The morphology of photophores in the garrick,cyclothone braueri (Family: Gonostomatidae): an ultrastructure study

A contribution to the knowledge of the photophore structure of the mesopelagic fish Cyclothone braueri (Gonostomatidae) from the central Mediterranean Sea (Strait of Messina) is given by means of a structural and ultrastructural study, to better identify and classify the real anatomical structures costituing these luminous organs. The photocytes exhibit numerous secretory granules, of different electron density, embedded in an extremely developed rough endoplasmic reticulum. The lens appear to be composed of tightly contiguous polyhedral cells. The reflector is made up of cells rich in guanine crystals, embedded in an amorphous matrix and is surrounded by a layer of connective tissue containing melanocytes. Unlike the present knowledge, it is shown that the bioluminescence emitted from C. braueri light organs has glandular nature, with photophores similar to type α from Bassot classification. The phenomenon of adaptive convergence, documenting how the morphology and physiology of the light organs of teleosts is similar in different species despite their taxonomic distance, is confirmed also for C. braueri.     

Pharmacomorphological study of denervation induced by 6-hydroxydopamine in Porichthys photophores

The effects of 6-hydroxydopamine (6-OHDA) on the bioluminescent response of Porichthys photophores were investigated as part of a pharmacological study of the neural control of luminescence in this fish. Subcutaneous injections of 6-OHDA induce a luminescent response similar to that of norepinephrine (NE), suggesting a sympathomimetic action. The luminescent response to electrical stimulation is almost completely and irreversibly abolished within 24 hours following low-dose treatment of the photophores with 6-OHDA, while the sensitivity of these organs to exogenous NE is increased significantly over the few days post-treatment. During this period the photophores continuously emitted a steady low-level glow. Electronmicroscopic studies of such photophores revealed progressive destruction of the nerve endings. Photophore luminescent sensitivity to NE subsequently became sub-normal, and at this stage electron microscopy revealed an increasingly larger number of damaged photocytes, supportive cells and, in one case, lens cells. From these results it is suggested that 6-OHDA initially impairs neuro-photocyte transmission by destroying catecholaminergic nerve endings. In turn, the transmitter reuptake mechanism is also impaired, thus accounting for development of supersensitive responses to exogenous NE. Subnormal luminescent responses to NE appear as a result of loss of photocyte competence due to structural deterioration. The latter are interpreted as the consequence of removal of trophic factors supplied by the photophore adrenergic innervation.Suppression of luminescent response to both electrical stimulation and exogenous NE in photophores treated with higher doses of 6-OHDA, may be due to a direct effect of this drug on the receptor sites of the photocytes.     

Reflector of the body photophore in lanternfish is mechanistically tuned to project the biochemical emission in photocytes for counterillumination

Lanternfish, a family Myctophidae, use ventro-lateral body photophores for camouflage of the ventral silhouette, a strategy called counterillumination. While other deep-sea fishes possess pigmented filters and silver reflectors to match sunlight filtering down through the depths, myctophids developed a blue-green reflector for this purpose. In this study, we showed in a lanternfish Diaphus watasei that the reflector comprised monolayered iridophores containing multilayered guanine crystals which enable high reflection with light interference colouration. Platelets shape in body photophores is an unique near-regular hexagonal, probably to allow the homogeneity of reflection angle of the luminescence from photocytes. Focus point of the parabola-like reflector is positioned on the photocytes that ensures the light produced from the photocytes is redirected to the ventral direction. In vitro luminescence reaction using purified luciferase and the substrate coelenterazine showed the light emission at λ_max 454 nm, while reflection spectra of the iridophores exhibit peaks at longer wavelength, which accomplish to alter the luminescence emitted from photocytes to longer wavelength to fit the mesopelagic light environment. Taken together, we revealed multiple mechanistic elaborations in myctophid body photophores to achieve effective control of biochemical luminescence for counterillumination.     

