Peritrophic Membrane Formation Research Articles

RNAi-mediated CHS-2 silencing affects the synthesis of chitin and the formation of the peritrophic membrane in the midgut of Aedes albopictus larvae

BackgroundMosquitoes are an important vector of viral transmission, and due to the complexity of the pathogens they transmit, vector control may be the most effective strategy to control mosquito-borne diseases. Chitin is required for insect growth and development and is absent in higher animals and plants, so regulating the chitin synthesis pathway can serve as a potentially effective means to control vector insects. Most of the current research on the chitin synthase (CHS) gene is focused on chitin synthase-1 (CHS-1), while relatively little is known about chitin synthase-2 (CHS-2).ResultsThe CHS-2 gene of Ae. albopictus is highly conserved and closely related to that of Aedes aegypti. The expression of CHS-2 in the third-instar larvae and pupal stage of Ae. albopictus was relatively high, and CHS-2 expression in adult mosquitoes reached the highest value 24 h after blood-feeding. In the fourth-instar larvae of Ae. albopictus, CHS-2 expression was significantly higher in the midgut than in the epidermis. Silencing CHS-2 in Ae. albopictus larvae had no effect on larval survival and emergence. The expression of four genes related to chitin synthesis enzymes was significantly upregulated, the expression level of three genes was unchanged, and only the expression level of GFAT was significantly downregulated. The expression of chitin metabolism-related genes was also upregulated after silencing. The level of chitin in the midgut of Ae. albopictus larvae was significantly decreased, while the chitinase activity was unchanged. The epithelium of the midgut showed vacuolization, cell invagination and partial cell rupture, and the structure of the peritrophic membrane was destroyed or even absent.MethodsThe expression of CHS-2 in different developmental stages and tissues of Aedes albopictus was detected by real-time fluorescence quantitative PCR (qPCR). After silencing CHS-2 of the fourth-instar larvae of Ae. albopictus by RNA interference (RNAi), the expression levels of genes related to chitin metabolism, chitin content and chitinaseactivity in the larvae were detected. The structure of peritrophic membrane in the midgut of the fourth-instar larvaeafter silencing was observed by paraffin section and hematoxylin–eosin (HE) staining.ConclusionCHS-2 can affect midgut chitin synthesis and breakdown by regulating chitin metabolic pathway-related genes and is involved in the formation of the midgut peritrophic membrane in Ae. albopictus, playing an important role in growth and development. It may be a potential target for enhancing other control methods.Graphical

Transcriptomic analysis of the poultry red mite, Dermanyssus gallinae, across all stages of the lifecycle

BackgroundThe blood feeding poultry red mite (PRM), Dermanyssus gallinae, causes substantial economic damage to the egg laying industry worldwide, and is a serious welfare concern for laying hens and poultry house workers. In this study we have investigated the temporal gene expression across the 6 stages/sexes (egg, larvae, protonymph and deutonymph, adult male and adult female) of this neglected parasite in order to understand the temporal expression associated with development, parasitic lifestyle, reproduction and allergen expression.ResultsRNA-seq transcript data for the 6 stages were mapped to the PRM genome creating a publicly available gene expression atlas (on the OrcAE platform in conjunction with the PRM genome). Network analysis and clustering of stage-enriched gene expression in PRM resulted in 17 superclusters with stage-specific or multi-stage expression profiles. The 6 stage specific superclusters were clearly demarked from each other and the adult female supercluster contained the most stage specific transcripts (2725), whilst the protonymph supercluster the fewest (165). Fifteen pairwise comparisons performed between the different stages resulted in a total of 6025 Differentially Expressed Genes (DEGs) (P > 0.99). These data were evaluated alongside a Venn/Euler analysis of the top 100 most abundant genes in each stage. An expanded set of cuticle proteins and enzymes (chitinase and metallocarboxypeptidases) were identified in larvae and underpin cuticle formation and ecdysis to the protonymph stage. Two mucin/peritrophic-A salivary proteins (DEGAL6771g00070, DEGAL6824g00220) were highly expressed in the blood-feeding stages, indicating peritrophic membrane formation during feeding. Reproduction-associated vitellogenins were the most abundant transcripts in adult females whilst, in adult males, an expanded set of serine and cysteine proteinases and an epididymal protein (DEGAL6668g00010) were highly abundant. Assessment of the expression patterns of putative homologues of 32 allergen groups from house dust mites indicated a bias in their expression towards the non-feeding larval stage of PRM.ConclusionsThis study is the first evaluation of temporal gene expression across all stages of PRM and has provided insight into developmental, feeding, reproduction and survival strategies employed by this mite. The publicly available PRM resource on OrcAE offers a valuable tool for researchers investigating the biology and novel interventions of this parasite.

