Autophagic activity in the midgut gland of the overwintering harvestmen Gyas annulatus (Phalangiidae,Opiliones)

Juvenile harvestmen Gyas annulatus overwinter in dormancy in hypogean habitats for 4–5 months. The ultrastructure of the autophagic structures in their midgut epithelium cells was studied by light microscopy, transmission electron microscopy (TEM) and immunofluorescence microscopy (IFM) during this non-feeding period. Before overwintering (November), autophagic structures were scarce. In the middle (January) and at the end of overwintering (March), phagophores, autophagosomes and autolysosomes were present in the cytoplasm of both the secretory and the digestive midgut epithelium cells, gradually increasing their abundance during overwintering. In addition, vacuolization of the cytoplasm intensified. Both processes are induced by starvation. Autophagic structures and cytoplasm vacuolization enable the reuse of the cell's own components required for the maintenance of vital processes during dormancy. While TEM is a much more convenient method for recognition of the autophagic structure types and their ultrastructure, IFM enables exact counting of these structures.     

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.     

Ultrastructure and morphology of midgut visceral muscle in early pupal Aedes aegypti mosquitoes

These studies focus on the pupal Aedes aegypti midgut muscularis for the first 26 h following larval-pupal transition. The midgut muscularis of Ae. aegypti pupae during this first half of the pupal stadium is a grid of both circularly and longitudinally oriented muscle bands, arranged in a manner resembling that of the larvae. While many muscle bands exhibit signs of degeneration during the time period studied, not all bands degrade, nor is this degradation simultaneous. Band deterioration involves destruction of internal elements while the muscle fiber plasma membrane remains intact. Deterioration of contractile elements may involve proteosome-like structures and associated enzymes. Many features of the larval muscularis including cruciform cells, bifurcating circular bands, and bifurcating longitudinal bands of muscle are retained during the time period investigated. Neuromuscular junctions along some muscle bands are retained through at least 16 h into the pupal stadium. The selective nature of muscle fiber degradation, coupled with the retention of larval features and neural input, may allow for limited functionality of the muscularis during metamorphosis. Evidence of sexual dimorphism in the midgut muscularis of male and female Ae. aegypti pupae was not observed during the time period studied.     

The haemoxisome: a haem-iron containing structure in the Rhodnius prolixus midgut cells

Rhodnius prolixus midgut was analysed using transmission electron microscopy and electron spectroscopic imaging in order to localize the cellular structures involved in haem metabolism. In the posterior midgut, special cellular electron-dense structures were observed. These structures are here designated haemoxisomes. Haemoxisomes are present in the epithelial cells at various time points after a blood meal. Several days after the blood meal, some of them become less electron-dense. By electron spectroscopic imaging, large amounts of iron and oxygen were detected in these cellular structures. The iron is probably bound to the porphyrin ring as an iron-protoporphyrin IX complex, as detected using the diaminobenzidine technique. An interesting observation was the presence of endoplasmic reticulum surrounding the haemoxisomes during some special periods. Iron content was monitored in the posterior midgut epithelium and was found to be constant at the initial days after a blood meal, but slightly higher at the end of the digestive process (from 13th up to 20th day). These results are in agreement with the observation that the appearance of the haemoxisomes changes at the end of the digestive process. The ability to degrade haem seems to depend on the presence of endoplasmic reticulum as observed using a haem degradation assay in the presence of an endoplasmic reticulum-enriched fraction. Taken together these results suggest that haemoxisomes may play a role in intracellular haem detoxification.     

Effects of soybean trypsin inhibitor on hypopharyngeal gland protein content, total midgut protease activity and survival of the honey bee (Apis mellifera L.)

Insecticidal properties of protease inhibitors have been established in transgenic plants. In the wake of continuous research and rapid development of protease inhibitors it is important to assess possible effects on beneficial insects like the honey bee (Apis mellifera L.). In this study, newly emerged caged bees were fed pollen diets containing three different concentrations (0.1%, 0.5% and 1% w:w) of soybean trypsin inhibitor (SBTI). Hypopharyngeal gland protein content, total midgut proteolytic enzyme activity of these bees, and survival were measured. Bees fed 1% SBTI had significantly reduced hypopharyngeal gland protein content and midgut proteolytic enzyme activity. There were no significant differences between control, 0.1% and 0.5% SBTI treatments. Bees fed a diet containing 1% SBTI had the lowest survival, followed by 0.5% and 0.1%, over a 30-day period. We concluded that nurse bees fed a pollen diet containing at least 1% SBTI would be poor producers of larval food, potentially threatening colony growth and maintenance.     

