Primary culture of insect midgut cells

This protocol describes the preparation of primary cell cultures from Lepidopteran midgut. These cultures have been used to identify factors that control midgut growth and differentiation, cell responses to these factors, effects of toxins on midgut growth, and the regulation of cell physiology. The protocol is divided into (1) procedures for cell collection, (2) composition of the culture, and (3) assay methods used for cell health, proliferation, and differentiation. Collection and setup require 4–6 h. Once established, a culture can survive several months at 25°C, be kept a year or longer at 4°C, or be frozen for indefinite storage.     

Active transport of potassium by insect midgut

Midguts isolated from fifth-instar larvae of the insert Hyalophora cecropia actively transport potassium in the hemolymph to lumen direction. No specific co- or counter-ion is required and other alkali ions are actively transported in the same direction as potassium. No specific inhibitor of K+ active transport has been found although most metabolic inhibitors reduce the net K+ flux, potential difference, and short-circuit current to zero. The site of the epithelial active transport of potassium has been identified by microelectrode measurements of intracellular resistance as the goblet cell, one of the two major cell types in the single-layered midgut. Under certain external conditions, the neighboring columnar cells are added to the goblet cell transport route through intercellular electrical coupling that occurs after application of external depolarizing current. Tracer influx kinetics were used to establish that the fraction of exchangeable K involved in the transport route under open-circuit conditions is small, corresponding to a goblet cell pathway. Under depolarizing current conditions, virtually all of the exchangeable midgut K is involved in the transport route, corresponding to a goblet and columnar cell pathway. These results and others are used to construct a model for rheogenic active transport of potassium in insect midgut.     

Resolution of the potassium pool size controversy in insect midgut

The isolated midgut of Lepidopteran larvae actively transports potassium from the hemolymph side to the lumen side when chamber-mountedin vitro. Active potassium transport is not affected by ouabain and is not dependent on sodium. A long controversy has existed over the fraction of the midgut cells that participate in active transport of potassium. One set of investigators demonstrated that only a small fraction of the tissue potassium was involved in the pool of potassium involved in active transport whereas a separate set of workers found that virtually all of the exchangeable potassium was thus involved. The results presented in this paper show that the insect's diet affects whether some or all the cells are in the pool, known formally as a transport pool. Leaf-reared insects are characterized by a small pool whereas diet-reared insects are characterized by a large pool. These results are shown to correlate with the pool size results of previous investigators.     

Haemozoin formation in the midgut of the blood-sucking insect Rhodnius prolixus

Malaria parasites digest haemoglobin and detoxify the free haem by its sequestration into an insoluble dark-brown pigment known as haemozoin (Hz). Until recently, this pigment could be found only in Plasmodium parasites. However, we have shown that Hz is also present in the midgut of the blood-sucking insect Rhodnius prolixus [Oliveira et al. (1999) Nature 400, 517-518]. Here we show that Hz synthesis in the midgut of this insect is promoted by a particulate fraction from intestine lumen. Haem aggregation activity is heat-labile and is inhibited in vitro by chloroquine (CLQ). Inhibition of Hz formation in vivo by feeding insects with CLQ leads to increased levels of haem in the haemolymph of the insect, which resulted in increased lipid peroxidation. Taken together, these results indicate that a factor capable of promoting Hz crystallisation is present in R. prolixus midgut and that this activity represents an important physiological defence of this insect against haem toxicity.     

A simple mechanical method to isolate the basal lamina of insect midgut epithelial cells

In mealworms (Tenebrio molitor) the midgut epithelium is surrounded by a 1.6mum thick basal lamina of low electron density with a framework of high electron density imbedded in a part of it. The lamina can be isolated by ultrasonication followed by repeated filtrations and high-speed centrifugations, making large-scale preparation of the lamina for further analyses possible. The isolation of the basal lamina is confirmed by electron microscopy.     

