It is well known that high potassium ion concentrations depolarize nervous tissue and it has been suggested that the nerve sheath surrounding the peripheral nerves of insects serves as a protective barrier for the exclusion of potassium ions, in the haemolymph, from the immediate environment of axons. Further it is known that the concentration of potassium ions in the haemolymph of phytophagous insects is far higher than that in predatory forms; this has led to the suggestion that the nerve sheath in plant feeding insects should be more highly developed than that of entomophagous insects. In this work the structure of the nerve sheath in phytophagous and predatory insects has been studied and this assumption has been shown to be groundles. However, preliminary experiments on the effects of ions and drugs on the peripheral nerves of phytophagous and predatory insects have shown that there is a definite difference in susceptibility between the nerves of these two forms and this has led to the postulate of a diffusion barrier beneath the level of the nerve sheath, which is more highly developed in phytophagous than in predatory forms. The properties of this second barrier are discussed. Part 1. / During the course of the work which has been described in Part 1- an outbreak of large saturniid moths Nudaurelia cytherea capensis Stoll.) occurred in the Grahamstown area. It was felt that an investigation into the properties of the flight motor of this moth, which has an extremely low wing beat frquency, might be rewarding as our knowledge of the flight motor in insects is limited to those with very much higher wing beat frequencies than that of this moth. The anatomy, innervation and histology of the flight muscles of Nudaurelia are described and it is shown that the flight motor of this moth is functionally different to that of other insects which have been investigated. Further, Nudaurelia shows a characteristic warm-up fluttering of the wings prior to flight - this phenomenon has also been examined in the following investigation. This study has yielded information about the location of a warm-up centre in the central nervous system of this moth. Part 2.

The secretion of the segmented Malpighian tubules of Cenocorixa bifida (Hungerford) was studied in vitro to determine if the Malpighian tubules of an aquatic predator function in a similiar manner to those of terrestrial insects, and to determine the importance of the different morphological segments in the ion transport of the whole tubule. Fluid secretion in the Malpighian tubules of C. bifida appears to be governed by the same in vitro factors found important in other insects. As in other insects, the secreted fluid is isosmotic to the bathing medium, while the potassium is hypertonic and sodium hypotonic over a wide range of bathing medium sodium-potassium ratios. At bathing medium potassium concentrations close to that of the insect's haemolymph, potassium and sodium are isotonic in the secreted fluid. The Malpighian tubules of C. bifida produce alkaline secretion when rate of secretion is increased by addition of cyclic AMP. Differences in ion and fluid transport between the segments of the Malpighian tubules of C. bifida are statistically significant, but only slight. This correlates with the lack of morphological differences in apical and basal infoldings between the segments. The major exceptions to this trend are (1) the high pH of Segment II fluid, (2) the transport of dyes by Segment III and (3) the production of 'secretory granules' by Segment III. Segment III is the most distinct ultrastructurally. / Science, Faculty of / Zoology, Department of / Graduate

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

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1. The patterns of projection of some of the perpendicular neurones between the retina and medulla of the optic lobes of various insects have been studied. Axon paths have been studied from consecutive semi-thin plastic sections cut transversely and stained with toluidine blue. The termination positions and the paths of axons are both highly ordered and predictable. 2. In all insects with fused-rhabdomere eyes the axons of one ommatidium project to one cartridge of the lamina and the array of cartridges duplicates the array of ommatidia. In insects with open-rhabdomere eyes visual information is distributed amongst a number of lamina cartridges so that each cartridge receives information originating from one visual axis. 3. In both open- and fused-rhabdomere types the cartridge, array of the lamina is exactly duplicated in the medulla but by the intervention of the chiasma is reversed about a dorso-ventral plane. The axons of one lamina cartridge contribute to one medulla cartridge. Thus, in all insects studied, the visual field is projected exactly from retina to medulla. 4. Most of the retinula axons from one ommatidium terminate in the lamina but usually one pair passes directly to the medulla. These are from the central retinula cells (open-rhabdomere eyes) or from the small retinula cells (apposition type fused-rhabdomere eyes). Retinal responses are known mainly only for the short retinula axons so that visual information delivered to the medulla cartridge is still largely unresolved. 5. The lamina neuropile probably contains the elements responsible for the lateral correlation between parallel receptor inputs which is necessary for movement perception, but units with long lasting responses which could act as the delay circuit of movement perception are unknown. 6. The occurrence of errors in termination of the first projection of the optic lobe of the fly, which are reported for the first time in this work, provide no direct clues to the developmental processes by which such a morphologically complex system arises. Nevertheless errors may arise within a sequence of growth processes which are fundamentally quite simple and not obvious from knowledge of the generalized perfect pattern of connections.

