The use of variance in enzyme activity as an indicator of long-term exposure to toxicant-stressed environments in Culex pipiens mosquitoes

1. The identification of easy to use and cheap biomarkers is important as a means of determining whether animals are developing under stressful environmental conditions. Previous studies have indicated that the variance about the mean esterase activity in toxic environments increases in the absence of a significant shift in the mean value, suggesting that variance levels may have potential as a biomarker of toxicant-stressed or otherwise stressful environments.
2. Several field and laboratory populations of the mosquito, Culex pipiens , were examined for esterase activities using a colorimetric assay and levels of polymorphism using polyacrylamide gel electrophoresis. Some of these populations had been exposed to environmental toxicants (organophosphorus (OP) insecticides).
3. The OP-stressed field population had lower levels of polymorphism as indicated by fewer electromorphs than the populations that had not been exposed to OPs. However, the mean level of esterase activity was higher in the OP-stressed populations.
4. Despite having lower genetic variation, the OP-stressed populations showed much higher levels of variation about the mean enzyme activity, at least two orders of magnitude higher, than the unstressed populations. Knowledge of the genetics of OP resistance in these populations confirmed that the increase in variance was not due to the general switching on of genes in response to stress.
5. One field population that had been exposed to heavy metal pollution had similar levels of esterase activity and variation about the mean as the unstressed populations, suggesting that variation only increases in characters directly affected by the environmental pollutant.
6. The probable factors causing the increase in variance and the potential of this type of variation as a biomarker of stressful environments are discussed.     

Comparing grazing by Dreissena polymorpha on phytoplankton in the presence of toxic and non-toxic cyanobacteria

SUMMARY 1. The feeding behaviour of the zebra mussel ( Dreissena polymorpha ) was studied in the laboratory on different combinations of food, including a green alga ( Chlamydomonas reinhardtii ) and toxic and non-toxic strains of the cyanobacterium Microcystis aeruginosa .
2. The highest clearance rate of phytoplankton by zebra mussels was found when the mussels were feeding on a mixture of Chlamydomonas and non-toxic Microcystis , the lowest on a mixture of Chlamydomonas and toxic Microcystis .
3. The differences found in the clearance rates between food combinations can be partly explained by the production of pseudofaeces containing live phytoplankton cells. Zebra mussels expelled significantly more live phytoplankton cells in the presence of toxic Microcystis than in the presence of non-toxic Microcystis . The pseudofaeces contained predominantly live Chlamydomonas cells. Proportionately much less live Microcystis cells were encountered in the pseudofaeces.
4. Consequently, grazing of zebra mussels on a combination of Chlamydomonas and Microcystis may finally result in a dominance of Chlamydomonas over Microcystis . The presence of toxic Microcystis may even strengthen this shift.     

CONDITION DEPENDENCE OF A SEXUALLY SELECTED TRAIT IN A CRUSTACEAN SPECIES COMPLEX: IMPORTANCE OF THE ECOLOGICAL CONTEXT

The genic capture model offers a promising solution to the lek paradox. Heightened condition dependency of sexually selected traits is a prerequisite of this model. Condition dependency is empirically inferred by the sensitivity of traits to stressors. The magnitude of ecological stress (e.g., competition and predation) experienced by populations varies considerably. Thus, condition dependence should manifest more in populations experiencing higher levels of stress. We experimentally assessed the sensitivity of a sexually selected trait (posterior gnathopod) to food resource stress in an amphipod species. We found that gnathopod size variation was 59% higher under food stress, with no corresponding effect on nonsexually selected traits. In addition, we assessed levels of gnathopod variation and the allometry of gnathopods for males sampled from natural populations for two amphipod species that experience different levels of stress (driven by contrasting size‐selective predation and associated life‐history trade‐offs). Populations that experience higher resource stress had both steeper allometries and greater gnathopod size variation. These results suggest that the magnitude of ecological stress experienced by natural populations strongly impacts condition dependency of sexually selected traits, and could play an important role in shaping trait variation and thus the opportunity for sexual selection.     

