Studies on water and ion transport in homopteran insects: ultrastructure and cytochemistry of the cicadoid and cercopoid Malpighian tubules and filter chamber

The filter chamber is a complex junction of anterior and posterior extremities of the midgut and Malpighian tubules. The sac-like anterior extremity, or filter chamber proper, comprises two cell types. These are large cuboidal cells which secrete a mucoprotein, and extremely thin cells which have regular tubular invaginations of the basal plasma membrane. The posterior extremity of the midgut and the internal Malpighian tubules coil round the filter chamber proper. They consist of thin epithelial cells identical in ultrastructure. The basal plasma membrane in these cells is formed into leaflets. A thin cellular sheath and thick muscle layers surround the filter chamber. The filter chamber proper is lined by the mucoprotein secretion of the cuboidal cells. This secretion appears to bind potassium ions. ATPase and alkaline phosphatase cannot be detected in the filter chamber epithelia. The structure and cytochemistry of the filter chamber suggests that water flows from filter chamber proper to midgut and Malpighian tubules by passive osmosis. This may be facilitated by ion binding in the filter chamber proper and by hydrostatic pressure engendered by contraction of the muscular coat. The Malpighian tubules appear to be structurally and chemically adapted for ion secretion by active transport and possibly for reabsorption in the Malpighian duct segment.     

Anatomy and fine structure of the alimentary canal of the spittlebug Lepyronia coleopterata (L.) (Hemiptera: Cercopoidea)

The alimentary canal of the spittlebug Lepyronia coleopterata (L.) differentiates into esophagus, filter chamber, midgut (conical segment, tubular midgut), and hindgut (ileum, rectum). The filter chamber is composed of the anterior extremity of the midgut, posterior extremity of the midgut, proximal Malpighian tubules, and proximal ileum; it is externally enveloped by a thin cellular sheath and thick muscle layers. The sac-like anterior extremity of the midgut is coiled around by the posterior extremity of the midgut and proximal Malpighian tubules. The tubular midgut is subdivided into an anterior tubular midgut, mid-midgut, posterior tubular midgut, and distal tubular midgut. Four Malpighian tubules run alongside the ileum, and each terminates in a rod closely attached to the rectum. Ultrastructurally, the esophagus is lined with a cuticle and enveloped by circular muscles; its cytoplasm contains virus-like fine granules of high electron-density. The anterior extremity of the midgut consists of two cellular types: (1) thin epithelia with well-developed and regularly arranged microvilli, and (2) large cuboidal cells with short and sparse microvilli. Cells of the posterior extremity of the midgut have regularly arranged microvilli and shallow basal infoldings devoid of mitochondria. Cells of the proximal Malpighian tubule possess concentric granules of different electron-density. The internal proximal ileum lined with a cuticle facing the lumen and contains secretory vesicles in its cytoplasm. Dense and long microvilli at the apical border of the conical segment cells are coated with abundant electron-dense fine granules. Cells of the anterior tubular midgut contain spherical secretory granules, oval secretory vesicles of different size, and autophagic vacuoles. Ferritin-like granules exist in the mid-midgut cells. The posterior tubular midgut consists of two cellular types: 1) cells with shallow and bulb-shaped basal infoldings containing numerous mitochondria, homocentric secretory granules, and fine electron-dense granules, and 2) cells with well-developed basal infoldings and regularly-arranged apical microvilli containing vesicles filled with fine granular materials. Cells of the distal tubular midgut are similar to those of the conical segment, but lack electron-dense fine granules coating the microvilli apex. Filamentous materials coat the microvilli of the conical segment, anterior and posterior extremities of the midgut, which are possibly the perimicrovillar membrane closely related to the nutrient absorption. The lumen of the hindgut is lined with a cuticle, beneath which are cells with poorly-developed infoldings possessing numerous mitochondria. Single-membraned or double-membraned microorganisms exist in the anterior and posterior extremities of the midgut, proximal Malpighian tubule and ileum; these are probably symbiotic.     

