The complex interplay between macronutrient intake, cuticular hydrocarbon expression and mating success in male decorated crickets.

The condition dependence of male sexual traits plays a central role in sexual selection theory. Relatively little, however, is known about the condition dependence of chemical signals used in mate choice and their subsequent effects on male mating success. Furthermore, few studies have isolated the specific nutrients responsible for condition-dependent variation in male sexual traits. Here, we used nutritional geometry to determine the effect of protein (P) and carbohydrate (C) intake on male cuticular hydrocarbon (CHC) expression and mating success in male decorated crickets (Gryllodes sigillatus). We show that both traits are maximized at a moderate-to-high intake of nutrients in a P:C ratio of 1 : 1.5. We also show that female precopulatory mate choice exerts a complex pattern of linear and quadratic sexual selection on this condition-dependent variation in male CHC expression. Structural equation modelling revealed that although the effect of nutrient intake on mating success is mediated through condition-dependent CHC expression, it is not exclusively so, suggesting that other traits must also play an important role. Collectively, our results suggest that the complex interplay between nutrient intake, CHC expression and mating success plays an important role in the operation of sexual selection in G. sigillatus.

Constrained evolution of the sex comb in Drosophila simulans.

Male fitness is dependent on sexual traits that influence mate acquisition (precopulatory sexual selection) and paternity (post-copulatory sexual selection), and although many studies have documented the form of selection in one or the other of these arenas, fewer have done it for both. Nonetheless, it appears that the dominant form of sexual selection is directional, although theoretically, populations should converge on peaks in the fitness surface, where selection is stabilizing. Many factors, however, can prevent populations from reaching adaptive peaks. Genetic constraints can be important if they prevent the development of highest fitness phenotypes, as can the direction of selection if it reverses across episodes of selection. In this study, we examine the evidence that these processes influence the evolution of the multivariate sex comb morphology of male Drosophila simulans. To do this, we conduct a quantitative genetic study together with a multivariate selection analysis to infer how the genetic architecture and selection interact. We find abundant genetic variance and covariance in elements of the sex comb. However, there was little evidence for directional selection in either arena. Significant nonlinear selection was detected prior to copulation when males were mated to nonvirgin females, and post-copulation during sperm offence (again with males mated to nonvirgins). Thus, contrary to our predictions, the evolution of the D. simulans sex comb is limited neither by genetic constraints nor by antagonistic selection between pre- and post-copulatory arenas, but nonlinear selection on the multivariate phenotype may prevent sex combs from evolving to reach some fitness maximizing optima.

Sexual Selection on male cuticular hydrocarbons via male-male competition and female choice.

Traditional views of sexual selection assumed that male-male competition and female mate choice work in harmony, selecting upon the same traits in the same direction. However, we now know that this is not always the case and that these two mechanisms often impose conflicting selection on male sexual traits. Cuticular hydrocarbons (CHCs) have been shown to be linked to both social dominance and male attractiveness in several insect species. However, although several studies have estimated the strength and form of sexual selection imposed on male CHCs by female mate choice, none have established whether these chemical traits are also subject to sexual selection via male-male competition. Using a multivariate selection analysis, we estimate and compare sexual selection exerted by male-male competition and female mate choice on male CHC composition in the broad-horned flour beetle Gnatocerus cornutus. We show that male-male competition exerts strong linear selection on both overall CHC abundance and body size in males, while female mate choice exerts a mixture of linear and nonlinear selection, targeting not just the overall amount of CHCs expressed but the relative abundance of specific hydrocarbons as well. We discuss the potential implications of this antagonistic selection with regard to male reproductive success.

Macronutrient intake regulates sexual conflict in decorated crickets.

Sexual conflict results in a diversity of sex-specific adaptations, including chemical additions to ejaculates. Male decorated crickets (Gryllodes sigillatus) produce a gelatinous nuptial gift (the spermatophylax) that varies in size and free amino acid composition, which influences a female's willingness to fully consume this gift. Complete consumption of this gift maximizes sperm transfer through increased retention of the sperm-containing ampulla, but hinders post-copulatory mate choice. Here, we examine the effects of protein (P) and carbohydrate (C) intake on the weight and amino acid composition of the spermatophylax that describes its gustatory appeal to the female, as well as the ability of this gift to regulate sexual conflict via ampulla attachment time. Nutrient intake had similar effects on the expression of these traits with each maximized at a high intake of nutrients with a P : C ratio of 1 : 1.3. Under dietary choice, males actively regulated their nutrient intake but this regulation did not coincide with the peak of the nutritional landscape for any trait. Our results therefore demonstrate that a balanced intake of nutrients is central to regulating sexual conflict in G. sigillatus, but males are constrained from reaching the optima needed to bias the outcome of this conflict in their favour.