Identification and Molecular Characterization of a Chitin Deacetylase from Bombyx mori Peritrophic Membrane

The insect midgut epithelium is generally lined with a unique chitin and protein structure, the peritrophic membrane (PM), which facilitates food digestion and protects the gut epithelium. PM proteins are important determinants for PM structure and formation. In this study, the silkworm Bombyx mori midgut PM protein BmCDA7 was identified by proteomic tools. The full-length BmCDA7 cDNA is 1357 bp; the deduced protein is composed of 379 amino acid residues and includes a 16 amino acid residue signal peptide, a putative polysaccharide deacetylase-like domain and 15 cysteine residues present in three clusters. The heterologously expressed proteins of the BmCDA7 gene in yeast displayed chitin deacetylase activity. Expression of B. mori BmCDA7 was detected in the midgut at both the transcriptional and translational levels. The BmCDA7 gene was expressed by the newly hatched silkworm larvae until day seven of the fifth instar and was expressed at a high level in the newly exuviated larvae of different instars. The functions and regulatory mechanism of BmCDA7, however, need further investigation.

COPI-mediated blood meal digestion in vector mosquitoes is independent of midgut ARF-GEF and ARF-GAP regulatory activities

We have previously shown that defects in COPI coatomer proteins cause 80% mortality in blood fed Aedes aegypti mosquitoes by 96 h post-feeding. In this study we show that similar deficiencies in COPII and clathrin mediated vesicle transport do not disrupt blood meal digestion and are not lethal, even though both COPII and clathrin functions are required for ovarian development. Since COPI vesicle transport is controlled in mammalian cells by upstream G proteins and associated regulatory factors, we investigated the function of the orthologous ADP-ribosylation factor 1 (ARF1) and ARF4 proteins in mosquito tissues. We found that both ARF1 and ARF4 function upstream of COPI vesicle transport in blood fed mosquitoes given that an ARF1/ARF4 double deficiency is required to phenocopy the feeding-induced mortality observed in COPI coatomer deficient mosquitoes. Small molecule inhibitors of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) are often transitory, and therefore, we investigated the role of five Ae. aegypti ARF-GEF and ARF-GAP proteins in blood meal digestion using RNA interference. Surprisingly, we found that ARF-GEF and ARF-GAP functions are not required for blood meal digestion, even though both vitellogenesis and ovarian development in Ae. aegypti are dependent on GBF1 (ARF-GEF) and GAP1/GAP2 (ARF-GAPs) proteins. Moreover, deficiencies in orthologous COPI regulating genes in Anopheles stephensi mosquitoes had similar phenotypes, indicating conserved functions in these two mosquito species. We propose that based on the need for rapid initiation of protein digestion and peritrophic membrane formation, COPI vesicle transport in midgut epithelial cells is not dependent on ARF-GEF and ARF-GAP regulatory proteins to mediate vesicle recycling within the first 48 h post-feeding.