Brush-border formation in the midgut of an insect,Calliphora erythrocephala meigen

Summary The embryonic development of the brush-border of anterior midgut cells of Calliphora was studied by electron microscopy. Dense surface-forming vesicles, as described by Bonneville (1970), are found prior to microvillus formation. These dense vesicles provide membranous and coating material for the moulding of the microvilli. The number of dense vesicles increases rapidly to a maximum just before brush-border formation, after which it decreases very rapidly, accompanied by an increase in the number of microvilli. Formation of microvilli proceeds in essentially the same way as in Xenopus. First, some of the vesicles fuse with the apical cell membrane, resulting in an increase of the cell surface, part of which is coated with filamentous material deriving from the dense vesicles. This in turn leads to bulging, and short irregular microvilli appear. These are erected and elongated.Prefabricated tubular elements are believed to play a part in this erection and elongation, probably due to the unwinding of spirally coiled strands.Microvillus formation proper lasts 2 to 3 hours in Calliphora. Almost the entire amount of membranous and coating material is prefabricated prior to the formation of microvilli.     

Ultrastructural study of cholecystokinin-like immunoreactivity in endocrine cells of the insect midgut

Summary Electron-microscopic immunocytochemistry for the demonstration of CCK-like material in basal endocrine cells of the midgut of Aeschna cyanea, Locusta migratoria, Carausius morosus and Periplaneta americana was performed by use of the peroxidase-antiperoxidase procedure and the colloidal gold method. Immunoreactive cells appeared scattered among digestive and regenerative cells of the epithelium. Immunoreactivity was specifically detected over round to oval electron-dense granules whose size appeared rather different from species to species. Thus, the average size of 30% (d30) of the largest granules ranged from 312 nm in Periplaneta americana to 159 nm in Carausius morosus with intermediate values in Aeschna cyanea (d30=195 nm) and Locusta migratoria (d30=225 nm).     

The molecular architecture of an insect midgut brush border cytoskeleton.

The cytoskeletal apparatus of the vertebrate intestinal brush border (BB) has served as a model system for the actin-based cytoskeleton of nonmuscle cells. In this study, we examine the structural organization and molecular architecture of the BB cytoskeleton expressed in the midgut of lepidopteran larvae, Manduca sexta. Electron microscopy of the midgut of the 5th instar larvae revealed enterocytes with an apical BB surface comparable to that in the vertebrate intestine, with both microvillar (MV) and terminal web (TW) domains, the latter defined by a zone of organelle exclusion directly beneath the MV. As reported previously for the larval dragon fly, the MV contain a bundle of actin filaments, as determined by staining with rhodamine phalloidin (Kukulies, J., et al., Protoplasma 121, 157-162 (1984)) and heavy meromyosin decoration (Komnick, H., J. Kukulies, Zoomorphology 107, 241-253 (1987)). Two-dimensional gel analysis revealed the presence of multiple isoelectric variants of actin with the major isoform corresponding to the non-muscle actin isoform II, expressed in Drosophila. Like the vertebrate BB, the Manduca BB can be isolated intact from enterocytes by mechanical shear. Immunochemical analysis of isolated BB fractions or whole homogenates of midgut revealed proteins of appropriate molecular weight immunoreactive with antibodies to the MV core proteins: BB myosin I, villin and fimbrin, and the TW components: spectrin, myosin II and tropomyosin. Immunocytochemical localization of a subset of these proteins at the light microscopic (spectrin) and electron microscopic (actin, villin, spectrin, myosin II, and tropomyosin) level reveals that the molecular architecture of the Manduca BB cytoskeleton is homologous to that found in vertebrates.     

DNA-RNA bodies in midgut cells of the stick insect, Bacillus rossius.

In the anterior part of the midgut and in the Malpighian tubules of the stick insect Bacillus rossius, about 10% of the epithelial cells develop endonuclear bodies which appear as DNA-RNA masses; in these cells the usual nucleoli are no longer evident. The DNA-RNA bodies are first formed in third instar larvae, become numerous in the fourth instar and persist in adults. In all larval instars and adults a different kind of DNA-body has been noticed in the epithelial cells of the posterior midgut. The DNA-RNA bodies of the anterior midgut and of the Malpighian tubules have been interpreted as the result of somatic gene amplification, whereas the DNA masses of the posterior midgut are likely due to a virus infection.     

