DNA barcodes to identify species and explore diversity in the Adelgidae (Insecta: Hemiptera: Aphidoidea).

The Adelgidae are relatively small, cryptic insects, exhibiting complex life cycles with parthenogenetic reproduction. Due to these characteristics, the taxonomy of the group is problematic. Here, we test the effectiveness of the standard 658-bp barcode fragment from the 5'-end of the mitochondrial cytochrome c oxidase 1 gene (COI) in differentiating among 17 species of Adelgidae, in associating life-cycle stages, and in assessing patterns of geographical variation in selected species. Species of Adelgidae are well-differentiated by DNA barcodes, enabling the identification of different morphological forms, immature stages and individuals on different hosts and at different periods of the life cycle. DNA barcodes have uncovered cryptic diversity within taxa and, in other cases, a lack of sequence divergence in species pairs previously separated by life-cycle characteristics, indicating a need for further taxonomic analysis.

Activity and residues of imidacloprid applied to soil and tree trunks to control hemlock woolly adelgid (Hemiptera: Adelgidae) in forests.

We studied imidacloprid application methods and timing to control the hemlock woolly adelgid, Adelges tsugae Annand (Hemiptera: Adelgidae), in forests. The methods compared were 1) soil injection near the trunk; 2) soil injection dispersed throughout the area under the canopy; 3) soil drench near the base of the trunk; and trunk injection with the 4) Arborjet, 5) Wedgle, and 6) Mauget systems. The applications were made in the fall and the following spring. Adelgid populations on the hemlocks (Tsuga spp.) were assessed in the fall of two successive years after the treatments. Relative to the untreated control trees, all the soil applications resulted in population reductions, but none of the trunk injections resulted in reductions. Fall and spring treatment efficacy did not differ. Reductions by the soil treatments were between 50 and 100% (avg 80%) by the first fall and 83-100% (avg 98.5%) by the second fall. Analysis of imidacloprid residues using enzyme-linked immunosorbent assay found residues in sap, needles, and twigs 1 mo to 3-yr after application. A laboratory dose-response bioassay using excised, adelgid-infested hemlock branches with cut ends immersed in serial dilutions of imidacloprid determined the LC50 value to be 300 ppb, based on an exposure of 20 d. A high degree of suppression of the adelgid on forest trees was associated with residues in hemlock tissue > 120 ppb 2 yr after soil treatment. Although precise relationships between residues and efficacy are elusive, it is clear that soil application of imidacloprid resulted in chronic residues of imidacloprid in tissues and suppression of adelgid populations for > 2 yr.

A field trial to evaluate a Mycoplasma hyopneumoniae vaccine: effects on lung lesions and growth rates in swine.

A killed Mycoplasma hyopneumoniae vaccine was evaluated in a single swine herd in which the farrowing barn and weaner rooms were on one Mycoplasma-free farm, while the growing and finishing barn was on a separate farm on which Mycoplasma was present. The study was carried out in a cohort of pigs born in a 12-week period. Pigs born in 6 of the 12 wk were vaccinated and the rest were left as controls. The vaccine was administered twice at approximately 3 and 6 wk of age. Carcass characteristics, lung lesions, and growth rates were recorded on 893, 390, and 220 pigs, respectively. The vaccine reduced the prevalence of pneumonic lesions in slaughter hogs from 69% to 36% (P < 0.001). It also appeared to reduce the prevalence of pleuritis from 20% to 13%, but the difference was only statistically significant at P = 0.07. The vaccine had no effects on carcass characteristics except that carcasses of vaccinated pigs were, on average, 1 kg heavier than those of nonvaccinated pigs, and a smaller percentage of vaccinated pigs were shipped "light" (carcass weight < 70 kg). Two methods were used to estimate the effect of the vaccine on growth rates (as measured by days to 80 kg carcass weight) resulting in estimates of 11 and 2 d reduction attributable to vaccination, respectively. The latter estimate was probably an underestimate for reasons discussed in the paper.

Effects of maternal nutrition and egg provisioning on parameters of larval hatch, survival and dispersal in the gypsy moth, Lymantria dispar L.