The saltwater mosquito larva, Aedes dorsal is, is one of the only organisms capable of inhabiting hypersaline lakes composed almost entirely of high concentrations of NaHC03 and Na2C03 salts. Under laboratory conditions larvae survived and developed normally in saline media with pH values up to 10.5, HC0₃⁻ concentrations up to 250 mM, or C0₃²- concentrations up to 100 mM. Despite ingestion of these alkaline media at rates equivalent to 130% of larval body weight per day, these insects regulated hemolymph pH (7.55-7.70) and HCO₃⁻ concentrations (8.0 - 18.5 mM) within narrow physiological limits. Micropuncture and microcannulation studies on the rectal salt gland demonstrated that this organ was an important site of pH and HC0₃⁻ regulation. Microcannulated salt glands secreted a strongly hyperosmotic fluid containing 402 mM HCO₃⁻ and 41 mM C0₃²⁻ at a rate of 38 nl-h⁻¹. Lumen-to-bath HC0₃⁻ and C0₃²⁻ gradients of 21:1 and 241:1, respectively, were generated by the salt gland epithelium against a transepithelial potential of -25 mV (lumen negative) demonstrating clearly the active nature of HCO₃⁻ secretion. To study the mechanisms of HCO₃⁻ transport, an in vitro microperfused rectal salt gland preparation was developed. Net total CO₂ transport (J[sup=JCo₂; sub=net]) as measured by microcalorimetry in perfused salt glands was unaffected by bilateral Na⁺ or K⁺ and serosal Cl⁻ substitutions, or by serosal addition of 1.0 mM ouabain, 2.0 mM amiloride or 0.5 mM SITS. Removal of luminal Cl⁻ inhibited (J[sup=JCo₂; sub=net] by 80%, while serosal addition of 1.0 mM acetazolamide or 0.5 mM DIDS inhibited by (J[sup=JCo₂; sub=net] 80% and 40%, respectively. Perfusion of the anterior and posterior rectal segments demonstrated clearly that the anterior rectum was the site of CO₂ secretion in the microperfused salt gland. Net Cl⁻ reabsorption in the anterior segment was measured by electron microprobe analysis and was equivalent to the rate of CO₂ secretion. In addition, Cl⁻ reabsorption in the anterior segment was completely inhibited by bilaterally replacing C0₂ and HC0₃⁻ with a phosphate or HEPES buffered saline. These data provide strong quantitative evidence for the presence of a 1:1 Cl⁻/HCO₃⁻ exchange mechanism located in the anterior rectal salt gland segment. Microcannulation studies on the individual salt gland segments demonstrated that both rectal segments are capable of secreting a hyperosmotic fluid containing Na⁻, Cl⁻ and HCO₃⁻. Based on these results and the results of studies in which the effects of serosal ion substitutions on salt gland fluid secretion were examined, it has been suggested tentatively that both segments secrete a NaCl-rich fluid and that fluid secretion is driven by coupled NaCl transport. It is further suggested that once this fluid enters the salt gland lumen its composition is modified by ion exchange and reabsorptive processes which are dependent upon the ionic regulatory needs of the animal. In larvae inhabiting low Cl⁻, NaHCO₃-CO₃ lakes, this modification involves a 1:1 exchange of luminal Cl⁻ for serosal HCO₃⁻. The cellular mechanisms of anterior salt gland HCO₃⁻ and Cl⁻ transport were examined using ion and voltage-selective microelectrodes in conjunction with a microperfused anterior segment preparation which allowed complete changes in serosal and mucosal saline composition to be made in <5-10 seconds. Addition of DIDS or acetazolamide to or removal of CO₂ and HCO₃⁻ from the serosal bath caused large, 20-50 mV hyperpolarizations of Va and had little effect on . Rapid changes in luminal Cl⁻ concentration altered Va in a rapid, step-wise manner. The slope of the relationship between Va and luminal Cl⁻ activity was 42.2 mV/decalog a[sup=l; sub=Cl⁻ (r = 0.992). Intracellular Cl⁻ activity was 23.5 mM and was approximately 10 mM lower than that predicted for a passive distribution at the apical membrane. Changes in serosal Cl⁻ concentration had no effect on indicating an electrically silent basolateral Cl⁻ exit step. Intracellular pH in anterior rectal cells was 7.67 and the calculated a[sup=C;sub=HCO₃] - was 14.4 mM. These results show that under control conditions HC0₃⁻ enters the anterior rectal cell by an active mechanism against an electrochemical gradient of 77.1 mV and exits the cell at the apical membrane down a favorable electrochemical gradient of 27.6 mV. Based on these results, a tentative cellular model has been proposed in which Cl⁻ enters the apical membrane of the anterior rectal cells by passive, electrodiffusive movement through a Cl⁻-selective channel, and HCO₃⁻ exits the cell by an active or passive electrogenic transport mechanism. The electrically silent nature of basolateral Cl⁻ exit and HC0₃⁻ entry, and the effects of serosal addition of Cl⁻/HCO₃⁻ exchange inhibitor DIDS on (J[sup=JCo₂; sub=net] and V[sub=te] suggest strongly that the basolateral membrane is the site of a direct coupling between Cl⁻ and HCO₃⁻ movements via a Cl⁻/HC0₃⁻ exchange mechanism. / Science, Faculty of / Zoology, Department of / Graduate

