Fluoreszenzmikroskopischer Nachweis des Schreckstoffes in den Schreckstoffzellen der Elritze,Phoxinus phoxinus (L.) (Cyprinidae,Ostariophysi, Pisces)

Zusammenfassung Die Schreckstoffzellen der Elritze,Phoxinus phoxinus (L.), zeigen nach Gefriertrocknung der Haut eine Eigenfluoreszenz. Diese wird bei 360–380 nm maximal angeregt; das Emissionsmaximum liegt bei ca. 515 nm. Die Fluoreszenz einer Schreckstoffzelle wird mit der Fluoreszenz des isolierten Schreckstoffes mikrospektralphotofluorimetrisch verglichen: die Emissions-maxima liegen nur 10 nm voneinander entfernt. Die Kurven relativer Fluoreszenzintensität verlaufen weitgehend gleichartig. Dies zeigt, daß der Schreckstoff tatsächlich den Schreckstoffzellen entstammt.

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Fluorescence microscopical demonstration of the alarm substance in the alarm substance cells of the European minnow,Phoxinus phoxinus (L.) (cyprinidae, ostariophysi, pisces)

Summary The alarm substance cells of the European minnow,Phoxinus phoxinus (L.), are autofluorescent after freeze-drying of the skin (Fig. 1). Their autofluorescence is maximally excited at 360–380 nm; the maximum of emission lies at about 515 nm. The fluorescence of an alarm substance cell is compared with the autofluorescence of the isolated alarm substance by means of a microspectro-photofluorometer. The maxima of emission are only about 10 nm apart. The curves of relative fluorescence intensity are almost identical (Fig. 3). These results show that the alarm substance actually comes from the alarm substance cells.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Population differences in the reaction of minnows to alarm substance*

Experiments showed that minnows, Phoxinus phoxinus, sympatric with pike, Esox lucius, responded more vigorously to alarm substance than minnows from a population with no experience of pike predation in the wild. Minnows from the pike-sympatric (Dorset) population were more likely to hide and less likely to risk feeding than their pike-allopatric (Gwynedd) counterparts. The reaction to alarm substance in the pike-sympatric population was further increased when it was presented along with the visual stimulus of a ‘stalking’ model pike. When the Dorset minnows experienced both alarm substance and the pike model together they reduced their inspection behaviour to a level below that of the Gwynedd minnows. Minnows from the Gwynedd (pike-allopatric) population displayed increased levels of shoaling in the treatments in which alarm substance was used.     

Reaction to Alarm Substance in Cave Populations of Astyanax fasciatus (Characidae,Pisces)1

The effect of species-specific skin-extract containing alarm substance has been studied in two epigean and three hypogean populations of the Mexican-tetra Astyanax fasciatus. In all populations tested the feeding rate at the water surface decreased significantly after introducing the alarm substance and the fish avoided this surface area. Additionally epigean fish and some individuals from the Chica cave showed zigzag movements, rapid swimming and hiding. The fish did not respond to a skinextract of a species without alarm substance.     

The Influence of Alarm Substance on Feeding in Zebra Danio Fish (Brachydanio rerio)

The effects of alarm substance on feeding behaviour of zebra danio fish (Brachydanio rerio) were tested by offering them high and low densities of enclosed waterfleas (Daphnia magna). Normally the fish attacked high densities of prey, but when exposed to alarm substance they preferred lower and presumably less confusing prey densities — also lowering their feeding rate.     

Fright Reaction in Gymnocharacinus bergi (Pisces, Characidae), A Relic Fish from Patagonia

Fright reaction, mediated by pheromones, is an antipredatory mechanism in the scale-less mojarra Gymnocharacinus bergi (Pisces, Characidae). The aim of this study was to characterise and quantify the fright reaction of this species and to analyse the alarm response to different doses of alarm substance. The alarm response consisted in a general decrease in the swimming speed and a tendency to remain at the bottom of the test aquarium. A threshold effect was induced by a very low dose of alarm substance, and the intensity of the fright reaction was positively correlated to the dose concentration. The main characteristics of the alarm response appear to be consistent with G. bergi's environmental conditions such as: a higher predation risk in the upper stratum, the abundant pond-weeds of the stream and the great flow and fast speed of the clear waters.     