A biochemical characterization of the photophore lenses of the midshipman fish,Porichthys notatus Girard

The present study is a biochemical characterization of the photophore lenses of the midshipman fish, Porichthys notatus, a species that bears 800 photophores distributed over the body surface. The biochemical properties of the photophore lenses were compared with those of the eye lens with which they share a similar developmental origin and analogous function. To achieve a high refractive index, the vertebrate eye lens has a relatively high concentration of structural proteins (20–50%, depending on species) and a simple protein composition, that is, relatively few proteins are synthesized in comparison to other tissues. Similarly, the photophore lenses of P. notatus had a relatively high protein concentration (average = 29%, n = 5) and approximately 60% of the total soluble protein was represented by two subunit species of 33 kD and 35 kD on denaturing polyacrylamide gels. The structural proteins of the eye lens are of two principle types: 1) and polypeptides which belong to vertebrate lens-specific crystallin families, and, 2) enzymes recruited into the lens which take on the function of structural proteins. Here, we report that the two major photophore lens subunits of 33 kD and 35 kD are biochemically similar to each other, but are clearly distinct from any of the previously characterized crystallins. Therefore, we propose that photophore lenses appear to recruit a novel protein.     

The photophore morphology of Selenoteuthis scintillans Voss and other lycoteuthids (Cephalopoda: Lycoteuthidae)

Females and juveniles of Selenoteuthis scintillans have photophores of several structural types, distributed on the tentacles and eyeballs, and within the mantle cavity and tail. Three distinct photophore types can be recognized on the basis of their accessory structures, though their photocytes are identical. The tail and some tentacular photophores (Type 1) lack any accessory optical structures; other tentacular and abdominal photophores (Type 2) have collagenous diffusing fibres; the anal and ocular photophores (Type 3) have a variety of iridosomes but no collagen. The distal tentacular organ is a double structure composed of a unit each of Type 1 and Type 2. Ocular photophores 1 and 5 are also double structures, composed of two Type 3 units. The photophores closely resemble in structure those of Lycoteuthis diadema. The photocytes have a marked fluorescence and luminesce on treatment with dilute hydrogen peroxide. The bio-luminescence intensity of the tail organ may be modified by chromatophore movements and has a blue-green spectral emission.
The photophores of juvenile Lampadioteuthis megaleia are similar in structure to those of Selenoteuthis but somewhat less complex. A comparison between the morphology of the photophores of lycoteuthid and enoploteuthid squids emphasizes the close similarity between the two families. At the ultrastructural level, certain photophores of both families have very characteristic microvillous blood vessels associated with the photocytes.     

Heterogeneity,position, and functional capability of the macrophages in Peyer's patches

Summary Radioenzymatic assays and light microscope radioautographic studies performed on photophores of Porichthys notatus demonstrated (1) significant amounts of catecholamines (dopamine, noradrenaline, adrenaline) and 5-hydroxytryptamine (serotonin) in these organs; (2) selective uptake and storage of [³H]noradrenaline ([³H]NA) by axon terminals innervating the photocytes, and (3) strong accumulation of [³H]5-hydroxytryptamine ([³H]5-HT) within the photocytes. Uptake and storage of [³H]NA in the nerve fibers were seemingly unaffected by the addition of ten-fold molar concentrations of unlabelled serotonin. Accumulation of [³H]5-HT by the photocytes was dose-dependent and diminished markedly in the presence of ten-fold molar concentrations of non-radioactive noradrenaline. Neither neuronal uptake of [³H]5-HT or [³H]A, nor photocytic accumulation of [³H]A were detectable under the conditions of the present experiments. This information should provide a framework for further investigations of the regulation of photophore luminescence by the biogenic amines.Supported by grants from the National Research Council and Medical Research Council of CanadaJacques de Champlain and Lise Farley provided facilities and expertise with the radioenzymatic techniques. The technical assistance of Sylvia Garcia and Marie-Hélène Parizeau was also appreciated     

Comparative innervation of cephalic photophores of the loosejaw dragonfishes (Teleostei: Stomiiformes: Stomiidae): Evidence for parallel evolution of long‐wave bioluminescence

