Segmental pairs of dermal secretory cells release proteins into the hemolymph at the larval-pupal molt.

At each molt of Manduca, the large dermal secretory cells expel the protein contents of their vacuoles into the hemocoel. The constellation of proteins expelled at the last larval-pupal molt, however, differs qualitatively from those proteins released at earlier larval-larval molts. Secretory cells at the two stages not only have different lectin staining properties but also have different proteins that separate on two-dimensional gels. Numerous physiological changes accompany the termination of the last larval instar, including increased chitin synthesis, diminished oxygen delivery, and reduced humoral immunity. Secretion of trehalase that is essential for chitin synthesis and the release of hypoxia up-regulated protein to ameliorate oxygen deprivation help ensure normal transition from larva to pupa. Proteins released by dermal secretory cells at this last molt could supplement the diminished immune defenses mediated by fat body and hemocytes at the end of larval life. Additional immune defenses provided by dermal secretory cells could help ensure a safe transition during a period of increased vulnerability for the newly molted pupa with its soft, thin cuticle and reduced mobility.

Biocompliant Composite Au/pHEMA Plasmonic Scaffolds for 3D Cell Culture and Noninvasive Sensing of Cellular Metabolites.

The field of 3D printing is an area of active research, with a substantial focus given to the design and construction of customized tools for applications in technology. There exists a particular need in these developing areas of opportunity for new multi-functional soft materials that are biologically compatible for the growth and directed culturing of cells. Herein, a composite material consisting of gold nanoparticles with useful plasmonic properties embedded within a highly hydrophilic poly-2-hydroxyethylmethacrylate matrix is described and characterized. This composite material serves dual functions as both host framework scaffold for cell lines such as pre-osteoblasts as well as a plasmonic biosensor for in situ measurements of living cells. The plasmonic properties of this system are characterized as a function of the material properties and related to compositional features of the material through a proposed light-directed mechanism. This chemistry provides a tunable, 3D printable plasmonic composite material of encapsulated gold nanoparticles in a biologically-compliant, acrylate-based hydrogel matrix. Surface-enhanced Raman scattering studies of 3D-microcultures supported by the scaffolds are carried out and the strong influence of perm-selective molecular diffusion in its analytical responses is established. Most notably, specific, largely hydrophilic, cellular metabolites are detected within the supported live cultures.

Interfaces between microbes and membranes of host epithelial cells in hemipteran midguts.

Certain families of plant-feeding insects in the order Hemiptera (infraorder Pentatomomorpha) have established symbiotic relationships with microbes that inhabit specific pouches (caeca) of their midgut epithelium. The placement of these caeca in a well-delineated region at the most posterior end of the midgut bordering the hindgut is conserved in these families; in situ the convoluted midgut is predictably folded so that this caecal region lies adjacent to the anterior-most region of the midgut. Depending on the hemipteran family, caeca vary in their number and configuration at a given anterior-posterior location. At the host-microbe interface, epithelial plasma membranes of midgut epithelial cells interact with nonself antigens of microbial surfaces. In the different hemipteran species examined, a continuum of interactions is observed between microbes and host membranes. Bacteria can exist as free living cells within the midgut lumen without contacting host membranes while other host cells physically interact extensively with microbial surfaces by extending numerous processes that interdigitate with microbes; and, in many instances, processes completely envelope the microbes. The host cells can embrace the foreign microbes, completely enveloping each with a single host membrane or sometimes enveloping each with the two additional host membranes of a phagosome.

Luminal membranes in the midgut of the lace bug Corythucha ciliata.

The inordinately long midgut of hemipterans is devoid of peritrophic membranes described for many other insects. These membranes separate apical microvilli of midgut cells from contents of the lumen. In hemipterans, by contrast, contents of the lumen are separated from apical surfaces of midgut epithelia by secretion of additional plasma membranes (perimicrovillar membranes) containing digestive enzymes. In the lace bug Corythucha ciliata, precursors for these perimicrovillar membranes arise in smooth endoplasmic reticula (SER) as stacked, coiled membranes and are continually expelled into the lumen along the entire length of the midgut as stacked, tubular membranes; these membranes undergo changes in form as they pass from the SER to the midgut lumen. Rather than adopting the double membrane configuration in the gut lumen that was first described for hemipteran perimicrovillar membranes, these modified perimicrovillar membranes of the Corythucha gut line apical surfaces of midgut apical lamellae and intermix with the contents of the lumen; foregut and hindgut epithelial cells are devoid of vesicles containing coiled membranes observed abundantly in midgut epithelia. Rather than achieving renewal of adult midgut epithelial cells through the divisions of regenerative cells as observed in many adult insects, prolific generation of perimicrovillar membranes apparently maintains the integrity of this lengthy hemipteran midgut epithelium.

