Major differences in the larval anatomy of the digestive and excretory systems of three Oestridae species revealed by micro-CT.

Oestrid flies (Diptera: Oestridae) do not feed during the adult stage, so they depend on an efficient assimilation and storage of nutrients during their parasitic larval stage. We describe the general morphology and provide volumetric data for the digestive and excretory organs of the three larval instars of the nasal bot fly Oestrus ovis L., using micro-computed tomography. The size of the digestive and excretory organs greatly increased across larval instars. In all instars, the two salivary glands were remarkably large and formed a 'glandular band' by coming together, but without lumina uniting, at their posterior ends. The distal region of the anterior Malpighian tubules was greatly enlarged and full of highly radio-opaque concretions. Moreover, the anatomy of O. ovis third-instar larva was compared to that of two species of, respectively, similar and different feeding habits: Cephenemyia stimulator (Clark) and Hypoderma actaeon Brauer. Whereas the general morphology and arrangement of the digestive and excretory systems of C. stimulator was similar to that of O. ovis, some differences were observed in H. actaeon: a swollen anterior region of the midgut, salivary glands shorter and not forming a 'band' and anterior Malpighian tubules narrowly uniform throughout their entire length.

Excretion in the mother's body: modifications of the larval excretory system in the viviparous dermapteran, Arixenia esau.

The vast majority of Dermaptera are free-living and oviparous, i.e., females lay eggs within which embryonic development occurs until the larva hatches. In contrast, in the epizoic dermapteran Arixenia esau, eggs are retained within mother's body and the embryos and first instar larvae develop inside her reproductive system. Such a reproductive strategy poses many physiological challenges for a mother, one of which is the removal of metabolic waste generated by the developing offspring. Here, we examine how the Arixenia females cope with this challenge by analyzing features of the developing larval excretory system. Our comparative analyses of the early and late first instar larvae revealed characteristic modifications in the cellular architecture of the Malpighian tubules, indicating that these organs are functional. The results of the electron probe microanalyses suggest additionally that the larval Malpighian tubules are mainly involved in maintaining ion homeostasis. We also found that the lumen of the larval alimentary track is occluded by a cellular diaphragm at the midgut-hindgut junction and that cells of the diaphragm accumulate metabolic compounds. Such an organization of the larval gut apparently prevents fouling of the mother's organism with the offspring metabolic waste and therefore can be regarded as an adaptation for viviparity.

Differential expression of putative sodium-dependent cation-chloride cotransporters in Aedes aegypti.

The yellow fever mosquito, Aedes aegypti, has three genes that code for proteins with sequence similarity to vertebrate Na+-K+-Cl- cotransporters (NKCCs) of the solute-linked carrier 12 superfamily of cation-chloride cotransporters (CCCs). We hypothesized that these mosquito NKCC orthologues have diverged to perform distinct roles in salt secretion and absorption. In phylogenetic analyses, one protein (aeNKCC1) groups with a Drosophila melanogaster NKCC that mediates salt secretion whereas two others (aeCCC2 and aeCCC3) group with a Drosophila transporter that is not functionally characterized. The aeCCC2 and aeCCC3 genes probably result from a tandem gene duplication in the mosquito lineage; they have similar exon structures and are consecutive in genomic DNA. Predicted aeCCC2 and aeCCC3 proteins differ from aeNKCC1 and vertebrate NKCCs in residues from the third transmembrane domain known to influence ion and inhibitor binding. Quantitative PCR revealed that aeNKCC1 and aeCCC2 were approximately equally expressed in larvae and adults, whereas aeCCC3 was approximately 100-fold more abundant in larvae than in adults. In larval tissues, aeCCC2 was approximately 2-fold more abundant in Malpighian tubules compared to anal papillae. In contrast, aeCCC3 was nearly 100-fold more abundant in larval anal papillae compared to Malpighian tubules, suggesting a role in absorption. Western blots with polyclonal antibodies against isoform-specific peptides revealed stronger aeCCC2 immunoreactivity in adults versus larvae, whereas aeCCC3 immunoreactivity was stronger in larvae versus adults. The differential expression pattern of aeCCC2 and aeCCC3, and their sequence divergence in transmembrane domains, suggests that they may have different roles in transepithelial salt transport.

Multicopper oxidase-1 is required for iron homeostasis in Malpighian tubules of Helicoverpa armigera.

