Physiological compartmentation in gonadal tissue of the common carp (Cyprinus carpio L.)

Summary Physiological compartmentation in carp (Cyprinus carpio L.) gonads was investigated after intracardial injection of horseradish peroxidase (HRP) and two mouse anti-carp-sperm monoclonal antibodies.Immunohistochemistry revealed that a physiological barrier exists in carp testis for HRP and mouse IgG monoclonal antibody around the central lumina of the tubules in which the spermatozoa are located, but not around the cysts containing the precursor germ cells. The results with HRP were confirmed by electron microscopy. Mouse IgM monoclonal antibody did not penetrate the spermatogenic cysts. Probably because of its large size, it was almost exclusively located inside blood capillaries and only sparsely in the interstitial tissue.In the ovary, HRP was regularly distributed in the gonadal tissue, whereas the IgG antibody was predominantly localised on oogonia and early prophase oocytes. The results indicate that in contrast with the testis, no barrier around germ cells exists in the carp ovary.     

Retinal projections in the electric catfish (Malapterurus electricus)

Summary The poorly developed visual system of the electric catfish was studied with silver-degeneration methods. Retinal projections were entirely contralateral to the hypothalamic optic nucleus, the lateral geniculate nucleus, the dorsomedial optic nucleus, the pretectal nuclei including the cortical nucleus, and the optic tectum. The small size and lack of differentiation of the visual system in the electric catfish suggest a relatively small role for this sensory system in this species.     

The lymphatic capillaries of the middle ear mucosa in the guinea pig ultrastructure and permeability

Summary The middle ear mucosa has an extensive lymphatic capillary network. Horseradish peroxidase passes through the lymphatic intercellular clefts and is taken up into intracellular vesicles.     

Inputs to the torus semicircularis in the electric fish Eigenmannia virescens

Summary The posterior lateral-line lobe, contrary to present belief, projects bilaterally to the torus semicircularis, although the contralateral projection is considerably more extensive. The torus also receives bilateral inputs from the medial octavo-lateralis nuclear complex, the reticular formation, a sublemniscal nucleus, and the nucleus prae-eminentialis. Unilateral inputs to the torus were found originating from the ipsilateral mesencephalic tectum and the contralateral lobus caudalis of the cerebellum. Extensive commissural systems between the right and left torus are also described for the first time.     

A combined horseradish peroxidase and Golgi study on the afferent connections of the ventrobasal complex of the thalamus in the cat

Summary Afferent connections to the ventrobasal complex (VB) of the thalamus were studied by means of retrograde transport of horseradish peroxidase (HRP) and by the Golgi-method. After HRP-injection into the VB, peroxidase-positive cells were observed contralaterally in the dorsal column nuclei (DCN), in the trigeminal nuclei and in the lateral cervical nucleus (LCN), and ipsilaterally in the somatosensory I (SI) and II (SII) cortical areas. Labeled cells of different shape and size were compared with neurons impregnated by the Golgi-technique. On the basis of HRP-labeling it is concluded that cells projecting to the VB are different in size and shape even within one region and that they correspond to the relay or efferent neurons observed in the Golgimaterial.     

New member of the protein disulfide isomerase (PDI) family identified in Amblyomma variegatum tick

Ticks belonging to arthropoda are blood feeding, geographically widespread ectoparasites of mammals, reptiles and birds. Their saliva contains active substances that protect them from host immune attack and allow for transmission of various pathogens during the feeding process. Characterization of tick saliva components can therefore contribute to the development of effective methods for the control of tick-borne diseases.

Here we describe the identification and basic characterization of a gene encoding a 55 kDa protein found in the salivary glands (SG) of Amblyomma variegatum tick. Based on the primary structure and homology to the family of protein disulfide isomerases (PDI; EC 5.3.4.1) the gene was named AvPDI. The 1461 nt long AvPDI open reading frame codes for a 487 amino acid protein. In vitro expressed AvPDI was exclusively localized in the endoplasmic reticulum. RT-PCR and Western blot analysis revealed that AvPDI expression is not restricted to the SG of the tick. More detailed analysis on tissue slides from SG detected an AvPDI specific signal in granular cells of the acini type II and III. Finally, reductase activity of AvPDI was confirmed in an insulin assay. The structural and functional characteristics suggest that AvPDI is another member of the PDI protein family and represents the first more closely characterized PDI in the ticks.     