Midgut proteins released by microapocrine secretion in Spodoptera frugiperda

Microapocrine vesicles bud from the lepidopteran midgut microvilli as double membrane vesicles. To identify the proteins secreted by this process, antibodies raised against isolated microapocrine vesicles from Spodoptera frugiperda were used for screening a midgut cDNA expression library. Positive clones were sequenced, assembled and N blasted against S. frugiperda sequences obtained by pyrosequencing midgut mRNA. This procedure led to the extension of microapocrine sequences that were annotated. A similar procedure was used to identify midgut microvillar proteins that necessarily are part of the microapocrine vesicle. Forty-eight proteins were associated with microvillar membranes. They pertain to 8 functional groups: digestive enzymes, peritrophic membrane, protection, transporters, receptors, secretory machinery, cytoskeleton and signaling, and unknown. Twenty-eight proteins are putatively secreted by microapocrine secretion. Most of them are digestive enzymes, but the list also includes proteins involved in protection and in peritrophic membrane formation. Among the identified digestive enzymes, aminopeptidases are typically microvillar and group into the classes 1, 2, 3, 5, and 6. There are two amylases secreted by microapocrine secretion: one is a digestive enzyme and the other is a transporter-like amylase with no clear function. One lipase has a predicted transmembrane loop, whereas the others are supposed to be secreted by microapocrine secretion and be digestive. Trypsin is membrane bound and is delivered by microapocrine secretion, but has no predicted features to bind membranes. It may remain bound through the signal peptide till be delivered into the midgut lumen. Proteins supposed to be involved in the microapocrine secretory machinery were: calmodulin, annexin, myosin 7a, and gelsolin 1. Their putative roles are discussed, but more research is necessary to settle this subject.

Description of the irradiated and nonirradiated midgut of Ceratitis capitata Wiedemann (Diptera: Tephritidae) and Anastrepha ludens Loew (Diptera: Tephritidae) used for sterile insect technique

Midguts from adult sterile male Ceratitis capitata Wiedemann and Anastrepha ludens Loew, the Mediterranean fruit fly and Mexican fruit fly, respectively, were examined microscopically to determine if radiation used in sterile insect technique (SIT) affected this non target tissue and/or the microorganisms associated with the midgut. Scanning and transmission electron microscopy were used to compare midgut tissues and microorganisms from irradiated and nonirradiated mass-reared adult flies. Observations for both fruit fly species were similar. Our comparisons revealed that newly emerged and two-day-old irradiated flies exhibited signs of damage to midgut tissue, cellular organelles, and gut microbiota not observed in nonirradiated flies of the same ages. Peritrophic membrane formation and bacterial growth appeared diminished in the midguts of irradiated flies compared to nonirradiated flies. Cellular damage of midgut tissue from irradiated flies included distorted, small nuclei that lacked nuclear material, and mitochondria that were dilated and/or vacuolated. No visual evidence of cellular damage was observed in nonirradiated flies. The impact of radiation used in SIT on fly competitiveness, referred to herein as the capability of adult flies to perform a function, is discussed, including the potential use of probiotic diets to improve damaged midgut tissue and restore midgut microbiota.

High levels of human chitotriosidase hinder the formation of peritrophic membrane in anopheline vectors

In the Anopheles midgut, Plasmodium falciparum produces a specific chitinase able to penetrate the blood meal surrounding the chitin-containing peritrophic membrane (PM). High levels of an analogous chitinase, chitotriosidase (CHIT), may be found in human blood, being the markers of macrophage activation. To verify the hypothesis that CHIT present in malaria patient blood could help parasite to overcome PM, we carried out a bioassay by feeding Anopheles stephensi females on an artificial apparatus that contained human blood from four different sources and with different chitinase concentrations: (1) healthy donors, as negative controls; (2) patients with malaria; (3) patients with Gaucher disease; and (4) whole blood enriched with commercial P. falciparum chitinase, as positive controls. After 16, 20 and 24 h of bloodfeeding, mosquitoes were dissected to extract the midgut and assess the effect of the different chitinases on membrane structure. Optical microscopy showed that formation of PM was clearly complete after 16 h in the posterior midgut from Anopheles already fed with healthy donor bloods. By contrast, PM formation was visible after 16 h in the posterior midgut of mosquitoes fed with malaria and Gaucher patient bloods but appeared clearly damaged at 20 and 24 h. At the same time, the PM formation was almost completely inhibited in the midgut of Anopheles fed with P. falciparum chitinase-enriched bloods. These alterations were clearly confirmed by transmission electronic microscopy. In the present paper, we demonstrate that human CHIT from different sources is active on anophelines' PM.

Immunolocalization of chitin synthase in the tobacco hornworm.

