Chloroplast/thylakoid-rich material: A possible alternative to the chemically synthesised flow enhancer polyglycerol polyricinoleate in oil-based systems.

Chloroplasts are abundant organelles in a diverse range of plant materials; they are predominantly composed of multicomponent thylakoid membranes which are lipid and protein rich. Intact or unravelled thylakoid membranes should, in principle, have interfacial activity, but little has been published on their activity in oil-in-water systems, and nothing on their performance on an oil continuous system. In this work different physical methods were used to produce a range of chloroplast/thylakoid suspensions with varying degrees of membrane integrity. Transmission electron microscopy revealed that pressure homogenisation led to the greatest extent of membrane and organelle disruption compared to less energy intensive preparation methods The ability of the derived materials to modulate the flow behaviour of a chocolate model system (65% (w/w) sugar/ sunflower oil (natural amphiphiles removed) suspension) was investigated by acquiring rheological parameters. All chloroplast/thylakoid preparations reduced yield stress, apparent viscosity, tangent flow point and cross over point in a concentration-dependent fashion, although not as significantly as polyglycerol polyricinoleate applied at a commercially relevant concentration in the same chocolate model system. Confocal laser scanning microscopy confirmed presence of the alternative flow enhancer material at the sugar surfaces. This research reveals that low-energy processing methods that do not extensively disrupt thylakoid membranes are applicable to generating materials with marked capacity to affect the flow behaviour of a chocolate model system. In conclusion, chloroplast/thylakoid materials hold strong potential as natural alternatives to synthetic rheology modifiers for lipid-based systems such as PGPR.

Vegetable oils composition affects the intestinal lymphatic transport and systemic bioavailability of co-administered lipophilic drug cannabidiol.

Although natural sesame oil has been shown to facilitate the lymphatic delivery and oral bioavailability of the highly lipophilic drug cannabidiol (CBD), considerable variability remains an unresolved challenge. Vegetable oils differ substantially in composition, which could lead to differences in promotion of intestinal lymphatic transport of lipophilic drugs. Therefore, the differences in composition of sesame, sunflower, peanut, soybean, olive and coconut oils and their corresponding role as vehicles in promoting CBD lymphatic targeting and bioavailability were investigated in this study. The comparative analysis suggests that the fatty acids profile of vegetable oils is overall similar to the fatty acids profile in the corresponding chylomicrons in rat lymph. However, arachidonic acid (C20:4), was introduced to chylomicrons from endogenous nondietary sources. Overall, fatty acid composition of natural vegetable oils vehicles affected the intestinal lymphatic transport and bioavailability of CBD following oral administration in this work. Olive oil led to the highest concentration of CBD in the lymphatic system and in the systemic circulation in comparison to the other natural vegetable oils following oral administration in rats.

Bioaccessibility of essential lipophilic nutrients in a chloroplast-rich fraction (CRF) from agricultural green waste during simulated human gastrointestinal tract digestion.

An in vitro gastrointestinal human digestion model, with and without additional rapeseed oil, was used to measure the bioaccessibility of the major lipophilic nutrients enriched in chloroplasts: ß-carotene; lutein; α-tocopherol; and α-linolenic acid. Chloroplast-rich fraction (CRF) material for this work was prepared from post-harvest pea vine field residue (pea vine haulm, or PVH), an abundant source of freely available, underutilised green biomass. PVH was either steam sterilised (100 °C for 4 min) and then juiced (heat-treated PVH, or HPVH), or was juiced fresh and the juice heated (90 °C for 3 min) (heat-treated juice, or HJ); the CRF from all biomass treatments was recovered from the juice by centrifugation. The impact of postharvest heat treatment of the biomass (HPVH), or of heat treatment of the juice (HJ) derived from the biomass, on the retention and bioaccessibility of the target nutrients was determined. The results showed that both heat treatments increased the apparent retention of ß-carotene, lutein, α-tocopherol, and α-linolenic acid in the CRF material during digestion. The presence of edible oil during digestion did not dramatically affect the retention of these nutrients, but it did increase the bioaccessibility of ß-carotene, lutein, and α-tocopherol from CRF material derived from heated biomass or juice. The presence of oil also increased the bioaccessibility of ß-carotene, but not of lutein, α-tocopherol, or α-linolenic acid, from fresh CRF material.

