Evidence for CRF-like and kinin-like peptides as neurohormones in the blood-feeding bug,Rhodnius prolixus

In Rhodnius prolixus, the rapid post-feeding diuresis is under neurohormonal control. While serotonin has been demonstrated to be a diuretic neurohormone [J Exp Biol 156 (1991) 557], a peptide is also known to be involved. Previously, we have demonstrated the presence of corticotropin releasing factor (CRF)-like and kinin-like peptides in the central nervous system (CNS) of 5th instar Rhodnius [J Exp Biol 202 (1999) 2017; Peptides 22 (2001) 161]. These peptides are present in neurohemal sites of the corpus cardiacum and are co-localized in neurohemal sites on abdominal nerves. While various CRF-like peptides have been demonstrated to increase Rhodnius Malpighian tubule secretion the kinin-like peptides do not [Peptides 23 (2002) 671]. The kinin-like peptides do however, increase hindgut contraction which may contribute to the rapid post feeding diuresis by the mixing of hemolymph and/or hindgut contents and the removal of wastes. The presence of these peptides in neurohemal sites suggests that they could be released into the hemolymph and act as neurohormones.We have used immunohistochemical techniques and radioimmunoassay (RIA) to demonstrate qualitative and quantitative changes of CRF-like and kinin-like peptides in the CNS associated with feeding. As well we have examined Malpighian tubule secretion in response to assays of hemolymph from unfed and fed insects. Hemolymph was also partially purified by Sep-Pak and HPLC and the fractions assayed for kinin-like immunoreactivity and the ability to stimulate Malpighian tubule secretion. The results suggest that both kinin-like and CRF-like peptides are neurohormones in Rhodnius, released in response to feeding.     

The distribution of a kinin-like peptide and its co-localization with a CRF-like peptide in the blood-feeding bug, Rhodnius prolixus

Rhodnius prolixus, a blood-feeding hemipteran insect, ingests large meals which are followed by rapid diuresis to eliminate excess water and salt. In Rhodnius, serotonin and an unidentified peptide(s) [33,34] have been shown to act as neurohormonal diuretic factors. In other insects, two families of diuretic peptides, the corticotropin releasing factor (CRF)-like, and kinin peptides [9], have been identified and sequenced. Recently, we demonstrated the presence of a CRF-like diuretic peptide in the CNS and digestive system of Rhodnius [47] using immunohistochemistry and bioassay.In this study, combining immunohistochemistry and radioimmunoassay (RIA) techniques, we show the presence of leucokinin-like peptide(s) in the CNS and digestive system of Rhodnius 5th instar. Additionally, double-label immunohistochemistry demonstrates that the leucokinin-like and CRF-like peptides are co-localized in the posterior lateral neurosecretory cells of the mesothoracic ganglionic mass (MTGM) and in neurohaemal areas on abdominal nerves one and two, suggesting the possibility of co-release of the peptides into the hemolymph.Partially purified extracts of the CNS and neurohaemal tissue were tested in vitro on Malpighian tubule secretion and cAMP assays. The factors eluting with increasing acetonitrile percentages from Sep-Pak cartridges were assayed in the presence or absence of ketanserin, a serotonin antagonist which blocks the effects of serotonin on Malpighian tubules. The results indicate activity of serotonin and a CRF-like diuretic peptide on Rhodnius Malpighian tubules, but fail to demonstrate activity of the leucokinin-like peptide(s).The rapid diuresis following feeding is a highly coordinated event, requiring the movement of water and salt across the epithelial cells of the crop into the hemolymph, and from the hemolymph across the cells of the Malpighian tubules. The urine then travels along the Malpighian tubules into the hindgut in order to be expelled. The presence of a leucokinin-like peptide(s) in the CNS and digestive system, which co-localizes with a CRF-like peptide(s), suggests that kinins may play a role in the rapid diuresis, although possibly not directly on the Malpighian tubules.     

