Ultrastructure of osmoregulatory organs in larvae of the brackish-water mosquito,Culiseta inornata (Williston)

The ultrastructure of the Malpighian tubules, ileum, rectum, anal canal, and anal papillae of larvae of the mosquito Culiseta inornata was examined. The Malpighian tubules, rectum, and anal papillae have many of the ultrastructural features characteristic of ion transport tissues, i.e., elaboration of the basal and apical membranes and a close association of these membranes with mitochondria. The Malpighian tubules possess two cell types, primary and stellate. The larval rectum of C. inornata is composed of a single segment containing a homogenous population of cells. In this respect, the larval rectum of C. inornata is distinct from that of saline-water species of Aedes. The cells in the larval rectum of C. inornata, however, closely resemble those of one cell type, the anterior rectal cells, of the saline-water mosquito Aedes campestris with regard to cell and nuclear size, the percentage of the cell occupied by apical folds, and mitochondrial density and distribution. No similarities can be found between the rectum of C. inornata and the posterior segment of the saline-water Aedes, which functions as a salt gland. On this basis, we have postulated that the rectum of C. inornata does not function as a site of hyperosmotic fluid secretion. The ultrastructure of the anal papillae of C. inornata is consistent with a role in ion transport. The significance of these findings to comparative aspects of osmoregulatory strategies in mosquito larvae is discussed.     

Cellular differentiation in relation to water vapour absorption in the rectal complex of the mealworm, Tenebrio molitor

Larvae of the mealworm beetle Tenebrio molitor, are able to absorb water vapour from subsaturated air, but adults cannot. This functional difference is paralleled by structural differences in the cryptonephridial rectal complex, the site of vapour absorption in the larva. Very distinctive differences occur in the rectal pad epithelium and these are reported in detail for the first time. The cells of the larval rectal pad are very closely apposed, forming a structural, and presumably functional, unit, whereas the cells of the adult rectal pad are more clearly separate. Intracellular organization also shows clear differences. These differences indicate that the rectal epithelium may play a more important role in vapour absorption than recently ascribed to it. Other, less striking, differences in the cryptonephric Malpighian tubules and perinephric membrane as previously recorded have largely been confirmed. Morphometric analysis suggests that diffusion alone could account for the observed absorption of water vapour across the larval system from rectal lumen to the lumen of the cryptonephric tubules, but this does not rule out the possibility that other transporting mechanisms are also involved. Radial diffusion and antero-posterior gradients may be facilitated by the predominently radial and circumferential arrangement of the rectal pad cells and the surrounding cryptonephric tubules. Reinvestigation of the isolating perinephric membrane and its insertion onto the rectal cuticle supports the conclusions that insertion occurs only posteriorly. The model incorporating anterior as well as posterior insertion does not apply. The membrane posteriorly encloses a single perirectal space between rectum and tubules and in this region no perinephric or peritubular space is found between inner and outer regions of the membrane. This is the region where maximal gradients occur across the system.     

Rectal water absorption in seawater-adapted Japanese eel Anguilla japonica

Marine teleosts drink large amounts of seawater to compensate for continuous osmotic water loss. We investigated a possible significant role of the rectum in water absorption in seawater-adapted eel. In rectal sacs filled with balanced salt solution (BSS) and incubated in isotonic BSS, water absorption was greater in seawater-adapted eel than in freshwater eel. Since rectal fluid osmolality was slightly lower than plasma osmolality in seawater-adapted eel, effects of rectal fluid osmolality on water absorption were examined in rectal sacs filled with artificial rectal fluid with different osmolality. Rectal water absorption was greater at lower rectal fluid osmolality, suggesting that an osmotic gradient between the blood and rectal fluid drives the water movement. Ouabain, a specific inhibitor of Na⁺/K⁺-ATPase, inhibited water absorption in rectal sacs, indicating that an osmotic gradient favorable to rectal water absorption was created by ion uptake driven by Na⁺/K⁺-ATPase. Expression levels of aquaporin 1 (AQP1), a water-selective channel, were significantly higher in the rectum than in the anterior and posterior intestines. Immunoreaction for Na⁺/K⁺-ATPase was detected in the mucosal epithelial cells in the rectum with more intense staining in the basal half than in the apical half, whereas AQP1 was located in the apical membrane of Na⁺/K⁺-ATPase-immunoreactive epithelial cells. The rectum is spatially separated from the posterior intestine by a valve structure and from the anus by a sphincter. Such structures allow the rectum to swell as intestinal fluid flows into it, and a concomitant increase in hydrostatic pressure may provide an additional force for rectal water absorption. Our findings indicate that the rectum contributes greatly to high efficiency of intestinal water absorption by simultaneous absorption of ions and water.     