North American gypsy moths disperse as newly hatched larvae on wind currents in a behavior called ballooning. Because ballooning occurs before neonates begin to feed, resources used in dispersal are limited to those carried over from the egg. We show that nutritional experience of the maternal parent can influence the tendency of offspring to disperse, and that resource provisioning of eggs by the maternal parent affects the duration of the window for disperal. Offspring of females from defoliated sites had a lower tendency to balloon in a wind tunnel than larvae from females which had not experienced nutritional stress associated with host defoliation. The number of eggs in an egg mass, a reflection of the maternal parent's nutritional experience, also contributed to the predictive model for dispersal that included defoliation level. Egg weight and the levels of two yolk proteins, vitellin (Vt) and glycine-rich protein (GRP), however, had no influence of the proportion of ballooning larvae. The length of survival without food, and thus the maximum period of time for dispersal, was correlated with levels of Vt and GRP, but not with egg weight. The level of defoliation at the site from which the maternal parent was collected was not related to the longevity of offspring, nor did it have a significant effect on the levels of Vt, GRP or egg weight. Levels of hemolymph proteins arylphorin and vitellogenin in the maternal parent during the prepupal stage had no influence on levels of yolk proteins, larval longevity, or tendency to balloon.

Modelling problems in conservation genetics using Drosophila: consequences of fluctuating population sizes.

Many natural populations fluctuate widely in population size. This is predicted to reduce effective population size, genetic variation, and reproductive fitness, and to increase inbreeding. The effects of fluctuating population size were examined in small populations of Drosophila melanogaster of the same average size, but maintained using either fluctuating (FPS) or equal (EPS) population sizes. FPS lines were maintained using seven pairs and one pair in alternate generations, and EPS lines with four pairs per generation. Ten replicates of each treatment were maintained. After eight generations, FPS had a higher inbreeding coefficient than EPS (0.60 vs. 0.38), a lower average allozyme heterozygosity (0.068 vs. 0.131), and a much lower relative fitness (0.03 vs. 0.25). Estimates of effective population sizes for FPS and EPS were 3.8 and 7.9 from pedigree inbreeding, and 4.9 vs. 7.1 from changes in average heterozygosities, as compared to theoretical expectations of 3.3 vs. 8.0. Results were generally in accordance with theoretical predictions. Management strategies for populations of rare and endangered species should aim to minimize population fluctuations over generations.

Measurement of amorphous ferric phosphate as an assessment of iron bioavailability.

A method for measuring amorphous ferric phosphate in complex salt mixtures and animal diets is described. The procedure uses citrate solutions for extraction of salt mixtures and tartrate solution for extraction of prepared diets. Iron in the solution is then determined colorimetrically. Crystalline ferric phosphate, which has no iron bioavailability, is not extracted by either solution. Thus, the procedure can determine if the amorphous form, which has a high iron bioavailability, is present. The procedure was tested on gypsy moth artificial diet and Wesson salt mixture, which is a salt supplement of the diet.

Effects of host switching on gypsy moth (Lymantria dispar (L.)) under field conditions.

Effects of various single and two species diets on the performance of gypsy moth (Lymantria dispar (L.)) were studied when this insect was reared from hatch to population on intact host trees in the field. The tree species used for this study were red oak (Quercus rubra L.), white oak (Q. alba L.), bigtooth aspen (Populus grandidentata Michaux), and trembling aspen (P. tremuloides Michaux). These are commonly available host trees in the Lake States region. The study spanned two years and was performed at two different field sites in central Michigan. Conclusions drawn from this study include: (1) Large differences in gypsy moth growth and survival can occur even among diet sequences composed of favorable host species. (2) Larvae that spent their first two weeks feeding on red oak performed better during this time period than larvae on all other host species in terms of mean weight, mean relative growth rate (RGR), and mean level of larval development, while larvae on a first host of bigtooth aspen were ranked lowest in terms of mean weight, RGR, and level of larval development. (3) Combination diets do not seem to be inherently better or worse than diets composed of only a single species; rather, insect performance was affected by the types of host species eaten and the time during larval development that these host species were consumed instead of whether larvae ate single species diets or mixed species diets. (4) In diets composed of two host species, measures of gypsy moth performance are affected to different extents in the latter part of the season by the two different hosts; larval weights and development rates show continued effects of the first host fed upon while RGRs, mortality, and pupal weights are affected strongly by the second host type eaten. (5) Of the diets investigated in this study, early feeding on red oak followed by later feeding on an aspen, particularly trembling aspen, is most beneficial to insects in terms of attaining high levels of performance throughout their lives.

Nutritional indices in the gypsy moth (Lymantria dispar (L.)) under field conditions and host switching situations.