Sexual selection and 'species recognition' revisited: serial processing and order-of-operations in mate choice.

Following the modern synthesis, mating signals were thought of principally as species recognition traits, a view later challenged by a burgeoning interest in sexual selection-specifically mate choice. In the 1990s, these different signal functions were proposed to represent a single process driven by the shape of female preference functions across both intra- and interspecific signal space. However, the properties of reliable 'recognition' signals (stereotyped; low intraspecific variation) and informative 'quality' signals (condition dependent; high intraspecific variation) seem at odds, perhaps favouring different signal components for different functions. Surprisingly, the idea that different components of mating signals are evaluated in series, first to recognize generally compatible mates and then to select for quality, has never been explicitly tested. Here I evaluate patterns of (i) intraspecific signal variation, (ii) female preference function shape and (iii) phylogenetic signal for male cricket call components known to be processed in series. The results show that signal components processed first tend to have low variation, closed preference functions and low phylogenetic signal, whereas signal components processed later show the opposite, suggesting that mating signal evaluation follows an 'order-of-operations'. Applicability of this finding to diverse groups of organisms and sensory modalities is discussed.

Minimal prezygotic isolation between ecologically divergent sibling species.

Divergence in mating signals typically accompanies speciation. We examine two ecologically divergent sibling species of crickets to assess the degree and timing of the evolution of prezygotic reproductive isolation. Gryllus saxatilis occurs in rocky habitats throughout western North America with long-winged individuals capable of long-distance dispersal; Gryllus navajo is endemic to red-rock sandstone areas of south-eastern Utah and north-eastern Arizona and has short-winged individuals only capable of limited dispersal. Previous genetic work suggested some degree of introgression and/or incomplete lineage sorting is likely. Here we: (1) use restriction site associated DNA sequencing (RAD-seq) genetic data to describe the degree of genetic divergence among species and populations; (2) examine the strength of prezygotic isolation by (i) quantifying differences among male mating songs, and (ii) testing whether females prefer G. saxatilis or G. navajo calling songs. Our results show that genetically distinct "pure" species populations and genetically intermediate populations exist. Male mating songs are statistically distinguishable, but the absolute differences are small. In playback experiments, females from pure populations had no preference based on song; however, females from a genetically intermediate population preferred G. navajo song. Together these results suggest that prezygotic isolation is minimal, and mediated by female behaviour in admixed populations.

Effect of steam sterilisation on lipophilic nutrient stability in a chloroplast-rich fraction (CRF) recovered from postharvest, pea vine field residue (haulm).

Postharvest, pea vine field residue (haulm) was steam-sterilised and then juiced; a chloroplast-rich fraction (CRF) was recovered from the juice by centrifugation. The stability of selected nutrients (ß-carotene, lutein, and α-tocopherol) in the freeze-dried CRF material was measured over 84 days; the impact of temperature (-20 °C, 4 °C, 25 °C and 40 °C), light and air on nutrient stability was established. All three nutrients were stable at -20 °C and 4 °C in the presence or absence of air; this stability was lost at higher temperatures in the presence of air. The extent and rate of nutrient breakdown significantly increased when the CRF samples were exposed to light. ß-Carotene appeared to be more susceptible to degradation than lutein and α-tocopherol at 40 °C in the presence of air, but when CRF was exposed to light all three nutrients measured were significantly broken down during storage at 25 °C or 40 °C, whether exposed to air or not.

Signaler-receiver-eavesdropper: Risks and rewards of variation in the dominant frequency of male cricket calls.