Grazing rates on toxic and non-toxic strains of cyanobacteria by Hypophthalmichthys molitrix and Oreochromis niloticus

The tilapia Oreochromis niloticus and the silver carp Hypophthalmichthys molitrix were exposed to toxic and non-toxic strains of the cyanobacterium Microcystis aeruginosa in order to determine if cells of the toxic strain were ingested and, if not, by what mechanism they were excluded. Enumeration of cyanobacterial particles before and after exposure to fish showed that there were no significant differences (P<0.05) at the end of the trial between the toxic treatment and the control consisting of toxic M. aeruginosa with no fish. Fish exposed to the non-toxic strain increased opercular beat rate, elevating the volumes of water and food material passed over the gills whereas those that were held in the toxic strain did not. Of the cyanobacterial toxins (microcystins) presented to the fish, most were in the cyanobacterial cells, toxin levels in the water being below the level of detectability (<250 ng l⁻¹), The ability of the fish to differentiate between toxic and non-toxic cyanobacterial strains may thus be determined by very low levels of extracellular microcystins or/and other features which distinguish toxic from non-toxic M. aeruginosa strains, such as cell surface components.     

Glutathione transferase activity and oocyte development in copepods exposed to toxic phytoplankton

Organisms present a series of cellular mechanisms to avoid the effects of toxic compounds. Such mechanisms include the increase in activity of detoxification enzymes [e.g., 7-ethoxyresorufin-O-deethylase (EROD) and glutathione S-transferase (GST)], which could explain the low retention of ingested toxins generally observed in copepods. In addition, decreasing gross growth efficiency (GGE) of copepods with increasing concentration of toxic diets could be caused either by a high expenditure coping with toxins (e.g., increase in the activity of detoxification enzymes) or by a deterioration of reproductive tissues. To assess the effect of toxic phytoplankton on the activity of detoxification enzymes and on oocyte maturation of Acartia tonsa and Temora longicornis, feeding and egg production experiments were carried out with a variety of toxic diets and an adequate non-toxic food control (Rhodomonas spp.) all provided as single species diets. Toxic diets included the nodularin-producing cyanobacterium Nodularia spumigena, the dinoflagellates Alexandrium minutum, and A. tamarense, which contained Paralytic Shellfish Poisoning (PSP) toxins, the dinoflagellate Prorocentrum lima with Diarrhetic Shellfish Poisoning (DSP) toxins and the haptophyte Prymnesium parvum, which produces ichtyotoxins with haemolytic activity. Feeding on toxic diets was lower than on Rhodomonas spp., except for A. minutum and A. tamarense. In addition, toxic diets negatively affected reproduction in both copepod species with the production of oocytes and oocyte development impaired with A. minutum and N. spumigena. While the negative effect of N. spumigena seemed to be connected to gonad atresia likely caused by severe food limitation (starvation), the negative effect of A. minutum could have been either caused by a direct effect of saxitoxins or nutritional inadequacy on oocyte production. We could not detect EROD activity in the copepods, while the activity of GST was generally higher with the non-toxic food control and positively related to the feeding and egestion rates, suggesting relation to feeding conditions rather than to exposure to toxic diets. No relationship was found between GGE and GST activity. Our results refute the hypothesis that toxic diets, provided at ecologically relevant levels, would induce cellular mechanisms in copepods regarding GST activity. GST activity thus seems to play no role in detoxification of copepods confronted with toxic phytoplankton. Toxin detoxification and its cost for copepods still remain an open question.     

Dietary protein constraint on age at maturity: an experimental test with wild deer mice

1. Five populations of Peromyscus maniculatus borealis (Mearns) were live-trapped in the Kananaskis Valley, Alberta, throughout the breeding seasons of 1996 and 1997, to test the hypothesis that growth and maturation of young-of-the-year (YY) females are limited by dietary protein.
2. In 1996, two populations received a high protein (30%) food supplement, one received a high energy, but low protein (14%) food supplement and two others were unsupplemented. In 1997, three populations received the protein (30%) supplement and two served as controls.
3. In 1996, control populations had high nestling growth rates and many (43%) YY females bred. In 1997, nestling growth rates were lower in control populations and no YY females bred.
4. Supplementation of high protein food resulted in consistently high nestling growth rates in both 1996 and 1997, and a significantly higher proportion of YY females bred than controls in both years.
5. Supplementation of high energy, but low protein food had no effect on YY growth or maturation.
6. Neither protein nor energy supplementation had any effects on the number of litters conceived per season, litter success, litter size or sex ratio of litters born to over-wintered females.
7. We conclude that growth and maturation of YY female Peromyscus maniculatus in this area are limited by the availability of dietary protein.     

