Functional significance of seminal receptacle length in Drosophila melanogaster.

Despite its central role in post-copulatory sexual selection, the female reproductive tract is poorly understood. Here we provide the first experimental study of the adaptive significance of variation in female sperm-storage organ morphology. Using populations of Drosophila melanogaster artificially selected for longer or shorter seminal receptacles, we identify relationships between the length of this primary sperm-storage organ and the number of sperm stored, pattern of progeny production, rate of egg fertilization, remating interval, and pattern of sperm precedence. Costs and benefits of relatively short or long organs were identified. Benefits of longer receptacles include increased sperm-storage capacity and thus progeny production from a single insemination. Results suggest that longer receptacles have not naturally evolved because of developmental time costs and a correlated reduction in longevity of mated females. This latter cost may be a consequence of sexual conflict mediated by ejaculate toxicity. Receptacle length did not alter the pattern of sperm precedence, which is consistent with data on the co-evolution of sperm and female receptacle length, and a pattern of differential male fertilization success being principally determined by the interaction between these male and female traits.

Quantitative genetics of seminal receptacle length in Drosophila melanogaster.

The length of the female's primary sperm-storage organ, the seminal receptacle, has undergone rapid divergence within the Drosophila genus. Quantitative genetic analysis of seminal receptacle length was carried out on two laboratory strains of Drosophila melanogaster that had undergone artificial selection for both increased and decreased organ length. Realized heritabilities were 0.176 and 0.270 for the two experiments. Parental strains, F1, F1r (reciprocal), F2, backcross, and backcross reciprocal generations were used in a line-cross (generation means) analysis. This analysis revealed that additive, dominance, and additive-by-dominance epistasis contributed significantly to the means. No significant maternal effects were found. Variance analysis indicated that a completely additive model was adequate to explain the variances observed in these lines. Castle-Wright minimal estimates of 5.25 and 1.91, segregating loci responsible for mean differences, were found for the two respective experiments. There were significant positive correlations between additive effects of seminal receptacle length and thorax length in both experiments. The correlated evolution of sperm and seminal receptacle length is discussed.

Pest response in packed table grapes to low temperature storage combined with slow-release sulfur dioxide pads in basic and large-scale tests.

The effect of low temperature storage combined with slow release sulfur dioxide pads was determined in basic laboratory and large-scale commercial tests on western flower thrips, Frankliniella occidentalis Pergande; grape mealybug, Pseudococcus maritimus (Ehrhorn); Pacific spider mite, Tetranychus pacificus McGregor; twospotted spider mite, Tetranychus urticae Koch; and omnivorous leafroller, Platynota stultana Walshingham. Temperatures within the foam containers among the packed clusters decreased from ambient to 2 degrees C within approximately 1 d and ranged from 0.4 to 1.7 degrees C in all tests. Sulfur dioxide concentrations in the foam containers ranged between 0.2 and 1.6 ppm during the 1- to 6-wk storage period in basic tests and 0.5-1.1 ppm during the 1- to 8-wk storage period in the large-scale test. Western flower thrips was completely controlled by a > or =1-wk exposure. Grape mealybug mortality was > or =93% after 2-5 wk exposures and 100% after a 6-wk exposure in basic tests. Pacific spider mite and twospotted spider mite mortality was 98.0 and 99.6%, respectively, after a 6-wk exposure. Mortality of grape mealybug and twospotted spider mite increased significantly at > or =3-wk exposures and Pacific spider mite mortality increased significantly at > or =4-wk exposures. Mortality of the spider mites in general was directly related to the duration of exposure. An 8-wk exposure to low temperature storage combined with slow release sulfur dioxide pads in the large-scale test resulted in 100% mortality of western flower thrips, twospotted spider mite, and omnivorous leafroller. The treatment resulted in <8% survival of grape mealybug and <1% survival of Pacific spider mite in the large-scale test. The combination treatment offers an economical method to attain quarantine control of certain insects and mites.

Males' evolutionary responses to experimental removal of sexual selection.