Perception of breath components by the tropical bont tick,Amblyomma variegatum Fabricius (Ixodidae)

Wall-pore olfactory sensilla located in the capsule of Haller's organ on the tarsus of Amblyomma variegatum ticks bear cells responding to vertebrate breath: one of these sensilla contains a CO2-excited receptor and a second sensillum has a CO2-inhibited receptor. Each of these antagonistic CO2-receptors, which display typical phasic-tonic responses, monitors a different CO2-concentration range. The CO2-inhibited receptor is very sensitive to small concentration changes between 0 and ca. 0.2%, but variations of 0.01% around ambient (ca. 0.04%) induce the strongest frequency modulation of this receptor. An increase of just 0.001-0.002% (10-20 ppm) above a zero CO2-level already inhibits this receptor. By contrast, the CO2-excited receptor is not so sensitive to small CO2 shifts around ambient, but best monitors changes in CO2 concentrations above 0.1%. This receptor is characterized by a steep dose-response curve and a fast inactivation even at high CO2-concentrations (greater than 2%). In a wind-tunnel, Amblyomma variegatum is activated from the resting state and attracted by CO2 concentrations of 0.04 to ca. 1%, which corresponds to the sensitivity range of its CO2-receptors. The task of perceiving the whole concentration range to which this tick is attracted would thus appear to be divided between two receptors, one sensitive to small changes around ambient and the other sensitive to the higher concentrations normally encountered when approaching a vertebrate host.     

Perception of breath components by the tropical bont tick,Amblyomma variegatum Fabricius (Ixodidae)

Summary Wall-pore sensilla in the capsule of Haller's organ on foreleg tarsi of the tick, Amblyomma variegatum, show multicellular responses upon stimulation with human and bovine breath. Filtering breath through charcoal removes the stimulant for some of these receptors. Analysis by gas chromatography coupled with olfactory sensillum electrophysiological recordings indicates that an ethanol extract of the breath components trapped on charcoal contains a major stimulant eluting at the same retention time as H₂S. Two types of H₂S-sensitive receptors have been identified. They are housed in separate sensilla, and are called sulfide-receptor 1 and 2.Although, both receptor types are characterized by a high sensitivity to H₂S with an estimated threshold of ca. 0.1 ppb and a response range covering 5–6 log orders of magnitude, their overall response to sulfides and mercaptans is nevertheless dissimilar. The type 1 receptor fires slightly more upon stimulations with H₂S than type 2, whereas ethylmercaptan induces a stronger response from type 2, and dimethyl sulfide activates only receptor 2.In a bioassay, H₂S tested at concentrations of ca. 0.02 ppm and 1 ppm equally arouses 60% of resting ticks. Two-thirds of these ticks quest the air with their first pair of legs, and the remainder start active search. By contrast, H₂S at ca. l ppm in a mixture with CO₂ severely diminishes the locomotor stimulating effect of CO₂.     

Ultrastructure of Haller's organ in the tick Amblyomma americanum (L.)

Summary Haller's organ on the tarsus of the tick Amblyomma americanum (L.) (Acarina: Ixodidae; nymphal stage) was studied by scanning and transmission electron microscopy. It consists of a distal bristle group, (the anterior pit), and a proximal capsule which encloses several sensilla. The seven sensilla of the anterior pit (A1–A7) are all thick-walled and multi-innervated (2–9 neurons), but at least three different types can be differentiated. Sensilla A1 and A2 possess large, plugged pores (>1000 Å) and are the only sensilla with branching dendrites. A3 and A5 are characterized by a spoke-wheel arrangement of the cuticle wall and very fine pores (100–200 Å) penetrating the spokes centrally; A4, A6, and A7 do not exhibit any pore system but a single opening at the bristle tip is assumed.The capsule contains seven thin-walled, blunt-tipped sensilla, and several non-sensory cuticular projections (pleomorphs). All of these sensilla possess large plugged pores in the cuticle wall and numerous dendritic branches of several neurons (3–5) in the lumen. Glandular openings were found inside the capsule; their significance is discussed.The fine structure of Haller's organ supports the functions postulated by Lees (1948), namely olfaction for the capsule and humidity reception (among others) for the anterior pit.This research was supported in part by the Office of Naval Research, and by NIH Training grant ES 00069. Paper no. 3459 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh.     