Presence and activity of a Dippu-DH31-like peptide in the blood-feeding bug, Rhodnius prolixus

The blood-feeding bug, Rhodnius prolixus, ingests large blood meals, then undergoes a period of rapid diuresis which is under neurohormonal control. In both cockroach (Diploptera punctata) and fruit fly (Drosophila melanogaster) a calcitonin-like DH31 neuropeptide has been identified [Coast GM, Webster SG, Schegg KM, Tobe SS, Schooley DA. The Drosophila melanogaster homologue of an insect calcitonin-like diuretic peptide stimulates V-ATPase activity in fruit fly Malpighian tubules. J Exp Biol 2001;204:1795-804; Furuya K, Milchak RJ, Schegg KM, Zhang J, Tobe SS, Coast GM, et al. Cockroach diuretic hormones: characterization of a calcitonin-like peptide in insects. Proc Natl Acad Sci USA 2000;97:6469-74] and demonstrated to be active on Malpighian tubule secretion [Coast GM, Webster SG, Schegg KM, Tobe SS, Schooley DA. The Drosophila melanogaster homologue of an insect calcitonin-like diuretic peptide stimulates V-ATPase activity in fruit fly Malpighian tubules. J Exp Biol 2001;204:1795-804; Furuya K, Milchak RJ, Schegg KM, Zhang J, Tobe SS, Coast GM, et al. Cockroach diuretic hormones: characterization of a calcitonin-like peptide in insects. Proc Natl Acad Sci USA 2000;97:6469-74]. Using an antibody raised against D. punctata (Dippu) DH31, we demonstrate the presence of Dippu-DH31-like immunoreactivity in the CNS, salivary glands, hindgut and neurohemal sites of 5th instar Rhodnius. Double-label immunohistochemistry for Dippu-DH31-like and serotonin-like immunoreactivity demonstrates some co-localization of these factors in cells of the mesothoracic ganglionic mass (MTGM) and in neurohemal sites on the abdominal nerves. When tested on Rhodnius 5th instar Malpighian tubules, Dippu-DH31 stimulated minor increases in rate of secretion. Dippu-DH31 tested in combination with serotonin resulted in increases in the rate of secretion which were at least additive.     

The distribution and function of serotonin in the large milkweed bug,Oncopeltus fasciatus. a comparative study with the blood-feeding bug,Rhodnius prolixus

The blood-feeding hemipteran, Rhodnius prolixus, ingests a large blood meal at the end of each larval stage. To accommodate and process this meal, its cuticle undergoes plasticisation, and its gut and Malpighian tubules respectively absorb and secrete a large volume of water and salts for rapid diuresis. Serotonin has been found to be integral to the feeding process in this animal, along with a diuretic peptide(s). The large milkweed bug, Oncopeltus fasciatus, tends to feed in a more continuous and abstemious manner, and therefore may have different physiological requirements than the blood feeder. Unlike R. prolixus, O. fasciatus is lacking serotonin-like immunoreactive dorsal unpaired median neurons in the mesothoracic ganglionic mass, and lacks serotonin-like immunoreactive neurohaemal areas and processes on the abdominal nerves, integument, salivary glands, and anterior junction of the foregut and crop. The salivary glands and crop do, however, respond to serotonin with increased levels of cAMP, while the integument and Malpighian tubules do not. In addition, O. fasciatus Malpighian tubules respond to both O. fasciatus and R. prolixus partially purified CNS extracts, which are likely to contain any native diuretic peptides. Thus, while serotonin and diuretic peptides may be involved in tubule control in R. prolixus, the latter may be of greater importance in O. fasciatus.     

The biological activity of diuretic factors in Rhodnius prolixus

The rapid post-feeding diuresis of Rhodnius prolixus is under neurohormonal control and involves the integrated activity of the crop, Malpighian tubules and hindgut. One of the factors which is involved in this rapid diuresis is serotonin, however a peptide(s) is also considered to be involved. In other insects, corticotropin releasing factor (CRF)-like and kinin-like, calcitonin-like peptides and CAP(2b) have been demonstrated to be diuretic factors/hormones.In the present study, serotonin and CRF-like peptides increased secretion rate and cAMP content of Rhodnius Malpighian tubules, while the kinin-like peptides tested did not increase secretion rate or cAMP content of the tubules. Extracts of the CNS were processed and several HPLC fractions revealed kinin-like immunoreactivity but these fractions did not increase secretion rate when tested on Malpighian tubules. However, these same fractions did possess activity when tested on the hindgut contraction assay. In addition, material eluting at higher acetonitrile concentrations from the HPLC increased secretion and cAMP content of Rhodnius Malpighian tubules. This material eluted at concentrations of acetonitrile consistent with the elution time of CRF-like peptide standards.Synergism was demonstrated using the pharmacological agent forskolin and serotonin, tested on the rate of secretion of Rhodnius Malpighian tubules, in agreement with data of Maddrell et al. As well, synergism could be demonstrated using mesothoracic ganglionic mass (MTGM) homogenates and serotonin at some concentrations of serotonin. However, combinations of CRF-like material and serotonin increased secretion additively, not synergistically. Kinin-like peptides, tested along with CRF-like material and serotonin, at low concentrations, did not increase secretion above that of those factors tested alone.     