To start investigation of chitin synthesis and peritrophic membrane formation in the midgut of Manduca sexta, we have cloned a cDNA fragment encoding chitin synthase. Northern blots with a corresponding RNA probe revealed a single transcript of 4.7 kb, which was most prominent in poly(A) RNA isolated from the anterior and median midgut as well as from tracheal cells. In situ hybridization showed that the amount of chitin synthase transcripts in the cytoplasm of columnar cells decreased from the anterior to the posterior midgut. Moreover, in the anterior midgut they were localized in the apical region of columnar cells. Southern blots suggested more than one gene locus for chitin synthase in the Manducagenome. To analyze the distribution of chitin synthases on the protein level, we expressed a polymerase chain reaction (PCR) fragment of 119 amino acids in Escherichia coli and generated polyclonal antibodies to the purified recombinant protein. In immunoblots of crude extracts derived from the anterior midgut as well as from partially purified brush border membranes of columnar cells the affinity-purified anti-chitin synthase antiserum labeled a single protein with an apparent molecular mass of 150-200 kDa. Immunohistochemistry showed intense labeling in midgut brush border membranes. Immunofluorescence was restricted to the apical ends of microvilli. Apical membranes of salivary glands and tracheal cells were labeled as well, but not those of Malpighian tubules. This is the first time that chitin synthase expression has been visualized in insect tissues on the level of proteins.

Molecular structure of the peritrophic membrane (PM): identification of potential PM target sites for insect control.

Peritrophic membranes (PMs) are an invertebrate-unique structure that lines the digestive tract, playing important roles in facilitating food digestion and providing protection to the gut epithelium. The importance of PMs in insects has been recognized ever since its presence was identified 200 years ago. In the last 5 years, significant progress towards understanding the PM molecular structure and the mechanism for PM formation has been made. Recent studies on Type 1 PMs from lepidopteran larvae have suggested a model for the PM molecular structure and formation. The important physiological functions of the PM suggest that PMs can be a significant structural target for insect control and the current understanding of the structure of lepidopteran larval PMs has provided us with potential opportunities for targeting the PM by various mechanisms.

Lepidopteran peritrophic membranes and effects of dietary wheat germ agglutinin on their formation and structure.

Peritrophic membrane (PM) structure and the effects of dietary wheat germ agglutinin (WGA) on PM formation were studied in larvae of the European corn borer (ECB), Ostrinia nubilalis, and the tobacco hornworm (THW), Manduca sexta. Growth of ECB was strongly inhibited by low amounts of WGA in the diet (0.05%), whereas THW was not affected by amounts of up to 2%. In ECB larvae, chitin microfibrils were secreted to form an orthogonal network within the apical region of the anterior midgut microvilli. The network then moved to the tips of the microvilli where proteinacious matrix was added prior to delamination of a single PM into the lumen to enclose the food bolus. Multiple PMs rapidly appeared as the food moved posteriorly and some of these became greatly thickened in the middle and posterior regions of the midgut. WGA in the diet caused hypersecretion of unorganized PM in the anterior midgut lumen, disintegration of microvilli, and cessation of feeding. It was also shown to bind to both the chitinous network and to several PM proteins, perhaps causing voids in the PM and sparse matrix material. This allowed the passage of food particles through a defective PM into the ectoperitrophic space and penetration into the microvillar brush border. Stimulation of PM secretion and cessation of feeding may have been a response to damage to the brush border. Unlike ECB, the chitinous network of THW is a randomly organized felt-like structure embedded in a proteinaceous matrix. This PM is secreted as a thin multilayered structure in the anterior region of the midgut, but multiple and thickened PMs occur in the middle and posterior lumens of the midgut. THW tolerated high amounts of WGA in its diet with no disruption of PM formation or inhibition of growth. WGA did accumulate as large masses embedded in the PM, but caused no voids that would allow the penetration of food particles and subsequent damage to the brush border. Therefore, differences in PM formation and structure between ECB and THW appeared to affect how WGA interacts with chitinous and proteinaceous components of the PM and subsequent effects on larval feeding and growth.