We evaluated the influence of pre- and post-copulatory sexual selection upon male reproductive traits in a naturally promiscuous species, Drosophila melanogaster. Sexual selection was removed in two replicate populations through enforced monogamous mating with random mate assignment or retained in polyandrous controls. Monogamous mating eliminates all opportunities for mate competition, mate discrimination, sperm competition, cryptic female choice and, hence, sexual conflict. Levels of divergence between lines in sperm production and male fitness traits were quantified after 38-81 generations of selection. Three a priori predictions were tested: (i) male investment in spermatogenesis will be lower in monogamy-line males due to the absence of sperm competition selection, (ii) due to the evolution of increased male benevolence, the fitness of females paired with monogamy-line males will be higher than that of females paired with control-line males, and (iii) monogamy-line males will exhibit decreased competitive reproductive success relative to control-line males. The first two predictions were supported, whereas the third prediction was not. Monogamy males evolved a smaller body size and the size of their testes and the number of sperm within the testes were disproportionately further reduced. In contrast, the fitness of monogamous males (and their mates) was greater when reproducing in a non-competitive context: females mated once with monogamous males produced offspring at a faster rate and produced a greater total number of surviving progeny than did females mated to control males. The results indicate that sexual selection favours the production of increased numbers of sperm in D. melanogaster and that sexual selection favours some male traits conferring a direct cost to the fecundity of females.

Solution conformation of the Met 61 to His 61 mutant of Pseudomonas stutzeri ZoBell ferrocytochrome c-551.

The gene encoding for bacterial cytochrome c-551 from Pseudomonas stutzeri substrain ZoBell has been mutated to convert the invariant sixth ligand methionine residue into histidine, creating the site-specific mutant M61H. Proton NMR resonance assignments were made for all main-chain and most-side chain protons in the diamagnetic, reduced form at pH 9.2 and 333 K by two-dimensional NMR techniques. Distance constraints (1074) were determined from nuclear Overhauser enhancements and main-chain torsion-angle constraints (72) from scalar coupling estimates. Solution conformations for the protein were computed by the simulated annealing approach. For 28 computed structures, the root mean squared displacement from the average structure excluding the terminal residues 1, 2, 81, and 82 was 0.52 A (sigma = 0.096) for backbone atoms and 0.90 A (sigma = 0.122) for all heavy atoms. The global folding of the mutant protein is the same as for wild type. The biggest changes are localized in a peptide span over residues 60-65. The most striking behavior of the mutant protein is that at room temperature and neutral pH it exists in a state similar to the molten globular state that has been described for several proteins under mild denaturing conditions, but the mutant converts to a more ordered state at high pH and temperature.

Evolution of female remating behaviour following experimental removal of sexual selection.

The relatively small number of ova produced by a female can be fertilized by a single ejaculate in most species. Why females of many species mate with multiple males is therefore enigmatic, especially given that costs associated with remating have been well documented. Recently, it has been argued that females may remate at a maladaptive rate as an outcome of sexually antagonistic coevolution: the evolutionary tug-of-war between manipulation by one sex and resistance to being manipulated by the other sex. We tested this hypothesis experimentally for the evolution of the female remating interval in a naturally promiscuous species, Drosophila melanogaster. In two replicate populations, sexual selection was removed through enforced monogamous mating with random mate assignment, or retained in polyandrous controls. Monogamy constrains the reproductive success of mates to be identical, thereby converting prior conflicts between mates into opportunities for mutualism. Under these experimental conditions, the sexually antagonistic coevolution hypothesis generates explicit predictions regarding the direction of evolutionary change in female remating behaviour. These predictions are contingent upon the mechanism of male manipulation, which may be mediated biochemically by seminal fluids or behaviourally by courtship. Levels of divergence in female remating interval across lines, and in male ejaculatory and courtship effects on female remating, were quantified after 84 generations of selection. Data refute the hypothesis that the evolutionary change in female remating behaviour was due to sexually antagonistic coevolution of courtship signal and receiver traits. The data were, however, consistent with a hypothesis of sexual conflict mediated through ejaculate manipulation. Monogamy-line males evolved ejaculates that were less effective in inducing female non-receptivity and monogamy-line females evolved to remate less frequently, symptomatic of lowered resistance to ejaculate manipulation. The consistency of the results with alternative hypotheses to explain female promiscuity are discussed.

