Experimental confinement of the transient receptor potential to the peripheral retinula cells in the trp mutant Drosophila: What it tells us about the mutation and visual function

By the use of appropriate light intensities the expression of the transient nature of the receptor potential observed in the trp mutant of Drosophila melanogaster can be confined to the peripheral retinula cells in which the visual pigment can also be manipulated predictably, affording an experimental means to probe in these receptors the relationship of the visual pigment to the “electrogenic membrane”. Repeated blue light exposures cause w;trp flies to respond in a manner like cn;bw flies in which the dark-adapted rhodopsin fraction is reduced to 0.5% of the normal level by vitamin A deprivation: this comparable response behaviour, since the amount of visual pigment in w;trp flies is normal, implies that only some subfraction of the photoequilibrium value of rhodopsin may be available. Recovery of the peripheral receptors' sensitivity in ambient light conditions which would render them insensitive by expression of the phenotype is paradoxical and allows a “wavelength effectivity” curve to be constructed which identifies the involvement of the rhodopsin. Resolution of the paradox is discussed.     

Sensitivity and adaptation in the Drosophila phototransduction and photoreceptor degeneration mutants trp and rdgB

We studied rdgB, a retinal degeneration mutant, and trp, a phototransduction mutant, separately and in combination in Drosophila. First we showed that trp did not block degeneration in white-eyed rdgB mutants. Thus, rdgB was useful in determining the defects which trp caused in the compound eye receptors R7 and R8; this is because rdgB selectively eliminates R1-6 photoreceptors which would, if present, dominate the compound eye responses. R7 and R8 both express the trptransient receptor potential phenotype in trp mutants. The trp mutation does not change receptor spectral sensitivities, nor does it alter the dark stability of R1-6's and R7's metarhodopsins as judged by dark adaptation studies. The dark adaptation is not significantly affected by trp. However, trp slows the dark adaptation of R8 considerably and seems to make the blue-induced inactivation of R1-6 less stable.     

The latency of the light response is modulated by the phosphorylation state of Drosophila TRP at a specific site

Drosophila photoreceptors respond to oscillating light of high frequency (～100 Hz), while increasing the oscillating light intensity raises the maximally detected frequency. Recently, we reported that dephosphorylation of the light-activated TRP ion channel at S936 is a fast, graded, light-, and Ca²⁺-dependent process. We further found that this process affects the detection limit of high frequency oscillating light. Accordingly, transgenic Drosophila, which do not undergo phosphorylation at the S936-TRP site (trp^S936A), revealed a short time-interval before following the high stimulus frequency (oscillation-lock response) in both dark- and light-adapted flies. In contrast, the trp^S936D transgenic flies, which mimic constant phosphorylation, showed a long-time interval to oscillation-lock response in both dark- and light-adapted flies. Here we extend these findings by showing that dark-adapted trp^S936A flies reveal light-induced current (LIC) with short latency relative to trp^WT or trp^S936D flies, indicating that the channels are a limiting factor of response kinetics. The results indicate that properties of the light-activated channels together with the dynamic light-dependent process of TRP phosphorylation at the S936 site determine response kinetics.     

Visual signals in the courtship of Drosophila melanogaster

Drosophila melanogaster carrying either of the mutations sev or dipp⁶ show defective phototactic behaviour owing to deficiencies in the processing of visual information perceived by the central retinula cells (R7, R8). Mutant females show increased time to mating because the deficient visual input via this subsystem has an inhibitory effect on female receptivity. Similarly, deficient input through the peripheral retinula cells (R1–R6) also makes females sexually unreceptive. Thus females require appropriate visual stimulation through both subsystems to become maximally sexually receptive. One major source of this stimulation is the red eye of the male.     