Beetle diuretic peptides: the response of mealworm (Tenebrio molitor) Malpighian tubules to synthetic peptides, and cross-reactivity studies with a dung beetle (Onthophagus gazella)

This paper reports the effects of different diuretic factors on the Malpighian tubules of beetles. Calcitonin (CT)-like peptides from silkmoth and mosquito increase fluid secretion in a dose-dependent manner in the tubules of Tenebrio molitor, but the cockroach CT-like peptide, Dippu-DH(31), has no effect. Thapsigargin induces a small but significant increase in tubule secretion rates. The interactions between different factors in mealworm tubules were explored by testing CT-like peptides, thapsigargin and the mealworm CRF-related diuretic factor Tenmo-DH(37) in various combinations, but no synergistic effects were observed. C-terminal fragments of the CRF-related diuretic peptides Locmi-DH(46) and Dippu-DH(46) fail to increase fluid secretion in mealworm tubules, unlike their corresponding whole peptides. Cross-reactivity of factors between beetle species was investigated using the scarabaeid Onthophagus gazella. Tenmo-DH(37) increases fluid secretion in isolated tubules of O. gazella in a dose-dependent manner, revealing a high degree of cross-reactivity in this distantly related beetle species. However, homogenates of O. gazella brains inhibited fluid secretion in mealworm tubules.     

Invitro effect of Canavalia ensiformis urease and the derived peptide Jaburetox-2Ec on Rhodnius prolixus Malpighian tubules

Ureases are metalloenzymes that are widespread among plants, fungi and bacteria. Urease isoforms (jack bean urease-JBU and canatoxin) from Canavalia ensiformis seeds are toxic to insects and fungi, suggesting a role in plant defense. The entomotoxic effect is due to the release of a 10-kDa peptide by cathepsin-like enzymes in the insect's midgut. Urease causes a decrease in post-feeding weight loss in Rhodnius prolixus, suggesting an effect on water balance. To investigate how this impairment occurs, we have evaluated the action of JBU and the urease-derivated peptide Jaburetox-2Ec on R. prolixus Malpighian tubules and also investigated the involvement of second messengers. JBU and Jaburetox-2Ec affect serotonin-induced secretion from Malpighian tubules. This effect is not cAMP-dependent, but the Jaburetox-2Ec effect is cGMP-dependent. Eicosanoid metabolites and calcium ions appear to be involved in JBU effect on diuresis, but are not involved in the action of Jaburetox-2Ec. Jaburetox-2Ec, but not JBU, causes a change in the transepithelial potential of the tubules. Canatoxin has a similar effect on tubules secretion, decreasing the secretion rate, but the urease from Helicobacter pylori has no significant effect. These data are helpful in our understanding of the actions of ureases and derived peptides on insects, and also reinforces the potential use of these proteins as biopesticides.     

The synthesis and biological activity of linear and cyclic analogs of the two diuretic peptides of Diploptera punctata

We have investigated the effect of analogs of the two Dippu diuretic hormones, Dippu-DH(46) and Dippu-DH(31), on fluid secretion by Malpighian tubules of male Diploptera punctata. We synthesized analogs containing the amino acid methyl-homoserine, to replace methionine residues, to render these modified peptides less subject to oxidation. We have also synthesized C-terminal fragments and their corresponding cyclic analogs to determine their effect on fluid secretion in D. punctata. Our results indicate that the modified peptides retain significant activity in the Ramsay secretion assay. The linear fragments displayed no activity or some inhibitory activity whereas the cyclic analog fragments showed stimulatory activity, in the case of DH(46), or slight inhibitory activity, in the case of DH(31).     