X-ray microanalysis of the filter chamber of the cicada,Cyclochila australasiae Don.

X-ray microanalysis of frozen-hydrated specimens of the cicada filter chamber shows that there are concentration changes in K+ and Cl- in the various compartments of the filter chamber from feeding to non-feeding states. These changes are consistent with an osmotic transfer of water between the anterior midgut and the posterior midgut and Malpighian tubules. K+ and Cl- account for a large proportion of the osmotically active solutes in these compartments, and it is suggested that the osmotic gradients are generated by secretion of K+ and Cl- into the posterior midgut and Malpighian tubule lumina. Analysis of freeze-substituted sections of filter chamber supports the analysis of frozen-hydrated specimens and indicates that Na+ may also be secreted into the posterior midgut and the Malpighian tubules. The system functions as a water-shunting complex.     

家蚕中的5-氧脯氨酸酶及保幼激素类似物对它的影响

本工作首次在家蚕Bombyx mori的马氏管、中肠、丝腺及脂肪体等组织中测到了γ-谷氨酰循环中一个关键酶——5-L-氧脯氨酸酶的活力.该酶以马氏管中活力最高,在蚕的中肠、血淋巴中均存在有游离的5-氧脯氨酸.观察了保幼激素类似物(JHA)处理后,家蚕中肠、丝腺和脂肪体中5-L-氧脯氨酸酶活力的变化,同时观察了血淋巴中5-氧脯氨酸含量的变化.对该酶及γ-谷氨酰循环在蚕体中氨基酸转运上的可能作用进行了讨论.     

The digestive system of the “stick bug” Cladomorphus phyllinus (Phasmida,Phasmatidae): A morphological,physiological and biochemical analysis

This work presents a detailed morphofunctional study of the digestive system of a phasmid representative, Cladomorphus phyllinus. Cells from anterior midgut exhibit a merocrine secretion, whereas posterior midgut cells show a microapocrine secretion. A complex system of midgut tubules is observed in the posterior midgut which is probably related to the luminal alkalization of this region. Amaranth dye injection into the haemolymph and orally feeding insects with dye indicated that the anterior midgut is water-absorbing, whereas the Malpighian tubules are the main site of water secretion. Thus, a putative counter-current flux of fluid from posterior to anterior midgut may propel enzyme digestive recycling, confirmed by the low rate of enzyme excretion. The foregut and anterior midgut present an acidic pH (5.3 and 5.6, respectively), whereas the posterior midgut is highly alkaline (9.1) which may be related to the digestion of hemicelluloses. Most amylase, trypsin and chymotrypsin activities occur in the foregut and anterior midgut. Maltase is found along the midgut associated with the microvillar glycocalix, while aminopeptidase occurs in the middle and posterior midgut in membrane bound forms. Both amylase and trypsin are secreted mainly by the anterior midgut through an exocytic process as revealed by immunocytochemical data.     

Morphology and ultrastructure of the alimentary canal of the cicada Platypleura kaempferi (Hemiptera: Cicadidae)

The alimentary canal of cicada Platypleura kaempferi is described. It comprises the oesophagus, filter chamber, external midgut section and hindgut. The elongate oesophagus expands posteriorly, with its posterior end constricting to become a bulb. The filter chamber consists of two parts: a very thin sheath and a filter organ. The filter organ is composed of the anterior and posterior ends of the midgut (internal midgut section), and the internal proximal ends of the Malpighian tubules. The external midgut section differentiates into a collapsed sac and a midgut loop. The latter is divided into three distinct segments. The hindgut contains a dilated rectum and a long narrow ileum. The distal portions of the four Malpighian tubules are enclosed in a peritoneal sheath together with the distal ileum before reaching to the rectum. Ultrastructurally, the oesophagus and the hindgut are lined with a cuticle. The filter chamber sheath consists of cells with large irregular nuclei. Filamentous substances coat the microvilli of the cells of the internal midgut section. The posterior end of the midgut comprises two types of cells, with the first type of cells containing many vesicles and scattered elements of rough endoplasmic reticulum. The anterior and posterior segments of the midgut loop cells have ferritin‐like granules. The ileum cells have well‐developed apical leaflets associated with mitochondria. Accumulations of virus‐like particles enclosed in the membrane are observed in the esophagus, conical segment, mid‐ and posterior segments of the midgut loop.     