Characterization and comparison of midgut proteases of Bacillus thuringiensis susceptible and resistant diamondback moth (Plutellidae: Lepidoptera)

The midgut proteases of the Bacillus thuringiensis resistant and susceptible populations of the diamondback moth, Plutella xylostella L. were characterized by using protease specific substrates and inhibitors. The midgut contained trypsin-like proteases of molecular weights of 97, 32, 29.5, 27.5, and 25 kDa. Of these five proteases, 29.5 kDa trypsin-like protease was the most predominant in activation of protoxins of Cry1Aa and Cry1Ab. The activation of Cry1Ab protoxin by midgut protease was fast (T(1/2) of 23-24 min) even at a protoxin:protease ratio of 250:1. The protoxin activation appeared to be multi-step process, and at least seven intermediates were observed before formation of a stable toxin of about 57.4 kDa from protoxin of about 133 kDa. Activation of Cry1Aa was faster than that of Cry1Ab on incubation of protoxins with midgut proteases and bovine trypsin. The protoxin and toxin forms of Cry proteins did not differ in toxicity towards larvae of P. xylostella. The differences in susceptibility of two populations to B. thuringiensis Cry1Ab were not due to midgut proteolytic activity. Further, the proteolytic patterns of Cry1A protoxins were similar in the resistant as well as susceptible populations of P. xylostella.     

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.     

Foxh1 and Foxa2 are not required for formation of the midgut and hindgut definitive endoderm

The definitive endoderm forms during gastrulation and is rapidly transformed into the gut tube which is divided along the anterior-posterior axis into the foregut, midgut, and hindgut. Lineage tracing and genetic analysis have examined the origin of the definitive endoderm during gastrulation and demonstrated that the majority of definitive endoderm arises at the anterior end of the primitive streak (APS). Foxh1 and Foxa2 have been shown to play a role in specification of the APS and definitive endoderm. However, prior studies have focused on the role of these factors in specification of foregut definitive endoderm, while their role in the specification of midgut and hindgut definitive endoderm is less understood. Furthermore, previous analyses of these mutants have utilized definitive endoderm markers that are restricted to the anterior endoderm, expressed in extraembryonic endoderm, or present in other germ layers. Here, we characterized the expression of several novel definitive and visceral endoderm markers in Foxh1 and Foxa2 null embryos. In accordance with previous studies, we observed a deficiency of foregut definitive endoderm resulting in incorporation of visceral endoderm into the foregut. Interestingly, this analysis revealed that formation of midgut and hindgut definitive endoderm is unaffected by loss of Foxh1 or Foxa2. This finding represents a significant insight into specification and regionalization of mouse definitive endoderm.     

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.     

Organization, ultrastructure, and development of midgut visceral muscle in larval Aedes aegypti

The midgut muscularis of larvae of the mosquito Aedes aegypti takes the form of a grid of longitudinal and circular muscle bands. The longitudinal and circular bands overlap at near right angles at many areas of intersection. The longitudinal bands run the length of the midgut. However, some bands of circular muscle, located in the anterior midgut, pass only partway around the gut. An unusual feature was observed at some regions where longitudinal and circular bands of muscle intersect: filaments oriented at near right angles to one another were present in the same membrane-bound fiber. These cruciform regions send contractile elements into both circular and longitudinal bands. The muscularis was fixed in a contracted state, so most of the sarcomeres are represented by complete overlap of myosin and lighter staining actin filaments. Features characteristic of supercontracting muscle, including perforated Z-lines, were seen in sarcomeres of circular muscle bands. Small invaginations resembling transverse tubules were present but a sarcoplasmic reticulum was not observed. While occasional cells that may be neurons or neurosecretory cells were observed, a network that might serve to coordinate the segmentation and peristaltic movement of the muscularis was not apparent.     