Four genera of the teleost family Stomiidae, the loosejaw dragonfishes, possess accessory cephalic photophores (AOs). Species of three genera, Aristostomias, Malacosteus, and Pachystomias, are capable of producing far‐red, long‐wave emissions (>650nm) from their AOs, a character unique among vertebrates. Aristostomias and Malacosteus posses a single far‐red AO, while Pachystomias possesses anterior and posterior far‐red AOs, each with smaller separate photophores positioned in their ventral margins. The purpose of this study was to establish the primary homology of the loosejaw AOs based on topological similarity of cranial nerve innervation, and subject these homology conjectures to tests of congruence under a phylogenetic hypothesis for the loosejaw dragonfishes. On the basis of whole‐mount, triple‐stained specimens, innervation of the loosejaw AOs is described. The AO of Aristostomias and the anterior AO of Pachystomias are innervated by the profundal ramus of the trigeminal (Tpr), while the far‐red AO of Malacosteus and a small ventral AO of Pachystomias are innervated by the maxillary ramus of the trigeminal (Tmx). The largest far‐red AO of Pachystomias, positioned directly below the orbit, and the short‐wave AO of Photostomias are innervated by a branch of the mandibular ramus of the trigeminal nerve. Conjectures of primary homology drawn from these neuroanatomical similarities were subjected to tests of congruence on a phylogeny of the loosejaws inferred from a reanalysis of a previously published morphological dataset. Optimized for accelerated transformation, the AO innervated by the Tpr appears as a single transformation on the new topology, thereby establishing secondary homology. The AOs innervated by the Tmd found in Pachystomias and Photostomias appear as two transformations in a reconstruction on the new topology, a result that rejects secondary homology of this structure. The secondary homology of AOs innervated by the Tmx found in Malacosteus and Pachystomias is rejected on the same grounds. Two short‐wave cephalic photophores present in all four genera, the suborbital (SO) and the postorbital (PO), positioned in the posteroventral margin of the orbit and directly posterior to the orbit, respectively, are innervated by separate divisions of the Tmd. The primary homologies of the loosejaw PO and SO across loosejaw taxa are proposed on the basis of similar innervation patterns. Because of dissimilar innervation of the loosejaw SO and SO of basal stomiiforms, primary homology of these photophores cannot be established. Because of similar function and position, the PO of all other stomiid taxa is likely homologous with the loosejaw PO. Nonhomology of loosejaw long‐wave photophores is corroborated by previously published histological evidence. The totality of evidence suggests that the only known far‐red bioluminescent system in vertebrates has evolved as many as three times in a closely related group of deep‐sea fishes. J. Morphol., 2010. © 2009 Wiley‐Liss, Inc.     

Distribution of [3H]noradrenaline and [3H]serotonin in photophores of Porichthys notatus

Summary Photophores of Porichthys notatus were examined by electron-microscopic radioautography following incubation in tritiated noradrenaline ([³H]NA) or serotonin ([³H]5-HT). Nerve varicosities surrounding the photocytes were found to accumulate [³H]NA but not [³H]5-HT, providing compelling evidence for the catecholaminergic nature of the monoaminergic innervation of photophores. The photocytes themselves appeared selectively labelled with both tracers, but the intensity of labelling after [³H]5-HT incubation was considerably greater than after [³H]NA. Stereological sampling of organelle content in photocytes showed ultrastructural differences between [³H]NA- and [³H]5-HT-labelled cells, probably related to light emission induced by NA. The main changes noted after incubation with [³H]NA were mitochondrial swelling and disorganization, increased coalescence of photocytic vesicles and extrusion of vesicular material into the extracellular matrix. With respect to the subcellular localization of [³H]NA and [³H]5-HT within the photocytes, statistical analysis of the distribution of silver grains disclosed a preferential affinity of both labels for appositional zones between mitochondria and coalescent vesicles. Moreover, in the case of 5-HT, selective affinity was also exhibited by sites comprising vesicular membrane and adjacent cytoplasm, suggesting binding of this biogenic amine to the entire membrane of photocytic vesicles.Supported by grants from the Natural Sciences and Engineering Research Council (M.A.), and Medical Research Council of Canada (L.D.). Dr. Pierre Legendre kindly provided advice on statistical methods     

Few But Relatively Large Prey: Trophic Ecology of <Emphasis Type="Italic">Chauliodus sloani</Emphasis> (Pisces: Stomiidae) in Deep Waters of the Central Mediterranean Sea