A large proportion of gypsy moths (Lymantria dispar (L.)) are likely to experience multiple species diets in the field due to natural wandering and host switching which occurs with these insects. Nutritional indices in fourth and fifth instar gypsy moth larvae were studied in the field for insects that were switched to a second host species when they were fourth instars. The tree species used as hosts were northern pin oak (Quercus ellipsoidalis E. J. Hill), white oak (Q. alba L.), big-tooth aspen (Populus grandidentata Michx.), and trembling aspen (P. tremuloides Michx.). Conclusions of this study include: 1) Insects which fed before the host switch on northern pin oak performed better after the host switch than did insects with other types of early dietary experience. While the northern pin oak-started insects had very low relative food consumption rates on their second host species immediately after the switch, one instar later they had the highest ranked consumption rates. During both instars they had the second highest efficiencies of converting ingested and digested food to body mass. High food consumption rates and relatively high efficiency of food conversion helped these insects to obtain the highest ranked mean relative growth rates in the fifth instar compared to the relative growth rates obtained by insects from any of the other first host species. 2) Among the four host species examined, a second host of trembling aspen was most advantageous for the insects. Feeding on this species after the switch led to higher larval weights and higher relative growth rates for insects than did any of the other second host species. The insects on trembling aspen attained excellent growth despite only mediocre to low food conversion efficiencies. The low efficiencies were offset by high relative food consumption rates. 3) Low food consumption rates often tend to be paired with high efficiency of conversion and vice versa. 4) There is no discernable tendency for the first plant species eaten to cause long-term inductions which affect the ability of gypsy moths to utilize subsequent host plants. Insects did not tend to consume more, grow faster, or be more efficient if their second host plant was either the same as their rearing plant or congeneric to it. Methods are delineated which allow values of nutritional indices to be obtained for insects on intact host plants under field conditions. These methods are useful for the purpose of answering questions about the relative effects that different diet treatments have on insect response.

Reproductive performance of dairy cows following treatment with cloprostenol 26 and/or 40 days postpartum: a field trial.

One hundred and seventy Holstein Friesian cows were randomly assigned to receive either 500 ug cloprostenol or saline placebo on Day 26 postpartum followed by 500 ug cloprostenol or saline on Day 40 postpartum. Four treatment groups were formed: Group 1-saline (Day 26)/saline (Day 40); Group 2-cloprostenol/(Day 26) saline (Day 40); Group 3-saline (Day 26)/cloprostenol (Day 40); Group 4-cloprostenol (Day 26)/cloprostenol (Day 40). Double blind techniques were used in administering treatments and in assessing the response to treatment. Palpation of the reproductive tract per tectum and uterine biopsies were performed on 92 cows prior to each treatment at Day 26 and Day 40 postpartum. Progesterone concentrations were determined on milk samples collected prior to treatment. There were no significant differences among treatment groups with respect to services per conception, number of heats detected before first service and culling for infertility. Cloprostenol treatment at Day 26 appeared to delay the first estrus, but it reduced the number of days to conception after the first service. Cows receiving cloprostenol at Days 26 and/or 40 had a decreased calving-to-conception interval compared to controls (P=0.01). Sequential therapy with two doses of cloprostenol resulted in slightly better reproductive performance than either treatment on Day 26 or 40 alone. Treatment with cloprostenol resulted in a decrease in the subsequent incidence of pyometra (P<0.05). It is concluded that in the herd studied, cloprostenol therapy at Day 26 and/or 40 postpartum was beneficial to reproductive performance. Although it was anticipated that cloprostenol would be more effective in cows with elevated progesterone levels, the opposite was observed at the Day 26 cloprostenol treatment. Uterine biopsy at Days 26 and/or 40 had a detrimental effect on subsequent reproductive performance.

A comparison of discriminant analysis and logistic regression for the prediction of coliform mastitis in dairy cows.

Results from discriminant analysis and logistic regression were compared using two data sets from a study on predictors of coliform mastitis in dairy cows. Both techniques selected the same set of variables as important predictors and were of nearly equal value in classifying cows as having, or not having mastitis. The logistic regression model made fewer classification errors. The magnitudes of the effects were considerably different for some variables. Given the failure to meet the underlying assumptions of discriminant analysis, the coefficients from logistic regression are preferable.

The resemblance of clinical attributes between mastitic cows with no growth on bacterial milk cultures and those with gram-positive bacteria cultured.