Liang Bua Homo floresiensis mandibles and mandibular teeth: a contribution to the comparative morphology of a new hominin species

In 2004, a new hominin species, Homo floresiensis, was described from Late Pleistocene cave deposits at Liang Bua, Flores. H. floresiensis was remarkable for its small body-size, endocranial volume in the chimpanzee range, limb proportions and skeletal robusticity similar to Pliocene Australopithecus, and a skeletal morphology with a distinctive combination of symplesiomorphic, derived, and unique traits. Critics of H. floresiensis as a novel species have argued that the Pleistocene skeletons from Liang Bua either fall within the range of living Australomelanesians, exhibit the attributes of growth disorders found in modern humans, or a combination of both. Here we describe the morphology of the LB1, LB2, and LB6 mandibles and mandibular teeth from Liang Bua. Morphological and metrical comparisons of the mandibles demonstrate that they share a distinctive suite of traits that place them outside both the H. sapiens and H. erectus ranges of variation. While having the derived molar size of later Homo, the symphyseal, corpus, ramus, and premolar morphologies share similarities with both Australopithecus and early Homo. When the mandibles are considered with the existing evidence for cranial and postcranial anatomy, limb proportions, and the functional anatomy of the wrist and shoulder, they are in many respects closer to African early Homo or Australopithecus than to later Homo. Taken together, this evidence suggests that the ancestors of H. floresiensis left Africa before the evolution of H. erectus, as defined by the Dmanisi and East African evidence.     

Structure and expression of glutathione S-transferase genes from the midgut of the Common cutworm, Spodoptera litura (Noctuidae) and their response to xenobiotic compounds and bacteria

Glutathione S-transferases (GSTs) play a pivotal role in detoxifying endogenous and xenobiotic compounds and oxidative stress resistance in cells. In this study, five GST genes, including three Sigma GSTs (SlGSTs1, SlGSTs2, and SlGSTs3), one Omega GST (SlGSTo1) and one un-classified GST (SlGSTu1) were identified from the midgut of the Common cutworm, Spodoptera litura. Structure analyses of the eight (including the previously identified Epsilon GST genes, SlGSTe1, SlGSTe2 and SlGSTe3 from the same insect) SlGSTs genes showed that the Epsilon SlGSTe genes do not contain any intron, while the Sigma SlGSTs contain three introns and the Omega SlGSTo1 and the un-classified SlGSTu1 contain five introns. Analysis of the spatial and temporal expression of these eight SlGSTs indicated that SlGSTe1, SlGSTs2 and SlGSTo1 expressed in all stages of development from the egg to the adult stages. SlGSTe2, SlGSTe3, SlGSTs1, SlGSTs3 and SlGSTu1 had higher expression levels in the larval stages than in other stages and their expression levels in the midgut were higher than in other tissues. SlGSTs1 was expressed in the larval midgut but not in the fat body and could be induced by bacterial infections. The expression of SlGSTe1, SlGSTe3, SlGSTs1 and SlGSTs3 was increased by chlorpyrifos to various degrees, while the expression of SlGSTe1, SlGSTe3, SlGSTs1, SlGSTs3 and SlGSTo1 was increased by xanthotoxin. Levels of malonaldehyde, an indicator of oxidative stress, were higher in the larval midgut than in the pupal midgut. Chlorpyrifos induced the malonaldehyde content in the larvae, whereas xanthotoxin did not. It is hypothesized that high expression levels of the midgut SlGSTs might be due to the increased levels of oxidative stress caused by feeding, bacterial infection and xenobiotic compounds.     