The trophic ecology of Chauliodus sloani (Stomiidae) was thoroughly investigated by stomach content analysis for the first time in the Mediterranean. Overall 206 individuals (64.0–260.0 mm SL) were collected from 2013 to 2015. C. sloani can be defined a specialist predator which feeds exclusively on mesopelagic fish belonging to Gonostomatidae, Myctophidae, Paralepididae, Phosichthyidae, Sternoptychidae and Stomiidae. Gonostomatidae (%IRI = 26.02), Myctophidae (%IRI = 24.77) and Sternoptychidae (%IRI = 24.35) were important food items for C. sloani. Maurolicus muelleri (%IRI = 36.43), Cyclothone braueri (%IRI = 28.26) and Vinciguerria attenuata (%IRI = 12.97) were the most important prey. Cases of cannibalism were also observed. The examination of food size spectrum, in relation to predator length, demonstrated that C. sloani developed a feeding strategy aimed to maximizing energy input and based on the capture of few and relatively large prey: more than 50% of prey items exceeded the value of 20% for ratio between prey and predator size (SL) and the 5.4% of prey measured more than 50% of predator size. Differences in food composition across seasons were found; C. braueri and M. muelleri were more abundant in autumn, V. attenuata during spring. Prey items mainly belongs to weakly vertical migrating fauna, usually concentrated at 400 m Deep Scattering Layer.     

Interaction between Connexin 43 and nitric oxide synthase in mice heart mitochondria

Connexin 43 (Cx43), which is highly expressed in the heart and especially in cardiomyocytes, interferes with the expression of nitric oxide synthase (NOS) isoforms. Conversely, Cx43 gene expression is down‐regulated by nitric oxide derived from the inducible NOS. Thus, a complex interplay between Cx43 and NOS expression appears to exist. As cardiac mitochondria are supposed to contain a NOS, we now investigated the expression of NOS isoforms and the nitric oxide production rate in isolated mitochondria of wild‐type and Cx43‐deficient (Cx43^{Cre‐ER(T)/fl}) mice hearts. Mitochondria were isolated from hearts using differential centrifugation and purified via Percoll gradient ultracentrifugation. Isolated mitochondria were stained with an antibody against the mitochondrial marker protein adenine‐nucleotide‐translocator (ANT) in combination with either a neuronal NOS (nNOS) or an inducible NOS (iNOS) antibody and analysed using confocal laser scanning microscopy. The nitric oxide formation was quantified in purified mitochondria using the oxyhaemoglobin assay. Co‐localization of predominantly nNOS (nNOS: 93 ± 4.1%; iNOS: 24.6 ± 7.5%) with ANT was detected in isolated mitochondria of wild‐type mice. In contrast, iNOS expression was increased in Cx43^{Cre‐ER(T)/fl} mitochondria (iNOS: 90.7 ± 3.2%; nNOS: 53.8 ± 17.5%). The mitochondrial nitric oxide formation was reduced in Cx43^{Cre‐ER(T)/fl} mitochondria (0.14 ± 0.02 nmol/min./mg protein) in comparison to wild‐type mitochondria (0.24 ± 0.02 nmol/min./mg). These are the first data demonstrating, that a reduced mitochondrial Cx43 content is associated with a switch of the mitochondrial NOS isoform and the respective mitochondrial rate of nitric oxide formation.     

UVB radiation and 17‐hydroxygeranyllinalool diterpene glycosides provide durable resistance against mirid (Tupiocoris notatus) attack in field‐grown Nicotiana attenuata plants

Depending on geographical location, plants are exposed to variable amounts of UVB radiation and herbivore attack. Because the role(s) of UVB in the priming and/or accumulation of plant defence metabolites against herbivores are not well understood, we used field‐grown Nicotiana attenuata plants to explore the effects of UVB on herbivore performance. Consistent with previous reports, UVB‐exposed plants accumulated higher levels of ultraviolet (UV)‐absorbing compounds (rutin, chlorogenic acid, crypto‐chlorogenic acid and dicaffeoylspermidine). Furthermore, UVB increased the accumulation of jasmonic acid, jasmonoyl‐L‐isoleucine and abscisic acid, all phytohormones which regulate plant defence against biotic and abiotic stress. In herbivore bioassays, N. attenuata plants experimentally protected from UVB were more infested by mirids in three consecutive field seasons. Among defence metabolites measured, 17‐hydroxygeranyllinalool diterpene glycosides (HGL‐DTGs) showed strongly altered accumulation patterns. While constitutive HGL‐DTGs levels were higher under UVB, N. attenuata plants exposed to mirid bugs (Tupiocoris notatus) had still more HGL‐DTGs under UVB, and mirids preferred to feed on HGL‐DTGs‐silenced plants when other UVB protecting factors were eliminated by UVB filters. We conclude that UVB exposure not only stimulates UV protective screens but also affects plant defence mechanisms, such as HGL‐DTGs accumulation, and modulates ecological interactions of N. attenuata with its herbivores in nature.     