3D-Printed Hydrogel Composites for Predictive Temporal (4D) Cellular Organizations and Patterned Biogenic Mineralization.

Materials chemistries for hydrogel scaffolds that are capable of programming temporal (4D) attributes of cellular decision-making in supported 3D microcultures are described. The scaffolds are fabricated using direct-ink writing (DIW)-a 3D-printing technique using extrusion to pattern scaffolds at biologically relevant diameters (≤ 100 µm). Herein, DIW is exploited to variously incorporate a rheological nanoclay, Laponite XLG (LAP), into 2-hydroxyethyl methacrylate (HEMA)-based hydrogels-printing the LAP-HEMA (LH) composites as functional modifiers within otherwise unmodified 2D and 3D HEMA microstructures. The nanoclay-modified domains, when tested as thin films, require no activating (e.g., protein) treatments to promote robust growth compliances that direct the spatial attachment of fibroblast (3T3) and preosteoblast (E1) cells, fostering for the latter a capacity to direct long-term osteodifferentiation. Cell-to-gel interfacial morphologies and cellular motility are analyzed with spatial light interference microscopy (SLIM). Through combination of HEMA and LH gels, high-resolution DIW of a nanocomposite ink (UniH) that translates organizationally dynamic attributes seen with 2D gels into dentition-mimetic 3D scaffolds is demonstrated. These analyses confirm that the underlying materials chemistry and geometry of hydrogel nanocomposites are capable of directing cellular attachment and temporal development within 3D microcultures-a useful material system for the 4D patterning of hydrogel scaffolds.

A novel arrangement of midgut epithelium and hepatic cells implies a novel regulation of the insulin signaling pathway in long-lived millipedes.

Nutrients absorbed by the epithelial cells of the millipede midgut are channeled to a contiguous population of hepatic cells where sugars are stored as glycogen. In insects and other arthropods, however, nutrients absorbed by midgut epithelia are first passed across the epithelial basal surface to the hemolymph before storage in fat body. The inter-digitation of cellular processes at the interface of hepatic and midgut epithelial cells offers a vast surface area for exchange of nutrients. At this interface, numerous small vesicles with the dimensions of exosomes (∼30nm) may represent the mediators of nutrient exchange. Longevity and the developmental arrest of diapause are associated with reduced insulin signaling. The long lifespans for which millipedes are known may be attributable to a novel pathway with reduced insulin signaling represented by the novel arrangement of hepatic storage cells and midgut epithelial absorbing cells.

Segmental pairs of giant insect cells discharge presumptive immune proteins at each larval molt.

A pair of massive secretory cells exists within each thoracic and the nine abdominal segments of Manduca larvae. Each of these cells is nestled between the dorsal integument and underlying muscles. Contents of large vacuoles in these cells are abruptly discharged at each molt and have always been considered to contribute to shedding and/or formation of cuticle. Peanut agglutinin is a specific lectin label for these secretory vacuoles; vacuoles label intensely immediately before each molt as vacuoles attain their maximal size. Contents of vacuoles are restored after each molt and throughout most of each intermolt. During the molt cycle these cells secrete contents of their vacuoles into the interior hemocoel rather than onto the exterior cuticle. Vacuoles discharge via a distinctive mechanism involving partitioning of contents into numerous vesicles that move to the cell surface. Dermal secretory cells were dissected from larvae before and after the 4th-5th instar molt. Proteins from pre-molt and post-molt secretory cells were separated by two-dimensional electrophoresis to establish which proteins are discharged at the molt. While secreted proteins are novel, all have presumptive roles in immune responses. Dermal secretory cells may represent a new, unsuspected component of the innate immune system that release their proteins during the vulnerable molting period of an insect's life.

Melanin-independent accumulation of turgor pressure in appressoria of Phakopsora pachyrhizi.

Appressoria of some plant-pathogenic fungi accumulate turgor pressure that produces a mechanical force enabling the direct penetration of hyphae through the epidermis. Melanin functions as an impermeable barrier to osmolytes, which allows appressoria to accumulate high turgor pressure. Deficiency of melanin in appressoria reduces turgor pressure and compromises the infection process. In Phakopsora pachyrhizi, the soybean rust pathogen, the appressoria are hyaline. Our objective was to ensure the absence of a melanin layer specifically between the appressorial cell wall and plasma membrane, as well as to determine the turgor pressure of P. pachyrhizi appressoria. We demonstrated that two melanin biosynthesis inhibitors neither reduced turgor pressure nor compromised the infection process. Transmission electron microscopy also showed the absence of a melanin layer between the appressorial cell wall and plasma membrane. In addition, the turgor pressure of P. pachyrhizi appressoria was 5 to 6 MPa, based on extracellular osmolytes used to simulate different osmotic pressures. This is the first report showing that turgor pressure accumulation of P. pachyrhizi appressoria was independent of melanin.