Repeatability of a cold stress test to assess cold sensitization.

BACKGROUND: Non-freezing cold injury (NFCI) is a syndrome in which damage to peripheral tissues occurs without the tissues freezing following exposure to low ambient temperatures. AIMS: To assess the test-retest reliability of a cold stress test (CST) used to assess cold sensitization. METHODS: Volunteers with no self-reported history of NFCI undertook the CST on three occasions. Thermal images were taken of the foot and hand before, immediately after and 5min after immersion of the limb in cold water for 2min. Cold sensitization was graded by the two clinicians and the lead author. Spot temperatures from the toe and finger pads were recorded. RESULTS: There were 30 white and 19 black male participants. The ratings indicated substantial agreement [a Cohen's kappa (κ) value of 0.61-0.8] to within ± one grading category for the hands and feet of the white volunteers and the hands of the black volunteers. Limits of agreement (LoA) analysis for toe and finger pad temperatures indicated high agreement (absolute 95% LoA < 5.5°C). Test-retest reliability for the feet of the black volunteers was not supported by the gradings (κ = 0.38) and toe pad temperatures (absolute 95% LoA = 9.5°C and coefficient of variation = 11%). CONCLUSIONS: The test-retest reliability of the CST is considered adequate for the assessment of the cold sensitization of the hands and feet of white and the hands of black healthy non-patients. The study should be repeated with patients who have suffered a NFCI.

Environmental heterogeneity, multivariate sexual selection and genetic constraints on cuticular hydrocarbons in Drosophila simulans.

Sexual selection is responsible for the evolution of many elaborate traits, but sexual trait evolution could be influenced by opposing natural selection as well as genetic constraints. As such, the evolution of sexual traits could depend heavily on the environment if trait expression and attractiveness vary between environments. Here, male Drosophila simulans were reared across a range of diets and temperatures, and we examined differences between these environments in terms of (i) the expression of male cuticular hydrocarbons (CHCs) and (ii) which male CHC profiles were most attractive to females. Temperature had a strong effect on male CHC expression, whereas the effect of diet was weaker. Male CHCs were subject to complex patterns of directional, quadratic and correlational sexual selection, and we found differences between environments in the combination of male CHCs that were most attractive to females, with clearer differences between diets than between temperatures. We also show that genetic covariance between environments is likely to cause a constraint on independent CHC evolution between environments. Our results demonstrate that even across the narrow range of environmental variation studied here, predicting the outcome of sexual selection can be extremely complicated, suggesting that studies ignoring multiple traits or environments may provide an over-simplified view of the evolution of sexual traits.

The genetics of primary and secondary sexual character trade-offs in a horned beetle.

When structures compete for shared resources, this may lead to acquisition and allocation trade-offs so that the enlargement of one structure occurs at the expense of another. Among the studies of morphological trade-offs, their importance has been demonstrated primarily through experimental manipulations and comparative analyses. Relatively, a few studies have investigated the underlying genetic basis of phenotypic patterns. Here, we use a half-sibling breeding design to determine the genetic underpinnings of the phenotypic trade-off between head horns and the male copulatory organ or aedeagus that has been found in the dung beetle Onthophagus taurus. Instead of the predicted negative genetic covariance among characters that trade-off, we find positive genetic covariance between absolute horn and aedeagus length and zero genetic covariance between relative horn and aedeagus length. Therefore, although the genetic covariance between absolute horn and aedeagus length would constrain the independent evolution of primary and secondary sexual characters in this population, there was no evidence of a trade-off. We discuss alternative hypotheses for the observed patterns of genetic correlation between traits that compete for resources and the implications that these have for selection and the evolution of such traits.

Females benefit from multiple mating but not multiple mates in the burying beetle Nicrophorus vespilloides.