Signals are important for communication and mating, and while they can benefit an individual, they can also be costly and dangerous. Male field crickets call in order to attract female crickets, but gravid females of a parasitoid fly species, Ormia ochracea, are also attracted to the call and use it to pinpoint male cricket hosts. Conspicuousness of the call can vary with frequency, amplitude, and temporal features. Previous work with this system has only considered temporal variation in cricket calls, both large scale, that is, amount of calling and at what time of evening, and small scale, that is, aspects of chirp rate, pulse rate, and numbers of pulses per chirp. Because auditory perception in both crickets and flies relies on the matching of the peak frequency of the call with the peripheral sensory system, peak frequency may be subject to selection both from female crickets and from female flies. Here, we used field playbacks of four different versions of the same male Gryllus lineaticeps calling song that only differed in peak frequency (3.3, 4.3, 5.3, and 6.3 kHz) to test the relative attractiveness of the calls to female crickets and female flies. Our results clearly show that lower frequency calls enhance male safety from fly parasitism, but that the enhanced safety would come at a cost of reduced attraction of female crickets as potential mates. The results imply that eavesdropper pressure can disrupt the matched coevolution of signalers and receivers such that the common concept of matched male-female signaler-receiver coevolution may actually be better described as male-female-predator signaler-receiver-eavesdropper coevolution.

Multilocus phylogeny of Gryllus field crickets (Orthoptera: Gryllidae: Gryllinae) utilizing anchored hybrid enrichment.

We present the first comprehensive molecular phylogeny of Gryllus field cricket species found in the United States and Canada, select additional named Gryllus species found in Mexico and the Bahamas, plus the European field cricket G. campestris Linnaeus and the Afro-Eurasian cricket G. bimaculatus De Geer. Acheta, Teleogryllus, and Nigrogryllus were used as outgroups. Anchored hybrid enrichment was used to generate 492,531 base pairs of DNA sequence from 563 loci. RAxML analysis of concatenated sequence data and Astral analysis of gene trees gave broadly congruent results, especially for older branches and overall tree structure. The North American Gryllus are monophyletic with respect to the two Old World taxa; certain sub-groups show rapid recent divergence. This is the first Anchored Hybrid Enrichment study of an insect group done for closely related species within a single genus, and the results illustrate the challenges of reconstructing the evolutionary history of young rapidly diverged taxa when both incomplete lineage sorting and probable hybridization are at play. Because Gryllus field crickets have been used extensively as a model system in evolutionary ecology, behavior, neuro-physiology, speciation, and life-history and life-cycle evolution, these results will help inform, interpret, and guide future research in these areas.

Molecular biogeography and host relations of a parasitoid fly.

Successful geographic range expansion by parasites and parasitoids may also require host range expansion. Thus, the evolutionary advantages of host specialization may trade off against the ability to exploit new host species encountered in new geographic regions. Here, we use molecular techniques and confirmed host records to examine biogeography, population divergence, and host flexibility of the parasitoid fly, Ormia ochracea (Bigot). Gravid females of this fly find their cricket hosts acoustically by eavesdropping on male cricket calling songs; these songs vary greatly among the known host species of crickets. Using both nuclear and mitochondrial genetic markers, we (a) describe the geographical distribution and subdivision of genetic variation in O. ochracea from across the continental United States, the Mexican states of Sonora and Oaxaca, and populations introduced to Hawaii; (b) demonstrate that the distribution of genetic variation among fly populations is consistent with a single widespread species with regional host specialization, rather than locally differentiated cryptic species; (c) identify the more-probable source populations for the flies introduced to the Hawaiian islands; (d) examine genetic variation and substructure within Hawaii; (e) show that among-population geographic, genetic, and host song distances are all correlated; and (f) discuss specialization and lability in host-finding behavior in light of the diversity of cricket songs serving as host cues in different geographically separate populations.