Helical carbon nanotubes enhance the early immune response and inhibit macrophage-mediated phagocytosis of Pseudomonas aeruginosa.

Aerosolized or aspirated manufactured carbon nanotubes have been shown to be cytotoxic, cause pulmonary lesions, and demonstrate immunomodulatory properties. CD-1 mice were used to assess pulmonary toxicity of helical carbon nanotubes (HCNTs) and alterations of the immune response to subsequent infection by Pseudomonas aeruginosa in mice. HCNTs provoked a mild inflammatory response following either a single exposure or 2X/week for three weeks (multiple exposures) but were not significantly toxic. Administering HCNTs 2X/week for three weeks resulted in pulmonary lesions including granulomas and goblet cell hyperplasia. Mice exposed to HCNTs and subsequently infected by P. aeruginosa demonstrated an enhanced inflammatory response to P. aeruginosa and phagocytosis by alveolar macrophages was inhibited. However, clearance of P. aeruginosa was not affected. HCNT exposed mice depleted of neutrophils were more effective in clearing P. aeruginosa compared to neutrophil-depleted control mice, accompanied by an influx of macrophages. Depletion of systemic macrophages resulted in slightly inhibited bacterial clearance by HCNT treated mice. Our data indicate that pulmonary exposure to HCNTs results in lesions similar to those caused by other nanotubes and pre-exposure to HCNTs inhibit alveolar macrophage phagocytosis of P. aeruginosa. However, clearance was not affected as exposure to HCNTs primed the immune system for an enhanced inflammatory response to pulmonary infection consisting of an influx of neutrophils and macrophages.

Sperm Cells of a Primitive Strepsipteran.

The unusual life style of Strepsiptera has presented a long-standing puzzle in establishing its affinity to other insects. Although Strepsiptera share few structural similarities with other insect orders, all members of this order share a parasitic life style with members of two distinctive families in the Coleoptera-the order now considered the most closely related to Strepsiptera based on recent genomic evidence. Among the structural features of several strepsipteran families and other insect families that have been surveyed are the organization of testes and ultrastructure of sperm cells. For comparison with existing information on insect sperm structure, this manuscript presents a description of testes and sperm of a representative of the most primitive extant strepsipteran family Mengenillidae, Eoxenos laboulbenei. We compare sperm structure of E. laboulbenei from this family with that of the three other families of Strepsiptera in the other strepsipteran suborder Stylopidia that have been studied as well as with members of the beetle families Meloidae and Rhipiphoridae that share similar life histories with Strepsiptera. Meloids, Rhipiphorids and Strepsipterans all begin larval life as active and viviparous first instar larvae. This study examines global features of these insects' sperm cells along with specific ultrastructural features of their organelles.

Cell renewal in adjoining intestinal and tracheal epithelia of Manduca.

Cell renewal continuously replaces dead or dying cells in organs such as human and insect intestinal (midgut) epithelia; in insects, control of self-renewal determines insects' responses to any of the myriad pathogens and parasites of medical and agricultural importance that enter and cross their midgut epithelia. Regenerative cells occur in the midgut epithelia of many, if not all, insects and are probably derived from a distinctive population of stem cells. The control of proliferation and differentiation of these midgut regenerative cells is assumed to be regulated by an environment of adjacent cells that is referred to as a regenerative cell niche. An antibody to fasciclin II marks cell surfaces of tracheal regenerative cells associated with rapidly growing midgut epithelia. Tracheal regenerative cells and their neighboring midgut regenerative cells proliferate and differentiate in concert during the coordinated growth of the midgut and its associated muscles, nerves and tracheal cells.

Stem cells of the beetle midgut epithelium.

At the completion of metamorphosis, adult insect cells have traditionally been assumed to halt cell divisions and terminally differentiate. While this model of differentiation holds for adult ectodermal epithelia that secrete cuticular specializations of exoskeletons, adult endodermal epithelia are populated by discrete three-dimensional aggregates of stem cells that continue to divide and differentiate after adult emergence. Aggregates of these presumptive adult stem cells are scattered throughout larval and pupal midgut monolayers. At the beginning of adult development (pupal-adult apolysis), the number of cells within each aggregate begins to increase rapidly. Dividing cells form three-dimensional, coherent populations that project as regenerative pouches of stem cells into the hemocoel surrounding the midgut. Stem cell pouches are regularly spaced throughout endodermal monolayers, having adopted a spacing pattern suggesting that each incipient pouch inhibits the formation of a similar pouch within a certain radius of itself-a process referred to as lateral inhibition. At completion of adult development (pupal-adult ecdysis), a distinct basal-luminal polarity has been established within each regenerative pouch. Dividing stem cells occupying the basal region are arranged in three-dimensional aggregates. As these are displaced toward the lumen, they transform into two-dimensional monolayers of differentiated epithelial cells whose apical surfaces are covered by microvilli. This organization of stem cell pouches in insect midguts closely parallels that of regenerative crypts in mammalian intestines.