Multicopper oxidases (MCOs) are enzymes that contain 10 conserved histidine residues and 1 cysteine residue. MCO1 has been extensively investigated in the midgut because this MCO is implicated in ascorbate oxidation, iron homeostasis and immune responses. However, information regarding the action of MCO1 in Malpighian tubules is limited. In this study, Helicoverpa armigera was used as a model to investigate the function of MCO1 in Malpighian tubules. Sequence analysis results revealed that HaMCO1 exhibits typical MCO characteristics, with 10 histidine and 1 cysteine residues for copper ion binding. HaMCO1 was also found to be highly abundant in Malpighian tubules. Temporal expression patterns indicated that HaMCO1 is mainly expressed during larval molting stages. Hormone treatments [the molting hormone 20-hydroxyecdysone (20E) and juvenile hormone (JH)] revealed that 20E inhibits HaMCO1 transcript expression via its heterodimer receptor, which consists of ecdysone receptor (EcR) and ultraspiracle (USP), and that JH counteracts the action of 20E to activate HaMCO1 transcript expression via its intracellular receptor methoprene-tolerant (Met). HaMCO1 knockdown caused a significant decrease in iron accumulation and also significantly reduced transferrin and ferritin transcript expression. Therefore, HaMCO1 is coordinately regulated by 20E and JH and is required for iron homeostasis in Malpighian tubules.

A comprehensive transcriptomic view of renal function in the malaria vector, Anopheles gambiae.

Renal function is essential to maintain homeostasis. This is particularly significant for insects that undergo complete metamorphosis; larval mosquitoes must survive a freshwater habitat whereas adults are terrestrial, and mature females must maintain ion and fluid homeostasis after blood feeding. To investigate the physiological adaptations required for successful development to adulthood, we studied the Malpighian tubule transcriptome of Anopheles gambiae using Affymetrix arrays. We assessed transcription under several conditions; as third instar larvae, as adult males fed on sugar, as adult females fed on sugar, and adult females after a blood meal. In addition to providing the most detailed transcriptomic data to date on the Anopheles Malpighian tubules, the data provide unique information on the renal adaptations required for the switch from freshwater to terrestrial habitats, on gender differences, and on the contrast between nectar-feeding and haematophagy. We found clear differences associated with ontogenetic change in lifestyle, gender and diet, particularly in the neuropeptide receptors that control fluid secretion, and the water and ion transporters that impact volume and composition. These data were also combined with transcriptomics from the Drosophila melanogaster tubule, allowing meta-analysis of the genes which underpin tubule function across Diptera. To further investigate renal conservation across species we selected four D. melanogaster genes with orthologues highly enriched in the Anopheles tubules, and generated RNAi knockdown flies. Three of these genes proved essential, showing conservation of critical functions across 150 million years of phylogenetic separation. This extensive data-set is available as an online resource, MozTubules.org, and could potentially be mined for novel insecticide targets that can impact this critical organ in this pest species.

The molecular correlates of organ loss: the case of insect Malpighian tubules.

Malpighian tubules play an essential role in excretion, osmoregulation and immunity of most insects. Exceptionally, aphids lack Malpighian tubules, providing the opportunity to investigate the fate of genes expressed in an organ that has undergone evolutionary reduction and loss. Making use of the sequenced genomes of Drosophila melanogaster and the pea aphid Acyrthosiphon pisum, we demonstrated that more than 50% of Drosophila genes expressed specifically in the Malpighian tubules had orthologues in the pea aphid genome and that most of the pea aphid orthologues with detectable expression were identified in the gut transcriptome. Relative to the whole genome, genes functioning in amino acid metabolism are significantly over-represented among the pea aphid orthologues of Malpighian tubule genes, likely reflecting the central importance of amino acid acquisition and metabolism in aphids. This study demonstrates that the evolutionary loss of a key insect organ, the Malpighian tubules, has not been associated with the coupled loss of molecular functions.

Malpighian tubule development in the red flour beetle (Tribolium castaneum).