Development of the Malpighian tubules in Cloeon dipterum (Ephemeroptera)

The development of the Malpighian tubules is studied in Cloeon dipterum through all stages from the youngest larva to the adult. The Malpighian tubules are found to be outgrowths of the posterior part of the endodermal midgut and not of the ectodermal hindgut. In the adult the part of the intestine with the tubule openings becomes separated by an ingrowing fold of the epithelium from the anterior main part of the midgut that forms a large thin-walled and air-filled bladder. The characteristics of the developmental stages, which served to determine the age of the animals, are given.     

The influence of ouabain on the elimination of injected and orally applied dyes in Drosophila hydei

The sour dyes azocarmine and indigocarmine are excreted through the Malpighian tubules and the midgut after injection into the body cavity of third instar Drosophila hydei larvae. After injection, the other organs are free of dyes. The epithelium of the midgut does not allow orally applied dyes to pass into the haemolymph. Ouabain diminishes significantly the content of dyes in the cavity of the Malpighian tubules and of the midgut. The maximal concentration of azocarmine decreases in the Malpighian tubules to about 65 per cent and in the midgut to about 70 per cent. Indigocarmine decreases in the Malpighian tubules to about 55 per cent. The content of indigocarmine of the midgut does not change significantly after ouabain injections. As ouabain inhibits active ion transport, the decrease of the concentration of dyes is seen as proof of the coupling of active ion transport processes and of excretion of the dyes. Moreover, this decrease points to an ouabain-sensitive transport mechanism, which is localized in the epithelia of the Malpighian tubules and midgut.     

Membrane-bound γ-glutamyl transpeptidase and its rôle in phenylalanine absorption-reabsorption in the larva of Musca domestica

Most of the γ-glutamyl transpeptidase (γ-GTP) activity of actively feeding third instar housefly larvae is located on the brush border of the proximal half of the Malpighian tubules and the brush border of epithelial cells of the anterior and posterior portions of the midgut. It is concluded that these membranes are the major sites of synthesis of the dipeptide, γ-l-glutamyl-l-phenyl-alanine (γ-glu-phe).In effect, γ-GTP and γ-glu-phe form a highly specific system for the absorption and reabsorption of phenylalanine from the lumen of the midgut and Malpighian tubules. Thus, membrane-bound γ-GTP combines with phenylalanine and glutathione and the resulting γ-glu-phe is translocated across the cell membrane and released within the cell. The dipeptide then enters the blood, presumably by simple diffusion in response to the concentration gradient generated by its build-up within the cell. It accumulates in the blood during larval growth and finally is consumed upon the onset of puparium tanning.Puparium formation was accompanied by an abrupt, ecdysone-induced appearance of intense γ-GTP activity on the epidermal cell membrane at the epidermis-cuticle interface. Epidermal cell γ-GTP activity was maximal 1 to 2 hr after puparium formation, after which time it began to diminish rapidly. It became virtually undectectable by the larval-pupal apolysis. Functionally, this hormonally induced new γ-GTP may catalyse reaction(s) which result in a rapid liberation of phenylalanine from γ-glu-phe for its subsequent conversion to tyrosine and quinones for tanning the puparium.The possibility that γ-GTP may also function in the transport of other amino acids in the housefly and in other insect genera is considered in terms of the Orlowski-Meister concept of a ‘γ-glutamyl cycle’.     