Teamwork. University of Miami uses competition to sharpen EMS team performance.

Many argue that experience is the best teacher. However, it's often dangerous for the patient and impractical for an EMS system to assess prehospital providers in their actual working environment. Simulated scenario competition fosters clearer thinking and translates into more effective action and enhanced patient outcomes during true emergencies.

Large-scale, on-site confirmatory, and varietal testing of a methyl bromide quarantine treatment to control codling moth (Lepidoptera: Tortricidae) in nectarines exported to Japan.

In total, 30,491 codling moth, Cydia pomonella (L.), 1-d-old eggs on May Grand nectarines in two large-scale tests, and 17,410 eggs on Royal Giant nectarines in four on-site confirmatory tests were controlled with 100% mortality after fumigation with a methyl bromide quarantine treatment (48 g3 for 2 h at > or = 21 degrees C and 50% volume chamber load) on fruit in shipping containers for export to Japan. Ranges (mean +/- SEM) were for percentage sorption 34.7 +/- 6.2 to 46.5 +/- 2.5, and for concentration multiplied by time products 54.3 +/- 0.9 to 74.5 +/- 0.6 g.h/m3 in all tests. In large-scale tests with May Grand nectarines, inorganic bromide residues 48 h after fumigation ranged from 6.8 +/- 0.7 to 6.9 +/- 0.5 ppm, which were below the U.S. Environmental Protection Agency tolerance of 20 ppm; and, organic bromide residues were < 0.01 ppm after 1 d and < 0.001 ppm after 3 d in storage at 0-1 degree C. After completion of larger-scale and on-site confirmatory test requirements, fumigation of 10 nectarine cultivars in shipping containers for export to Japan was approved in 1995. Comparison of LD50s developed for methyl bromide on 1-d-old codling moth eggs on May Grand and Summer Grand nectarines in 1997 versus those developed for nine cultivars in the previous 11 yr showed no significant differences in codling moth response among the cultivars.

Response of omnivorous leafroller (Lepidoptera: Tortricidae) and onion thrips (Thysanoptera: Thripidae) to low-temperature storage.

Eggs and first-fifth instars of omnivorous leafroller, Platynota stultana Walshingham, had a mean percentage survival to the adult stage of 60.7-95.2% for nonexposed immatures and 14.5-54.3% for immatures exposed to 1 wk at 0-1 degree C. Exposures of 2-5 wk resulted in 0-6.7% survival, and a 6-wk exposure resulted in < 1% survival of all stages tested. A significant reduction in survival of all larval stages occurred between exposures of 0 and 1 wk and between 1 wk and 2-6 wk. Survival of eggs after exposures of 0 and 1 wk was significantly different than survival after exposures of 2-6 wk. The second instar was the stage least susceptible to low-temperature storage. Adults that were exposed to low temperature for 1 wk in the third through fifth instars laid a mean of 120-289 eggs per female, and the mean percentage viability of the eggs ranged from 56.2 to 71.4%. Mean percentage survival of adults and nymphs of onion thrips, Thrips tabaci Lindeman, was inversely related to the duration of exposure from 1 through 3-6 wk at 0-1 and 5 degrees C and was lower at 0-1 (0.2-52.5%) than at 5 degrees C (17.6-66.6%). Exposure to 0-1 degree C for 4 wk attained 91.2% control, which increased to 99.8% after 6 wk. Low-temperature storage has potential to control omnivorous leafroller in table grapes, Vitis vinifera L., and onion thrips in onions, Allium cepa L.

Converting a c-type to a b-type cytochrome: Met61 to His61 mutant of Pseudomonas cytochrome c-551.