The identity of the Bahama endemic field cricket Gryllus bryanti Morse (Orthoptera: Gryllidae; Gryllinae; Gryllini).

The Bahama endemic field cricket, Gryllus bryanti Morse, 1905, previously known from only the lost female holotype, is redescribed, including designation of a neotype. Known only from Andros and Eleuthera Islands in The Bahamas, where it occurs with G. assimilis, it can be separated from the latter by song, tegmen file characteristics, head wider than pronotum, and genetics.

Impact of the partial replacement of fish meal with a chloroplast rich fraction on the growth and selected nutrient profile of zebrafish (Danio rerio).

Increasing global aquaculture production, is putting pressure on fishmeal and fish oil supply. There is therefore a growing search for more sustainable sources of proteins and polyunsaturated fatty acids as fish feed ingredients. Chloroplasts are the organelles in the leaves of plants where many of the valuable nutrients, fatty acids (FAs), amino acids, vitamins and pigments, are synthesised. Chloroplasts could be incorporated into fish diets either retained in, or liberated from, plant cells. In this study zebrafish were fed with seven different diets individually; fish were fed with diets reducing fishmeal levels (10, 20 or 50%) using either spinach leaf powder (SLP) or a chloroplast rich fraction (CRF) prepared by an established method to recover chloroplasts. Both SLP and CRF had a positive impact on the growth, taste response, whole fish FA composition, and carotenoid profile. Fish fed with CRF diets showed significantly (P ≤ 0.05) greater α-linolenic (C18:3 n-3) and hexadecatrienoic (C16:3) acid contents than those of SLP and the control. Hexadecanoic acid (C16:3) is a unique FA in the galactolipids of the chloroplast; its presence in zebrafish tissues proves that zebrafish digest and absorb chloroplast galactolipids. The lutein profile of eggs produced by zebrafish fed with the CRF diet was significantly (P ≤ 0.05) higher than those of SLP and the control. Alterations in egg colour were also noted, warranting further investigations of the diet impact on fish fecundity, embryo fertility, hatch rate and larval survival.

Crickets of the genus Gryllus in the United States (Orthoptera: Gryllidae: Gryllinae).

Gryllus field and wood crickets of the United States, mostly west of the Mississippi River, are reviewed and revised. We validate the following 18 Gryllus cricket names: G. armatus, G. assimilis, G. brevicaudus, G. cayensis, G. cohni, G. firmus, G. fultoni, G. integer, G. lineaticeps, G. multipulsator, G. ovisopis, G. pennsylvanicus, G. personatus, G. rubens, G. texensis, G. veletis, G. vernalis, and G. vocalis. We synonymize G. alogus under G. vocalis. We designate a lectotype for G. armatus. We describe the following 17 new Gryllus species: G. chisosensis, G. leei, G. lightfooti, G. longicercus, G. makhosica, G. montis, G. navajo, G. planeta, G. regularis, G. saxatilis, G. sotol, G. staccato, G. thinos, G. transpecos, G. veintinueve, G. veletisoides, and G. vulcanus. We present biology, distribution, and genetic analysis of all taxa and discuss their nearest relatives.

Induced expression of a vestigial sexual signal.

Vestigial morphological traits are common and well known in a variety of taxa. Identification of vestigial genes has illustrated the potential for evolutionary reversals and the re-expression of atavistic traits. Here we induce expression of a behavioural sexual signal, male calling song, in a cricket species, Gryllus ovisopis, which lacks a functional calling song. We successfully used acetylcholine injections in the frontal space of the head of male crickets to activate cerebral command neurons for cricket calling, and we recorded calling songs with a temporal chirp pattern similar to that of G. ovisopis' close evolutionary relatives, G. firmus and G. pennsylvanicus, implying that the neural pattern generators that underlie cricket calling behaviour persist in a vestigial state in G. ovisopis To our knowledge, this is the first demonstration of the induced expression of a vestigial behaviour in any organism. The retention of latent neural capacity to express sexual behaviours could have important implications for rapid evolution, trait re-emergence and reproductive isolation.