Ultrastructure of the single-chamber stemmata of Arge pagana (Panzer, 1798) (Hymenoptera: Argidae)

Stemmata are peculiar visual organs of most larvae in holometabolous insects. In Hymenoptera, Symphyta larvae exclusively possess a pair of stemmata, whose cellular organizations have not been thoroughly elucidated to date. In this paper, the morphology and fine structure of stemmata were investigated in the large rose sawfly Arge pagana (Panzer, 1798) using light and electron microscopy. The larvae possess a pair of stemmata, which belong to the “unicorneal composite eye” or single-chamber stemmata. Each stemma is composed of a biconvex cornea lens, a layer of corneagenous cells, numerous pigment cells, and hundreds of retinula cells. According to the number of retinula cells forming a rhabdom, the stemma can be divided into two regions, the larger Region I and the smaller Region II. The former occupies the largest area of the stemma and contains the majority of rhabdoms, each of which is formed by the rhabdomeres of eight retinula cells. The latter occupies a narrow posterior margin, where each rhabdom consists of nine retinula cells. Based on the different cellular organizations of rhabdoms, the stemma of Argidae is likely developed by the fusion of two types of ommatidial units.     

Cycling expression and cooperative operator interaction in the trp operon of Escherichia coli

Oscillatory behaviour in the tryptophan operon of an Escherichia coli mutant strain lacking the enzyme-inhibition regulatory mechanism has been observed by Bliss et al. but not confirmed by others. This behaviour could be important from the standpoint of synthetic biology, whose goals include the engineering of intracellular genetic oscillators. This work is devoted to investigating, from a mathematical modelling point of view, the possibility that the trp operon of the E. coli inhibition-free strain expresses cyclically. For that we extend a previously introduced model for the regulatory pathway of the tryptophan operon in Escherichia coli to account for the observed multiplicity and cooperativity of repressor binding sites. Thereafter we investigate the model dynamics using deterministic numeric solutions, stochastic simulations, and analytic studies. Our results suggest that a quasi-periodic behaviour could be observed in the trp operon expression level of single bacteria.     

Blue adaptation: an experimental tool for the study of visual receptor mechanisms and behaviour of Drosophila

Physiological and behavioural studies with Drosophila to elucidate visual mechanisms have exploited the bi-stability of the visual pigment in the peripheral retinula cells R1-6, and the 'off-on switch' action of blue and orange light. Measurements of flicker fusion and response waveform from both receptor and lamina regions prior and subsequent to blue adaptation, which induces a prolonged depolarising afterpotential and loss of visual function in R1-6, show these retinula cells to have a high fusion frequency and R7/8, the central retinula cells, a lower fusion frequency. Such measurements also allow analysis of the extracellular response in terms of contributing cells, and its potential for studying the fly's ability to respond to various potential visual cues such as a rotating plane of polarised light. Blue adapted flies fail to fixate normally a black stripe, confirming a role for R1-6 in orientation behaviour requiring a competent degree of acuity.     

Blue adaptation: An experimental tool for the study of visual receptor mechanisms and behaviour of Drosophila

Physiological and behavioural studies with Drosophila to elucidate visual mechanisms have exploited the bi-stability of the visual pigment in the peripheral retinula cells R1–6, and the off-on switch action of blue and orange light. Measurements of flicker fusion and response waveform from both receptor and lamina regions prior and subsequent to blue adaptation, which induces a prolonged depolarising afterpotential and loss of visual function in R1–6, show these retinula cells to have a high fusion frequency and R7/8, the central retinula cells, a lower fusion frequency. Such measurements also allow analysis of the extracellular response in terms of contributing cells, and its potential for studying the fly's ability to respond to various potential visual cues such as a rotating plane of polarised light. Blue adapted flies fail to fixate normally a black stripe, confirming a role for R1–6 in orientation behaviour requiring a competent degree of acuity.Based on material presented at the European Neurosciences Meeting, Florence, September 1978     

Visual signals in the courtship of Drosophila melanogaster: Mutant analysis

Visual cues are necessary for optimal mating success in Drosophila melanogaster. The male's most important visually guided behaviour is tracking. It is shown here that tracking requires intact visual receptor cells R1–6 and the presence of screening pigments in the eye. Thus flies carrying the mutation ebony as well as wild type flies affected in receptor cell R1–6 are unable to use visual cues when they track females. A similar defect was obseved in white-eyed flies lacking screening pigments. Female receptivity depends on visual signals provided by the male flies. Most important cues are the light reflection from and the shape of the male's eyes. No influence of the light reflected from the thorax could be seen. Absence of eyes in the male, however, does not depress female receptivity as much as white eyes. Some evidence is provided that male courtship behaviour is evaluated visually by the female.     