Alarm reaction and alert state inGambusia Affinis (Pisces,Poeciliidae) in response to chemical stimuli from injured conspecifics

We studied the behavior of the Poeciliid fishGambusia affinis after the introduction of 3 substances into their tank: a homogenization ofGambusia affinis, a homogenization of the Anabantid fishBetta splendens, and a blank made of distilled water. The response of the fish was measured as a change in their spatial distribution in the tank after the introduction of the substance. Two sizes of fish were used, and theGambusia homogenization produced clear alarm reactions in both, the fish fleeing to the bottom of the tank. This is one of a few examples available of recognition of alarm substances in non-ostariophysian fish. In addition, we found that the small fishes that had recently been exposed to the alarm substance stayed in an ‘alert state’, in which they had an increased sensitivity to mechanical and visual fright stimuli.     

Predator Avoidance and Alarm-response Behaviour in Kin-discriminating Tadpoles (Rana cascadae)

When captured by predators, the tadpoles of some species of frogs and toads may release an alarm substance that alters the behaviour of conspecifics. Such ‘alarm response’ behaviour has been proposed as a potential mechanism whereby related conspecifics may ‘warn’ relatives of a predator's presence and thus, improve their inclusive fitness. We examined predator avoidance and alarm response behaviour in tadpoles of the Cascades frog (Rana cascadae) and tested whether such behaviour is influenced by kinship factors. Tadpoles reduced activity when in the presence of a predatory newt (Taricha granulosa). Individuals in sibling groups were more active than both solitary tadpoles and individuals in mixed groups of siblings and nonsiblings. However, we found no evidence of an alarm response in R. cascadae. Behaviour of tadpoles in groups exposed only to predators was not different from that of tadpoles in groups exposed to predators plus crushed conspecifics. Tadpoles in groups exposed to crushed tadpoles were as active as tadpoles in groups exposed to water controls, and some test individuals fed upon the dead tadpoles. Thus, while R. cascadae tadpoles reduce activity in response to newt predators, crushed tadpoles appear to initiate a feeding response rather than an alarm response as has been previously proposed.     

Untersuchungen zur Isolierung und Identifizierung des Schreckstoffes aus der Haut der Elritze,Phoxinus phoxinus (L.) (Cyprinidae,Ostariophysi, Pisces)

Zusammenfassung Es wurde versucht, den Schreckstoff aus der Haut der Elritze,Phoxinus phoxinus (L.) (Cyprinidae), zu isolieren und zu identifizieren. Hierfür wurden die einzelnen Fraktionen im Verhaltensexperiment an Schwärmen des Malabarbärblings,Danio malabaricus (Jerdon) auf ihren Schreckstoffgehalt geprüft. Nach der dünnschichtchromatographischen Auftrennung der reinsten Fraktionen löste nur das Eluat von zwei stark fluoreszierenden Flecken die Schreckreaktion aus. Die Ergebnisse von Vergleichsuntersuchungen an zwei weiteren Species, nämlich dem Malabarbärbling (Cyprinidae) und dem SalmlerAlestes longipinnis (Günther) (Characidae), stimmten mit den an der Elritze gewonnenen Befunden völlig überein. Beim Schreckstoff handelt es sich sehr wahrscheinlich um ein Pterin. Isoxanthopterin wurde als Vergleichssubstanz getestet und löste ebenfalls die Schreckreaktion aus.