Retinal projections in the catfish,Mystus vittatus (Bloch) as revealed by tracer studies with horseradish peroxidase

Summary The retinal efferents of the catfish, Mystus vittatus, were investigated with the use of the horseradish peroxidase (HRP) technique. Most retinal fibres extended contralateral to the eye that had received HRP label, while a few fascicles projected to the ipsilateral side without decussation in the optic chiasma. The contralateral fibres projected to the suprachiasmatic nucleus, the nucleus opticus dorsolateralis, the nucleus of the posterior commissure, the nucleus geniculatus lateralis, pretectal nuclear complex, and to two layers of the optic tectum, i.e., stratum fibrosum et griseum superficiale and stratum griseum centrale. The accessory optic tract arose from the inner area of the optic tract and extended ventromedially to the accessory optic nucleus. The ipsilateral fascicles projected to almost all the above mentioned nuclei, but these projections were comparatively sparse. The ipsilateral retinal projection was restricted to the rostral tectum.     

AvGI,an index of genes transcribed in the salivary glands of the ixodid tick Amblyomma variegatum

Random clones from a cDNA library made from mRNA purified from dissected salivary glands of feeding female Amblyomma variegatum ticks were subjected to single pass sequence analysis. A total of 3992 sequences with an average read length of 580 nucleotides have been used to construct a gene index called AvGI that consists of 2109 non-redundant sequences. A provisional gene identity has been assigned to 39% of the database entries by sequence similarity searches against a non-redundant amino acid database and a protein database that has been assigned gene ontology terms. Homologs of genes encoding basic cellular functions including previously characterised enzyme activities, such as stearoyl CoA saturase and protein phosphatase, of ixodid tick salivary glands were found. Several families of abundant cDNA sequences that may code for protein components of tick cement and A. variegatum proteins which may contribute to anti-haemostatic and anti-inflammatory responses, and, one with potential immunosuppressive activity, were also identified. Interference with the function of such proteins might disrupt the life cycle of A. variegatum and help to control this ectoparasite or to reduce its ability to transmit disease causing organisms. AvGI represents an electronic knowledge base, which can be used to launch investigations of the biology of the salivary glands of this tick species. The database may be accessed via the World Wide Web at http://www.tigr.org/tdb/tgi.shtml.     

Anteroposterior gradient during nymphal-adult moulting cycle of the tropical bont tick,Amblyomma variegatum (Acarina: Ixodidae)

Summary The structure of the extensible (alloscutum) and inextensible (scutum) integument of the nymph, Amblyomma variegatum was examined during the whole bloodmeal and the nymphal-adult moulting cycle. Integumental events were tentatively correlated with the ecdysteroid levels measured by radioimmunoassay. We observed that all the integumental events were realised along an anteroposterior gradient. During the 5 days corresponding to the bloodmeal, although the hormone concentration was low, a new endocuticle was deposited on both the alloscutum and scutum. Furthermore, mitoses were initiated in the capitulum. On days 1–2 after the meal, ecdysteroid titres began to increase and reached a first peak corresponding to 4.1 ng 20-hydroxyecdysone equivalents/tick on the 4th day after the ticks dropped off their host. At this time the epidermis of the capitulum was detached and the outline of the adult capitulum was already visible. Mitotic activity in the alloscutum was initiated. On day 6 post-drop, the frontal apolysis was achieved and the ecdysteroid titres declined to basal values. A second peak much higher than the first one (maximum value of 33.7 ng/tick) and identified principally as 20-hydroxyecdysone by HPLC/RIA was noted on the 13th day post-drop. During the period of increase in the ecdysteroid levels (days 9–10 post-drop), the mitotic phase ended in the alloscutum and the apolysis began. Epicuticle was deposited after day 12 postdrop. Then, while the titre fell to low values (about 1.6 ng/tick, days 16–20 post-drop), the exocuticle was deposited and the nymphal cuticle was digested. All adult structures were functional 3 days before ecdysis. In young male as in female adults the mean value of the ecdysteroid levels corresponded to about 2.5 ng/tick. Finally, hydrolysis of tick whole extracts with esterase demonstrated a low increase of RIA-positive material, demonstrating the probable presence of natural ecdysteroid fatty-acid conjugates in this species.     