The ultrastructural investigation of the midgut in the quill mite Syringophilopsis fringilla (Acari,Trombidiformes: Syringophilidae)

The midgut of the females of Syringophilopsis fringilla (Fritsch) composed of anterior midgut and excretory organ (=posterior midgut) was investigated by means of light and transmission electron microscopy. The anterior midgut includes the ventriculus and two pairs of midgut caeca. These organs are lined by a similar epithelium except for the region adjacent to the coxal glands. Four cell subtypes were distinguished in the epithelium of the anterior midgut. All of them evidently represent physiological states of a single cell type. The digestive cells are most abundant. These cells are rich in rough endoplasmic reticulum and participate both in secretion and intracellular digestion. They form macropinocytotic vesicles in the apical region and a lot of secondary lysosomes in the central cytoplasm. After accumulating various residual bodies and spherites, the digestive cells transform into the excretory cells. The latter can be either extruded into the gut lumen or bud off their apical region and enter a new digestive cycle. The secretory cells were not found in all specimens examined. They are characterized by the presence of dense membrane-bounded granules, 2–4 μm in diameter, as well as by an extensive rough endoplasmic reticulum and Golgi bodies. The ventricular wall adjacent to the coxal glands demonstrates features of transporting epithelia. The cells are characterized by irregularly branched apical processes and a high concentration of mitochondria. The main function of the excretory organ (posterior midgut) is the elimination of nitrogenous waste. Formation of guanine-containing granules in the cytoplasm of the epithelial cells was shown to be associated with Golgi activity. The excretory granules are released into the gut lumen by means of eccrine or apocrine secretion. Evacuation of the fecal masses occurs periodically. Mitotic figures have been observed occasionally in the epithelial cells of the anterior midgut.     

黄脸油葫芦与短额负蝗马氏管组织结构比较

采用组织学方法对直翅目剑尾亚目和锥尾亚目的两个物种--黄脸油葫芦Teleogryllus emma和短额负蝗Atractomorpha sinensis成虫的马氏管进行了观察,发现两者在着生位置、方式和细胞结构上存在明显不同.着生位置上,黄脸油葫芦的马氏管着生在后肠前端与后肠后端的交界处,短额负蝗的马氏管着生在中肠与后肠的交界处.着生方式上,黄脸油葫芦的马氏管是通过一根无色透明的公共管与肠道相通的,而短额负蝗的马氏管分为12丛,每一丛直接与肠道相连.细胞结构上,黄脸油葫芦的管壁由8个细胞构成,且集中在管的中央,与管壁有空隙;而短额负蝗的管壁由3～4个细胞组成,分散在管壁外围,有马氏管凸.     

Intestinal expression of H+ V-ATPase in the mosquito Aedes albopictus is tightly associated with gregarine infection

Vacuolar ATPase (V-ATPase) is a family of ATP-dependent proton pumps expressed on the plasma membrane and endomembranes of eukaryotic cells. Acidification of intracellular compartments, such as lysosomes, endosomes, and parasitophorous vacuoles, mediated by V-ATPase is essential for the entry by many enveloped viruses and invasion into or escape from host cells by intracellular parasites. In mosquito larvae, V-ATPase plays a role in regulating alkalization of the anterior midgut. We extracted RNA from larval tissues of Aedes albopictus, cloned the full-length sequence of mRNA of V-ATPase subunit A, which contains a poly-A tail and 2,971 nucleotides, and expressed the protein. The fusion protein was then used to produce rabbit polyclonal antibodies, which were used as a tool to detect V-ATPase in the midgut and Malpighian tubules of mosquito larvae. A parasitophorous vacuole was formed in the midgut in response to invasion by Ascogregarina taiwanensis, confining the trophozoite(s). Acidification was demonstrated within the vacuole using acridine orange staining. It is concluded that gregarine sporozoites are released by ingested oocysts in the V-ATPase-energized high-pH environment. The released sporozoites then invade and develop in epithelial cells of the posterior midgut. Acidification of the parasitophorous vacuoles may be mediated by V-ATPase and may facilitate exocytosis of the vacuole confining the trophozoites from the infected epithelial cells for further extracellular development.     