Evidence for hormonal control of diuresis after a blood meal in the mosquito Aedes aegypti

In previous studies we have presented evidence for the role of peptides, isolated from heads of the mosquito Aedes aegypti, in stimulating fluid secretion by isolated Malpighian tubules. In the present study we conducted experiments to investigate whether these peptides are involved in hormone-mediated diuresis after a blood meal. In vivo experiments showed that the head was required to maintain diuresis after the blood meal. Whereas feeding on blood triggered a prompt diuresis in the intact mosquito, subsequent decapitation caused a gradual, not an abrupt, decline in urine excretion rate. Hemolymph collected from mosquitoes fed blood significantly stimulated fluid secretion in vitro by isolated Malpighian tubules, whereas hemolymph from unfed or blood-fed decapitated mosquitoes did not. These results indicate that a diuretic factor was released into the hemolymph after a blood meal. This factor was not present in the hemolymph of decapitated females. We identified the head as a source of diuretic factors. Peptides isolated from a head extract by high-performance liquid chromatography, when injected into the hemocoel of blood-fed decapitated mosquitoes, triggered diuresis in vivo and also stimulated fluid secretion in isolated Malpighian tubules. These studies support the hypothesis that the head is a storage site for diuretic peptides that may be released after a blood meal to control diuresis.     

Variations induced by juvenile hormone and precocene II in the metabolic rate of 3 species of insects

The effects of juvenile hormone and precocoene II on the metabolic rate of Blattela germanica L. (Blattaria, Blattellidae), Spilostethus pandurus Scop. (Heteroptera: Lygaeidae) and Oncopeltus fasciatus Dallas (Heteroptera: Lygaeidae) 5th instar nymphs and adults have been investigated. Direct volumetric method was used to determine the oxygen consumption rate. JH produced a stage and/or sex dependent increase in the metabolic rate according to the species considered. On the other hand, PII decreased oxygen consumption in Blattella germanica and S. pandurus fifth instar and males, whereas O. fasciatus metabolic rate was not affected by the treatment.     

Hypothalamic peptides: central nervous system control of visceral functions

Thyrotropin-releasing factor (TRF), somatostatin, and bombesin-like peptide are present in the brain and may be involved in central nervous system (CNS) control of visceral functions. All three peptides exert potent actions to modify animal thermoregulation. TRF and somatostatin or somatostatin analogs act within the brain to influence parasympathetic and sympathetic outflow resulting in changes of adrenal epinephrine secretion, gastric acid secretion, heart rate, and blood pressure. Bombesin acts within the brain to increase adrenal epinephrine secretion and to inhibit gastric acid secretion without influencing other sympathetic or parasympathetic activities. These peptides and others may be important physiological regulators of CNS information processing related to a variety of visceral systems.     

Biostable insect kinin analogs reduce blood meal and disrupt ecdysis in the blood-gorging Chagas’ disease vector,Rhodnius prolixus

Rhodnius prolixus is a blood-gorging hemipteran that takes blood meals that are approximately 10 times its body weight. This blood meal is crucial for growth and development and is needed to ensure a successful molt into the next instar. Kinins are a multifunctional family of neuropeptides which have been shown to play a role in the control of feeding in a variety of insects. In this study, two biostable Aib-containing kinin analogs were tested to see if they interfere with blood-feeding and subsequent development into the next instar. One of the analogs, 1729 (Ac-R[Aib]FF[Aib]WGa), had no effect on the size of the blood meal or on the subsequent molting of the insect into the next instar. This analog also did not interfere with either short-term or long-term diuresis. The second analog, 1728 ([Aib]FF[Aib]WGa), appeared to be an antifeedant. Insects feeding on blood containing this analog (15 μM) only consumed 60% of the blood meal taken by insects fed on blood without analog. Insects feeding on blood containing 1728 had a slower rate of rapid diuresis (diuresis in the first 3–5 h after feeding) leading to less urine being excreted by 5 days post feeding. The consequence of these effects was that insects fed on 1728 did not molt. This data indicates that the biostable Aib-containing analog 1728 disrupts normal growth and development in the blood-feeding insect, R. prolixus.     