Vertical transmission of photosymbionts in the colonial ascidian Didemnum molle: the larval tunic prevents symbionts from attaching to the anterior part of larvae

Morphological processes in the vertical transmission of photosymbionts were investigated in the Prochloron-bearing ascidian Didemnum molle. Prochloron cells were found exclusively in the common cloacal cavity of the colony, attached mainly to the tunic lining of the cavity wall. Oocytes were found in the abdominal region of each zooid, but no Prochloron cells were associated with this stage. During embryogenesis, embryos moved into the tunic core of the colony and were always separated from Prochloron cells in the cloacal cavity by the tunic matrix, until they hatched out from the tunic core. In swimming larvae, Prochloron cells covered the surface of the posterior half of the larval trunk, whereas a thin larval tunic layer covered the anterior half, where no Prochloron cells were found. The tunic of the posterior half of the larval trunk had many folds that enfolded the Prochloron cells and may be adhesive in order to acquire Prochloron cells from the mother colony. The thin larval tunic layer is probably not adhesive and protects the anterior half of the trunk from interference by Prochloron cells with sensory receptors and adhesive organs.     

Surface ultrastructure of third-instar Megaselia scalaris (Diptera: Phoridae)

We describe some ultrastructure of the third-instar Megaselia scalaris (Diptera: Phoridae) using scanning electron microscopy, with the cephalic segment, anterior spiracle and posterior spiracle being emphasized. This study provides the taxonomic information of this larval species, which may be useful to differentiate from other closely-related species.     

Evidence for a frontal-organ homologue in the pineal complex of the salamander,Hynobius dunni

The pineal complex of larval and adult salamanders, Hynobius dunni, was examined by light and scanning electron microscopy. This pineal complex displays an anterior and a posterior portion, both of which possess a lumen. The anterior lumen is small and closed, whereas the posterior lumen is in open communication with the third ventricle. Cell processes of the photoreceptor cells and microvilli of the supportive cells are visible in both lumina. The anterior part of the complex is formed by an independent, second evagination from the common pineal anlage; this process takes place immediately after hatching. The anterior body of the pineal complex of H. dunni appears to be homologous to the frontal organ of anurans.     

The protocerebral neurosecretory system and associated cerebral neurohemal area of Acheta domesticus

Summary A comparison of rectal morphology and ultrastructure is made between a freshwater (A. aegypti) and salt water (A. campestris) species of mosquito larvae, and between A. campestris larvae producing hyper- and hyposmotic urine.The epithelium of A. aegypti contains one cell type characterized by infolding of both the apical and basal membranes, straight lateral borders, and evenly distributed mitochondria.The rectum of A. campestris contains distinct anterior and posterior regions, each made up of a single cell type. These two regions can be distinguished on the basis of cell thickness, depth of apical infolding and distribution of mitochondria. The anterior region is similar to the rectum of A. aegypti, while the posterior region is considered unique to the salt-water species and hence probably is associated with the formation of hyperosmotic urine.In A. campestris, the apical (rather than lateral or basal) membranes are probably the site of hyperosmotic urine production. Two possible mechanisms for this process are discussed.This work was supported by operating grants from the National Research Council of Canada.     