Retina and dioptric apparatus of the dung beetle Euoniticellus africanus

Retinal fine structure and optics of the eye of the dung beetle Euoniticellus africanus have been studied and compared with those of three other scarabaeid beetles: Repsimus manicatus, Anoplognathus pallidicollis and Sericesthis geminata. The eye of Euoniticellus, in common with that of the other three species, possesses a dioptric system in which light first passes through a thick optically homogeneous cornea, and then enters a non-homogeneous crystalline cone. The lens cylinder properties of the latter cause the light rays to become partially focused across the clear-zone upon the rhabdom layer. Rays traced through a large scale drawing of the eye, with refractive indices measured for each component, predict an acceptance angle of approximately 26°. Since no significant aperture changes, lengthening of crystalline thread, cell or pigment migrations appear to be associated with dark/light adaptation, the eye may be assumed to be permanently poorly focused. In optomotor experiments the beetles did not show their characteristic antennal following response to black and white stripes when the latter had repeat periods of <30°. Structurally the eye of Euoniticellus differs markedly from that of other scarabaeids. It is totally divided into dorsal and ventral eye which are of a different size (the dorsal eye is smaller), but whose structural organization is basically the same. Principal pigment cells (they do not fully surround the cone) as well as accessory pigment cells (they accompany the retinula cells in an extraordinarily regular fashion as far as to the basement membrane) exhibit some unusual features. On the proximal side of the clear-zone, at a level where all retinula cell membranes form complex meanders and convolutions, cell 1 is the first to possess a rhabdomere. In it, all microvilli run parallel. This rhabdomere becomes part of the rectangular proximal rhabdom over the upper 20% of its length. Below this level the rhabdom consists of 6 rhabdomeres, but throughout its length microvilli are oriented in 2 orthogonal directions. It is thought that polarization sensitivity in dung beetles generally is related to the rhabdom organization described for Euoniticellus. An eighth (basal) cell is present in each ommatidium, but it lacks a rhabdomere. A tracheal tapetum is not developed. Finally, the point is made not to regard all different eye structures in insects as perfect adaptations to a particular environment or way of living, for specializations of photoreceptors may either follow, parallel or precede any ecological adaptation.     

rgn gene is required for gut cell homeostasis after ingestion of sodium dodecyl sulfate in Drosophila

Resistance and resilience constitute the two complementary aspects of epithelial host defenses in Drosophila. Epithelial cell homeostasis is necessary for the recovery of damages caused by stress or infections. However, the genes responsible for gut epithelial homeostasis remain poorly understood. Here, we show that rgn^G4035 mutant flies have higher mortality than wild-type flies after ingestion of sodium dodecyl sulfate (SDS). Excessive melanization and increased necrotic cells in the gut contribute to the reduced survival of rgn^G4035 mutant flies following SDS ingestion. rgn mutant flies have a defect in the replenishment of intestinal stem cells (ISCs) following gut damage. The antimicrobial peptide (AMP) expression is affected in rgn^G4035 mutant fly guts. Together, our study provides evidence that rgn gene is essential for gut cell homeostasis following damage in Drosophila.     

Fine structure of the compound eyes of the midwater amphipod Phronima in relation to behavior and habitat

Pelagic amphipods belonging to the genus Phronima have four compound eyes; two lateral eyes and two large transparent medial eyes which comprise the entire top of the head. The eyes are structurally similar but the crystalline cones of the medial eyes are more than twenty times as long as those of the lateral eyes, reaching 5 mm in a large animal. The dioptric system of each ommatidium consists of an unfaceted cornea, a layer of hypodermal cells, two rudimentary cone cells, two cells which surround and form the crystalline cone, and the cone itself. The cone and its surrounding cells penetrate the layer of accessory pigment cells which surrounds the retina. The fused rhabdom is formed by the five retinula cells but is separated from them by an extracellular palisade which is crossed by bridges. The retinula cell nuclei lie proximal to the basement membrane. Further proximally the bundle of retinula cell axons is crossed by a second basement membrane, which surrounds each axon with a collar. Medial and lateral eyes on each side of the head share a common lamina. The medial eyes of Phronima appear to be a solution to the problem of remaining inconspicuous to predators while still maintaining sensitivity and resolution.     