Calcofluor disrupts the midgut defense system in insects

The insect midgut is generally lined with a unique protective chitin/protein structure, the peritrophic membrane (PM). We demonstrated that in Trichoplusia ni larvae, the majority of PM proteins were assembled with chitin as a consequence of their chitin binding properties. These proteins could be dissociated from the PM in vitro by Calcofluor, a well-known chemical with chitin binding properties. The chitin binding characteristics of PM proteins were confirmed by their high affinity binding in vitro to regenerated chitin. In vivo assays demonstrated that Calcofluor could inhibit PM formation in five lepidopteran insects tested. The inhibition of T. ni PM formation by Calcofluor, was accompanied by increased larval susceptibility to baculovirus infection. Continuous inhibition of PM formation by Calcofluor resulted in retarded larval development and mortality. The destructive effect of Calcofluor on PM formation was demonstrated to be transient and reversible depending on the presence of Calcofluor within the midgut. In addition, degradation of the insect intestinal mucin was observed concurrently with the inhibition of PM formation by Calcofluor. Our studies revealed a potential novel approach to develop strategies for insect control by utilizing chitin binding molecules to specifically target PM formation in a broad range of insect pest species.

The structure of the foregut and midgut of nymphs, subimagos and imagos of Cloeon dipterum (Ephemeroptera) and the functions of the gut of adult mayflies

AbstractThe anatomy of the gut and the fine structure of the foregut and midgut of the nymphal, subimaginal and imaginal stages of C. dipterum are described. A new description is given of the nymphal foregut–midgut junction and its relationship to peritrophic membrane formation. Although the mouthparts are lost at the end of the nymphal stage, and the structure of the gut undergoes extensive and often degenerative changes both during and after each of the moults to subimago and imago, the structure of the subimaginal and imaginal gut nevertheless suggests that the gut is still performing some active function during both adult stages. This is also suggested by the persistence in subimagos and imagos of rhythmical waves of midgut contraction, an energetically expensive process for an animal which has ceased to feed. Although a very occasional uptake of fluids by imagos has been observed, it has been shown, by the use of a fluorescent stain, that fluid which disappears from the mouth region is most frequently being spread as a very thin film over the body surface rather than entering the gut. Frequency of midgut contraction has been measured in resting subimagos and imagos of C. dipterum and C. simile, in imagos of C. dipterum during and after being held in a dry atmosphere, in ovipositing imagos of C. dipterum, in ejaculating male imagos of C. simile and Ecdyonurus venosus, and after successive bouts of flying by E. venosus. Previous claims that the midgut contractions either control or affect oviposition and sperm ejaculation have not been substantiated. The possibility of a key relationship between the midgut contractions and the need for rapid transport of metabolities is discussed.

Structure and formation of the peritrophic membrane in the larva of the southern corn rootworm, Diabrotica undecimpunctata

The peritrophic membrane (PM) in larvae of the southern corn rootworm Diabrotica undecimpunctata (Coleoptera:Chrysomelidae) forms along the full length of the midgut epithelium, defining D. undecimpunctata as a Type I insect with respect to PM formation. PM formation occurs in three phases: organization of a continuous lamella of matrix from material secreted into the interstices between the microvilli, maturation and apical movement of the lamella along the microvilli, and shedding of the lamella from the tips of the microvilli into the midgut lumen. Subsequent cycles of synthesis and shedding give rise to multiple, concentric lamellae which surround the food in the gut lumen. PM lamellae are 0.2 μm in profile width and consist of a core of bundles of 5 nm-diameter microfibers encased in a finely-granular homogeneous material. The microfiber bundles are arranged in an orthogonal grid-like array with dimensions consistent with formation around the microvilli. The homogeneous material separates from the PM lamellae to enclose food particles suggesting it may contain digestive enzymes. The PM, microvilli and intracellular vesicles in the midgut epithelium stain intensely with wheat germ agglutinin reflecting the presence and sites of secretion and synthesis of chitin.