A conserved role for the MEK signalling pathway in neural tissue specification and posteriorisation in the invertebrate chordate,the ascidian Ciona intestinalis

Ascidians are invertebrate chordates with a larval body plan similar to that of vertebrates. The ascidian larval CNS is divided along the anteroposterior axis into sensory vesicle, neck, visceral ganglion and tail nerve cord. The anterior part of the sensory vesicle comes from the a-line animal blastomeres, whereas the remaining CNS is largely derived from the A-line vegetal blastomeres. We have analysed the role of the Ras/MEK/ERK signalling pathway in the formation of the larval CNS in the ascidian, Ciona intestinalis. We show evidence that this pathway is required, during the cleavage stages, for the acquisition of: (1) neural fates in otherwise epidermal cells (in a-line cells); and (2) the posterior identity of tail nerve cord precursors that otherwise adopt a more anterior neural character (in A-line cells). Altogether, the MEK signalling pathway appears to play evolutionary conserved roles in these processes in ascidians and vertebrates, suggesting that this may represent an ancestral chordate strategy.     

Observations on first and second-instar larvae of Megaselia scalaris (Loew) (Diptera: Phoridae).

The ultrastructure of the first and second-instar larvae of Megaselia scalaris (Loew) was studied using scanning electron microscopy (SEM). Significant changes in morphological features were observed in the anterior and posterior spiracles, but only minimal changes in the labium and mouthhooks were seen. The ultrastructure of M. scalaris larvae not only provides chronological transformation of their larval instars, but it can also be used to explain their feeding behavior and mode of respiration. In addition, morphological structures useful for specific identification of first or second-instar larvae collected from human corpses may be used in forensic investigations.     

Morphological and ultrastructural characterization of the alimentary canal in larvae of Streltzoviella insularis (Staudinger) (Lepidoptera: Cossidae)

Streltzoviella insularis (Staudinger) is an important tree‐boring pest, that primarily damages Sophora japonica (Linnaeus) and Ginkgo biloba (Linnaeus), as well as other common species, at great economic cost to the urban landscape construction industry in China. In the present study, the alimentary canal morphology of S. insularis was observed using light microscopy, and its ultrastructure was investigated by scanning and transmission electron microscopy. The foregut of S. insularis can be divided into the pharynx, esophagus, crop, proventriculus, and cardiac valve. The well‐developed crop forms the longest section of the foregut. It is able to store large amounts of food and is lined with a monolayer of epithelial cells. Many sclerotized microspines occur on the surface of the anterior intima and there are dense spines on the posterior intima of the proventriculus. Epithelial cells of the midgut include columnar cells, goblet cells, and regenerative cells, but endocrine cells are absent. The hindgut consists of the pyloric valve, ileum, and rectum. There is no clear distinction between the ileum and colon. The intima surface of the pyloric valve carries many microspines, whereas the intestinal wall of the rectum is thin with well‐developed rectal pads. The rectal epithelial cells form a squamous monolayer. A cryptonephric excretory system is located in the hindgut. There are six spiral Malpighian tubules, in which a cellular layer on a basement membrane encloses a lumen. These results will provide the basis for further studies of the structure and function in S. insularis larvae.     

Electroneutral cation-Cl- cotransporters NKCC2β and NCCβ expressed in the intestinal tract of Japanese eel Anguilla japonica

In the present study, we aimed to elucidate the mechanisms of intestinal Na(+) and Cl(-) absorption in Japanese eel, focusing on electroneutral cation-Cl(-) cotransporters, NKCC2β and NCCβ, expressed in the intestinal tract. First, we cloned cDNAs encoding NKCC2β and NCCβ from the intestinal tract of Japanese eel. In both freshwater- and seawater-acclimated eels, quantitative PCR analysis showed that NKCC2β was predominantly expressed in the anterior and posterior intestines, and that NCCβ expression was specifically high in the rectum. According to immunohistochemistry with anti-eel NKCC2β (reacting with NKCC2β but not with NCCβ) and T4 antibody (reacting with both NKCC2β and NCCβ), NKCC2β was localized in the apical surface of the epithelial cells in the anterior and posterior intestines, whereas NCCβ was likely to be distributed to that in the rectum. Furthermore, a specific NCC inhibitor, hydrochlorothiazide, inhibited of Na(+) and Cl(-) absorption, as well as water absorption, in the rectal sac preparations from seawater eel, indicating the involvement of NCCβ in ion absorption in the rectum. Our findings indicate that NKCC2β expressed in the anterior and posterior intestines and NCCβ in the rectum are importantly involved in ion absorption to reduce osmolality of ingested seawater prior to water absorption in seawater-acclimated eel.     