Malpighian tubules (MpTs) are the major organ for excretion and osmoregulation in most insects. MpT development is characterised for Drosophila melanogaster, but not other species. We therefore do not know the extent to which the MpT developmental programme is conserved across insects. To redress this we provide a comprehensive description of MpT development in the beetle Tribolium castaneum (Coleoptera), a species separated from Drosophila by >315 million years. We identify similarities with Drosophila MpT development including: 1) the onset of morphological development, beginning when tubules bud from the gut and proliferate to increase organ size. 2) the tubule is shaped by convergent-extension movements and oriented cell divisions. 3) differentiated tip cells activate EGF-signalling in distal MpT cells through the ligand Spitz. 4) MpTs contain two main cell types - principal and stellate cells, differing in morphology and gene expression. We also describe development of the beetle cryptonephridial system, an adaptation for water conservation, which represents a major modification of the MpT ground plan characterised by intimate association between MpTs and rectum. This work establishes a new model to compare MpT development across insects, and provides a framework to help understand how an evolutionary novelty - the cryptonephridial system - arose during organ evolution.

Shaping up for action: the path to physiological maturation in the renal tubules of Drosophila.

The Malpighian tubule is the main organ for excretion and osmoregulation in most insects. During a short period of embryonic development the tubules of Drosophila are shaped, undergo differentiation and become precisely positioned in the body cavity, so they become fully functional at the time of larval hatching a few hours later. In this review I explore three developmental events on the path to physiological maturation. First, I examine the molecular and cellular mechanisms that generate organ shape, focusing on the process of cell intercalation that drives tubule elongation, the roles of the cytoskeleton, the extracellular matrix and how intercalation is coordinated at the tissue level. Second, I look at the genetic networks that control the physiological differentiation of tubule cells and consider how distinctive physiological domains in the tubule are patterned. Finally, I explore how the organ is positioned within the body cavity and consider the relationship between organ position and function.

Organ growth without cell division: somatic polyploidy in a moth, Ephestia kuehniella.

Organ growth depends on cell division and (or) cell growth. Here, we present a study on two organs whose growth depends entirely on cell growth, once they are formed in the embryo: Malpighian tubules and silk glands of the flour moth, Ephestia kuehniella . Between first and last larval instar, the volume of Malpighian tubule cells increases by a factor of ∼1800 and that of silk gland cells by a factor of ∼3100. We determined the number of endocyles required to reach these stages by Feulgen cytometry. Cells of Malpighian tubules were in the 2C stage in first instar larvae and reached 1024C after 9 endocycles in last instar larvae (1C = 0.45 pg DNA). Silk gland cells already reached a DNA content of 8C-16C in first instar larvae and attained up to 8192C in last instar larvae after a total of 12 endocycles. The nuclei were small and more or less spherical in first instar larvae, but they were huge, flat, and bizarrely branched in last instar larvae. We consider branching as a compensatory adaptation to improve molecular traffic between nucleus and cytoplasm in these excessively large and highly polyploid cells (i) by reducing the mean distance between nucleus and cytoplasm and (ii) by enlarging the surface-to-volume ratio of these nuclei.

Immune response and anti-microbial peptides expression in Malpighian tubules of Drosophila melanogaster is under developmental regulation.

Malpighian tubules (MT) of Drosophila melanogaster are osmoregulatory organs that maintain the ionic balance and remove toxic substances from the body. Additionally they act as autonomous immune sensing organs, which secrete antimicrobial peptides in response to invading microbial pathogens. We show that the antimicrobial peptides (AMP) diptericin, cecropinA, drosocin and attacinA are constitutively expressed and are regulated in developmental stage specific manner. Their developmental expression begins from 3(rd) instar larval stage and an immune challenge increases the expression several folds. Spatial variations in the level of expression along the MT tissue are observed. The mortality of 3(rd) instar larvae fed on bacterial food is much less than that of the earlier larval stages, coinciding with the onset of innate immunity response in MT. Ectopic expression of AMP imparts better resistance to infection while, loss of function of one of the AMP through directed RNAi reduces host survival after immune challenge. The AMP secreted from the MT exhibit bactericidal activity. Expression of the NF-κB transcription factor, Relish, also coincides with activation of immune responsive genes in MT, demonstrating that immune regulation in MT is under developmental control and is governed by the Imd pathway.

Non-apoptotic function of apoptotic proteins in the development of Malpighian tubules of Drosophila melanogaster.