The Role of the Mosquito Peritrophic Membrane in Bloodmeal Digestion and Infectivity of Plasmodium Species

Secretion and luminal formation of the peritrophic membrane (PM) were induced in female Anopheles stephensi and Aedes aegypti by feeding the mosquitoes on a warmed suspension of latex particles in Ringer's solution. The PM in A. stephensi was produced from apical secretion vesicles stored in the midgut epithelial cells and secreted into the lumen during feeding. In A. aegypti, the PM was formed de novo. When the latex feeding was followed 24 hr later by a meal of lyophilized pig blood, the 2 mosquito species exhibited very different modifications to their PM structure; in A. stephensi no PM was formed around the blood meal, whereas de novo synthesis of the PM in A. aegypti continued during the blood meal, with the resulting PM greatly thickened compared to the normal feeding. This artificial induction of PM formation was used as the basis to study the role of the PM in blood meal digestion and in infectivity of mosquitoes by the appropriate species of Plasmodium. The feeding of a latex suspension alone had no stimulatory effect on the 2 major midgut proteases, trypsin and aminopeptidase, in either species. After a blood meal alone, proteases rose to maximum activity at 30 hr and 24 hr after feeding in A. stephensi and A. aegypti, respectively. After double feeding, protease activities in both species were almost identical to those in blood-fed mosquitoes. Neither the absence of a PM (in A. stephensi) nor the presence of a thickened PM (in A. aegypti), therefore, has any effect on the ability of mosquitoes to digest a blood meal. Malaria infectivity, measured by oocyst counts, also was compared after normal and double feeding using infective blood meals. Infectivity of A. stephensi by Plasmodium berghei was unaffected by the presence or absence of the PM. The thickened PM produced by double feeding in A. aegypti caused a reduction of midgut infectivity by Plasmodium gallinaceum. These results suggest that the PM may act as a partial, but not an absolute, barrier to invasion of the midgut by the ookinete.

Peritrophic membrane structure and formation in the larva of a moth, Heliothis

The peritrophic membrane (PM) in tobacco budworm larvae ( Heliothis virescens, Lepidoptera: Noctuidae), is a continuous sac which encloses the food bolus in the midgut and hindgut. The PM is a single-walled structure 3–5 μm thick which is comprised of two main layers or laminae. The laminae may be fused into a single structure or remain separated by a space which may contain additional thin strands of matrix. Staining with an anti-PM antibody and wheat germ agglutinin (WGA) illustrate the laminar nature of the PM and suggest that protein and chitin have co-incident spatial distributions within the matrix. By transmission electron microscopy, the PM is composed of a loose network of fibrils and small granules, the only structural difference among laminae being a compaction of the matrix along the edges of the two limiting laminae facing the endoperitrophic and ectoperitrophic spaces. By scanning electron microscopy, the PM surface has a wrinkled, felt-like texture without pores or slits. Contrary to the classical view that lepidopterans are Type I insects with respect to PM formation in which the PM forms along the full length of the midgut, the PM in the tobacco budworm forms primarily from secretions of specialized midgut epithelial cells at the junction of the foregut and midgut. The secretory cells, their secretions and the nascent PM stain intensely with the anti-PM antibody but not with WGA suggesting that chitin is added more posteriorly. The PM may be supplemented by the addition of minor amounts of matrix material along the length of the midgut. PM synthesis begins during embryogenesis prior to the initiation of feeding. The PM in neonates is only about 0.1 μm thick but otherwise is structurally similar to that in older larvae.

Dynamic role of microvilli in peritrophic membrane formation

Although the peritrophic membrane (PM) is a common extracellular construction in many invertebrate groups, evidence of the location of its secretion has never been reported. In this study a specific marker for chitin has been developed, enabling a separate examination of secretion of the chitinous and proteinaceous components of the PM in the millipede, Glomeris marginata. Chitin appears first at the base of the microvilli (MV), synchronized in adjacent cells along the entire length of the midgut. Evidence showing that it originates at the plasma membrane is discussed. Proteinaceous components appear to be added from the MV to the chitinous sheet as it moves along the MV toward the lumen. Precedence for such a dynamic role for MV in formation of extracellular structures is reviewed. The completed PM extends around individual items in the gut contents as well as forming a multilayered envelope; this may enhance both its digestive and protective functions.