Dermal gland secretion improves the heat tolerance of the brown dog tick, Rhipicephalus sanguineus, allowing for their prolonged exposure to host body temperature

Dermal glands (sensilla sagittiformia) secrete when brown dog ticks, Rhipicephalus sanguineus, are mechanically disturbed, presumably as a defensive mechanism. Recently, we observed that these glands secrete due to the pressure stimulation of engorgement. In this study, we examine how dermal gland secretion alters the physiology of R. sanguineus, particularly if this secretion is an important mechanism during blood feeding. The ability of ticks to retain water was not modified by dermal gland secretion, but heat tolerance was enhanced. Short-term heat shock was improved slightly (1 h at 50 °C to 1 h at 56 °C) and featured reduced injury responses and greater recovery after heat shock. When exposed to their host body temperature (37 °C) for prolonged periods, individuals that had secreted survived over 1 week longer than individuals that did not secrete. Dorsal application of squalene, the main component of dermal gland secretion, did not increase temperature tolerance, suggesting that the act of secreting rather than the physical properties of the secretion itself is responsible for the increase in heat tolerance. Based on our results, dermal gland secretion may be an essential mechanism in certain tick species (Amblyomma, Dermacentor, Hyalomma, Rhipicephalus, but not Ixodes) for tolerating body temperature and not succumbing to heat stress during the extended time periods of feeding on a mammalian host, serving as a mechanism to prevent heat damage from the host during feeding.     

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.     

Understanding chilling responses in Arabidopsis seeds and their contribution to life history

Winter chilling is of central importance in the phenology of temperate annual and perennial plants. Chilling accelerates flowering through the process of vernalization and breaks both bud and seed dormancy, permitting the onset of growth in the spring. The quantitative effects of chilling in floral promotion in winter annual Arabidopsis accessions are well-documented, but very little is known about the basic physiology underlying summer annual responses to winter chilling, which acts on seeds within the soil seed bank. Here, we analyse the response of wild accessions to extended chilling in seeds, and explore the interaction between seed-maturation temperature and chilling responses. We show that two weeks of chilling induces secondary dormancy, and that this time period is not dependent on seed-maturation temperature. In addition, we found that seeds for most accessions set under simulated summer conditions in the laboratory are unable to overwinter in the soil seed bank, as they germinate without light during extended chilling treatments. This shows that these seeds are committed to re-establishment in the same growing season. Understanding how winter chilling affects the timing of Arabidopsis phenology will enable us to explore the genetics behind adaptation to changing climates, and inform rational approaches to breeding crops with improved performance under new climate scenarios and develop a systems ecology of Arabidopsis.     

Study of the Bacillus thuringiensis Vip3Aa16 histopathological effects and determination of its putative binding proteins in the midgut of Spodoptera littoralis

The bacterium Bacillus thuringiensis produces, at the vegetative stage of its growth, Vip3A proteins with activity against a broad spectrum of lepidopteran insects. The Egyptian cotton leaf worm (Spodoptera littoralis) is an important agricultural pest that is susceptible to the Vip3Aa16 protein of Bacillus thuringiensis kurstaki strain BUPM95. The midgut histopathology of Vip3Aa fed larvae showed vacuolization of the cytoplasm, brush border membrane destruction, vesicle formation in the apical region and cellular disintegration. Biotinylated Vip3Aa toxin bound proteins of 55- and 100-kDa on blots of S. littoralis brush border membrane preparations. These binding proteins differ in molecular size from those recognized by Cry1C, one of the very few Cry proteins active against the polyphagous S. littoralis. This result supports the use of Vip3Aa16 proteins as insecticidal agent, especially in case of Cry-resistance management.     

Cell death and regeneration in the midgut of the mosquito, Culex quinquefasciatus

Haematophagy, the utilization of blood as food, has evolved independently among insects such as mosquitoes, bedbugs, fleas, and others. Accordingly, several distinct biological adaptations have occurred in order to facilitate the finding, ingestion and digestion of blood from vertebrate sources. Although blood meals are essential for survival and reproduction of these insects, mechanical and chemical stresses are caused by the ingestion of a sizable meal (frequently twice or more times the weight of the insect) containing large amounts of cytotoxic molecules such as haem. Here we present data showing that the stresses caused by a blood meal induce cell death in the midgut epithelium of Culex quinquefasciatus mosquitoes. The process involves apoptosis, ejection of dead cells to the midgut lumen and differentiation of basal regenerative cells to replace the lost digestive cells. The basal cell differentiation in blood-fed mosquito midguts represents an additional mechanism by which insects cope with the stresses caused by blood meals. C. quinquefasciatus adult females are unable to replace lost cells following a third or fourth blood meal, which may have a significant impact on mosquito longevity, reproduction and vectorial capacity.