The in vitro, short-circuited locust rectum actively transports chloride. This electrogenic transport is energized by aerobic metabolism, based primarily upon proline oxidation. Isolated rectal mito-chondrialoxidize a variety of substrates, but the highest state 3 rate of 0₂ consumption occurs when proline is the stubstrate. An enzyme profile of the rectal tissue indicates that the rectum can oxidize amino acids and carbohydrates but has a limited capacity for lipid oxidation. Analysis of the two fluids which bathe the rectum in vivo, the hemolymph and the Malpighian tubule fluid, reveals that proline is present in bothethe hemolymph (12-15 mM) and tubule fluid (38-43 mM). Although glycine is also found in high concentration in the hemolymph (13-17 mM), far lower concentrations of this and other amino acids are found in the Malpighian tubule fluid. Glucose is also found in the hemolymph (2 mM) and Malpighian tubule fluid (4 mM). The high concentraion of proline in the tubule fluid indicated that this amino acid may be actively transported by tubules. This prediction was confirmed by experiments with in vitro tubules. This is the first evidence of aetive secretion of a metabolically useful compound by insect Malpighian tubules. Experiments involving the measurement of short-circuit current (Isc) across the locust rectum were also performed. The results of these experiments indicate that high levels (50 mM) of proline stimulate the Isc of substrate-depleted recta better than high levels of any other substrate tested. Physiological levels of proline also cause a large increase in the Isc of substrate-depleted recta, while physiological levels of glucose produce a much smaller stimulation. Over 90% of the Isc stimulation can be produced by adding proline (15 mM) solely to the lumen side of the tissue. Other studies were performed to estimate the metabolic cost of actively transporting chloride. The oxygen consumption of chloride-depleted recta were measured before and after reintroduction of chloride. From these data a Cl⁻/ATP ratio of 3-4 was obtained. Further calculations indicated that tissue proline oxidation is sufficient to energize active chloride transport. The results outlined here suggest that in vivo, the rectal lumen is bathed with a high concentration of proline which can be readily oxidized by the rectal mitochondria to support the work of actively transporting chloride. / Science, Faculty of / Zoology, Department of / Graduate

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