Ultrastructure of the hindgut of Drosophila larvae, with special reference to the domains identified by specific gene expression patterns

The hindgut of Drosophila larvae consists of nine domains that have been distinguished by specific gene expression patterns. In the present study, we examined the ultrastructure of the hindgut of Drosophila larvae, with special reference to the domains, in order to determine whether or not the domains are morphologically distinct functional units. Each domain showed specific ultrastructural features that suggested specific corresponding functions. According to the morphological features, terms are proposed for each domain: the imaginal ring; the "pylorus," which has a thick cuticular layer and well-developed sphincter muscles; the "large intestine," which occupies a major middle portion of the hindgut and has a unique dorsal and ventral subdivision; "border cells," which delineate the anterior and posterior borders of the large intestine and the border between the dorsal and ventral domains of the large intestine; and the "rectum," which is situated at the posterior end of the hindgut and has a thick cuticular layer and sphincter muscles. The morphological features indicate that the large intestine has active absorptive activities. The domains, which have been distinguished by gene expressions, were demonstrated to be functional tissue units of the gut.     

The microdistribution of the trichomycete Smittium culisetae in the hindgut of the black fly host Simulium vittatum

We examined the distribution of hyphae of the trichomycete fungus Smittium culisetae (Harpellales: Legeriomycetaceae) in the hindgut of a larval black fly (Simulium vittatum, cytospecies IS-7) by analyzing its prevalence and relative abundance. Hyphal prevalence was highest in the posterior colon (93.1%) and rectum (86.3%), with low prevalence (12.0%) in the anterior colon. Relative abundance of hyphae was highest in the posterior colon, followed by the rectum; relative abundance of hyphae in the anterior colon was lower. Hyphae of S. culisetae were not observed in the pylorus. We used a novel method of quantifying the relative abundance of S. culisetae in the host hindgut. The hindgut was observed with an ocular grid, and abundance was expressed as the ratio of grids occupied by hyphae to the number of grids occupied by hindgut.     

Studies on water and ion transport in homopteran insects: ultrastructure and cytochemistry of the cicadoid and cercopoid midgut

The midgut of cicadoid and cercopoid insects is differentiated at the anatomical, ultrastructural and cytochemical levels into a conical segment, anterior, mid, and posterior midgut. The cells of the conical segment and anterior midgut are cytochemically very similar. They differ in ultrastructure, the anterior midgut cells having a submicrovillar row of mitochondria and a very marked mucoprotein coat investing the microvilli. The mid-midgut contains mineral spherites, which are formed in cisternae in the endoplasmic reticulum, and ferritin. The posterior midgut differs cytochemically from the anterior midgut and the cells are characterized by deep narrow basal invaginations and the absence of a mucoprotein coat investing the microvilli. It is suggested that nutrient absorption occurs in the conical segment and anterior midgut. Ion absorption may also occur in the anterior midgut. Storage excretion of calcium, magnesium and phosphate occurs in the mid-midgut. Ferritin is also stored here but may be found in other regions of the midgut, particularly in the cicada. The posterior midgut may be involved in ion secretion which could be related to filter chamber function.     

Active muscle migration during insect metamorphosis

The R1 abdominal retractor muscles of the insect Tenebrio molitor change position during the course of metamorphosis. These muscles detach from the epidermal tendon cells at their anterior ends, and migrate in a posterior direction, parallel to the body axis, to form completely new attachments shortly before adult emergence. Movement is preceded by the loss of sarcomere structure, and the muscles migrate in a partially dedifferentiated condition, closely accompanied by satellite cells and haemocytes. Movement appears to result from the extension of muscle processes towards the epidermis posterior to the larval attachment sites, which contact reciprocal processes extended from the epidermis. Contacts at the new posterior sites are then reinforced, and relinquished at the anterior. This cycle is subsequently repeated. It is envisaged that migration ceases when the muscles encounter a contour in the epidermal gradient known to specify the position of the adult muscle attachment sites. This positional information may be encoded in the epidermal basal lamina. The muscles then redifferentiate, with concurrent differentiation of new epidermal tendon cells. Development of adult muscle attachments appears to require reciprocal morphogenetic interactions between muscle and epidermis.     