昆虫图像自动鉴别技术

昆虫是地球上物种多样性最为丰富的生物类群,其物种鉴定任务复杂而艰巨,可靠的物种鉴定是开展昆虫学工作的重要基础之一。当前,国内外的人工昆虫物种鉴定能力均不能满足实际需求,因而人们开始不断探索利用计算机自动鉴定昆虫的原理和方法。目前,模式识别技术的迅猛发展已为昆虫图像的自动鉴定提供可能。文章概述昆虫图像自动鉴定技术研究的历史与现状,总结主要原理和方法,介绍工作流程,并展望发展前景。     

Preliminary measurements of intracellular calcium in an insect salivary gland using a calcium-sensitive microelectrode

A calcium-sensitive microeletrode was used to measure free intracellular calcium in salivary gland cells of Calliphora during stimulation with 5-hydroxytryptamine (5-HT). The resting level of calcium was approximately 10^?7M or less but increased in a dose-dependent way sometimes to levels in excess of 10^?6M. The onset of the calcium signal was closely related to changes in membrane and transepithelial potential. This calcium response was greatly reduced when the extracellular calcium concentration was reduced from 10^?3 to 10^?4M. This dependence on external calcium is consistent with previous observations that 5-HT acts to increase the permeability of the basal plasma membrane to calcium. These observations indicate that an increase in the intracellular level of calcium is an early event associated with the onset of fluid secretion in this insect salivary gland.     

Expression of Cry1Ac cadherin receptors in insect midgut and cell lines

Cadherin-like proteins have been identified as putative receptors for the Bacillus thuringiensis Cry1A proteins in Heliothis virescens and Manduca sexta. Immunohistochemistry showed the cadherin-like proteins are present in the insect midgut apical membrane, which is the target site of Cry toxins. This subcellular localization is distinct from that of classical cadherins, which are usually present in cell-cell junctions. Immunoreactivity of the cadherin-like protein in the insect midgut was enhanced by Cry1Ac ingestion. We also generated a stable cell line Flp-InT-REX-293/Full-CAD (CAD/293) that expressed the H. virescens cadherin. As expected, the cadherin-like protein was mainly localized in the cell membrane. Interestingly, toxin treatment of CAD/293 cells caused this protein to relocalize to cell membrane subdomains. In addition, expression of H. virescens cadherin-like protein affects cell-cell contact and cell membrane integrity when the cells are exposed to activated Cry1Ab/Cry1Ac.     

The maximum likelihood identification method applied to insect morphometric data

To distinguish species or populations using morphometric data is generally processed through multivariate analyses,in particular the discriminant analysis.We explored another approach based on the maximum likelihood method.Simple statistics based on the assumption of normal distribution at a single variable allows to compute the chance of observing a particular data (or sample) in a given reference group.When data are described by more than one variable,the maximum likelihood (MLi) approach allows to combine these chances to fmd the best fit for the data.Such approach assumes independence between variables.The assumptions of normal distribution of variables and independence between them are frequently not met in morphometrics,but improvements may be obtained after some mathematical transformations.Provided there is strict anatomical correspondence of variables between unknown and reference data,the MLi classification produces consistent classification.We explored this approach using various input data,and compared validated classification scores with the ones obtained after the Mahalanobis distance-based classification.The simplicity of the method,its fast computation,performance and versatility,make it an interesting complement to other classification techniques.     

Digestion of bacteria and the role of midgut lysozyme in some insect larvae.

1. Lysozyme is absent from tissues other than the midgut in the drug-feeding larvae of Musca domestica (Diptera, Cyclorrhapha, Muscidae) and in the fruit-feeding larvae of Anastrepha fraterculus (Diptera, Cyclorrhapha, Tephritidae), whereas in the detritus-feeding larvae of Trichosia pubescens (Diptera, Nematocera, Sciaridae) lysozyme is only found in the hemolymph and in the fat body. 2. A. fraterculus larvae have a midgut region with a luminal pH of 3.4, and display a pepstatin-inhibited acid proteolytic activity which has a spec. act. (7.2 U/mg protein) similar to that of M. domestica. 3. The midgut lysozyme from M. domestica and A. fraterculus is more active (high ionic strength) at pH 3.5 than at pH 6.0, the contrary being true for a midgut chitinase. 4. The results suggest that the adaptations to digest bacteria in insects are similar to those in vertebrate foregut fermenters, and that these characteristics were probably present in the Cyclorrhapha ancestor, but not in the Diptera ancestor.     

