<Emphasis Type="Italic">Pinus radiata</Emphasis> bark extract induces caspase-independent apoptosis-like cell death in MCF-7 human breast cancer cells

In the present study, we investigated the anticancer activity of Pinus radiata bark extract (PRE) against MCF-7 human breast cancer cells. First, we observed that PRE induces potent cytotoxic effects in MCF-7 cells. The cell death had features of cytoplasmic vacuolation, plasma membrane permeabilization, chromatin condensation, phosphatidylserine externalization, absence of executioner caspase activation, insensitivity to z-VAD-fmk (caspase inhibitor), increased accumulation of autophagic markers, and lysosomal membrane permeabilization (LMP). Both the inhibition of early stage autophagy flux and lysosomal cathepsins did not improve cell viability. The antioxidant, n-acetylcysteine, and the iron chelator, deferoxamine, failed to restore the lysosomal integrity indicating that PRE-induced LMP is independent of oxidative stress. This was corroborated with the absence of enhanced ROS production in PRE-treated cells. Chelation of both intracellular calcium and zinc promotes PRE-induced LMP. Geranylgeranylacetone, an inducer of Hsp70 expression, also had no significant protective effect on PRE-induced LMP. Moreover, we found that PRE induces endoplasmic reticulum (ER) stress and mitochondrial membrane depolarization in MCF-7 cells. The ER stress inhibitor, 4-PBA, did not restore the mitochondrial membrane integrity, whereas cathepsin inhibitors demonstrated significant protective effects. Collectively, our results suggest that PRE induces an autophagic block, LMP, ER stress, and mitochondrial dysfunction in MCF-7 cells. However, further studies are clearly warranted to explore the exact mechanism behind the anticancer activity of PRE in MCF-7 human breast cancer cells.     

Evaluation of <Emphasis Type="Italic">Galleria mellonella</Emphasis> larvae as an in vivo model for assessing the relative toxicity of food preservative agents

Larvae of Galleria mellonella are widely used for evaluating the virulence of microbial pathogens and for measuring the efficacy of anti-microbial agents and produce results comparable to those that can be obtained using mammals. In this work, the suitability of using G. mellonella larvae to measure the relative toxicity of a variety of food preservatives was evaluated. The response of larvae to eight commonly used food preservatives (potassium nitrate, potassium nitrite, potassium sorbate, sodium benzoate, sodium nitrate, sodium chloride, sodium nitrite and sodium acetate) administered by feeding or by intra-haemocoel injection was measured. A significant correlation between the LD₅₀ (R ²?=?0.8766, p?=?0.0006) and LD₈₀ (R ²?=?0.7629, p?=?0.0046) values obtained due to oral or intra-haemocoel administration of compounds was established. The response of HEp-2 cells to the food preservatives was determined, and a significant correlation (R ²?=?0.7217, p?=?0.0076) between the LD₅₀ values of the compounds administered by feeding in larvae with the IC₅₀ values of the compounds in HEp-2 cells was established. A strong correlation between the LD₅₀ values of the eight food preservatives in G. mellonella larvae and rats (R ²?=?0.6506, p?=?0.0156) was demonstrated. The results presented here indicate that G. mellonella larvae may be used as a model to evaluate the relative toxicity of food preservatives, and the results show a strong positive correlation to those obtained using established cell culture and mammalian models.     

Cytotoxicity,apoptosis, DNA damage and methylation in mammary and kidney epithelial cell lines exposed to ochratoxin A

This study aimed to investigate the in vitro damage induced by ochratoxin A (OTA) in BME-UV1 and MDCK epithelial cells. Both cells lines were treated with OTA (0 up to 10 μg/mL), and cell viability (MTT assay), membrane stability (lactate dehydrogenase (LDH) release assay) and apoptotic cell rate (Tunel assay) were investigated. Further, the effect of the incubation with OTA has been evaluated at DNA level by the determination of DNA integrity, by the quantification of DNA adduct formation (8-hydroxy-2′-deoxyguanosine (8-OHdG)) and by the assessment of the global DNA methylation status (5-methyl-cytosine (5-mC)). The obtained results showed that after 24 h of OTA treatment, BME-UV1 cell viability was reduced in a dose-dependent way. OTA significantly (P?<?0.05) increased LDH release in BME-UV1 cells at all concentrations tested. OTA (1.25 μg/mL) induced 35 % LDH release in MDCK cells (P?<?0.05). A significant (P?<?0.05) change in percentages of apoptotic BME-UV1 (10?±?0.86) and MDCK (25?±?0.88) cells was calculated when the cells were co-incubated with OTA. The level of 8-OHdG adduct formation was significantly (P?<?0.05) increased in BME-UV1 cells treated with 1.25 μg/mL of OTA. The results of the present study suggest that a different mechanism of action may occur in these cell lines.

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Graphical abstract Study results overview

     

Taxon-specific prey handling by the loggerhead shrike (<Emphasis Type="Italic">Lanius ludovicianus</Emphasis>)

True Shrikes (Laniidae) are sit-and-wait predators that hunt prey from elevated perches and frequently impale them on sharp objects in conspicuous places. During the 2014 breeding season, we followed a pair of Loggerhead Shrike (Lanius ludovicianus) through their reproductive cycle and documented over 100 h of their behavior on video. On three occasions, the shrikes were observed hunting grasshoppers and in a sequence of very quick stereotypic behaviors, they eviscerated their prey. The shrike held the grasshopper in its feet and with its sharp beak, penetrated the body between the head and the shield-like pronotum, removed the digestive tract in one pull, and then ingested the prey. The removal of the alimentary tract appears to target the anterior portion, specifically the foregut or crop, along with its associated contents. This particular behavior was observed only when grasshoppers were caught but not with other invertebrates. The whole process lasted ca. 5 (±2.6 SD) s.     