In vivo binding of the Cry11Bb toxin of Bacillus thuringiensis subsp. medellin to the midgut of mosquito larvae (Diptera: Culicidae)

Bacillus thuringiensis subsp. medellin produces numerous proteins among which 94 kDa known as Cry11Bb, has mosquitocidal activity. The mode of action of the Cry11 proteins has been described as similar to those of the Cry1 toxins, nevertheless, the mechanism of action is still not clear. In this study we investigated the in vivo binding of the Cry11Bb toxin to the midgut of the insect species Anopheles albimanus, Aedes aegypti, and Culex quinquefasciatus by immunohistochemical analysis. Spodoptera frugiperda was included as negative control. The Cry11Bb protein was detected on the apical microvilli of the midgut epithelial cells, mostly on the posterior midgut and gastric caeca of the three mosquito species. Additionally, the toxin was detected in the Malpighian tubules of An. albimanus, Ae. aegypti, Cx. quinquefasciatus, and in the basal membrane of the epithelial cells of Ae. aegypti midgut. No toxin accumulation was observed in the peritrophic membrane of any of the mosquito species studied. These results confirm that the primary site of action of the Cry11 toxins is the apical membrane of the midgut epithelial cells of mosquito larvae.     

Allatotropin expression during the development of the fourth instar larvae of the kissing-bug Triatoma infestans (Klüg)

Allatotropin (AT) is a neuropeptide originally isolated from the brain of Manduca sexta and then characterized in several insect species. It acts as a neurohormone, as well as a neuromodulator. While it was primarily characterized on the basis of its ability to stimulate the secretion of juvenile hormones, it was also found that it acts as a cardioaccelerator and myostimulator. The presence of AT in IV instar larvae of T. infestans was previously described at the level of the Malpighian tubules. In the present study we report the presence of the peptide at the level of the brain, retrocerebral complex, as well as in the anterior midgut and aorta. The presence of AT at the corpora allata suggests that the peptide is acting on the gland during the first days of the moulting cycle. Neural processes at the level of the aorta and the anterior midgut suggests that, like in adults, the hormone is acting as a cardioaccelerator and myostimulator. The peptide was also found in open-type cells of the midgut. Finally the presence of allatotropic neurons in the optical lobe of the brain suggests that as in other species, the peptide is related with the control of circadian rhythms.     

CELLULAR SPECIALIZATION IN THE EXCRETORY EPITHELIA OF AN INSECT, Macrosteles fascifrons STÅL (HOMOPTERA)

An electron microscopic investigation of the Malpighian tubules of a leaf hopper, Macrosteles fascifrons, shows that these organs comprise three quite distinct cell types, and the structure of these and of the mid- and hindgut epithelial cells is described. In particular, a comparison is made between the organization of the basal and apical surfaces of cells in the Malpighian tubule and in the vertebrate kidney, and it is suggested that similarities between these excretory epithelia reflect functional parallels between them. While the midgut and one region of the Malpighian tubule bear a typical microvillar brush border, elsewhere in the tubule and in the hindgut the apical surface bears cytoplasmic leaflets or lamellae. The sole solid excretory material of these insects consists of the brochosomes, secreted by cells of one region of the Malpighian tubule. The structure, geometry, and development of these unusual bodies, apparently formed within specialized Golgi regions, has been investigated, and histochemical tests indicate that they contain lipid and protein components.     

庭疾灶螽中肠及马氏管结构

【目的】本研究旨在以庭疾灶螽Tachycines asynamorus为例探索驼螽消化系统和排泄系统在结构上与其生活环境的适应关系。【方法】运用解剖学方法、石蜡切片技术、冰冻切片技术及超薄切片技术对庭疾灶螽中肠及马氏管的结构进行研究。【结果】庭疾灶螽中肠向前延伸出3个胃盲囊包围着前胃。中肠上皮由再生细胞、柱状上皮细胞和内分泌细胞构成,具有典型的再生细胞龛;闭合型内分泌细胞紧贴在再生细胞龛的外围,基底区聚集大量的分泌颗粒。柱状上皮细胞内聚集有2类大的分泌颗粒：线团状颗粒和电子密度很高的球状颗粒;中肠管腔内有明显的围食膜结构,中肠基底部由基膜和肌肉层组成。马氏管着生在中后肠的交界处,从横切面看马氏管管壁具有3~5个细胞,细胞近管腔端部具有大量长微绒毛,细胞质内分布着电子致密的同心圆球晶体,基底膜内折形成膜迷路。【结论】庭疾灶螽中肠柱状上皮细胞的线团状颗粒由微丝包裹;内分泌细胞由再生细胞龛中的细胞分化而来,产生内分泌颗粒并将其排到血腔;中肠基膜发达,包含微丝与复合糖成分,基膜通过对中肠上皮细胞的支撑作用为肠道蠕动提供保障。庭疾灶螽马氏管细胞中可见大量颗粒和大量同心圆球晶体,推测可能是一种储存排泄。     