The clinical attributes of 40 dairy cows which had mastitis but no growth of bacteria from the milk were analyzed and compared to the attributes in 102 cows with only gram-positive and 61 cows with only gram-negative bacteria cultured from the milk. Cows with no bacteria cultured from the milk did not differ significantly from cows with gram-positive bacteria cultured, but 9 of 12 attributes were significantly different between cows with no bacteria cultured and cows with gram-negative bacteria cultured. Discriminant analysis was used to classify cows as members of the gram-positive or gram-negative culture groups. The discriminant equation was then applied to the cows with no bacteria cultured, and 78% of cows with no bacteria cultured were classified as members of the gram-positive group. Most mastitis in cows with no bacteria grown from the milk was probably due to gram-positive bacteria. If antibiotic therapy is used in cows with persistent mastitis and a negative culture in the belief that the culture is a false negative, treatment with antibiotics effective only against gram-negative organisms would not be appropriate.

Analysis of the clinical findings used to diagnose coliform mastitis in dairy cows, and comparison to a prediction model.

Logistic regression was used to analyze the clinical findings (attributes) which predicted coliform mastitis in 113 dairy cattle, 36 of which had coliforms cultured from milk. Weakness of the cow, swelling of the udder, decreased body temperature and watery consistency of the milk were selected for the model. An analysis was then done to find the attributes which clinicians used when predicting that a cow would have a coliform cultured. Clinicians appeared to use water consistency of the milk, shivering, firmness of the udder, pulse rate, elevated body temperature, and respiratory rate. In a final analysis the clinicians' predictions were forced into the model to determine which attributes might be used by clinicians to increase diagnostic accuracy. Inclusion of weakness of the cow, swelling of the udder, decreased temperature of the cow, and duration of mastitis of less than 24 hours increased accuracy over clinical prediction alone. Accuracy of cowside diagnosis might be increased if more attention were paid to these attributes when making a diagnosis of coliform mastitis.

Relationships between leaf age and the food quality of cottonwood foliage for the gypsy moth, Lymantria dispar.

The cottonwood tree, Populus deltoides, continues to produce leaves late into the growing season, exposing midseason herbivores to leaves of a wide range of maturity. Gypsy moth larvae preferred and grew best on the oldest cottonwood leaves and suffered higher mortality and 85% less growth when fed young, expanding leaves. Concentration of phenolics in the youngest leaves was 3 times that in the oldest leaves and was negatively correlated with caterpillar growth rate. The active phenolics were not identified; tannin was present but its concentration changed more with season than leaf age.

Discriminant analysis of the clinical indicants for bovine coliform mastitis.

We used discriminant analysis to assess the indicants most useful in predicting whether a cow had coliform bacterial mastitis. One hundred and twenty-nine mastitic cows were divided into two groups, namely those with milk cultures that yielded pure or mixed gram negative organisms, and cows with other organisms or negative culture. Of 21 indicants examined by discriminate analysis only a history of previous mastitis in the affected quarter, weakness, clear or white color of milk, swelling of the udder, water consistency of the milk, lack of previous mastitis in other quarters, lack of palpable udder abscesses, and elevated body temperature were significantly associated with coliform mastitis. Using these variables 78% of cases were correctly classified.

Accuracy of a discriminant analysis model for prediction of coliform mastitis in dairy cows and a comparison with clinical prediction.

We tested an equation, which had been developed previously using discriminant analysis, for predicting whether a cow has coliform mastitis. Variables indicating a high probability of coliform infection included history of previous mastitis in the affected quarter, weakness, clear or white color of milk, water consistency of the milk, swelling of the udder, lack of previous mastitis in other quarters, lack of palpable udder abscesses, and a high body temperature. Application of this predictive equation to 114 cows with mastitis to determine if they would have coliform organisms cultured from the affected quarters resulted in an accuracy of 71% (sensitivity = 0.42, specificity = 0.85), compared to an accuracy of 62% (sensitivity = .64, specificity = .61) for cowside prediction by the attending clinicians. Changing the cutoff score of the discriminant rule so that the sensitivity of the discriminant prediction was similar to that of the clinicians yielded an accuracy of 64% (sensitivity = .64, specificity = .64).

Effect of hydrogen peroxide on prostaglandin production and cellular integrity in cultured porcine aortic endothelial cells.

