region in Spodoptera littoralis larvae. Electron microscopy showed that the bacteria occupied the extracellular matrix of connective tissues within the muscle layers of the Spodoptera midgut. We confirmed the existence of such a specific infection site in the natural route of infection by infesting Spodoptera littoralis larvae with nematodes harboring GFP-labeled Xenorhabdus. When the infective juvenile (IJ) nematodes reached the insect gut, the bacterial cells were rapidly released from the intestinal vesicle into the nematode intestine. Xenorhabdus began to escape from the anus of the nematodes when IJs were wedged in the insect intestinal wall toward the insect hemolymph. Following their release into the insect hemocoel, GFP-labeled bacteria were found only in the anterior midgut region and hemolymph of Spodoptera larvae. Comparative infection assays conducted with another insect, Locusta migratoria, also showed early bacterial colonization of connective tissues. This work shows that the extracellular matrix acts as a particular colonization site for X. nematophila within insects.     

Microscopical techniques reveal the in situ microbial association inside Aplysina aerophoba, Nardo 1886 (Porifera, Demospongiae, Verongida) almost exclusively consists of cyanobacteria

Sponges (Porifera) are aquatic, sessile filter feeders. As such they are permanently exposed to bacteria in the seawater. Molecular data recovered from sponges by PCR shows a high diversity in bacterial DNA. Hence, sponges are considered to live in close association with a diverse and abundant bacterial community. To recover the spatial distribution of bacteria in sponges we retrieved histological sections of Aplysina aerophoba fixed in situ. By combining signals from fluorescence in situ hybridization (FISH), light microscopy and scanning electron microscopy we revealed a detailed histological picture of the spatial organization of the sponge microbial association within the sponges. Our histological results confirm a high abundance of cyanobacteria inside A. aerophoba while other living bacteria are almost absent. This detailed insight into sponge microbiology could only be achieved by the combination of careful sample preparation and different microscopical and histological methods. It also shows the need to confirm molecular datasets in situ and with a high spatial resolution.     

Morphological Changes in the Midgut of Aedes aegypti L. (Diptera: Culicidae) Larvae Following Exposure to an Annona coriacea (Magnoliales: Annonaceae) Extract

Bioinsecticides are important in the control of disease vectors, but data regarding their physiological effects on target insects are incomplete. This study describes morphological changes that occur in the midgut of third instar Aedes aegypti L. (Diptera: Culicidae) following treatment with a methanolic extract of Annona coriacea (Magnoliales: Annonaceae). Dissected midguts were subdivided into anterior and posterior regions and analyzed by light and scanning electron microscopy. Insects exposed to the extract displayed intense, destructive cytoplasmic vacuolization in columnar and regenerative midgut cells. The apical surfaces of columnar cells exhibited cytoplasmic protrusions oriented toward the lumen, suggesting that these cells could be involved in apocrine secretory processes and/or apoptosis. We report that A. coriacea extracts induced morphological alterations in the midgut of A. aegypti midgut larvae, supporting the use of plant extracts for control of the dengue vector.     

Rumen bacterial degradation of forage cell walls investigated by electron microscopy

The association of rumen bacteria with specific leaf tissues of the forage grass Kentucky-31 tall fescue (Festuca arundinacea Schreb.) during in vitro degradation was investigated by transmission and scanning electron microscopy. Examination of degraded leaf cross-sections revealed differential rates of tissue degradation in that the cell walls of the mesophyll and pholem were degraded prior to those of the outer bundle sheath and epidermis. Rumen bacteria appeared to degrade the mesophyll, in some cases, and phloem without prior attachment to the plant cell walls. The degradation of bundle sheath and epidermal cell walls appeared to be preceded by attachment of bacteria to the plant cell wall. Ultrastructural features apparently involved in the adhesion of large cocci to plant cells were observed by transmission and scanning electron microscopy. The physical association between plant and rumen bacterial cells during degradation apparently varies with tissue types. Bacterial attachment, by extracellular features in some microorganisms, is required prior to degradation of the more resistant tissues.     