Altitudinal and environmental variation in lifespan in the Copper butterfly Lycaena tityrus

1.  Variation in longevity within and between natural populations is widespread, and understanding the relative importance of environmental and genetic factors as well as their interactions in mediating such variation is crucial in longevity research.
2.  In this study lifespan of adult copper butterflies was examined in relation to altitude, temperature (20 and 27 °C), sex and adult feeding.
3.  As expected, longevity increased with decreasing temperature, and sucrose-fed butterflies had longer lifespans compared to water-fed and finally non-fed individuals. The impact of feeding, especially of having access to water or not, was larger at the higher compared to the lower temperature.
4.  Regarding altitudinal patterns, increased lifespan in high-altitude populations was largely restricted to beneficial feeding conditions, while under carbohydrate deprivation low-altitude animals lived longer, suggesting that low-altitude butterflies do better under food stress.
5.  Differences in longevity between sexes were small at 20 °C, while females lived substantially longer than males at the higher temperature. Consequently, females may be less susceptible to high temperature stress than males. Further, males suffered more from food stress than females, suggesting that females are generally more stress resistant than males.
6.  Using a full factorial design, this study demonstrates that variation in longevity is caused by several factors, and additionally by substantial interactive effects. Consequently, patterns of variation in longevity are complex, and one needs to be cautious when neglecting this source of variation, by focussing on individual factors only.     

Dynamics of food availability, body condition and physiological stress response in breeding Black-legged Kittiwakes

1. The seasonal dynamics of body condition (BC), circulating corticosterone levels (baseline, BL) and the adrenocortical response to acute stress (SR) were examined in long-lived Black-legged Kittiwakes, Rissa tridactyla , breeding at Duck (food-poor colony) and Gull (food-rich colony) Islands in lower Cook Inlet, Alaska. It was tested whether the dynamics of corticosterone levels reflect a seasonal change in bird physiological condition due to reproduction and/or variation in foraging conditions.
2. BC declined seasonally, and the decline was more pronounced in birds at the food-poor colony. BL and SR levels of corticosterone rose steadily through the reproductive season, and BL levels were significantly higher in birds on Duck Island compared with those on Gull Island. During the egg-laying and chick-rearing stages, birds had lower SR on Duck Island than on Gull Island.
3. The results suggest that, in addition to a seasonal change in bird physiology during reproduction, local ecological factors such as food availability affect circulating levels of corticosterone and adrenal response to acute stress.     

Response of two zooptankton grazers to an ichthyotoxic estuarine dinoflagellate

The dinoflagellate Pfiesteria piscicida (gen. et sp. nov.).a toxic ‘ambush predator’, has been implicated asa causative agent of major fish kills in estuanne ecosystemsof the southeastern USA. Here we report the first experimentaltests of interactions between P.piscicida and estuarine zooplanktonpredators. specifically the rotifer Brachionus plicatilis andthe calanoid copepod Acartia tonsa. Short-term (10 day) exposureof adult B.plicatilis to P.piscicida as a food resource, aloneor in combination with the non-toxic green algae Nannochlorisand Tetraselmis. did not increase rotifer mortality relativeto animals that were given only non-toxic greens Similarly,short-term (3 day) feeding trials using adult A.tonsa indicatedthat the copepods survived equally well on either P.piscicidaor the non-toxic diatom Thalassiosira pseudonana. Copepods giventoxic dinoflagellates exhibited erratic behavior, however, relativeto animals given diatom prey. The fecundity of B.plicatiliswhen fed the toxic dinoflagellate was comparable to or higherthan that of rotifers fed only non-toxic greens We concludethat, on a short-term basis, toxic stages of P.piscicida canbe readily utilized as a nutritional resource by these commonestuarine zooplankters. More long-term effects of P.piscicidaon zooplankton, the potential for toxin bioaccumulation acrosstrophic levels, and the utility of zooplankton as biologicalcontrol agents for this toxic dinoflagellate. remain importantunanswered questions.     