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On the isolation and identification of the alarm substance from the skin of the European minnow,Phoxinus phoxinus (L.) (cyprinidae, ostariophysi, pisces)

Summary The alarm substance from the skin of the European minnow,Phoxinus phoxinus (L.) (Cyprinidae), was isolated and identified. The amounts of alarm substance in various fractions of the skin were tested by means of behavioural experiments on schools of the giant danio,Danio malabaricus (Jerdon). From all thinlayer chromatograms tested, only two strongly fluorescent spots elicited the fright reaction. The results of comparative investigations on two other species, the giant danio (Cyprinidae) and the long-finned African tetraAlestes longipinnis (Günther) (Characidae), were in complete agreement with the results onPhoxinus. The results indicate that the alarm substance very probably is a pterin. Comparative tests with isoxanthopterin also elicited the fright reaction.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Role of olfaction and vision cues in feeding behavior and alarm reaction in the catfish pintado, Pseudoplatystoma corruscans

Experiments were performed to investigate senses that are essential for mediating fright reaction and food behavior in Pseudoplatystoma corruscans, pintado. The dilemma “to feed or to flee” was also analyzed in fishes with intact and sectioned olfactory tracts, stimulated by alarm substance extracts and food. Fishes were arranged into five groups: fish with intact lateral olfactory tracts (LOT), fish with intact medial olfactory tract (MOT), fish with tracts totally sectioned (TOTAL, both LOT and MOT), sham operated, and nonoperated fish. The five groups were submitted to either alarm substance extract and food stimulus or to distilled water (control) and food stimulus. Fish reacted to food independently of which tract (LOT, MOT or TOTAL) was sectioned; vision seems necessary and elemental to detect and deflagrate food response. Latency of the responses to each reaction was different between groups. None of the fish with sectioned tracts reacted to alarm substance extract, while sham- and nonoperated fish showed the typical alarm behavior response, leading to the conclusion that olfaction is essential for mediating alarm response. These results indicate that others sense systems (e.g., vision) are sufficient to trigger and elicit feeding behavior and that olfaction is not necessary to fully maintain food detection to qualitative and quantitative extent. However, olfactory tract integrity seems to be required for mediation of alarm reaction in P. corruscans.     

Die Schreckstoffzellen von Phoxinus phoxinus und Morulius chrysophakedion (Cyprinidae,Ostariophysi, Pisces)

Zusammenfassung Die Untersuchung der Histochemie und der Ultrastruktur der Schreckstoffzellen von Phoxinus phoxinus und Morulius chrysophakedion (Cyprininae, Cyprinidae, Cypriniformes, Ostariophysi) führte zu übereinstimmenden Ergebnissen. Unmittelbar perinucleär sind Ribosomen, Mitochondrien und ein Golgikomplex nachweisbar, von dem ein Netzwerk tubulärer Systeme ausgeht. Histochemisch sind in unmittelbarer Kernnähe gelegentlich Glykogengranula, stets ein deutlicher RNS-Gehalt und die Aktivitäten der Succinatdehydrogenase, der Lactatdehydrogenase, in einigen Zellen der Glucose-6-phosphat-dehydrogenase darstellbar. Im gesamten Cytoplasma der Schreckstoffzelle ist die Aktivität der Leucinaminopeptidase vorhanden, deren Maximum ebenfalls in Kernnähe vorliegt. Nur im kernfernen Cytoplasma wurden Proteine und diastaseresistente Polysaccharide nachgewiesen. Aus den Ergebnissen geht hervor, daß die Schreckstoffzellen sekretorisch tätig sind; vermutlich handelt es sich bei dem spezifischen Sekret um ein kleinmolekulares Protein.

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The alarm substance cells of Phoxinus phoxinus and Morulius chrysophakedion (Cyprinidae, Ostariophysi, Pisces)Histochemical and electron microscopical study

Summary The histochemical studies of the alarm substance cells from Phoxinus phoxinus and Morulius chrysophakedion (Cyprininae, Cyprinidae, Cypriniformes, Ostariophysi) are in total agreement with the results of the ultrastructural investigations. Perinuclear ribosomes, mitochondria, and a golgi complex are demonstrated. A tubular network radiates from the golgi complex. Histochemically, glycogen granula can occasionally be demonstrated in proximity to the nucleus. Consistently a strong RNA reaction as well as succinic-dehydrogenase activity, lactic-dehydrogenase activity, and in some cells glucose-6-phosphatedehydrogenase activity can be shown. In the cytoplasm of the alarm substance cell leucyl-aminopeptidase activity exists; its maximum lies in vicinity of the nucleus. Proteins and diastase-resistant polysaccharides are found only in cytoplasm not directly adjacent to the nucleus. The results indicate that the alarm substance cells show a secretory activity. It is assumed that the specific secrete is a protein of low molecular weight.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Predator Inspection Behaviour in a Characin Fish: an Interaction between Chemical and Visual Information?