Connections of the olfactory bulb in the piranha (Serrasalmus nattereri)

Summary The connections of the olfactory bulb were studied in the piranha using the Nauta and horseradish-peroxidase methods. Three olfactory tracts project to seven terminal fields in the telencephalon and one in the diencephalon, all of them bilaterally. The contralateral olfactory bulb also receives a small input. All contralateral projections decussate in the anterior commissure and are relatively weak compared to the ipsilateral projections. HRP-containing cells were found in all of the ipsilateral telencephalic aggregates receiving an olfactory tract projection; the contralateral side was free of labeled cell bodies. Although only about one fourth of the entire telencephalon receives a direct olfactory input, the high degree of differentiation of the olfactory system suggests that the piranha depends substantially on the sense of olfaction and that this species may be a good model for further studies on olfactory mechanisms.     

Identification of vertebrate volatiles stimulating olfactory receptors on tarsus I of the tick Amblyomma variegatum Fabricius (Ixodidae)

Bovine odour excites olfactory receptor(s) in a wall-pore olfactory sensillum on the anterior pit of Haller's organ in Amblyomma variegatum. Gas chromatography-coupled electrophysiology recordings from this sensillum reveal the presence of 4 active compounds in bovine odour. The two strongest stimulants were identified as 2-nitrophenol and 4-methyl-2-nitrophenol by gas chromatography-coupled mass spectrometry, and by matching electrophysiological activity of synthetic analogues. Synthetic analogues of known vertebrate-associated volatiles also stimulate other olfactory receptors in sensilla on the surface of tarsus I: a lactone receptor responding to -valerolactone and 6-caprolactone; different fatty acid receptor types responding best to either pentanoic acid, 2-methylpropanoic acid or to butanoic acid; three receptors responding to NH₃; and one receptor responding to 3-pentanone. Gas chromatography-coupled mass spectrometry analysis of vertebrate volatiles revealed presence of a number of these olfactory stimulants in concentrates of rabbit and steer odour, i.e. 2-methylpropanoic acid, butanoic acid, 3-methylbutanoic acid, pentanoic acid, and -valerolactone.Abbreviations GC-EL gas chromatography-coupled electrophysiological recording - GC-MS gas chromatography-coupled mass spectrometry     

Fine structural analysis of palpal receptors in the tick Amblyomma americanum (L.)

Summary Palps of the tick Amblyomma americanum (L.) (Acarina: Ixodidae; nymphal stage) were studied by scanning and transmission electron microscopy. The terminal palp segment (IV) bears the so-called palpal organ, a cluster of 10 short, blunt-tipped sensilla. All sensilla (except for the center sensillum) receive a dual innervation: 2 mechanoreceptive dendrites which terminate in the socket membrane plus several chemoreceptive dendrites (4–12) which enter the lumen. The thick-walled cuticular shaft possesses 2–3 small pore openings (100 Å) below the tip, thus establishing communication between dendrites and environment. Two structurally different types of palpal sensilla exist: The A-type has a characteristic doublelumen and always contains 4 dendrites, the B-type features a single lumen and a specially layered cuticular shaft with 6–12 dendrites. The fine structure of the tick palpal receptors corresponds closely to that of known contact chemoreceptors in insects.This research was supported in part by a contract with the Office of Naval Research (R. C. Axtell, principal investigator), and by NIH Training grant ES 00069. Paper no. 3700 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh.     