Demography and selection shape transcriptomic divergence in field crickets.

Gene flow, demography, and selection can result in similar patterns of genomic variation and disentangling their effects is key to understanding speciation. Here, we assess transcriptomic variation to unravel the evolutionary history of Gryllus rubens and Gryllus texensis, cryptic field cricket species with highly divergent mating behavior. We infer their demographic history and screen their transcriptomes for footprints of selection in the context of the inferred demography. We find strong support for a long history of bidirectional gene flow, which ceased during the late Pleistocene, and a bottleneck in G. rubens consistent with a peripatric origin of this species. Importantly, the demographic history has likely strongly shaped patterns of genetic differentiation (empirical FST distribution). Concordantly, FST -based selection detection uncovers a large number of outliers, likely comprising many false positives, echoing recent theoretical insights. Alternative genetic signatures of positive selection, informed by the demographic history of the sibling species, highlighted a smaller set of loci; many of these are candidates for controlling variation in mating behavior. Our results underscore the importance of demography in shaping overall patterns of genetic divergence and highlight that examining both demography and selection facilitates a more complete understanding of genetic divergence during speciation.

Component analysis of nutritionally rich chloroplasts: recovery from conventional and unconventional green plant species.

A study of the literature indicates that chloroplasts synthesise a range of molecules, many of which have nutritional value for humans, but the nutritional credentials of chloroplasts recovered from plant cells are not established. Chloroplast-rich-fractions (CRFs) were prepared from green plant species and the macro- and micro-nutrient composition compared with the whole leaf materials (WLMs). The results indicated that, on a dry weight basis, CRF material from a range of green biomass was enriched in lipids and proteins, and in a range of micronutrients compared with the WLM. Vitamins E, pro-vitamin A, and lutein were all greater in CRF preparations. Of the minerals, iron was most notably concentrated in CRF. Spinach CRFs possessed the highest α-tocopherol [62 mg 100 g-1, dry weight (DW)], ß-carotene (336 mg 100 g-1 DW) and lutein (341 mg 100 g-1 DW) contents, whilst grass CRFs had the highest concentration of alpha-linolenic acid (ALA) (69.5 mg g-1). The higher concentrations of α-tocopherol, ß-carotene, lutein, ALA and trace minerals (Fe and Mn) in CRFs suggested their potential use as concentrated ingredients in food formulations deficient in these nutrients.

Crystal structure determination as part of an undergraduate laboratory experiment: 1',3',3'-tri-methyl-spiro-[chromene-2,2'-indoline] and 1',3',3'-trimethyl-4-[(E)-(1,3,3-tri-methyl-indolin-2-yl-idene)meth-yl]spiro-[chroman-2,2'-indoline].

The crystal structures of the title compounds, C19H19NO and C31H34N2O, were determined as part of an experiment in an undergraduate teaching laboratory that demonstrates the relationship between mol-ecular structure and function. 1',3',3'-Tri-methyl-spiro-[chromene-2,2'-indoline] is both a photoswitch and thermochromic mol-ecule. Students synthesized it and a bis-indoline adduct and compared the crystallographically determined structures to computed gas-phase models.

How females of chirping and trilling field crickets integrate the 'what' and 'where' of male acoustic signals during decision making.