Fine structure of the intestine development in cultured sea bream larvae

At hatching, the gut cells of Sparus aurata are quite undifferentiated; however, slight ultrastructural differences can already be distinguished between the presumptive intestinal regions. The hindgut cells are more differentiated than midgut cells and the rectal cells show rather particular ultrastructural features. During days 1 (D1) and 2 (D2) after hatching, major changes occur that lead to full differentiation of the epithelial cells. Shortly before the onset of exogenous feeding (D3), the anterior intestine enterocytes can synthesize lipoprotein particles (LP) from endogenous lipids. The posterior intestine enterocytes show morphological features indicating a role in absorption and intracellular digestion of nutrients, whereas the rectal cells do not. Transient ciliated cells occur at hatching (D0) in the presumptive intestine, except in the caudal rectum, and disappear at the start of the late endotrophic phase about 3 days after hatching (D3). At hatching, very scarce enteroendocrine and leucocyte-like cells are found at the base of the gut epithelium. Their number increases throughout development. At D3 (late endotrophic phase), LP synthesized mainly in the periblast invade the circulatory system, interstitial spaces of the subepithelial tissue and intercellular spaces of the gut epithelium. When the endo-exotrophic phase begins (D4), the enterocytes can absorb exogenous food. Acid phosphatase activity was detected in microvilli, pical vacuoles and Golgi complex in both anterior and posterior enterocytes, as well as in supranuclear vacuoles (SNV) of posterior enterocytes, but not in the apical tubulovesicular system (TVS). During the exotrophic phase, large lipid droplets (LD) are found in anterior enterocytes, and the SNV occupy a large cell volume in posterior enterocytes. LP accumulate first in extracellular spaces and then are transferred to the circulatory system. Mucous and rodlet cells appear in the intestinal epithelium during the exotrophic phase, from D15.     

Morphogenesis and organogenesis in the regenerating planktotrophic larvae of asteroids and echinoids

In a previous study, we described complete body regeneration (with organogenesis) following surgical bisection in the planktotrophic larvae of the asteroids Luidia foliolata and Pisaster ochraceus. Here we present further detailed observations of these unique regenerative processes not presented in the previous paper. Furthermore, we describe for the first time complete regeneration following surgical bisection of planktotrophic larvae of the regular echinoid Lytechinus variegatus and the irregular echinoid Dendraster excentricus. Larvae of both asteroids and echinoids displayed a capacity for rapid regeneration regardless of their developmental stage. Within 48 h after bisection, aggregations of mesenchyme cells with pseudopodia were observed at the site of surgical bisection. These cellular aggregations were similar in appearance to the mesenchymal blastemas that form in adult echinoderms prior to their arm regeneration, and to those described in other deuterostomes that undergo regeneration. When asteroid larvae were surgically bisected in the early stages of their development, clusters of mesenchyme cells developed into completely new pairs of coelomic pouches located anterior to the newly regenerated digestive tract. This indicates that cell fate in regenerating asteroid larvae remains indeterminate during early development. In the larvae of P. ochraceus, regardless of the developmental stage at the time of bisection, both the anterior and posterior portions regenerated all their missing organs and tissues. However, the larvae of L. foliolata displayed differential regenerative capacity in bisected larval halves at the late bipinnaria stage. The differences observed may be due to differences in larval development (L. foliolata has no brachiolaria stage), and may have evolutionary implications. In the regular echinoid L. variegatus, both larval portions regenerated into morphologically and functionally normal larvae that were indistinguishable from non-bisected control larvae. The regenerative processes were similar to those we observed in planktotrophic asteroid larvae. Regenerating larvae readily metamorphosed into normal juveniles. In the irregular echinoid D. excentricus, posterior portions of larvae completed regeneration and metamorphosis, but anterior portions regenerated only partially during the 2-week study. Our observations confirm that asteroid and echinoid larvae provide excellent models for studies of regeneration in deuterostomes.     