Do colours that deter birds affect cereal bait acceptance by possums (

Poisonous baits used for pest control in New Zealand commonly contain green dye and cinnamon oil to make them less attractive to birds. Research aimed at reducing the impact of poison based pest control on birds has shown that some birds are initially deterred from feeding on blue or, to a lesser extent, green coloured food and are attracted to yellow or red food. We determined whether colours that deter or attract birds affected the acceptance of non-toxic and toxic cereal baits by captive brushtail possums (Trichosurus vulpecula). Individual possums were offered, daily, a choice between a standard green dyed non-toxic cereal bait and either a blue dyed(17 possums) or yellow dyed non-toxic bait (16 possums) for 10 days. Following this, for the first group of 17 possums, 1080 toxin was added to either the green bait (9 possums) or blue bait (8 possums) and possums were offered the green versus blue choice again. Two additional groups that had not previously been fed cereal baits were also given a choice between blue and green baits, one of which was toxic. All possums offered non-toxic bait ate less on the first day of presentation than on subsequent days. There was no difference in acceptance of either blue or yellow coloured non-toxic bait compared to the standard green non- toxic bait on any days. Bait colour appeared to be unimportant in cereal bait choice and did not deter possums from eating any of the baits. The addition of toxin to baits did not significantly alter bait choice in any groups, although some individuals which had no previous experience with baits ate more toxic than non-toxic bait. These data suggest that adding a stronger bird deterring colour (i.e., blue) to poisonous baits is unlikely to adversely affect the acceptance of baits by possums.     

Regulation of Arabidopsis thaliana (L.) Heynh Arginine decarboxylase by potassium deficiency stress.

Arginine decarboxylase (ARGdc) is the first enzyme in one of the two pathways to putrescine in plants. ARGdc enzyme activity has been shown to be induced by many environmental factors, including potassium deficiency stress. We investigated the mechanism for induction of ARGdc activity during potassium deficiency stress in Arabidopsis thaliana (L.) Heynh. We show that A. thaliana responds to potassium deficiency stress by increasing ARGdc activity by up to 10-fold over unstressed plants with a corresponding increase in putrescine levels of up to 20-fold. Spermidine and spermine levels do not increase proportionately. Northern analysis showed no increase in ARGdc mRNA levels correlated with the increase in ARGdc enzyme activity. Western analysis revealed that there was no difference between ARGdc protein levels in stressed plants compared with controls. The increase in ARGdc enzyme activity due to potassium deficiency stress does not appear to involve changes in mRNA or protein abundance.     

Horizontal transfer of non-conjugative plasmid in colony biofilm of Escherichia coli on food-based media

We previously showed that non-conjugative, non-viral lateral plasmid transfer occurs in a colony biofilm of mixed Escherichia coli strains cultured on common laboratory media, such as LB agar. In this report, to investigate the possibility of this plasmid transfer under conditions possible outside the laboratory, we examined the activities of foodstuffs and mixed food extracts, which are possible nutrients for bacteria in human environments, for supporting lateral plasmid transfer. Lateral plasmid transfer occurred in colony biofilms grown on several foodstuffs (roasted meats) and on agar media containing mixed food extracts, which consisted of sugar, milk, and extracts of several foodstuffs (vegetables, fruits, and meats). Lateral plasmid transfer did not occur in liquid culture consisting of the same mixed food extracts, suggesting the importance of colony-biofilm formation. These results suggest the possibility that lateral transfer of non-conjugative plasmid between bacterial cells occurs in biofilms grown with foods or food-like nutrients in the environment.     

Na2CO3胁迫下青海星星草种子萌发过程中淀粉酶活性等的变化

对Na2CO3胁迫下青海星星草种子萌发过程中淀粉酶活性、可溶性糖含量、呼吸强度的变化进行了研究。结果表明在Na2CO3胁迫下,种子萌发过程中淀粉酶活性、可溶性糖含量、呼吸强度与空白对照相比均下降,当Na2CO3浓度大于5.00%时,青海星星草种子在播种后至测量结束时,淀粉酶活性、可溶性糖含量、呼吸强度几乎都没有变化,三者与Na2CO3胁迫浓度的负相关关系均极其显著。在播种后的1～8d可溶性糖含量的变化与淀粉酶活性的变化基本趋于一致,两者呈显著的正相关关系。第8天以后,可溶性糖含量开始降低,而淀粉酶活性继续升高,说明可溶性糖已被利用、转化或合成新物质,Na2CO3胁迫下可溶性糖含量减少主要是因为淀粉酶活性受到了抑制。水解酶活性降低、储藏物质不能动员导致其不能水解、呼吸代谢受抑制是Na2CO3胁迫下青海星星草种子萌发受抑制的主要原因。     