Male reproductive success generally increases with number of mates but this need not be true for females. If females are the limiting sex, as few as one mate can be optimal. Despite the theoretical differences driving multiple mating in the sexes, multiple mating is the norm rather than the exception. Empirical investigations are therefore required to determine why females mate with multiple males. Both nonadaptive (correlated responses to selection on males, given the mean mating rates have to be the same) and adaptive (direct or indirect fitness benefits) can drive the evolution of multiple mating in females. Females of the burying beetle Nicorphorus vespilloides often mate repeatedly with the same male, but this appears to be a correlated response to selection on males rather than reflecting direct benefits to females for multiple mating. However, an unexamined alternative to this nonadaptive explanation is that females benefit by mating with multiple different males and therefore are selected for general promiscuity. Here we examine if mating polyandrously provides fitness benefits by examining the effects of number of mates (1, 2 or 3), mating system (monogamous, polyandrous) and their interaction. The only significant influence was mating more than once. This did not depend on type of mating. We suggest that unlike most other species examined, in N. vespilloides mating with the same male repeatedly or with several different males reflects an indiscriminate willingness to mate as a result of correlated selection on males for high rates of mating.

Casein kinase II beta-subunit inhibits the activity of the catalytic alpha-subunit in the absence of salt.

Casein kinase II (CKII) has a subunit structure of alpha 2 beta 2 where alpha is the catalytic subunit. Recombinant Drosophila casein kinase II alpha-subunit expressed in insect cells is inhibited by NaCl, thermally labile and inactivated by binding to plastic. In the presence of detergent (Tween 80) recombinant alpha-subunit has a kcat of 249 min-1 (Km 170 microM) for the peptide substrate RRRDDDSDDD-NH2, compared to recombinant Drosophila CKII with a kcat of 71 min-1 (per mol alpha) (Km 42 microM) and bovine CKII with a kcat of 123 min-1 (per mol alpha) (Km 45 microM) when measured in the absence of NaCl. The kcat values of bovine CKII and recombinant Drosophila CKII measured in the presence of 150 mM NaCl were 429 min-1 (per mol alpha) (Km 82 microM) and 204 min-1 (per mol alpha) (Km 51 microM), respectively. Since the kcat for the Drosophila alpha-subunit is approx. 3-fold greater than the Drosophila CKII measured in the absence of added salt these results indicate that the beta-subunit acts primarily as an inhibitory subunit.

Regulation and crystallization of phosphorylated and dephosphorylated forms of truncated dimeric phenylalanine hydroxylase.

Phenylalanine hydroxylase is regulated in a complex manner, including activation by phosphorylation. It is normally found as an equilibrium of dimeric and tetrameric species, with the tetramer thought to be the active form. We converted the protein to the dimeric form by deleting the C-terminal 24 residues and show that the truncated protein remains active and regulated by phosphorylation. This indicates that changes in the tetrameric quaternary structure of phenylalanine hydroxylase are not required for enzyme activation. Truncation also facilitates crystallization of both phosphorylated and dephosphorylated forms of the enzyme.

FHA domain boundaries of the dun1p and rad53p cell cycle checkpoint kinases.

Dun1p and Rad53p of the budding yeast Saccharomyces cerevisiae are members of a conserved family of cell cycle checkpoint protein kinases that contain forkhead-associated (FHA) domains. Here, we demonstrate that these FHA domains contain 130-140 residues, and are thus considerably larger than previously predicted by sequence comparisons (55-75 residues). In vivo, expression of the proteolytically defined Dun1p FHA domain, but not a fragment containing only the predicted domain boundaries, inhibited the transcriptional induction of repair genes following replication blocks. This indicates that the non-catalytic FHA domain plays an important role in the transcriptional function of the Dun1p protein kinase.

Heated socks maintain toe temperature but not always skin blood flow as mean skin temperature falls.

INTRODUCTION: This study was conducted to examine whether toe skin blood flow (Q(toe)) could be maintained in subjects exposed to cold air by warming the foot using a heated sock. METHODS: Four male subjects were exposed to air at 4 degrees C. They wore aramid shirt and trousers, a coverall made from metalized spun bonded polyethylene ("space blanket" fabric), arctic mittens, a 4-ply aramid balaclava, and loose fitting padded boot liners. The subjects rested recumbent on a couch with a pair of heated socks turned on for 120 min and with socks turned off for a further 60 min. Q(toe) was measured using laser Doppler flowmetry. RSULTS: Although the heated socks maintained the skin temperature of the feet above 37 degrees C, Q(toe) was not maintained in two of the four male subjects for the 120 min that the socks were turned on. DISCUSSION: Heated socks cannot be relied on to maintain Q(toe) or to prevent non-freezing cold injuries during prolonged exposure to cold conditions.