肺内调节肽对兔支气管上皮细胞分泌白介素的影响

为探讨肺内调节肽对气管上皮细胞（brochial epithelial cells,BECs）分泌功能的影响,实验观察了兔BECs在未受应激与臭氧应激两种条件下白细胞介素（interleukin,ILs）的分泌。结果发现：血管活性肠肽（vasoac-tive intestinal peptide,VIP）以未受应激BECs存在抑制作用,并使用臭氧应激BECs分泌ILs下降;表皮生长因子（epi-dermal growth factor,EGF）使未受应激BECs IL-1、IL-8分泌增加,使臭氧应激的BECs ILs分泌降低;内皮素-1（endothelin-1,ET-1）、降钙素基因相关肽(calctionin gene-related peptide,CGRP）可使未受应激的BECs分泌ILs增加,CGRP还可使臭氧应激的BECs ILs分泌增加。结果提示：肺内调节肽可调控BECs ILs的分泌,在调控气道炎症损伤信号传递方面具有一定的作用。     

AedesCAPA-PVK-1 displays diuretic and dose dependent antidiuretic potential in the larval mosquito Aedes aegypti (Liverpool)

This study reveals that AedesCAPA-PVK-1 (GPTVGLFAFPRV-NH(2)) inhibits basal and serotonin stimulated fluid secretion in the Malpighian tubules of larval Aedes aegypti at femtomolar concentrations. Conversely 10(-4)moll(-1) of the peptide stimulated fluid secretion rates. The diuretic effects of 10(-4)moll(-1)AedesCAPA-PVK-1 and antidiuretic effects of 10(-15)moll(-1)AedesCAPA-PVK-1 were abolished by protein kinase A (PKA) and protein kinase G (PKG) inhibition, respectively. Similar to the peptide, 10(-3)moll(-1) cGMP stimulated fluid secretion but doses in the micromolar to nanomolar range inhibited fluid secretion of the Malpighian tubules. Stimulatory effects of cGMP were abolished by PKA inhibition and inhibitory effects of cGMP were abolished by PKG inhibition. Furthermore, the nitric oxide synthase inhibitor l-NAME attenuated the inhibitory effects of AedesCAPA-PVK-1 but did not affect inhibition by cGMP. Based on the results we propose that AedesCAPA-PVK-1 inhibits fluid secretion rates of larval Malpighian tubules via the NOS/cGMP/PKG pathway and that high doses of the peptide lead to diuresis through the cGMP mediated activation of PKA.     

5-Hydroxytryptamine stimulates fluid secretion in locust malpighian tubules independently of cAMP

5-Hydroxytryptamine (5-HT) stimulates fluid secretion by semi-isolated Malpighian tubules of Locusta in a dose-dependent manner. The threshold of stimulation is between 10(-8) and 10(-7) M 5-HT; maximal activation occurs at doses greater than 10(-6) M. Relative to the activation induced by diuretic hormone (storage lobe extracts), 5-HT increases the rate of fluid secretion by only 65%. Phentolamine, the alpha-adrenergic blocker, failed to inhibit either DH or 5-HT stimulated secretion. Diuretic hormone raises the levels of intracellular of cAMP, and activates adenylate cyclase in plasma membrane preparations of Locusta Malpighian tubules. 5-HT (10(-4) M) has no effect in either assay system. Thus 5-HT can stimulate fluid secretion independently of cAMP. A hypothetical model for hormone stimulated fluid secretion by Locusta Malpighian tubules, involving dual-receptor activation, is proposed. Other biogenic amines, including octopamine, adrenalin, dopamine, synephrine and the formamidine chlordimeform were tested for their ability to stimulate fluid secretion. Only dopamine showed a weakly stimulatory effect.     

The control of Malpighian tubule secretion in a predacious hemipteran insect, the spined soldier bug Podisus maculiventris (Heteroptera, Pentatomidae)

Spined soldier bugs, Podisus maculiventris, are heteropteran insects that feed voraciously on other insects, particular the soft bodied larval forms of Lepidoptera and Coleoptera. The response of P. maculiventris Malpighian tubules (MTs) to serotonin and known diuretic and antidiuretic peptides has been investigated, and is compared with that of MT from the hematophagous and phytophagous heteropteran bugs Rhodnius prolixus and Acrosternum hilare, respectively. A CRF-related peptide diuretic hormone (DH) from the termite Zootermopsis nevadensis (Zoone-DH) stimulated MT secretion, which was reversed by a member of the CAP_2b family of peptides from A. hilare (Acrhi-CAP_2b-2), an antidiuretic effect. Serotonin had no effect on secretion, neither did a representative calcitonin-like DH, kinin, tachykinin-related peptide, and an antidiuretic factor from the mealworm Tenebrio molitor (Tenmo-ADFb) in both P. maculiventris or A. hilare. Serotonin is a DH in R. prolixus, and its lack of effect on MT from P. maculiventris and A. hilare suggests this is an adaptation to hematophagy. On the other hand, the antidiuretic activity of members of the CAP_2b family in all three bugs is consistent with this being a heteropteran feature rather than a specialism for hematophagy.     