Transneuronal uptake of horseradish peroxidase in the central nervous system of dipterous insects

Summary Horseradish peroxidase (HRP) applied to lesioned neurons in the retina and thoracic ganglia of the flies Musca, Calliphora and Drosophila labeled axon terminals, dendrites and perikarya of the severed neurons after anterograde or retrograde passage. In addition, HRP reaction product secondarily labeled intact neurons that are contiguous with injured nerve cells. In many cases labeling of optic lobe neurons remote from primarily filled ones was also seen (here called tertiary labeling). HRP labeling was extensive and both primarily and transneuronally filled neurons could be resolved in almost as much detail as Golgi-impregnated or cobalt-silver-labeled cells. Electron microscopy showed that in both primarily and secondarily filled neurons, reaction product was distributed diffusely in the cytoplasm.Transneuronal uptake of HRP was specific to certain types of neurons in the brain and thus displayed certain pathways. The pathways resolved by transneuronal labeling with HRP extend from the optic lobes to the thoracic ganglia and include visual neurons previously identified electrophysiologically and anatomically.Transneuronal HRP uptake, although believed to occur in vivo, could not be shown to be dependent on synaptic activity. Three other heme peptides tested were taken up by injured neurons, but showed no transneuronal labeling: lactoperoxidase, cytochrome c, and microperoxidase.     

Retrograde labeling of regenerated electromotor neurons with HRP in a teleost fish,Sternarchus albifrons: Relation to cell death

Summary Back-labeling of regenerated electromotor neurons in the teleost Sternarchus albifrons was performed to test the hypothesis that, in regenerated spinal cord, incorrectly located electromotor neurons are eliminated because their axons do not reach the correct target area (electric organ). In each cross section examined, all of the regenerated electromotor neurons ipsilateral to the implantation site were labeled with horseradish peroxidase, including those ectopic cells located at the edge of the cord, which are later eliminated by selective cell death. Retrograde labeling of these ectopic neurons demonstrates that their axons do extend into the correct target area (the regenerated electric organ). Thus total misdirection of the axons cannot be the cause of their subsequent cell death. We conclude that selective neuronal death in this system does not reflect the absence of axonal projection to the correct target area.A preliminary report on this work has been presented in Soc. Neurosci. Abstracts 10:48 (1984)     

Distributions of the vectors of heartwater, Amblyomma hebraeum and Amblyomma variegatum (Acari: Ixodidae), in Zimbabwe

The tick vectors of heartwater (Cowdria ruminantium infection) in Zimbabwe, Amblyomma hebraeum and Amblyomma variegatum, historically were believed to be confined to the low-lying regions of the south and north-west of the country. However, country-wide surveys performed in 19751980 and 19881991 demonstrated that both species were also established in western parts of the highveld plateau and had started to encroach on the predominantly heartwater-free central and eastern highveld regions. To determine the current distributions of both the vectors and evaluate the potential threat of heartwater to animals in the highveld, a survey of ticks infesting cattle was performed in 1996 at 2994 locations in small-holder and large-scale commercial farming areas throughout Zimbabwe. Amblyomma hebraeum was collected at 1329 locations, A. variegatum at 72 locations and both A. hebraeum and A. variegatum at 13 locations. The results demonstrated that A. hebraeum was present, as previously recorded, throughout the southern half of the country and appeared to have undergone further limited spread into the central and eastern highveld regions. Only the northern-most region of the country appeared to be free of this species. Amblyomma variegatum was collected mainly in the north-west, as previously recorded, but was also found at isolated locations across the central highveld region and along the eastern border with Mozambique. This species was, however, still absent from the southern half and the northern-most regions of the country. An overlap of the distributions of the two species existed within a zone along the southern and eastern regions of the distribution of A. variegatum. These results suggest that the vectors of heartwater are spreading and threaten to introduce heartwater into intensive livestock-producing regions of the country.Exp Appl Acarol 22: 725740 © 1998 Kluwer Academic Publishers