Tackling the toxics in plastics packaging

The widespread use of plastic packaging for storing, transporting, and conveniently preparing or serving foodstuffs is significantly contributing to the global plastic pollution crisis. This has led to many efforts directed toward amending plastic packaging’s end of life, such as recycling, or alternative material approaches, like increasingly using paper for food packaging. But these approaches often neglect the critical issue of chemical migration: When contacting foodstuffs, chemicals that are present in packaging transfer into food and thus unwittingly become part of the human diet. Hazardous chemicals, such as endocrine disrupters, carcinogens, or substances that bioaccumulate, are collectively referred to as “chemicals of concern.” They can transfer from plastic packaging into food, together with other unknown or toxicologically uncharacterized chemicals. This chemical transfer is scientifically undisputed and makes plastic packaging a known, and avoidable, source of human exposure to synthetic, hazardous, and untested chemicals. Here, I discuss this issue and highlight aspects in need of improvement, namely the way that chemicals present in food packaging are assessed for toxicity. Further, I provide an outlook on how chemical contamination from food packaging could be addressed in the future. Robust innovations must attempt systemic change and tackle the issue of plastic pollution and chemical migration in a way that integrates all existing knowledge.

The widespread use of plastic packaging for storing, transporting, and conveniently preparing or serving foodstuffs is significantly contributing to the global plastic pollution crisis. This Essay exhorts us to change the conversation about plastic packaging and address the chemicals that migrate into food.     

Effects of changes in environmental lighting upon levels of hydroxyindole O-methyltransferase activity in the developing-chick gland.

The level of hydroxyindole O-methyltransferase (HIOMT) activity in the pienal gland of developing chicks raised under constant illumination rose more rapidly and to higher values than in the gland of birds maintained in constant darkness. Rates of net increase in activity, and levels of activity attained, for birds raised under a diurnal cycle of illumination were intermediate between those maintained in constant light or darkness. Under each of the lighting conditions, the course of increase in enzymic activity was markedly affected by variations in an unidentified factor, the source of which appeared to be the hatching eggs. Birds transferred from constant light to the dark showed either an arrest of increase in enzyme activity or a loss of activity until the levels equalled that observed for chicks of the same age raised in constant darkness. Chicks transferred from constant darkness to constant illumination showed marked increases in levels of enzyme activity at rates comparable with the maximal values observed with birds maintained under constant illumination, regardless of age and without delay. No diurnal cycle in level of HIOMT activity was observed in the pineals of 15-day birds.     

Can ingested cyanobacteria be harmful to the signal crayfish (Pacifastacus leniusculus)?

1. A sample of adult signal crayfish were taken from a pond with a hepatotoxic bloom of the benthic cyanobacterium Oscillatoria sancta . Cyanobacteria were found in the stomachs of thirty‐one out of thirty‐two crayfish examined.
2. To examine the effect of hepatotoxic cyanobacteria on crayfish a 14‐day feeding trial was carried out with thirty‐six animals. There were three treatments: (i) hepatotoxic and (ii) non‐toxic Planktothrix agardhii; and (iii) crayfish pellets as a control.
3. High‐performance liquid chromatography (HPLC) showed that microcystins (the toxins of P. agardhii ) had accumulated in the hepatopancreas of 50% of the animals in the toxic treatment.
4. The cyanobacteria did not appear to have any negative impact on the crayfish. All crayfish survived, remained motile and ate throughout the experiment.
5. During the experiment blood samples were taken and the total number of haemocytes counted. At the end of the experiment glucose concentration and relative wet weight of the hepatopancreas were measured. No differences between crayfish fed on toxic and non‐toxic P. agardhii and the controls were found.
6. The fact that microcystin accumulates in the crayfish hepatopancreas indicates that the toxin may be transferred further up the food chain.     