Will prior hyperventilation reduce cerebral blood flow during escape from a submarine?

This study was undertaken to determine if hyperventilation would reduce cerebral blood velocity (CBV) and thereby the risk of decompression illness (DCI) during escape from a submarine and increase the depth from which escape can be made. CBV was measured in eight subjects using Doppler ultrasound as they completed a mock submarine escape exercise. The exercise involved climbing a ladder followed by immersion in cold water-in a real escape the escapee would be exposed to increased pressure and at risk of DCI during the immersion phase. Immediately before the escape exercise the seated subjects either rested or hyperventilated at a controlled rate for2 min. There was a third condition in which the subjects hyperventilated for 2 min and then sat and rested. The three conditions were each undertaken twice. Hyperventilation reduced mean CBV by 45%. In the first session during the first 90s of immersion, CBV was 10% lower (P < 0.05) when the escape procedure followed hyperventilation than when following rest. In the second session CBV was similar for the two escape conditions. Following hyperventilation the restoration of CBV was more rapid during the escape condition than when the subjects rested--the reasons for this are unclear. It is concluded that, although hyperventilation effectively reduces CBV, the reduction is neither sustained during the escape procedure nor sufficiently consistent to recommend that it should be used before escape from a submarine.

Findings from a simulated disabled submarine survival trial.

Eleven volunteer submariners were exposed to simulated disabled submarine conditions for a maximum of 7 days to determine if the limited clothing and rations provided in escape compartments would compromise survival prospects. Daily rations were 0.568 liters of water (none on Day 1) and 100 g of barley sugar. The subjects wore working rig and the liner from the Mark 10 submarine escape and immersion equipment throughout, and slept in the outer dry suit. Air temperature fell from 22 degrees to 4.4 degrees C over 2 days and then remained at 4.4 degrees C. Although the subjects felt cold they were able to maintain their deep body temperature. The greatest threat to survival in this situation would be dehydration, one subject was withdrawn on Day 4 as his urine production over the previous 24 h was 130 ml and if not withdrawn and rehydrated this may have led to renal failure. Other medical problems suffered by the subjects during the 7 days included diarrhea, vomiting, hypoglycemia, headaches, and back pains, and, following the trial, non-freezing cold injuries to their feet. It is concluded that the rations are not adequate and could compromise the submariners ability to survive for 7 days in these conditions and during a subsequent escape procedure.

Inhibition of Siah ubiquitin ligase function.

Tumor hypoxia induces the upregulation of hypoxia-inducible factor 1alpha (Hif-1alpha), which in turn induces the expression of genes including VEGF to recruit new blood vessel outgrowth, enabling tumor growth and metastasis. Interference with the Hif-1 pathway and neoangiogenesis is an attractive antitumor target. The hydroxylation of Hif-1alpha by prolyl-hydroxylase (PHD) proteins during normoxia serves as a recognition motif for its proteasomal degradation. However, under hypoxic conditions, hydroxylation is inhibited and furthermore, PHD proteins are themselves polyubiquitylated and degraded by Siah ubiquitin ligases. Our data demonstrate for the first time that inhibition of the interaction between Siah and PHD proteins using a fragment derived from a Drosophila protein (phyllopod) interferes with the PHD degradation. Furthermore, cells stably expressing the phyllopod fragment display reduced upregulation of Hif-1alpha protein levels and Hif-1-mediated gene expression under hypoxia. In a syngeneic mouse model of breast cancer, the phyllopod fragment reduced tumor growth and neoangiogenesis and prolonged survival of the mice. In addition, levels of Hif-1alpha and its target Glut-1 are reduced in tumors expressing the phyllopod fragment. These data show, in a proof-of-principle study, that Siah protein, the most upstream component of the hypoxia pathway yet identified, is a viable drug target for antitumor therapies.

No evidence for condition-dependent expression of male genitalia in the dung beetle Onthophagus taurus.