The intracellular pathway and kinetics of digestive enzyme secretion in an insect midgut cell

The opaque zone cells of the midgut of the stablefly, Stomoxys calcitrans display a cyclical series of ultrastructural events in response to feeding, which it has been suggested are related to the synthesis and secretion of digestive enzymes. These cells have been studied in vivo using a combination of biochemical, morphometric and electron microscopical autoradiographic techniques. The cyclical nature, timing and relationship of the ultrastructural events to enzyme secretion has been confirmed. The autoradiographic data presented is in good agreement with the classical synthetic pathway for exported proteins. The kinetics of the cellular process have been described: transfer of newly synthesized product from the rough endoplasmic reticulum to Golgi apparatus begins ca. 13-14 min after labelling of the fly and from the Golgi apparatus to secretory granules after ca. 24-26 min. Secretion of this newly synthesized material begins before 60 min and possibly as early as 30 min after labelling of the fly. The data are discussed in relation to the comparable studies in other tissues.     

Developmental and hormonal regulation of midgut remodeling in a lepidopteran insect, Heliothis virescens

Midgut tissue undergoes remodeling during metamorphosis in insects belonging to orders Lepidoptera and Diptera. We investigated the developmental and hormonal regulation of these remodeling events in lepidopteran insect, Heliothis virescens. In H. virescens, programmed cell death (PCD) of larval midgut cells as well as proliferation and differentiation of imaginal cells began at 108 h after ecdysis to the final larval instar (AEFL) and proceeded through the pupal stages. Expression patterns of pro- cell death factors (caspase-1 and ICE) and anti-cell death factor, Inhibitor of Apoptosis (IAP) were studied in midguts during last larval and pupal stages. IAP, Caspase-1 and ICE mRNAs showed peaks at 48 h AEFL, 96 h AEFL and in newly formed pupae, respectively. Immunohistochemical analysis substantiated high caspase-3 activity in midgut at 108 h AEFL. Application of methoprene, a juvenile hormone analog (JHA) blocked PCD by maintaining high levels of IAP, downregulating the expression of caspase-1, ICE and inhibiting an increase in caspase-3 protein levels in midgut tissue. Also, the differentiation of imaginal cells was impaired by methoprene treatment. These studies demonstrate that presence of JHA during final instar larvae affects both midgut remodeling and larval-pupal metamorphosis leading to larval/pupal deformities in lepidopteran insects, a mechanism that is different from that in mosquito, Ae. aegypti where JHA uncouples midgut remodeling from metamorphosis.     

Barium modifies the concentration dependence of active potassium transport by insect midgut

Summary The rate of active K⁺ transport by the isolated lepidopteran midgut shows a rectangular hyperbolic relation to [K⁺] over the range 20 to 70mm K⁺ in the absence of any divalent cation. Addition of Ba⁺⁺ to the hemolymph (K⁺ uptake) side introduces a linear component to the concentration dependence, such that active K^– transport is decreased at [K⁺] of 55mm or less, but increased transiently at higher [K⁺]. As [Ba⁺⁺] is increased over the range 2 to 8mm the linear component increases and the saturating component decreases; in 8mm Ba⁺⁺ the concentration dependence is dominated by the linear component. The effect of Ba⁺⁺ cannot easily be accounted for by simple competition with K⁺ for basal membrane uptake sites. Similar effects might be exercised by other alkali earth cations, since the concentration dependence of active K⁺ transport possesses a substantial linear component in solutions containing 5mm Ca⁺⁺ and 5mm Mg⁺⁺ (the alkali earth metal concentrations of standard lepidopteran saline).