Defective gut function in drop-dead mutant Drosophila

Mutation of the gene drop-dead (drd) causes adult Drosophila to die within 2 weeks of eclosion and is associated with reduced rates of defecation and increased volumes of crop contents. In the current study, we demonstrate that flies carrying the strong allele drdlwf display a reduction in the transfer of ingested food from the crop to the midgut, as measured both as a change in the steady-state distribution of food within the gut and also in the rates of crop emptying and midgut filling following a single meal. Mutant flies have abnormal triglyceride (TG) and glycogen stores over the first 4 days post-eclosion, consistent with their inability to move food into the midgut for digestion and nutrient absorption. However, the lifespan of mutants was dependent upon food presence and quality, suggesting that at least some individual flies were able to digest some food. Finally, spontaneous motility of the crop was abnormal in drdlwf flies, with the crops of mutant flies contracting significantly more rapidly than those of heterozygous controls. We therefore hypothesize that mutation of drd causes a structural or regulatory defect that inhibits the entry of food into the midgut.     

Subunit structure of anthranilate synthase from Neurospora crassa: Preparation and characterization of a protease-free form

The multifunctional enzyme complex anthranilate synthase from Neurospora crassa has been purified to homogeneity by a new procedure which yields a stable preparation of the enzyme. Unlike earlier preparations of the enzyme, anthranilate synthase prepared by this technique is not degraded during incubation at 37 °C or during freeze-thaw treatment. Purified anthranilate synthase contains two subunits of M_r 84,000 (β-subunit) and 76,000 (α-subunit), which are shown, by partial proteolysis, to be unrelated in sequence. Immunoprecipitation studies demonstrate that freshly prepared crude extracts of Neurospora contain anthranilate synthase subunits identical in size with those of the purified enzyme. The β-subunit is shown to be the product of the trp1 gene, and the a-subunit, of the trp2 gene.     

Light-initiated responses of retinula and eccentric cells in the Limulus lateral eye

The relationship between retinula and eccentric cells in the lateral eye of Limulus polyphemus was studied using a double electrode technique which permitted simultaneous recording of light-initiated responses in two sense cells and the labeling of the cells for subsequent histological examination and identification. The following results were obtained: (a) light-initiated slow responses with and without superimposed spike potentials were recorded from retinula cells and from eccentric cells (only one eccentric cell yielded responses without superimposed spike potentials); (b) spike potentials recorded in different cells within the same ommatidium were always synchronous; (c) a complete absence of spike potentials was observed in two experiments in which no eccentric cells could be found in the ommatidia containing the labeled retinula cells; (d) the greatest differences in the characteristics of responses recorded simultaneously occurred in those recorded from retinula-eccentric combinations. The results indicate that there is only one source of spike potential activity within an ommatidium (presumably the eccentric cell) and that the light-initiated response of retinula cells may be independent of the eccentric cell response. The suggestion is advanced that the response of the retinula cell may "trigger" the eccentric cell response.     

The reversion of trp frameshift mutations in mut,polA, lig and dnaE mutant strains of Escherichia coli

Mutator mutations mutL25, mutR34, and mutU4 had similar effects on the reversion of 4 trp frameshift mutations of known sequence. The mutation trpE9777, which resulted from the addition of an A–T base-pair to a run of 5 A–T base-pairs, was most strongly reverted by the 4 mutators. Reversion of trpE9777 was also increased by mutation polA1 (DNA polymerase I) and dnaE486 and dnaE511 (DNA polymerase III). No effect was found with the ligase mutations, lig-4 or lig-ts7. Mutations polAex1 and polA107, both deficient in the 5′ → 3′ exonuclease activity of DNA polymerase I, had different mutator effects; the factor increase in reversion of trpE9777 was 28-fold for polAex1, 6-fold for polA107, and 21-fold for polA1. The trpE9777 mutation is a useful indicator of frameshift mutator activity.     