The gene nirM, coding for cytochrome c-551 in Pseudomonas stutzeri substrain ZoBell, was engineered to mutate Met61, the sixth ligand to the heme c, into His61, thereby converting the typical Met-His coordination of a c-type cytochrome into His-His, typical of b-type cytochromes. The mutant protein was expressed heterologously in Escherichia coli at levels 3-fold higher than in Pseudomonas and purified to homogeneity. The mutant retained low-spin visible spectral characteristics, indicating that the strong field ligand His 61 was coordinated to the iron. The physiochemical properties of the mutant were measured and compared to the wild-type properties. These included visible spectra, ligand binding reactions, stability to temperature and chemical denaturant, oxidation-reduction potentials, and electron-transfer kinetics to the physiological nitrite reductase of Pseudomonas. Despite a change in potential from the normal 260 mV to 55 mV, the mutant retained many of the properties of the c-551 family.

Correlated response in reproductive and life history traits to selection on testis length in Drosophila hydei.

Flies in the genus Drosophila have undergone striking evolutionary divergence in the size and number of sperm produced. Based on comparative studies of sperm length, testis length, and other reproductive and life history traits, including body size, age at first reproduction, and the number of sperm produced, macroevolutionary trade-offs resulting from the need to produce high-investment testes have been postulated. To understand better the microevolutionary processes underlying these interspecific patterns, we imposed replicated bidirectional selection for testis length for 11-12 generations on D. hydei, a species with 23.5 mm-long sperm and 30 mm-long testes. Testis length exhibited realized heritabilities ranging from 0.45 to 0.72. Following selection, traits were assayed for correlated responses. Thorax length, testis mass, sperm length, egg-to-adult development time, and posteclosion maturation time showed consistent positive correlated responses. Numbers of sperm produced and transferred to females, male longevity, female egg productivity, and seminal receptacle length did not show consistent correlated responses to selection on testis length. The pattern of correlated responses to testis length reveal the potential for the evolution of reproductive strategies to alter important life history attributes.

Anthrax explodes in an Australian summer.

Anthrax occurred on 83 properties in an area of north central Victoria between 26 January and 26 March in the summer of 1997. Anthrax had not been recorded in the outbreak area since records were initiated in 1914, although anthrax did occur in the general area in the 1880s to 1890s. Standard Australian control measures were applied to the properties, including quarantine, tracing movements of animals on and off affected properties, secure disposal of carcases by burning, enhanced surveillance of stock generally in the area and the use of local disaster control procedures including an alert of health authorities. As affected property numbers began to increase dramatically from 8 February, it was decided to use blanket area vaccination to control the disease. By 26 February, the epidemic curve had returned to the base line and a buffer vaccination zone of 457 farms holding 78,649 cattle was formed by early March 1997. Between 26 January and 26 March when the outbreak was declared over, 202 cattle and 4 sheep were confirmed to have died of anthrax. Between 27 March and early November a further 26 cattle were confirmed as dying due to anthrax and 14 of these had not had previous vaccination, including four young calves and one horse. One new property within the vaccination buffer zone had an anthrax case in a cow in early November 1997. By mid-November 1997, all previously infected and all neighbouring properties within 1 km were compulsorily re-vaccinated, as were all calves when two months of age and all introduced cattle. In 1998, only two confirmed cases of anthrax were diagnosed; both were vaccinated calves on farms which had had multiple cases during the outbreak. The public reaction and attention fueled by unprecedented media attention led to intense international scrutiny from countries where anthrax is a particular zoonotic problem. Very strong representations had to be made about the safety of livestock and livestock products that came from Victoria. This event has demonstrated that there is a need to review OIE and other requirements and recommendations covering anthrax where strict restrictions are placed on livestock and livestock products to protect livestock and human populations against anthrax infection.

Host status of fresh prunes by potential quarantine pests in laboratory tests and evaluation of packinghouse culls.