Microbiota Associated with the Gastrointestinal Tract of the Common House Cricket, Acheta domestica

The location and morphology of the bacteria associated with the gastrointestinal tract of Acheta domestica were studied, and these bacteria were partially characterized. Bacteria were associated with the peritrophic membrane in the midgut and with the gut wall and cuticular structures of the hindgut. No bacteria were associated with the fat bodies. Colony-forming unit determinations indicated that there were three times more cultivatable bacteria in the hindgut than in the midgut. Of these bacteria, 40 to 85% cleared uric acid anaerobically, and 90 to 100% cleared uric acid aerobically. Of the 25 isolates obtained, 21 belonged to the genera Citrobacter, Klebsiella, Yersinia, Bacteroides, and Fusobacterium.     

Morphological specializations of the digestive tract of Zacryptocerus rohweri (Hymenoptera: Formicidae)

Light, scanning, and transmission electron microscopy are used to examine the morphology and ultrastructure of the peculiar digestive tract of the turtle ant, Zacryptocerus rohweri. The proventriculus is heavily sclerotized and covered with clusters of small spines. Narrow spine-lined channels converging at the opening to the midgut act as a fine filter of food; particles >12.5 μm are unable to pass through the proventriculus. In the midgut, ultrastructural study reveals bacteria among the microvilli of midgut epithelial cells. The hindgut of Z. rohweri consists of an enlarged, dark-colored pouch filled with masses of bacteria of three major morphotypes. A thick layer of circular muscle and deep infoldings of the epithelium greatly increase surface area for absorption. Newly emerged individuals appear to acquire these microorganisms by soliciting material from the abdomen tip of other older workers in the colony. Whether or not the hindgut bacteria are true symbionts is unknown; their acquisition and presence suggest that they may supplement the ants' limited, liquid diet by supplying essential amino acids and other nutrients. J. Morphol. 234:253–262, 1997. © 1997 Wiley-Liss, Inc.     

Ultrastructure of the digestive system of the Le fhopper Euscelidius variegatus Kirshbaum (Homoptera : Cicadellidae), with and without congenital bacterial infections

In the digestive system of Euscelidius variegatus Kirshbaum (Homoptera : (Cicadellidae), the close apposition of the anterior midgut with its posterior tabular midgut forms a filter chamber, which shunts excess water in the imbibed plant sap to the hindgut. Leafhoppers congenitally infected with a parasitic enteroform bacterium (designated BEV) had slightly atrophied digestive systems. There were numerous bacteria within the cells of the filter chamber, conical segment, and tubular midgut. Bacteria within the epithelium cells were usually enclosed within lysosomes. Epithelium cells swollen with large numbers of bacteria, had deteriorated cell membranes, and bacteria had erupted into the gut lumen. Leafhoppers not infected by BEV, harbored bacteria in the gut lumen, but not intracellularly within gut cells.     

A putative kinase-related protein (PKRP) from Plasmodium berghei mediates infection in the midgut and salivary glands of the mosquito

The completion of the Plasmodium (malaria) life cycle in the mosquito requires the parasite to traverse first the midgut and later the salivary gland epithelium. We have identified a putative kinase-related protein (PKRP) that is predicted to be an atypical protein kinase, which is conserved across many species of Plasmodium. The pkrp gene encodes a RNA of about 5300 nucleotides that is expressed as a 90 kDa protein in sporozoites. Targeted disruption of the pkrp gene in Plasmodium berghei, a rodent model of malaria, compromises the ability of parasites to infect different tissues within the mosquito host. Early infection of mosquito midgut is reduced by 58-71%, midgut oocyst production is reduced by 50-90% and those sporozoites that are produced are defective in their ability to invade mosquito salivary glands. Midgut sporozoites are not morphologically different from wild-type parasites by electron microscopy. Some sporozoites that emerged from oocysts were attached to the salivary glands but most were found circulating in the mosquito hemocoel. Our findings indicate that a signalling pathway involving PbPKRP regulates the level of Plasmodium infection in the mosquito midgut and salivary glands.