Drosophila metamorphosis is characterized by the histolysis of larval structures by programmed cell death, which paves the way for the establishment of adult-specific structures under the influence of the steroid hormone ecdysone. Malpighian tubules function as an excretory system and are one of the larval structures that are not destroyed during metamorphosis and are carried over to adulthood. The pupal Malpighian tubules evade destruction in spite of expressing apoptotic proteins, Reaper, Hid, Grim, Dronc and Drice. Here we show that in the Malpighian tubules expression of apoptotic proteins commences right from embryonic development and continues throughout the larval stages. Overexpression of these proteins in the Malpighian tubules causes larval lethality resulting in malformed tubules. The number and regular organization of principal and stellate cells of Malpighian tubules is disturbed, in turn disrupting the physiological functioning of the tubules as well. Strikingly, the localization of beta-tubulin, F-actin and Disclarge (Dlg) is also disrupted. These results suggest that the apoptotic proteins could be having non-apoptotic function in the development of Malpighian tubules.

The ultrastructure of malpighian tubules and the chemical composition of the cocoon of Aeolothrips intermedius Bagnall (Thysanoptera).

The secretory activity of the two branched malpighian tubules (MTs) of the second-instar larva in Aeolothrips intermedius is described. MTs of adult thrips have the typical ultrastructure of excretory epithelium with apical microvilli containing long mitochondria and a rich system of basal membrane infoldings. In the second-instar larva just before pupation, the ultrastructure of MT epithelial cells is dramatically different, and there are numerous huge Golgi systems in the cytoplasm. These cells are involved in an intense secretory activity to produce an electron-dense product which is released into the MTs lumen. This secretion is extruded from the hindgut and used by the second-instar larva to build an elaborate protective cocoon for pupation. Electron-spray-ionization mass spectrometry analysis of the cocoon revealed the presence of a beta-N-acetyl-glucosamine, the main component of chitin, which is also present in the cocoons of Neuroptera and some Coleoptera.

Developmental expression of Manduca shade, the P450 mediating the final step in molting hormone synthesis.

The ecdysone 20-monooxygenase (E20MO; 20-hydroxylase) is the enzyme that mediates the conversion of ecdysone (E) to the active insect molting hormone, 20-hydroxyecdysone (20E), which coordinates developmental progression. We report the identification and developmental expression of the Halloween gene shade (shd; CYP314A1) that encodes the E20MO in the tobacco hornworm, Manduca sexta. Manduca Shd (MsShd) mediates the conversion of E to 20E when expressed in Drosophila S2 cells. In accord with the central dogma, the data show that Msshd is expressed mainly in the midgut, Malpighian tubules, fat body and epidermis with very low expression in the prothoracic gland and nervous system. Developmental variations in E20MO enzymatic activity are almost perfectly correlated with comparable changes in the gene expression of Msshd in the fat body and midgut during the fifth instar and the beginning of pupal-adult development. The results indicate three successive and overlapping peaks of expression in the fat body, midgut and Malpighian tubules, respectively, during the fifth larval instar. The data suggest that precise tissue-specific transcriptional regulation controls the levels, and thereby the activity, of the Manduca E20MO.

The signals that drive kidney development: a view from the fly eye.

PURPOSE OF REVIEW: Development of the mammalian kidney is a complex process involving numerous signals and signaling pathways. Other complex tissues have benefited enormously from studies in lower, simpler organisms. The present review provides an update on what we have learned from the fruitfly Drosophila melanogaster, and argues that Drosophila is an important but under-utilized organism for study of renal development. RECENT FINDINGS: The Malpighian tubules provide renal function to the fly. These require a number of signaling pathways for their development that are also seen in vertebrate kidney development, including the Notch, Ras, and Wnt signaling pathways, as well as nuclear factors such as Krüppel and Cut/Cux-1. Many of these factors are shared between early Malpighian tubule development and ureteric bud formation. The Ret signaling receptor, which is central to mammalian renal development, is poorly understood in flies, although its expression pattern is intriguing. Surprisingly, other signaling factors such as Neph-1, Pax2, and Wilms' tumor suppressor-1 appear to work within later fly retinal development, providing a surprising link between these two disparate tissues. SUMMARY: Drosophila offers a powerful palate of tools for dissecting developmental processes. Importantly, these tools can often be examined at the level of single cells, permitting us to address issues of differentiation with high resolution. If we are to take full advantage of Drosophila, however, then we must target specific issues and gain a better understanding of the details of Malpighian tubule development.