In crickets acoustic communication serves mate selection. Female crickets have to perceive and integrate male cues relevant for mate choice while confronted with several different signals in an acoustically diverse background. Overall female decisions are based on the attractiveness of the temporal pattern (informative about the 'what') and on signal intensity (informative about the 'where') of male calling songs. Here, we investigated how the relevant cues for mate choice are integrated during the decision process by females of five different species of chirping and trilling field crickets. Using a behavioral design, female preferences in no-choice and choice situations for male calling songs differing in pulse rate, modulation depth, intensities, chirp/trill arrangements and temporal shifts were examined. Sensory processing underlying decisions in female field crickets is rather similar as combined evidence suggested that incoming song patterns were analyzed separately by bilaterally paired networks for pattern attractiveness and pattern intensity. A downstream gain control mechanism leads to a weighting of the intensity cue by pattern attractiveness. While remarkable differences between species were observed with respect to specific processing steps, closely related species exhibited more similar preferences than did more distantly related species.

Divergence in male cricket song and female preference functions in three allopatric sister species.

Multivariate female preference functions for male sexual signals have rarely been investigated, especially in a comparative context among sister species. Here we examined male signal and female preference co-variation in three closely related, but allopatric species of Gryllus crickets and quantified male song traits as well as female preferences. We show that males differ conspicuously in either one of two relatively static song traits, carrier frequency or pulse rate; female preference functions for these traits also differed, and would in combination enhance species discrimination. In contrast, the relatively dynamic song traits, chirp rate and chirp duty cycle, show minimal divergence among species and relatively greater conservation of female preference functions. Notably, among species we demonstrate similar mechanistic rules for the integration of pulse and chirp time scales, despite divergence in pulse rate preferences. As these are allopatric taxa, selection for species recognition per se is unlikely. More likely sexual selection combined with conserved properties of preference filters enabled divergent coevolution of male song and female preferences.

The role of plant cell wall encapsulation and porosity in regulating lipolysis during the digestion of almond seeds.

Previous studies have provided evidence that the physical encapsulation of intracellular nutrients by cell walls of plant foods (i.e. dietary fibre) plays a predominant role in influencing macronutrient bioaccessibility (release) from plant foods during human digestion. One unexplored aspect of this is the extent to which digestive enzymes can pass through the cell-wall barrier and hydrolyse the intracellular lipid in almond seeds. The purpose of the present study was to assess the role played by cell walls in influencing the bioaccessibility and digestibility of almond lipid using a range of techniques. Digestibility experiments were performed on raw and roasted almond cells as well as isolated almond oil bodies using in vitro gastric and duodenal digestion models. Residual triacylglycerols and lipolysis products were extracted after 1 h of incubation and analysed by thin layer chromatography. The lipolysis kinetics of almond cells and oil bodies were also investigated using the pH-stat technique. Finally, the potential penetration of pancreatic lipase through the cell wall matrix was investigated using confocal microscopy. Differences in the rates and extent of lipolysis were clearly seen between almond cells and oil bodies, and these differences were observed regardless of the lipase(s) used. These results also showed that almond cell walls that are completely intact limit lipid digestibility, due to an encapsulation mechanism that hinders the diffusion of lipase into the intracellular environment and lipolysis products out of the cells.

Male Armaments and Reproductive Behavior in "Nutcracker" Camel Crickets (Rhaphidophoridae, Pristoceuthophilus).

Males of many species possess striking weaponry used in intrasexual competition for access to females. Until recently, there were no known cases of male weaponry being used against females in sexual coercion. However, in the camel cricket, Pristoceuthophilus marmoratus, males use modified hind legs to fight with each other and also to trap females and force them to copulate. To determine whether hind leg armaments serve similar fighting and mating functions in morphologically similar congeners, we performed a comparative survey of armament use in intra- and inter-sexual interactions in four additional species of Pristoceuthophilus (P. arizonae and three undescribed species: P. 'Huachuca summer,' P. 'Madera' and P. 'Mt. Pinos'). Intrasexual leg fighting occurred in all species for which trials were performed, and hints of sexual coercion occurred in two species (P. 'Huachuca summer' and P. 'Mt. Pinos'), suggesting additional cases of a uniquely dual-purpose armament. These findings suggest an evolutionary exaptation of hind leg armaments in this genus, wherein an intrasexual fighting weapon took on a secondary function of sexual coercion.