Recent evidence suggests that predator inspection behaviour by Ostariophysan prey fishes is regulated by both the chemical and visual cues of potential predators. In laboratory trials, we assessed the relative importance of chemical and visual information during inspection visits by varying both ambient light (visual cues) and predator odour (chemical cues) in a 2 × 2 experimental design. Shoals of glowlight tetras (Hemigrammus erythrozonus) were exposed to a live convict cichlid (Archocentrus nigrofasciatus) predator under low (3 lux) or high (50 lux) light levels and in the presence of the odour of a cichild fed tetras (with an alarm cue) or swordtails (Xiphophorus helleri, with an alarm cue not recognized by tetras). Tetras exhibited threat‐sensitive inspection behaviour (increased latency to inspect, reduced frequency of inspection, smaller inspecting group sizes and increased minimum approach distance) towards a predator paired with a tetra‐fed diet cue, regardless of light levels. Similar threat‐sensitive inspection patterns were observed towards cichlids paired with a swordtail‐fed diet cue only under high light conditions. Our data suggest that chemical cues in the form of prey alarm cues in the diet of the predator, are the primary source of information regarding local predation risk during inspection behaviour, and that visual cues are used when chemical information is unavailable or ambiguous.     

Red Squirrels (Sciurus vulgaris) Respond to Alarm Calls of Eurasian Jays (Garrulus glandarius)

Recognition of heterospecific (interspecific) alarm calls has been demonstrated in birds and mammals, but bird–mammal interactions have rarely been studied. Here, I tested the hypothesis that red squirrels (Sciurus vulgaris) are able to recognize alarm calls of a sympatric bird species, the Eurasian jay (Garrulus glandarius), and respond adequately with anti‐predator behaviour. Both animals are preyed upon by the same predators. To test whether squirrels would react to heterospecific alarm calls, I recorded squirrels behaviour during playbacks of jay alarm calls, control playbacks (territorial songs of sympatric songbirds) and during silence. Differences between the control treatment (songbirds) and silence were not significant. Seven of the 13 squirrels responded with escape after broadcasting alarm calls of jays. Further, squirrels spent less time in the patch, expressed a higher vigilance, and showed more rapid head and body movements. These results suggest that squirrels recognize heterospecific alarm vocalizations of jays and discriminate them from equally loud non‐threatening sounds.     

Some Characteristics of Red Light-induced Substance(s) against Botrytis cinerea Produced in Broad Bean Leaflets

The red light-induced antifungal substance(s) produced in broad bean was of relatively high molecular weight, water soluble, heat stable and fungi specific. Cellulose thin layer chromatography (TLC) of infection droplets of Botrytis cinerea or water droplets without spores of B. cinerea, recovered from inoculated broad bean leaflets kept under red light for 48 h, displayed inhibition zones at approximate Rf values of 0.0 and 0.6. Inhibition zones observed in cellulose TLC of water droplets were relatively faint compared to those of infection droplets. In a time-course study of accumulation of the antifungal substance(s), antifungal activity in both water and infection droplets recovered from red light irradiated broad bean leaflets occurred after 24 h irradiation. However, the antifungal activity in infection droplets was significantly higher than in water droplets. The antifungal substance(s) was less active against Botrytis fabae than B. cinerea.     