Larval anatomy and structure of absorbing epithelia in the aphid parasitoid Aphidius ervi Haliday (Hymenoptera, Braconidae)

The present work describes Aphidius ervi Haliday (Hymenoptera, Braconidae) larval anatomy and development, focusing on time-related changes of body structure and cell ultrastructure, especially of the epithelial layers involved in nutrient absorption. Newly hatched 1st instar larvae of A. ervi are characterised by gut absence and a compact cluster of cells makes up their body. As the parasitoid larva develops, the central undifferentiated cell mass becomes hollowed out, leading to the formation of gut anlage. This suggests that absorption of nutrients at that stage may take place through the body surface, as more directly demonstrated by the occurrence on the epidermis of proteins associated with transepithelial transport, such as Na(+)/K(+)-ATPase and alkaline phosphatase (ALP). Second instar larvae show the presence of the gut with a well-differentiated brush border and a peritrophic membrane. Gut cells are filled by masses of glycogen granules and lipid droplets. The tracheal system starts to be visible. The haemocoel becomes evident in late 2nd instar, and contains large silk glands. Mature 3rd instar larvae are typically hymenopteriform. The midgut accounts for most of the body volume and is actively involved in nutrient absorption, as indicated by the well developed brush border and by the presence of Na(+)/K(+)-ATPase and ALP on the basolateral and luminal membrane respectively. At this stage, large lipid droplets have gradually replaced the cellular glycogen stores in the midgut cells. The tracheae are completely differentiated, but their internal lumen still contains fibrillar material, suggesting that they are not functional as long as host fluids bath the parasitoid larva. In late 3rd instar larvae, silk glands, structurally similar to Malpighian tubules, show a very intense vesicular traffic toward the internal lumen, which, eventually, results in being filled by secretion products, suggesting the possible recycling of metabolic waste products during mummy formation.     

基于双元转基因CRISPR/Cas9系统的家蚕pax3基因功能分析

【目的】本研究旨在以家蚕Bombyx mori为研究模型探索pax3基因在鳞翅目昆虫中的生物学功能。【方法】利用PCR扩增验证家蚕Bmpax3外显子序列;利用qRT-PCR检测Bmpax3在5龄第3天家蚕幼虫头、表皮、脂肪体、中肠、马氏管、前部丝腺、中部丝腺、后部丝腺和生殖腺(包括精巢和卵巢)中的表达谱;利用双元转基因CRISPR/Cas9系统构建Bmpax3敲除突变体,分析Bmpax3突变对家蚕幼虫存活、体节分化及性别差异的影响。【结果】Bmpax3在家蚕5龄第3天幼虫头、中肠和丝腺中均有表达,其中在前部丝腺表达量最高。Bmpax3突变体的卵孵化率约为90%,但约有80%的突变体在1龄幼虫期死亡,有将近10%的突变个体能幸存并发育到成虫阶段,并且存活成虫数存在性别差异,雄性显著多于雌性。在幸存的成虫中,约有将近1/2的个体腹部末端体节分节异常,表皮条纹混乱,腹节腹板部分缺失,生殖器官及其周围的其他辅助器官出现发育缺陷。【结论】Bmpax3发生突变后会对家蚕的生存及形态发育产生较大的影响,提示Bmpax3可能参与了家蚕的生长发育过程。