Emc, a negative HLH regulator with multiple functions in Drosophila development.

Expression and functional analyses of Emc have demonstrated that it is a prototype for a protein required for multiple processes in development. Initially characterized as a negative regulator of sensory organ development, it was later found to regulate many other developmental processes and cell proliferation. Its ability to block the function of bHLH proteins by forming heterodimers, which are ineffective in DNA binding, accounts for the role of Emc in preventing the acquisition of several cell fates which are under the control of bHLH proteins. However, while maintaining this repressive molecular mechanism, emc also appears to act as a positive regulator of differentiation.

Morphometric changes associated with sex and development in the Malpighian tubules of Aedes aegypti.

The Malpighian tubules of Aedes aegypti showed significant differences in their diameters between male and female larvae, male and female pupae, male larvae and male adults and male pupae and male adults. In every case, female values were greater than in males. Measurements of mean nuclear areas of the principal and stellate cells from Malpighian tubules, taken in males and females during development, showed that this parameter in both types of cell was significantly greater in females than in male larvae, pupae and adult stages. In males, significant differences between developmental stages were observed only in comparison with the nuclear area of larvae and adults in the principal cells, but in females, every comparison between stages showed significant differences except between pupae and adults in stellate cells. The frequency distribution of nuclear area values, in development, for male stellate and principal cells, were mostly concentrated in the first seven classes among the 30 classes considered in every stage, while for females, the frequency dropped drastically in the same classes from larvae to pupae and adults, moving to classes of higher values. Considering the importance of Malpighian tubules in insect physiology, the meaning of the differences detected are discussed on the basis of different metabolic levels, between sexes and developmental stages.

Seven-up, the Drosophila homolog of the COUP-TF orphan receptors, controls cell proliferation in the insect kidney.

Morphogenesis of the insect kidney, the Malpighian tubules, is controlled in Drosophila by a single large cell, the tip cell. It has been postulated that this cell sends out a mitogenic signal that induces the division of neighboring cells. The signal and the molecules that receive it have remained elusive. We show that the COUP-TF-related nuclear orphan receptor Seven-up is a key component that becomes induced in response to mitogenic EGF receptor signaling activity emanating from the tip cell. Seven-up in turn is capable of regulating the transcription of cell cycle regulators.

Functional domains are specified to single-cell resolution in a Drosophila epithelium.

Specification of pattern is fundamental to the development of a multicellular organism. The Malpighian (renal) tubule of Drosophila melanogaster is a simple epithelium that proliferates under the direction of a single tip cell into three morphologically distinct domains. However, systematic analysis of a panel of over 700 P[GAL4] enhancer trap lines reveals unexpected richness for such an apparently simple tissue. Using numerical analysis, it was possible formally to reconcile apparently similar or complementary expression domains and thus to define at least five genetically defined domains and multiple cell types. Remarkably, the positions of domain boundaries and the numbers of both principal and secondary ("stellate") cell types within each domain are reproducible to near single-cell precision between individual animals. Domains of physiological function were also mapped using transport or expression assays. Invariably, they respect the boundaries defined by enhancer activity. These genetic domains can also be visualized in vivo, both in transgenic and wild-type flies, providing an "identified cell" system for epithelial physiology. Building upon recent advances in Drosophila Malpighian tubule physiology, the present study confirms this tissue as a singular model for integrative physiology.

Larval and imaginal pathways in early development of Drosophila.

In holometabolous development, higher insects have two different life forms, the larva and the imago. Both larval and imaginal cells are derived from cells of the blastoderm stage. After the final embryonic wave of mitosis, however, only the imaginal cells remain diploid, proliferate massively and do not differentiate until metamorphosis. The separation of these two pathways was described by many authors as a fundamental process that must take place at a very early stage of development, most probably the blastoderm stage. Mainly by using single cell transplantations at the blastoderm or early gastrula stages, respectively, we found common cell lineages between larval and imaginal structures by clones overlapping in the ectoderm (i.e. larval epidermal cells and imaginal discs within a segment, or larval and imaginal salivary gland cells), the mesoderm (i.e. larval somatic muscles and adepithelial cells), and the endoderm (i.e. larval and imaginal midgut cells). From these findings we conclude that it seems to be a principle in Drosophila embryogenesis that the separation of larval and imaginal pathways is postponed to a later developmental stage.