The ontogeny of soldiers in Prorhinotermes simplex (Isoptera, Rhinotermitidae)

The ontogeny of the soldier caste in Prorhinotermes simplex is studied with emphasis on the instar origin of soldiers, the duration of the presoldier stage, and changes in external anatomy during the soldier morphogenesis. The soldiers of different instar origin are compared morphometrically and by analysis of the soldier defensive secretion. The soldiers were found to develop from larvae of the second up to the eighth instar, via a short (13–17 days) presoldier stage. The early soldier instars were found exclusively in incipient colonies while the mature colony contains late instar soldiers only. The abrupt change in the external anatomy occurs in two steps. During the larva-presoldier moult, the head increases in length but only slightly in width. During the presoldier-soldier moult, both the length and width of the head increase markedly and the typical cordate shape is attained; the relative size of the pronotum increases considerably. The long falcate mandibles develop mainly during the larva-presoldier moult. One antennal segment is added during these two moults. With increasing instar age of soldiers a relative increase of the pronotum and the head size in its posterior region was observed. No functional differences in external anatomy were found among the six soldier instars. The composition of the frontal gland secretion is similar in the six soldier instars, an extraordinarily high amount of a defensive substance, (E)-1-nitropentadecene, was detected together with (Z,E)-α-farnesene, presumably an alarm substance. The strategy of soldier production is discussed. We conclude that the observed patterns of soldier production in P. simplex correspond well to its ecological strategy of an advanced single-site nester able to migrate and form foraging groups. Received 16 August 2005; revised 21 November and 6 February 2006; accepted 16 February 2006.     

Effects of steroid hormones and other substances on alarm substance cells and mucous cells in the epidermis of the European minnow, Phoxinus phoxinus (L.), and other Ostariophysi (Pisces)

The alarm substance cells and mucous cells of various species of Ostariophysi do not react uniformly to treatment with androgen and other steroid hormones. This indicates that the mechanism controlling formation of alarm substance is not uniform in the Ostariophysi and three exist at least two such mechanisms, one of which is operated by androgens and perhaps other steroid hormones of the gonads. Steroid hormones from the adrenal cortex proved ineffective. In the European minnow, anti-androgen (androcur®, cyproteroneacetate) had an efrect opposite to that of androgen, while androgen treatment upon this species led to an increase in the size of mucous cells and a reduction in the alarm substance cells. The epidermis of androgen-treated European minnows contained neither alarm substance cells nor alarm substance, and skin extracts of such fish proved ineffective in behaviour experiments upon fish schools. The effect of further hormone treatments are discussed and the reactions of the European minnow are compared to those of other species.     

An electron microscope study of intrusions into alarm substance cells of the channel catfish

Thin and ultrathin sections of the epidermis of two channel catfish Ictalurus punctatus were studied in light and electron microscopes, respectively, to learn more about intrusions of entire other cell types into alarm substance cells, first noted in 1981. Several degrees of intrusion and several stages in the process of total intrusion are described: microvillus-like projections, pseudopod-like projections, telophase-stage projections, total cell intrusion. In addition, several different cell types intrude: lymphocyte-like cells, general epidermal cells, virus-infected cells and other alarm substance cells. These findings indicate that the alarm substance cell is extraordinarily subject to invasion by other cell types. They suggest that its plasma membrane may have been modified so as to be somewhat less effective in preserving cell integrity (and thus more easily release its alarm pheromone on injury) than is the case with most other cell types or that the alarm substance acts as an attractant for other epidermal cells.     

Circadian Rhythm in Pineal N-Acetyltransferase Activity: Phase Shifting by Light Pulses (II)

Abstract: N-Acetyltransferase (NAT) is an enzyme whose rhythmic activity in the pineal gland and retina is thought responsible for melatonin circadian rhythms. The enzyme has properties of a circadian biological clock—its rhythm persists in constant conditions and it is precisely controlled by light and dark. Experiments are reported in which light pulses of 1 to 10 h duration were imposed on chicks during their dark-time. The effect of these pulses upon the NAT was measured and the effect of the pulses on subsequent NAT was also determined. The experiments support the conclusion that the amount and/or duration of dark-time NAT is limited. This finding is interpreted as supporting the idea that a fixed amount of some substance, an initiator, is synthesized during the subjective day.     