Early development of bioluminescence suggests camouflage by counter‐illumination in the velvet belly lantern shark Etmopterus spinax (Squaloidea: Etmopteridae)

The development of luminous structures and the acquisition of luminescence competence during the ontogeny of the velvet belly lantern shark Etmopterus spinax, a deep‐sea squalid species, were investigated. The sequential appearance of nine different luminous zones during shark embryogenesis were established, and a new terminology for them given. These zones form the complex luminous pattern observed in free‐swimming animals. The organogenesis of photophores (photogenic organs) from the different luminous zones was followed, and photophore maturation was marked by the appearance of green fluorescent vesicles inside the photocytes (photogenic cells). Peroxide‐induced light emissions as well as spontaneous luminescence analysis indicated that the ability of E. spinax to produce light was linked to the presence of these fluorescent vesicles and occured prior to birth. The size of photogenic organs, as well as the percentage of ventral body surface area occupied by the luminous pattern and covered by photophores increased sharply during embryogenesis but remained relatively stable in free‐swimming animals. All these results strongly suggest camouflage by counter‐illumination in juvenile E. spinax.     

‘Real time’ genetic manipulation: a new tool for ecological field studies

Field experiments with transgenic plants often reveal the functional significance of genetic traits that are important for the performance of the plants in their natural environments. Until now, only constitutive overexpression, ectopic expression and gene silencing methods have been used to analyze gene‐related phenotypes in natural habitats. These methods do not allow sufficient control over gene expression for the study of ecological interactions in real time, of genetic traits that play essential roles in development, or of dose‐dependent effects. We applied the sensitive dexamethasone (DEX)‐inducible pOp6/LhGR expression system to the ecological model plant Nicotiana attenuata and established a lanolin‐based DEX application method to facilitate ectopic gene expression and RNA interference‐mediated gene silencing in the field and under challenging conditions (e.g. high temperature, wind and UV radiation). Fully established field‐grown plants were used to silence phytoene desaturase and thereby cause photobleaching only in specific plant sectors, and to activate expression of the cytokinin (CK) biosynthesis gene isopentenyl transferase (ipt). We used ipt expression to analyze the role of CKs in both the glasshouse and the field to understand resistance to the native herbivore Tupiocoris notatus, which attacks plants at small spatial scales. By spatially restricting ipt expression and elevating CK levels in single leaves, damage by T. notatus increased, demonstrating the role of CKs in this plant–herbivore interaction at a small scale. As the arena of most ecological interactions is highly constrained in time and space, these tools will advance the genetic analysis of dynamic traits that matter for plant performance in nature.     

Patterns in the distribution of myctophid fish in the northern Scotia Sea ecosystem

The mesopelagic fish community of the northern Scotia Sea was investigated during the austral autumn using multi-frequency acoustics, opening and closing nets and pelagic trawls fished from the surface to 1,000 m. The Family Myctophidae (15 species in 5 genera) dominated the ichthyofauna, with larval notothenids caught over the South Georgia shelf and bathylagids and stomiids abundant in deeper hauls. The biomass of myctophids was estimated to be 2.93 g wet weight 1,000 m⁻³, with Electrona carlsbergi, E. antarctica, Protomyctophum bolini, P. choriodon, Gymnoscopelus braueri, G. fraseri, G. nicholsi and Krefftichthys anderssoni, being the most abundant species. Analysis of community structure indicated a high level of depth stratification within the myctophids, with evidence of diurnal vertical migration in some, but not all, species. Length-frequencies of G. braueri, G. nicholsi, E. antarctica and K. anderssoni were multimodal, suggesting that all life stages may be present in the northern Scotia Sea. In contrast, P. choriodon, P. bolini, G. fraseri and E. carlsbergi had unimodal distributions despite having multi-year lifecycles, indicating that they probably migrate into the region from warmer areas to the north.     