Midgut barrier as a possible factor in filarial worm vector competency in Aedes trivittatus

Studies were conducted on the ability of microfilariae (mff) of Brugia pahangi to penetrate midguts of Aedes trivittatus and Aedes aegypti (black-eyed Liverpool strain) in vitro. At 12–14 days postexposure (PE) to microfilaremic jirds, A. trivittatus exhibited a significantly lower prevalence and mean intensity of infection than A. aegypti. Nearly 50% of the mff ingested by A. aegypti, but only 7.5% of the mff ingested by A. trivittatus, penetrated through midguts within 2 hr PE. Examination of 4-μm plastic sections of midguts revealed few quantifiable differences in rate or extent of peritrophic membrane formation between the two mosquito species at 0, 30, 60, 90, or 120 min and 6, 12, or 24 hr PE. At 2 hr PE, blood meals of A. trivittatus appeared more coagulated than did blood meals from A. aegypti. Feeding both mosquito species on heparinized or citrated blood in membrane feeders resulted in significant increases in the percentage of mff penetrating the midgut of A. aegypti, but a significantly reduced percentage of mff penetrating the midgut of A. trivittatus. A significant decrease in percentage of mff penetrating midguts of both mosquito species resulted when mosquitoes were exposed to blood containing EDTA.

Peritrophic membrane formation in three temperate simuliids, Simulium ornatum, S. equinum and S. lineatum, with respect to the migration of onchocercal microfilariae.

Engorged females of three temperate simuliid species, Simulium ornatum, S. lineatum and S. equinum, were collected immediately after their bloodfeeds from a cow host. Midguts were dissected out at selected time intervals thereafter, from two minutes to 24 hours post engorgement (p.e.) and peritrophic membrane formation observed. There was an initial rapid secretion of peritrophic membrane (PM) material from the midgut epithelium. The peritrophic membrane produced is Type 1, being formed by delamination from the entire midgut epithelium. This secretory phase continued for approximately two hours p.e. in all three species and was followed by a period of organization within the membrane. At between 12 and 24 hours p.e., the PM appeared more organized and was associated with the formation of several distinct laminae in both S. ornatum and S. lineatum, although in S. equinum it consisted of only a single homogenous layer. Both the rate of secretion and the resulting morphological appearance of the PM appeared species-specific and there was great variation in its thickness in all three species at all time intervals examined. It is suggested that microfilariae penetrate the PM during the initial period of its secretion before it has condensed to form a distinct structure. Furthermore, the migration of microfilariae may be restricted to, or enhanced by, the presence of very thin areas within the membrane where erythrocytes lie almost adjacent to the midgut epithelium.

The peritrophic membrane and cocoon ribbons in larvae of Cionus scrophulariae

Feeding larvae of Cionus scrophulariae secrete a diffuse peritrophic membrane (PM). The principal site of production is the posterior mid-gut. At the pre-pupal stage the mid-gut switches its chitinogenic activity from PM production to synthesising ribbons of striking appearance. These ribbons, which are used for cocoon construction, are formed in deep crypts which occur only in the posterior mid-gut. Microfibrillar material for both PM and cocoon ribbons is formed at the tips of the microvilli. The e.m. observations of type I PM formation in at least Cionus indicate that there is not sufficient justification for the general acceptance of the theories of templating and delamination. Similar unsettling evidence obtained by other authors is discussed.

The fine structure of peritrophic membranes of the blowfly, Calliphora erythrocephala, grown in vitro under different conditions

The formation of peritrophic membranes (PM) in vitro was studied in a flow chamber in order to avoid the accumulation of metabolic substances during prolonged incubation. During the first 8 to 10 hr of incubation the production of PM was nearly constant—3·5 ± 1·4 mm PM/hr. After about 10 hr it decreased and stopped after about 35 hr. Between 1 and 8 hr after the beginning of incubation the width of the periodic crossband pattern reached or nearly reached the values found in PM which had formed in vivo; afterwards it decreased more and more. During the first 20 min of incubation a ‘disturbed zone’ of PM without any regular crossband pattern is formed. In the cardia of adult Calliphora erythrocephala there are three formation zones forming three PM of different fine structures. The fine structure of PM 1 to 3 formed in vitro during the first 6 to 8 hr of incubation in Leloup's medium 1, with an osmolarity of 340 mOsmol, a pH of 6·8, and a temperature of 27°C, does not differ from the PM grown in vivo. PM 1–3 grown in vitro in Tyrode's solution with added glutamine or in Leloup's medium with added β-ecdysone show a considerable increase in thickness and a disturbed formation of the electron dense layer of PM 1.