Differential expression of heat shock protein 70 in relation to stress type in the flatworm <Emphasis Type="Italic">Schmidtea polychroa</Emphasis>

Most heat shock proteins help to cope with stress in organisms ranging from bacteria to vertebrates. Many stress types acting on the intensity of intracellular protein can induce expression of heat shock proteins. Here, we studied changes in expression level of heat shock protein 70 (Hsp70), one of the best investigated stress proteins, in response to five potential stress factors in the planarian flatworm Schmidtea polychroa: (1) homogenized planarian tissue, which releases an alarm substance that signals predation injury, (2) physical damage by puncturing, (3) a simulation of ecological competition by adding a mixture of naturally co-occurring species: one Dendrocoelum and two Polycelis flatworms, one Asellus water louse and one leech, and (4) magnesium chloride, which inhibits regeneration ability. We found that alarm substance (1), physical harm (2), and magnesium chloride (4) led to increased expression of Hsp70, while interspecific competition (3) did not result in elevated Hsp70 expression. There was no difference between the experimental negative control and two temporal controls immediately after collection and just before the experiment. Results show that Schmidtea polychroa is not sensitive to sampling and lab maintenance. However, planarian homogenate, magnesium chloride and physical harm all caused Hsp70-inducing stress. We conclude that Hsp70 quantification is appropriate to study the current stress level in planaria in response to specific conditions.     

繁殖期4种淡水鱼类化学预警行为及其与性腺指数的关联

化学预警信息（Chemical alarm cue,CAC）在鱼类捕食者-猎物关系中发挥重要作用,并与二者生存适合度密切关联。繁殖期鱼类的繁殖生理投入和化学预警响应均具有较高的能量需求,因而可能存在某种程度上的生存-繁殖的权衡（Trade-off）。选取高体鳑鲏（Rhodeus ocellatus,卵生,繁殖投入较低）、斑马鱼（Danio rerio,卵生,繁殖投入较高）、孔雀鱼（Poecilia reticulata,卵胎生,繁殖投入较高）和皮球鱼（Poecilia latipinna,卵胎生,繁殖投入很高）等繁殖方式各异、能量投入不同的4种淡水鱼类为实验对象,测定了繁殖IV期实验鱼对不同浓度的化学预警信息（Damage-released chemical alarm cues,DCAC）的行为响应模式,并探究了4种实验鱼的化学预警响应与其性腺指数（Gonadosomatic index,GSI）的关联。结果表明：（1）DCAC对4种实验鱼的空间分布、活跃状态、逃逸行为等大部分化学预警响应参数均有不同程度的影响（P<0.05）;（2）4种实验鱼GSI与其化学预警响应参数的变化无显著相关性（P>0.05）（除高浓度DCAC处理组皮球鱼GSI与底栖时间的变化负相关外）。研究回答了长期以来有关繁殖期鱼类化学预警通讯是否缺失的问题,证实了繁殖期不同繁殖生理投入/繁殖对策的4种淡水鱼类均有显著的化学预警响应,提示鱼类GSI与其化学预警响应程度通常不相关（仅高风险环境下繁殖生理投入很高的物种中可能呈现负相关）。     

The ‘club’ cell and behavioural and physiological responses to chemical alarm cues in the Nile tilapia

The alarm response to skin extract has been well documented in fish. In response to skin extract, there is a decline in both locomotion activity and aggressive interactions. Our observation herein of these responses in the cichlid Nile tilapia, Oreochromis niloticus, confirmed the existence of the alarm response in this species. However, so far there has been a paucity of information on the autonomic correlates of this response. In this study, the ventilatory change in response to the chemical alarm cue was evaluated. This parameter was measured 4 min before and 4 min after exposure to 1 mL of either conspecific skin extract or distilled water (extract vehicle). Skin extract induced an increase in the ventilation rate, which suggested an anticipatory adjustment to potentially harmful stimuli. The chemical cue (alarm substance) also interfered with the prioritisation of responses to different environmental stimuli (stimuli filtering); this was suggested by the observation that the Nile tilapia declined to fight after exposure to a cue that indicates a risk of predation. Furthermore, histological analysis of the Nile tilapia skin revealed the presence of putative alarm substance-producing (club) cells.