Influence of environmental conditions on the ovarian cycle and serum chemistry of Cyprinus carpio in the Dal lake, Kashmir (India)

SUMMARY 1. The study presents adaptive and acclimatory changes observed in Cyprinus carpio communis L with respect to ovarian cycle and serum levels of electrolytes, calcium, total proteins and albumin as a consequence of wide temperature and sunshine variation in the natural environment of Dal lake in Srinagar, Kashmir (India).
2. The atmospheric temperatures in Srinagar show well-defined seasonal and diurnal variations. During the period of study, the mean monthly average temperature in summer (June-August) was 24°C whereas in winter (December-February) it fell to 3.6°C. The total hours of sunshine during the winter months were about half of those found in summer.
3. Under local thermal and photo conditions the fish was found to spawn once a year compared to biannual spawning in tropical chmates.
4. A significant decrease was found in serum sodium, potassium and chloride concentrations during the months of January and February when the mean minimum atmospheric temperatures reached −2.5°C and −l.2°C respectively.
5. The serum calcium levels showed a marked increase during the period corresponding to ovarian maturity and spawning of fish.
6. A significant rise in total serum protein concentration was induced by decreased environmental temperatures. Serum albumin levels were found to exhibit a sharp fall coinciding with the spawning period.     

Natural thermal stress and heat-shock protein expression in Drosophila larvae and pupae

1. Whether Drosophila larvae and pupae naturally experience temperatures that can cause heat damage or death is poorly understood, but bears directly on numerous investigations of the thermal biology and heat-shock response in Drosophila . Accordingly, the temperatures of necrotic fruit, which Drosophila larvae and pupae inhabit, the temperatures of larvae and pupae outside the laboratory, and the levels of the heat-shock protein hsp 70 expressed by larvae in nature were examined.
2. When necrotic fruit was sunlit, internal temperatures rose to levels that can harm indwelling insects. Fruit size and evaporative water loss affected these temperatures. Temperatures of larvae and pupae in the field commonly exceeded 35 °C, with living larvae recorded at >44°C and pupae at >41°C. Natural mortality was evident, presumably because of heat.
3. In the laboratory, these temperatures kill larvae rapidly, with LT₅₀s (time taken for half the sample to be killed) of 30 min at 39 °C, 15 min at 40 °C and 8·5 min at 41 °C. Gradual transfer from 25°C to these temperatures resulted in no lesser mortality than did direct transfer.
4. Hsp 70 levels in lysates of whole larvae were measured by ELISA (enzyme-link immunosorbent assay) with an hsp 70-specific antibody. For larvae within necrotic apples experimentally transferred from shade to sun and within necrotic fruit in situ , hsp 70 levels equalled or exceeded levels detected in parallel laboratory studies of whole larvae or cells in culture.
5. These data provide an ecological context for studies of thermal stress and the heat-shock response in Drosophila that has heretofore been lacking.     

Influence of toxic and non-toxic phytoplankton on feeding and survival of Dreissena polymorpha (Pallas) larvae

Grazing and survival of larvae of the zebra mussel, Dreissena polymorpha, on a green alga and cyanobacteria were studied in laboratory experiments. Clearance rates of the larvae were determined for Chlamydomonas reinhardtii (green alga), two non-toxic and two toxic Microcystis aeruginosa strains (Cyanobacteria). Clearance rates of larvae on non-toxic Microcystis were significantly higher than on toxic Microcystis. The clearance rate on Chlamydomonas reinhardtii was in between the clearance rates on toxic and non-toxic Microcystis strains and not significantly different from them. Effects of toxicity of Microcystis on the survival of zebra mussel larvae was investigated in a short-term experiment. Survival of larvae fed toxic Microcystis was lower than that of larvae fed non-toxic Microcystis, but higher than that of starved larvae. This may imply that, for survival of zebra mussel larvae, it is better to have bad quality (toxic) food than no food.     

A circadian rhythm influencing foraging behaviour in the saw-toothed grain beetle Oryzaephilus surinamensis

ABSTRACT. Adults of Oryzaephilus surinamensis (L.) (Coleoptera, Silvanidae) placed in an open arena containing a refuge showed a cyclic pattern of activity in light, dark (LD) cycles when food in the form of damaged wheat grains was placed in the arena outside the refuge. Placing food in the refuge reduced cyclic change and lowered the general activity of the beetles. Batches of fifty insects conditioned in LD 9:15 h or 15:9 h at 25°C , 65% r.h., and transferred to continuous darkness (DD) at the end of a photophase, showed a circadian rhythm of foraging activity of periodicity near 24 h. Those transferred to continuous light at the end of a scotophase showed a 6 h delay in the onset of the next peak of activity, but subsequent peaks, although damped, revealed a periodicity near that in DD. The mean number of beetles wandering in the arena ranged from about eight in LD 15:9 h with all food in the refuge to about twenty-one in LD 9:15 h with all food in the arena.