Digestive System of Trichodorus porosus

The onchiostyle of Trichodorus porosus has an anterior outer portion, a fine inner spear and a posterior onchiostyle extension. The extension has a ventral lumen and is fused to the pharynx wall. The inner spear enters the dorsal wall of the outer onchiostyle posterior to the guide ring and extends anteriorly inside the anterior portion of the onchiostyle. Muscle cells are absent in the basal position of the esophagus. The glandular portion of the basal part of the esophagus consists mainly of endoplasmic reticulum lined with ribosomes. A sinus empties into the lumen through the dorsal esophageal gland orifice. The configuration of the intesinal lumen is highly variable. The rectum is attached to the dorsal and ventral walls of the body cavity by striated rectal muscle cells.     

Trypanosoma cruzi: origin of metacyclic trypomastigotes in the urine of the vector Triatoma infestans

Population density and percentage of the different stages of an established infection of Trypanosoma cruzi were determined for two parts of the excretory system and for the rectum of fifth instars of Triatoma infestans unfed and 4 hr after feeding. These data were also evaluated for feces and urine of the fed bugs. In the first unfed group only small populations of the flagellate occurred in the Malpighian tubules and ampullae and not in all bugs. The three rectal populations (rectal lumen and anterior and posterior rectal wall) consisted of approximately equal numbers. About 10% were spheromastigotes and about 10% were stages intermediate to epimastigotes. Significantly fewer epimastigotes and more trypomastigotes were present on the rectal wall than in the lumen. Two intermediate forms leading to the trypomastigote stage occurred in similar numbers. In nearly all bugs the initial excretion (feces) contained the highest number of flagellates as compared to the following drops of urine. More flagellates were excreted through the urine than were contained in the excretory system of unfed bugs. The population in the feces reflected the percentage of forms present in the rectal lumen of unfed bugs, but in the urine the percentage of trypomastigotes increased up to 100%. Four hours after blood uptake, dissection of bugs still showed parasites in the Malpighian tubules and ampullae; the total number of parasites in the rectum was reduced by more than 50%. This reduction was more pronounced in the rectal lumen and on the posterior rectal wall. In stained smears from all three rectal populations there were rarely spheromastigotes but high percentages of epimastigotes. The intermediate stages leading to trypomastigotes mainly originated from short epimastigotes. Comparison of the T. cruzi populations before and after feeding demonstrates that the trypomastigotes in the urine should originate from the rectal wall, especially from the posterior part.     

Ultrastructure of the tail region of the second stage preparasitic larva of the root-knot nematode

Studies on the tail of second-stage infective larvae (L₂s) of Meloidogyne javanica, M. incognita and M. hapla from a region anterior to the rectal gland to the tail tip have revealed the presence of a previously undescribed sensory organ, the caudal sensory organ, in the posterior region of the tail.The extreme tip of the tail consists of solid cuticle and the different zones of this structure are described throughout the tail region. Longitudinal sections through the anus and rectum have revealed that the cortical (external cortical, epicuticle) layer gradually decreases in thickness until its outermost layer appears to merge with the plasma membrane of the rectal inflation or gland. This gland appears to be similar in all three species studied.The two phasmidial glands and their canals are described from transverse sections. Somatic muscle is first found in the region of the anus and it extends anteriorly throughout most of the length of the L₂. Depressor ani muscles which insert on the dorsal surface of the rectum are also described.The rectal gland in the dilated state occupies about three quarters of the diameter of the L₂. It contains a matrix which resembles that extruded from the adult female rectal gland cells. The rectal gland cells contain large amounts of rough endoplasmic reticulum and desmosomes are found close to the junction of these cells and the plasma membrane of the gland itself. More anteriorly in the L₂ most of the area is taken up by large lipid droplets which function as an energy reserve.It is suggested that the rectal gland should not be used as a taxonomic criterion for separating the La_2^S of Meloidogyne because it can vary so much in appearance within the same species.