Sexual aggression by intruders in hooded crow <Emphasis Type="Italic">Corvus cornix</Emphasis>

The hooded crow Corvus cornix is a west Palaearctic, solitary nesting, monogamous corvid. In the breeding season, populations are characterized by a social organization wherein breeding pairs are territorial and non-breeding individuals, called floaters, live in flocks. During a study of the breeding ecology of the hooded crow, conducted in a protected flooded area, we monitored nests with video cameras. We recorded two separate incidents when intruders attacked a female at the nest. We believe that she remained in the nest in order to prevent the strangers cannibalizing the nestlings by mantling over the brood. The spatio-temporal occurrence of these attacks suggests that the observed behaviour is intraspecific sexual aggression wherein non-breeding males mounted an immobilized female.     

Is hissing behaviour of incubating great tits related to reproductive investment in the wild?

Anti-predator behaviour of breeding animals is a complex trait that depends on current reproductive investment as well as individual differences in risk-taking propensities. In response to nest predation, many bird species produce specific sounds, such as the hissing calls in incubating great tits (Parus major), that may provoke an acoustic startle response in the predator. However, it is still unclear whether the propensity of incubating females to produce hissing calls towards nest predators depends on the reproductive investment. With our 3-year study, we show that response type (females that do not hiss versus females giving hissing calls) to a potential nest predator, the woodpecker, is a repeatable trait. We found no differences in the studied reproductive traits between hissing and non-hissing birds. Interestingly, among hissing birds, fast-responding females started egg-laying earlier than slow-responding ones. Among non-hissing birds, heavier birds initiated clutches earlier. We also revealed that hissing birds breed in areas with decreased nest-box occupancy, suggesting either that they potentially select different areas to breed or that territory size is larger as a result of hissing birds being more aggressive. These findings demonstrate that response type is not related to the early reproductive value of the brood across distinct behavioural groups. However, our results do suggest that non-hissing and hissing females differ in terms of individual quality or dominance or personality related aspects.     

Soybean Aphid Response to their Alarm Pheromone E-ß-Farnesene (EBF)

When attacked by natural enemies some insect pests, including many aphid species, alert neighboring conspecifics with alarm pheromones. Cornicle secretions with pheromones benefit the attacked aphid but are costly to produce, while alarm pheromone benefits probably fall largely on alerted conspecifics. Given these variable benefits, the likelihood of a secretion may change depending on aphid density. Thus, we first hypothesized that the common alarm pheromone in aphids, E-ß-farnesene (EBF), was present in soybean aphid (Aphis glycines Matsumura) cornicle secretions and would elicit an alarm response in aphids exposed to it. Second, since aphids other than the secretor also benefit from cornicle secretions, we hypothesized that the likelihood of secretion would increase concurrently with the density of neighboring clonal conspecifics. Third, because alarm reaction behavior (e.g. feeding cessation) is probably costly, we hypothesized that alarm reaction behavior would decrease as conspecific density (i.e. alternative prey for an attacking natural enemy) increased. We found that soybean aphids 1) produce cornicle secretions using EBF as an alarm pheromone, 2) are less likely to release cornicle secretions when alone than in a small group (~10 individuals), but that the rate of secretion does not increase further with additional conspecific density, and 3) also exhibit alarm reaction behavior in response to cornicle secretions independent of aphid density. We show that soybean aphids can use their cornicle secretions to warn their neighbors of probable attack by natural enemies, but that both secretion and alarm reaction behavior does not change as density of nearby conspecifics rises above a few individuals.     

The Mating Behavior of an Undescribed Species of <Emphasis Type="Italic">Rhamphomyia</Emphasis> (Diptera: Empididae)

An undescribed sex-role reversed species of an empidid fly in the genus Rhamphomyia exhibits several unusual behavioral features including the early morning and early evening formation of female swarms low in open woodlands, high in treetops, and over grassy hayfields. These swarms only last between 1 and 2 h each and are composed of females with highly ornamented legs that they hold around their narrow abdomens, which they do not inflate in the manner of R. longicauda and some other members of the genus. Males visit the swarms carrying small insects as nuptial gifts that they transfer to the partners they choose as do males of some other sex-role reversed empidids.     

Feeding Kinematics and Nectar Intake of the Honey Bee Tongue

Most flower-visiting insects employ highly-evolved organs to feed themselves rapidly and efficiently on the floral nectar. A honey bee drives its segmented tongue (glossa) covered by dense hairs reciprocatingly to load nectar. A high-speed camera system ameliorated by a microscope revealed morphological changes in glossal surfaces during live honey bees’ nectar dipping and surface configurations through the stretching of postmortem honey bees’ glossae. Both the in vivo and postmortem observations reveal that shortening and lengthening of the glossal segments perform high concordance with the erection of glossal hairs, which aids in developing deformable gaps between rows of glossal hairs during nectar trapping. A model was proposed to evaluate the nectar-intake volume considering the experimentally-measured average erection angle and tongue elongation length during nectar feeding. The theoretical results fit the experimental data well and disclose that these two factors contribute to an augmentation of nectar-intake observably. We also theoretically present that the extendible and deformable glossae have advantages for the polylectic feeding behavior.