The status of fresh prunes, Prunus domestica L., as a host for codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae); peach twig borer, Anarsia lineatella Zeller (Lepidoptera: Gelechiidae); omnivorous leafroller, Platynota stultana Walshingham (Lepidoptera: Tortricidae); oriental fruit moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae); navel orangeworm, Amyelois transitella (Walker) (Lepidoptera: Pyralidae); and walnut husk fly, Rhagoletis completa Cresson (Diptera: Tephritidae), was investigated in laboratory tests and by examination of packinghouse culls. In laboratory no-choice tests, the mean number of adults reared per fruit was 0.01 for codling moth, 0.08 for omnivorous leafroller, 0 for oriental fruit moth, and 1.6 for navel orangeworm. In choice tests the mean number of adults reared per apple or fresh prune was for codling moth, 0.78 and 0.02 (significantly different); for omnivorous leafroller, 0.05 and 0.02; and for oriental fruit moth, 2.07 and 0 (significantly different), respectively. Walnut husk fly oviposited in fresh prunes in no-choice tests but pupae did not develop from the fruit. In choice tests, walnut husk fly did not oviposit in fresh prunes when caged with its normal host, green walnuts, in which large numbers of pupae developed. Inspection of packinghouse culls for immature insects showed that fresh prunes with possible larval feeding sites in the form of frass or fruit gum extrusions were lighter in weight, significantly less firm, similar in color, and had significantly higher soluble solids than noninfested fruit. Based on packinghouse cull samples, 1 fresh prune per 133 harvested fruit would be expected to show possible insect damage. Eleven peach twig borer larvae were found in fresh prune cull samples (213.9 kg) removed from a 16,744.5-kg harvest. The calculated level of infestation was 1 infested fruit per 8,501.8 fruit harvested or per 21.7 cartons of medium-sized packed fruit. Based on our results, the risk of infestation of fresh prunes by the insects in this study would be minimal in fruit exported from the San Joaquin Valley of California.

Treatment of murine experimental autoimmune encephalomyelitis with a myelin basic protein peptide analog alters the cellular composition of leukocytes infiltrating the cerebrospinal fluid.

Experimental autoimmune encephalomyelitis (EAE) can be effectively treated during disease exacerbation by administration of a peptide corresponding to the major T cell epitope of myelin basic protein (MBP), but the mechanism by which T cell tolerance leads to clinical improvement is not well-defined. Acute exacerbations of EAE are accompanied by an infiltration of blood-borne leukocytes into the brain and spinal cord, where they mediate inflammation and demyelination. To investigate peptide effects on infiltrating cells, we collected cerebrospinal fluid (CSF) from (PL/JxSJL)F1 mice with MBP-induced EAE. Pleiocytosis by lymphocytes, neutrophils, and macrophages was seen throughout the course of relapsing-remitting disease. A single administration of the MBP peptide analog, Ac1-11[4Y], reduced disease severity, accompanied by a dramatic and selective loss of neutrophil pleiocytosis. A longer course of peptide therapy resulted in complete recovery from clinical signs of disease, and decreased pleiocytosis by all cell types. Clinical severity throughout the course of disease and therapy was directly related to the degree of infiltration by neutrophils and macrophages, and the clinical improvement following peptide therapy was accompanied by decreased central nervous system (CNS) expression of chemoattractants for these cell types. These observations support a model of disease exacerbation mediated by phagocytic cellular infiltration under the ultimate control of T cell-derived factors, amenable to treatment by down-regulation of the T cell activation state.

Amino acid residues required for binding of lymphocyte function-associated antigen 3 (CD58) to its counter-receptor CD2.

Efficient activation and regulation of the cellular immune response requires engagement of T cell accessory molecules as well as the antigen-specific T cell receptor. The lymphocyte function-associated antigen (LFA) 3 (CD58)/CD2 accessory pathway, one of the first discovered, has been extensively characterized in terms of structure and function of the CD2 molecule, which is present on all T lymphocytes and natural killer cells of the human immune system. The binding site of human CD2 for LFA-3 has been localized to two epitopes on one face of the first immunoglobulin (Ig)-like domain of this two-domain, Ig superfamily molecule. Human LFA-3 is genetically linked and is 21% identical in amino acid sequence to CD2, suggesting that this adhesive pair may have evolved from a single ancestral molecule. We have aligned the amino acid sequences of LFA-3 and CD2 and mutagenized selected amino acids in the first domain of LFA-3 that are analogous to those implicated in the binding site of CD2. The data show that K30 and K34, in the predicted C-C' loop, and D84, in the predicted F-G loop of LFA-3, are involved in binding to CD2, suggesting that two complementary sites on one face of the first domain of each molecule bind to each other.