The distribution of NADPH diaphorase activity and immunoreactivity to nitric oxide synthase in the nervous system of the pulmonate mollusc Helix aspersa

Enzyme histochemistry and immunocytochemistry were used to determine the distribution of neurons in the snail Helix aspersa which exhibited nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity and/or immunoreactivity to nitric oxide synthase (NOS). NADPH diaphorase-positive cells and fibres were distributed extensively throughout the central and peripheral nervous system. NADPH diaphorase-positive fibres were present in all neuropil regions of the central and peripheral ganglia, in the major interganglionic connectives and in peripheral nerve roots. NADPH diaphorase-positive cell bodies were found consistently in the eyes, the lips, the tentacular ganglia and the procerebral lobes of the cerebral ganglia; staining of cell bodies elsewhere in the nervous system was capricious. The distribution of NOS-like immunoreactivity differed markedly from that of NADPH diaphorase activity. Small clusters of cells which exhibited NOS-like immunoreactivity were present in the cerebral and pedal ganglia; fibres which exhibited NOS-like immunoreactivity were present in restricted regions of the neuropil of the central ganglia. The disjunct distributions of NADPH diaphorase activity and NOS-like immunoreactivity in the neurvous system of Helix suggest that the properties of neuronal NOS in molluscs may differ sigificantly from those described previously for vertebrate animals.     

Priming and filtering of antiherbivore defences among Nicotiana attenuata plants connected by mycorrhizal networks

Arbuscular mycorrhizal fungi (AMF) establish symbiotic associations with a majority of terrestrial plants to form underground common mycorrhizal networks (CMNs) that connect neighbouring plants. Because Nicotiana attenuata plants do not respond to herbivory‐elicited volatiles from neighbours, we used this ecological model system to evaluate if CMNs function in interplant transmission of herbivory‐elicited responses. A mesocosm system was designed to establish and remove CMNs linking N. attenuata plants to examine the herbivory‐elicited metabolic and hormone responses in CMNs‐connected “receiver” plants after the elicitation of “donor” plants by wounding (W) treated with Manduca sexta larval oral secretions (OS). AMF colonization increased constitutive jasmonate (JA and JA‐Ile) levels in N. attenuata roots but did not affect well‐characterized JAs‐regulated defensive metabolites in systemic leaves. Interestingly, larger JAs bursts, and higher levels of several amino acids and particular sectors of hydroxygeranyllinalool diterpene glycoside metabolism were elevated in the leaves of W + OS‐elicited “receivers” with CMN connections with “donors” that had been W + OS‐elicited 6 hr previously. Our results demonstrate that AMF colonization alone does not enhance systemic defence responses but that sectors of systemic responses in leaves can be primed by CMNs, suggesting that CMNs can transmit and even filter defence signalling among connected plants.     

Trichobaris weevils distinguish amongst toxic host plants by sensing volatiles that do not affect larval performance

Herbivorous insects use plant metabolites to inform their host plant selection for oviposition. These host‐selection behaviours are often consistent with the preference–performance hypothesis; females oviposit on hosts that maximize the performance of their offspring. However, the metabolites used for these oviposition choices and those responsible for differences in offspring performance remain unknown for ecologically relevant interactions. Here, we examined the host‐selection behaviours of two sympatric weevils, the Datura (Trichobaris compacta) and tobacco (T. mucorea) weevils in field and glasshouse experiments with transgenic host plants specifically altered in different components of their secondary metabolism. Adult females of both species strongly preferred to feed on D. wrightii rather than on N. attenuata leaves, but T. mucorea preferred to oviposit on N. attenuata, while T. compacta oviposited only on D. wrightii. These oviposition behaviours increased offspring performance: T. compacta larvae only survived in D. wrightii stems and T. mucorea larvae survived better in N. attenuata than in D. wrightii stems. Choice assays with nicotine‐free, JA‐impaired, and sesquiterpene‐over‐produced isogenic N. attenuata plants revealed that although half of the T. compacta larvae survived in nicotine‐free N. attenuata lines, nicotine did not influence the oviposition behaviours of both the nicotine‐adapted and nicotine‐sensitive species. JA‐induced sesquiterpene volatiles are key compounds influencing T. mucorea females’ oviposition choices, but these sesquiterpenes had no effect on larval performance. We conclude that adult females are able to choose the best host plant for their offspring and use chemicals different from those that influence larval performance to inform their oviposition decisions.