Diuresis in a desert beetle? hormonal control of the malpighian tubules ofOnymacris plana (Coleoptera: Tenebrionidae)

Summary Malpighian tubules of a desert tenebrionid beetle,Onymacris plana, have been studied as isolated preparations. Under control conditions tubules of female beetles secreted fluid at an average rate of 3.3 nl/min, but this rate was increased 20–25 times by a diuretic hormone (DH).Homogenates of the brain, corpora cardiaca (CC) and prothoracic ganglion induced striking increases in tubule secretion rates, which sometimes exceeded 100 nl/min. The increased rates were sustained for 3 h without renewal of the medium. Diuretic activity was also present in the other thoracic ganglia. High K treatment caused release of DH from the CC only.Exogenous cyclic AMP (1 mM) stimulated the isolated tubules ofO. plana, but to a lesser extent than the DH. The cationic composition of the secreted fluid resembled that of most other insect tubules, with high K and low Na concentrations. Stimulation with DH doubled the Na concentration.The DH was not inactivated by the tubules themselves, but was destroyed by contact with the haemolymph. An inactivation mechanism is vital in the apparently contradictory situation of a desert beetle possessing a diuretic hormone. The role of the cryptonephric system during diuresis is unknown.Abbreviations DH diuretic hormone - cAMP adenosine 3:5-cyclic monophosphoric acid - CC corpora cardiaca     

小分子热激蛋白基因HaHSP19.8在棉铃虫抗逆反应中的作用

【目的】小分子热激蛋白(small heat shock protein, sHSP)在昆虫抵御外界环境压力中至关重要。本研究旨在探究小分子热激蛋白sHSP19.8基因在棉铃虫Helicoverpa armigera生长发育、抵御高温胁迫和对Cry1Ac杀虫蛋白抗性机制中的作用,为更深入探析该基因作用机理及棉铃虫的防治奠定基础。【方法】通过PCR结合RACE克隆棉铃虫sHSP19.8基因序列,利用生物信息学软件对该基因序列进行分析;通过qRT-PCR测定Cry1Ac敏感棉铃虫5龄幼虫在40℃高温下处理1 h和2 h及饲喂含30 μg/mL Cry1Ac的人工饲料1 h和2 h后该基因的表达量,并测定抗感Cry1Ac棉铃虫不同发育阶段(1-5龄幼虫、蛹及成虫)和5龄幼虫不同组织(前肠、中肠、后肠、马氏管及表皮)中该基因的表达模式。【结果】获得了棉铃虫sHSP19.8基因的全长cDNA序列,命名为HaHSP19.8(GenBank登录号: XP_021195228.1),长608 bp,开放阅读框长528 bp,编码175个氨基酸残基,具有小分子热激蛋白的典型α-晶体结构域(α-crystallin domain, ACD)。该基因受40℃高温和30 μg/mL Cry1Ac杀虫蛋白诱导时在Cry1Ac敏感棉铃虫5龄幼虫中均过量表达;在Cry1Ac敏感棉铃虫整个发育阶段和5龄幼虫各组织中均表达,其中在成虫和5龄幼虫以及5龄幼虫表皮、马氏管和中肠内表达量较高;但是该基因在Cry1Ac抗性品系各个发育阶段和5龄幼虫各组织中表达量相比敏感品系都显著较低。【结论】结果说明HaHSP19.8参与棉铃虫生长发育和生理生化的过程,帮助昆虫抵御外界环境压力,并可能参与到棉铃虫对Cry1Ac的抗性机制中。     

Natriuresis and diuretic hormone synergism in R. prolixus upper Malpighian tubules is inhibited by the anti-diuretic hormone, RhoprCAPA-α2