The larval alimentary canal of the Antarctic insect, Belgica antarctica.

On the Antarctica continent the wingless midge, Belgica antarctica (Diptera, Chironomidae) occurs further south than any other insect. The digestive tract of the larval stage of Belgica that inhabits this extreme environment and feeds in detritus of penguin rookeries has been described for the first time. Ingested food passes through a foregut lumen and into a stomodeal valve representing an intussusception of the foregut into the midgut. A sharp discontinuity in microvillar length occurs at an interface separating relatively long microvilli of the stomodeal midgut region, the site where peritrophic membrane originates, from the midgut epithelium lying posterior to this stomodeal region. Although shapes of cells along the length of this non-stomodeal midgut epithelium are similar, the lengths of their microvilli increase over two orders of magnitude from anterior midgut to posterior midgut. Infoldings of the basal membranes also account for a greatly expanded interface between midgut cells and the hemocoel. The epithelial cells of the hindgut seem to be specialized for exchange of water with their environment, with the anterior two-thirds of the hindgut showing highly convoluted luminal membranes and the posterior third having a highly convoluted basal surface. The lumen of the middle third of the hindgut has a dense population of resident bacteria. Regenerative cells are scattered throughout the larval midgut epithelium. These presumably represent stem cells for the adult midgut, while a ring of cells, marked by a discontinuity in nuclear size at the midgut-hindgut interface, presumably represents stem cells for the adult hindgut.

Distinctive features of the alimentary canal of a fungus-feeding hemipteran, Mezira granulata (Heteroptera: Aradidae).

Hemiptera (Insecta) have specialized mouthparts for fluid feeding as well as distinctive midgut epithelia. The gut epithelia of Mezira granulata, a member of an unusual family of Hemiptera - the Aradidae - are described in this manuscript. Species of this family are thought to feed on fungi instead of plant or animal materials, as is more typical of the Hemiptera. The midgut lumen is lined by perimicrovillar membranes rather than by the peritrophic membranes formed by specialized midgut cells of stomodeal valves found at foregut-midgut interfaces in many insects. However, a stomodeal valve also occurs at the foregut-midgut boundary in these aradid bugs, and certain midgut epithelial cells located at the interface are specialized for secretion of an electron-dense extracellular matrix that fills the midgut lumen in the vicinity of the stomodeal valve. In addition to the distinctive cellular architecture of the apical (luminal) surfaces of midgut epithelial cells, luminal surfaces of the aradid hindgut epithelia are regionally differentiated into three regions with very different cuticles.

Communities of microbes that inhabit the changing hindgut landscape of a subsocial beetle.

Microbes that have adopted endosymbiotic life styles not only have evolved to live in specialized habitats within living organisms, but the living habitats also have evolved to accommodate them. The hindgut of the passalid beetle (Odontotaenius disjunctus) is lined with a cuticle that undergoes dramatic topographic changes during the life cycle of the beetle. This manuscript addresses the changes that have been observed in time and space for the cuticular landscape of the hindgut as well as for the microbial communities within the hindgut. Microbial identity is based on morphology, culture, and extrapolation from previously reported passalid gut inhabitants.

Expression patterns of odorant-binding proteins in antennae of the moth Manduca sexta.

Monoclonal antibodies (MAbs) were generated to six recombinant proteins (odorant-binding proteins; OBPs) of Manduca sexta. The specificity of each MAb was demonstrated by labeling six immunoblots, each of which contained samples of all six recombinant OBPs. The expression patterns of the six OBPs could be grouped into three classes: (1) one (GOBP1) was expressed in sensilla located throughout each annulus; (2) two (ABPX and ABP2) were expressed in the long sensilla trichoidea bordering a zone that was arranged as an arch on the periphery of each annulus; (3) three (PBP2, PBP3, and GOBP2) were expressed in shorter sensilla occupying a wedge-shaped mid-annular zone of each annulus. In female antennae, sensilla expressing these OBPs were intermixed, and the distinct zonation observed in the male antenna was absent. In males, PBP2 was co-expressed in exactly the same cells of the mid-annular zone as those expressing PBP3 and most of the same cells expressing GOBP2, although its expression overlapped with no or only a few sensilla expressing OBPs of class 1 (GOBP1) or class 2 (ABPX, ABP2). This overlap of expression or lack of overlap between PBP2 and the other OBPs for male antennae was mirrored in female antennae. In view of the restricted spatial expression of OBPs within an annulus and the diversity of possible dimeric combinations of OBPs that arises from the co-expression of multiple OBPs in a given sensillum, OBPs could contribute to the specificity of the olfactory responses of insects.