Specific interaction of lymphocyte function-associated antigen 3 with CD2 can inhibit T cell responses.

Accessory cell surface molecules, such as T cell antigen CD2 and its ligand lymphocyte function-associated antigen 3 (LFA-3; CD58), are critical costimulatory pathways for optimal T cell activation in response to antigens. Interaction of CD2 with cell surface LFA-3 not only increases T cell/accessory cell adhesion, but also induces signal transduction events involved in the regulation of T cell responses. In this report, we show that specific interactions of LFA-3 with CD2 can result in T cell unresponsiveness to antigenic or mitogenic stimuli in vitro. By deletion of certain regions of the extracellular domain of LFA-3, we localized the CD2 binding site to the first domain of LFA-3. We then demonstrated that a soluble, purified first domain-LFA-3/IgG1 fusion protein (LFA3TIP) interacts with CD2 and binds to the same CD2 epitope as purified multimeric or cell surface-expressed LFA-3. LFA3TIP inhibits tetanus toxoid, hepatitis B surface antigen, anti-CD3 mAb, Con A, and phytohemagglutinin P-induced T cell proliferation, as well as xenogeneic and allogeneic mixed lymphocyte reactions (MLR). Unlike anti-LFA-3 or anti-CD2 monoclonal antibodies (mAbs) which inhibit T cell responses by blocking LFA-3/CD2 binding, LFA3TIP is capable of rendering T cells unresponsive to antigenic stimuli in situations where T cell activation is independent of CD2/LFA-3 interactions. Furthermore, LFA3TIP, but not blocking anti-CD2 mAbs, is capable of inducing T cell unresponsiveness to secondary stimulation in allogeneic MLR. This inhibition of T cell responses by LFA3TIP occurs through a different mechanism from that of mAbs to LFA-3 or CD2.

Failure of a single postpartum prostaglandin treatment to improve the reproductive performance of dairy cows.

A study was conducted to evaluate the effects of postpartum prostaglandin treatment on reproduction in 3 seasonal calving dairy herds. Recently calved lactating dairy cows were paired on herd, age, calving date and previous production index. One cow in each of the 196 pairs received a single intramuscular injection of 25 mg of the prostaglandin analogue, dinoprost, between 14 and 28 days after calving. Subsequent reproduction was monitored. Within each herd and overall, there was no significant effect of treatment on the intervals from calving to first service, mating start date to first service, calving to conception, mating start date to conception and first service to conception. Treatment also had no significant effect on 21-day submission and pregnancy rates, on the proportion of each group not pregnant at the end of mating, and on first service pregnancy rates. Responses to treatment did not vary between cows calving within 50 days of mating start date and earlier calving cows or between cows aged less than 5 years and older cows.

Five distinct calcium and phospholipid binding proteins share homology with lipocortin I.

We have purified two 35-kDa proteins from rat peritoneal lavages that inhibit phospholipase A2 activity. Both are calcium/phospholipid-dependent membrane binding proteins and share similar structural and biochemical properties with lipocortins I and II. By sequence analysis we confirmed that they are lipocortin-related, and we refer to the two inhibitors as lipocortins III and V. Using partial sequence information obtained from the purified rat proteins, full length cDNA clones for both proteins and for their human counterparts were isolated. As with lipocortins I and II, the amino acid sequences of lipocortins III and V which were deduced from the cDNA clones are highly conserved, sharing 50% identity with other family members. Related proteins were also purified from bovine intestinal mucosa and characterized by peptide mapping, sequence, and immunological analyses. In addition to lipocortins III and V the bovine preparation contained a third 35-kDa inhibitor and a 68-kDa inhibitor, extending the number of known lipocortins to six distinct proteins. While the various lipocortins are structurally similar, distinct differences in their cellular distribution indicate specialized roles for the individual proteins.