Insects contain an array of hormones that coordinate the actions of the excretory system to achieve osmotic and ionic balance. In the hematophagous insect, Rhodnius prolixus, two diuretic hormones have been identified, serotonin (5HT) and a corticotropin releasing factor-related peptide (RhoprDH), and both lead to an increase in fluid secretion by Malpighian tubules (MTs). However, only 5HT activates reabsorption by the lower MTs to recover K(+) and Cl(-). An anti-diuretic hormone (RhoprCAPA-α2) is believed to coordinate the cessation of the rapid diuresis following blood meal engorgement. However, the role of RhoprCAPA-α2 on fluid secretion by MTs stimulated by RhoprDH was previously unknown. Here we demonstrate that, unlike the inhibitory effect on 5HT-stimulated secretion by MTs, RhoprCAPA-α2 does not inhibit secretion stimulated by RhoprDH although it does abolish the synergism that occurs between the two diuretic hormones. In addition, we show that the natriuresis elicited by either diuretic hormone is reduced by RhoprCAPA-α2. Using electrophysiological tools, we investigate the possible mechanism by which this complex regulatory pathway is achieved. Analysis of the pH of secreted fluid as well as the triphasic response in transepithelial potential in MTs treated with diuretic hormones, suggests that RhoprCAPA-α2 does not inhibit the V-type H(+) ATPase. Taken together, these results indicate that RhoprCAPA-α2 functions to reduce the rapid diuresis following blood feeding, and in addition, it inhibits the natriuresis associated with diuretic hormone stimulated MTs. This may reflect an important regulatory mechanism related to the slow diuresis that occurs as the K(+)-rich blood cells are digested.     

胞质型肽聚糖识别蛋白RfPGRP-L2在维持红棕象甲肠道菌群稳态中的作用

【目的】明确入侵害虫红棕象甲Rhynchophorus ferrugineus胞质型肽聚糖识别蛋白RfPGRP-L2在肠道菌群稳态的维持和调控过程中的作用,将为靶向破坏肠道菌群稳态的害虫控制新策略研发提供新的科学依据和作用靶标。【方法】利用生物信息学方法分析RfPGRP-L2的序列特征。利用RT-qPCR分析RfPGRP-L2在健康红棕象甲4龄幼虫不同组织(头、脂肪体、表皮、前肠、中-/后肠、血淋巴)以及大肠杆菌Escherichia coli DH5α和金黄色葡萄球菌Staphylococcus aureus经注射（注射1 μL OD₆₀₀=1.6的菌液）和喂食（取食涂抹1 mL OD₆₀₀=1.6的菌液的甘蔗薄片）两种不同方式分别感染后红棕象甲4龄幼虫肠道和脂肪体中的表达量;进行RfPGRP-L2原核表达,利用体外孵育方法检测重组蛋白RfPGRP-L2对大肠杆菌DH5α和金黄色葡萄球菌的凝集和抑菌活性;RNAi干扰RfPGRP-L2后,检测红棕象甲4龄幼虫血淋巴和肠道中大肠杆菌菌落数的变化;利用RT-qPCR分析RNAi干扰RfPGRP-L2后红棕象甲4龄幼虫脂肪体和肠道中抗菌肽基因表达量的变化;利用基于细菌16S rRNA的高通量测序分析RNAi干扰RfPGRP-L2对健康红棕象甲4龄幼虫肠道菌群结构组成的影响。【结果】SMART预测发现红棕象甲RfPGRP-L2基因编码的蛋白中无跨膜结构域也无信号肽,这表明RfPGRP-L2是一种胞质型肽聚糖识别蛋白。RT-qPCR检测发现,RfPGRP-L2主要在健康红棕象甲4龄幼虫血淋巴、肠道和脂肪体等免疫组织中表达;被注射感染大肠杆菌和金黄色葡萄球菌6 h和12 h后,红棕象甲4龄幼虫脂肪体中RfPGRP-L2的表达量分别显著上调;被喂食感染大肠杆菌6 h后,红棕象甲4龄幼虫肠道中RfPGRP-L2的表达量显著增加。重组表达蛋白RfPGRP-L2能引起大肠杆菌和金黄色葡萄球菌发生凝集反应,这说明RfPGRP-L2能够识别这两种细菌。当RfPGRP-L2被干扰后,红棕象甲4龄幼虫对肠道和血淋巴中感染EGFP标记的大肠杆菌的清除能力显著弱于对照组;肠道中抗菌肽基因RfCecropin的表达量显著降低;健康红棕象甲4龄幼虫肠道中细菌的菌落数量显著高于对照组,而且肠道菌群结构组成也发生了明显的变化。【结论】红棕象甲体内胞质型肽聚糖识别蛋白RfPGRP-L2能够通过识别细菌并激活肠上皮细胞中相应的免疫信号通路促进抗菌肽基因的表达,从而介导对肠道菌群稳态的调控。