We examine the condition-dependence of male genitalia in the dung beetle Onthophagus taurus by manipulating the quality of dung provided for larval growth and development. We show that the influence of larval nutrition differed considerably across three different trait classes (sexual, nonsexual and genital). The size of all nonsexual traits varied with dung quality but their allometric slopes remained unchanged. Relative horn length and allometry, but not absolute horn length, showed a high degree of plasticity with differences in dung quality. In contrast, both absolute size and allometry of genitalia were largely unresponsive to changes in dung quality. Male genitalia exhibited intermediate levels of phenotypic variation and lower allometric slopes than both horns and nonsexual traits. Thus, our findings provide little support for good genes hypotheses of genital evolution. We use our findings to discuss a developmental mechanism and selection pressures that may prevent the condition-dependent expression of genitalia.

The evolution of male genitalia: patterns of genetic variation and covariation in the genital sclerites of the dung beetle Onthophagus taurus.

Three main hypotheses, have been invoked to explain divergent genital evolution, the lock and key, pleiotropy, and sexual selection hypotheses, each of which make different predictions about how genital traits are inherited. Here we used a half-sib breeding design to examine the patterns of genetic variation and covariation between male genital sclerites, and their covariance with general body morphology in the dung beetle Onthophagus taurus. We found CV(A)'s and CV(P)'s were similar for both genital and general morphological traits and that CV(R)'s were large for both trait types. We found that male genital sclerites were negatively genetically correlated with general morphological traits. Variation in male genital morphology has direct implications for a male's fertilization success and the resulting sexual selection acting on male genitalia is predicted to maintain high levels of additive genetic variance. Contrary to this prediction, we found that individual genital sclerites all had low levels of additive genetic variance and large maternal and environmental sources of variation. Our data suggest that the genital sclerites in O. taurus are not inherited independently but as a genetically integrated unit. More importantly, the way the different sclerites function to influence male fertilization success reflects this genetic integration. Even though levels of V(A) in individual genital sclerites may be low, there may still be sufficient V(A) in multivariate trait space for selection to generate evolutionary change in the overall morphology of male genitalia.

Polarographic study of oxaloacetate reduction by isolated pea chloroplasts.

Suspensions of pea chloroplasts, prepared by differential centrifugation, catalyzed oxaloacetate-dependent O(2) evolution (mean rate of 29 determinations 10.9 micromoles per milligram of chlorophyll per hour, sd 3.2) with the concomitant production of malate. At optimum concentrations of oxaloacetate, both reactions were light-dependent, inhibited by 3-(3,4- dichlorophenyl)-1, 1-dimethylurea and oxalate, and enhanced 2.5- to 4-fold by 10 millimolar NH(4)Cl. At concentrations of oxaloacetate (<50 micromolar), 10 millimolar NH(4)Cl was inhibitory. The ratio of O(2) evolved to malate produced was 0.39 to 0.58. The ratio of O(2) evolved to oxaloacetate supplied was commensurate with the theoretical value of 0.5.Chloroplast suspensions contained both NAD- and NADP-malate dehydrogenase activities. It was concluded from oxalate inhibition studies and the promotion of oxaloacetate-dependent O(2) evolution by shocked chloroplasts by NADPH (but not NADH) that the reaction was mediated via the NADP enzyme.

Polarographic Study of Dicarboxylic-Acid-dependent Export of Reducing Equivalents from Illuminated Chloroplasts.

Isolated pea chloroplasts, prepared by differential centrifugation, catalyzed O(2) evolution in the light in the presence of 0.03 to 3 millimolar malate, 0.12 to 1.2 millimolar NAD, 4 millimolar pyruvate and exogenous NAD-malate dehydrogenase and lactate dehydrogenase. The reaction, which did not occur in the absence of any one of these factors, was accompanied by the consumption of pyruvate; the ratio of O(2) evolved to pyruvate consumed was <0.5. When 0.1 millimolar [(14)C]malate was supplied most of the (14)C label was recovered as malate. At low concentrations of malate (<0.1 millimolar), the ratio of O(2) evolved to malate supplied was greater than 0.5.Chloroplasts catalyzed oxaloacetate-dependent O(2) evolution in approximate agreement with the theoretical stoichiometry for the light-coupled reduction of oxaloacetate to malate. Subsequent addition of NAD, pyruvate, NAD-malate dehydrogenase, and lactate dehydrogenase caused immediate resumption of O(2) evolution with the concomitant reduction of pyruvate to lactate externally to the chloroplasts. Resumption of O(2) evolution did not occur in the absence of oxaloacetate. The results are consistent with a mechanism in which oxaloacetate/malate cycles continuously between the chloroplasts and the external solution and serves as a carrier for the transfer of light-generated reducing equivalents for pyruvate reduction outside the chloroplast.