Coelomomyces dodgei: Establishment of an in vivo laboratory culture

An in vivo laboratory culture of the fungus Coelomomyces dodgei (Chytridiomycetes: Blastocladiales) was established, using the copepod Cyclops vernalis as an intermediate host and the larvae of the mosquito Anopheles quadrimaculatus as the definitive host. The culture was perpetuated by infecting copepods and mosquitoes using separate procedures. Copepods were infected by being combined with dehiscing sporangia. Patently infected copepods, which contained either light amber, bright orange, or both light amber and bright orange mycelia, were collected daily beginning 12 days later. Mosquitoes were infected by combining 100 first-instar larvae for 48 hr with a mixture of 12 infected copepods, four of each of the above types. The mean rate of infection for the first 100 trials was 41%. When groups of 100 first-, second-, third-, and fourth-instar larvae were exposed to a similar mixture of infected copepods for 48 hr, the mean rates of infection were 37.4, 27.0, 17.8, and 2.4%, respectively. Observations and experimental evidence suggest that the differentially pigmented mycelia found in infected copepods are gametophytes which develop into gametangia that subsequently release gametes of opposite mating types, the light amber gametangia producing female and the bright orange gametangia producing male gametes. Zygotes resulting from the fusion of these gametes lead to the infection of mosquito larvae. Thus, C. dodgei appears to have an Euallomyces type of life cycle with sporophyte and gametophyte generations alternating between mosquito and copepod hosts, respectively, with differentially pigmented sexual structures present in the gametophyte phase.     

First records of flower biology and pollination in African Annonaceae: Isolona, Piptostigma, Uvariodendron, Monodora and Uvariopsis

Seven species from five genera of Annonaceae were studied with regard to their flower biology and pollination in the Southwest Province of Cameroon, West Africa. They have protogynous hermaphroditic flowers, with exception of Uvariopsis species, which are monoecious. Fused petals of Isolona campanulata remain apically spreading and open during anthesis but form a deep basal urceolate tube around the reproductive organs. At anthesis the yellow pendent flowers emit a fruit-like scent and attracted small beetles, the likely pollinators. Piptostigma sp. flowers also emit a fruit-like scent but provide a closed pollination chamber formed by the three inner petals. Small staphylinid beetles attracted during the female stage of anthesis are released from the flowers at the end of the male stage 2-3 days later. Both species have diurnal anthesis, attracting and releasing the flower visitors during daytime. In contrast, Uvariodendron connivens and U. calophyllum have nocturnal anthesis with floral thermogenesis, produce spicy, aromatic and fruity scents and attract large Scarabaeidae beetles, the pollinators, along with many curculionid beetles, which were principally predators of the thick petals. The very large flowers of Monodora tenuifolia have yellowish petals which are spotted with dark red markings. Together with the sweetish, slightly disagreeable scent the flowers attract flies, principally dung flies. The two investigated Uvariopsis species are monoecious with pistillate and staminate flowers being functional at the same time. The violet red flowers of U. bakeriana visually seem to mimic the fruiting body of certain stinkhorn fungi (Phallaceae) although without producing their strong unpleasant carcass stench. Flower-visiting dung flies were rare. Conversely, U. congolana has a strong fungus-like scent, its flowers are presented at litter height and dung flies living in the litter were the flower visitors, albeit sporadic. The 4-5 days lasting anthesis of both Uvariopsis species appears to be an evolutionary consequence of their diffuse pollinator spectra. The studied African Annonaceae therefore have either cantharophilous or myiophilous/sapromyiophilous flowers with, in part, respectively, remarkably long anthesis, thermogenesis, and widely open, large flowers - all attributes unknown or rare in the hitherto better studied Neotropical Annonaceae.     

THE MICROSTRUCTURE OF THE COMPOUND EYES OF INSECTS

The apposition eyes of two diurnal insects, Sarcophaga bullata (Diptera) and Anax junius (Odonata), have been examined with the electron microscope. In the latter case only the rhabdom is described. The rhabdom of the fly consists of a central matrix and seven rhabdomeres, one for each retinula cell. The rhabdomeres show an ordered internal structure built up of transverse tubes, hexagonal in cross-section. These slender compartments running the width of the rhabdomere are 370 A in diameter. After fixation with osmium tetroxide the walls of the compartments are more electron dense than the interiors. The retinula cells contain mitochondria, and pigment granules smaller than those found in the pigment cells. These granules tend to cluster close behind the membranes which separate the retinula cells from their rhabdomeres. The rhabdom of the dragonfly is a single structure which appears to be composed of three fused "rhabdomeres," each similar to a rhabdomere of Sarcophaga. Reasons are given for believing that the rhabdom may be the site of photoreception, as well as the organ for analyzing plane-polarized light, as suggested by other workers.