Oxidative damage to the vascular endothelium may play an important role in the pathogenesis of atherosclerosis and aging, and may account in part for reduced vascular prostacyclin (PGI2) synthesis associated with both conditions. Using H2O2 to induce injury, we investigated the effects of oxidative damage on PGI2 synthesis in cultured endothelial cells (EC). Preincubation of EC with H2O2 produced a dose-dependent inhibition (inhibitory concentration [IC50] = 35 microM) of PGI2 formation from arachidonate. The maximum dose-related effect occurred within 1 min after exposure although appreciable H2O2 remained after 30 min (30% of original). In addition, H2O2 produced both a time- and dose-dependent injury leading to cell disruption, lactate dehydrogenase release, and 51Cr release from prelabeled cells. However, in dramatic contrast to H2O2 effects on PGI2 synthesis, loss of cellular integrity required doses in excess of 0.5 mM and incubation times in excess of 1 h. The superoxide-generating system, xanthine plus xanthine oxidase, produced a similar inhibition of PGI2 formation. Such inhibition was dependent on the generation of H2O2 but not superoxide in that catalase was completely protective whereas superoxide dismutase was not. H2O2 (50 microM) also effectively inhibited basal and ionophore A23187 (0.5 microM)-stimulated PGI2 formation. However, H2O2 had no effect on phospholipase A2 activity, because ionophore A23187-induced arachidonate release was unimpaired. To determine the effects on cyclooxygenase and PGI2 synthase, prostaglandin products from cells prelabeled with [3H]arachidonate and stimulated with ionophore A23187, or products formed from exogenous arachidonate were examined. Inhibition of cyclooxygenase but not PGI2 synthase was observed. Incubation of H2O2-treated cells with prostaglandin cyclic endoperoxide indicated no inhibition of PGI2 synthase. Thus, in EC low doses of H2O2 potently inhibit cyclooxygenase after brief exposure whereas larger doses and prolonged exposure are required for classical cytolytic effects. Surprisingly, PGI2 synthase, which is known to be extremely sensitive to a variety of lipid peroxides, is not inhibited by H2O2. Lipid solubility, enzyme location within the EC membrane, or the local availability of reducing factors may explain these results, and may be important determinants of the response of EC to oxidative stress.

The sensitivity and specificity of postbreeding plasma progesterone levels as a pregnancy test for dairy cows.

Plasma progesterone levels on day 4 and day 8 postbreeding were measured for one hundred and eighty-four dairy cows. These two parameters (PPD4, PPD8), their absolute difference (PPDIFF) and their ratio (PPRATIO) were assessed for their ability to identify cows not conceiving, using the principles of sensitivity and specificity. PPD4 was significantly higher (p less than 0.10) and PPD8, PPDIFF and PPRATIO were significantly lower (p less than 0.01) in cows remaining open than in pregnant cows. Evaluating each parameter separately, PPDIFF greater than 3.00 units had the highest specificity, 85.7%, but a low sensitivity (27.0%). Combining two parameters using series interpretation to increase specificity resulted in the best combination of specificity (87%) and sensitivity (27%). Maximum specificity was 97% for PPD4 less than or equal to 1.00 units and PPD8 greater than 4.00 units, and also for PPD4 less than or equal to 1.00 units and PPDIFF greater than 3.00 units, but sensitivity was very low (7% and 10% respectively). Predictive values of the test results with the best specificity were evaluated; given the population pregnancy rate of 54%, none exceeded 50%, indicating that the plasma progesterone parameters were not very useful for identifying open dairy cows.

Arachidonic acid metabolism in cultured aortic endothelial cells. Effect of cAMP and 3-isobutyl-1-methylxanthine.

To investigate the hypothesis that cyclic AMP (cAMP) regulates arachidonic acid metabolism in vascular tissue, we have studied the effects of forskolin (FSK), an activator of adenylate cyclase, and 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, on hormone-stimulated prostacyclin (PGI2) synthesis in porcine aortic endothelial cells grown in culture. In these experiments, bradykinin (1 microgram/ml) and A23187 (0.2 microM) potently stimulated PGI2 biosynthesis (9- and 10-fold respectively). However, prostaglandin synthesis in response to either of these agents was not affected by FSK even though FSK elevated intracellular levels of cAMP 10-fold. IBMX failed to elevate basal cAMP levels when incubated with unstimulated cells. Stimulation of IBMX-treated (0.1 but not 1.0 or 4.0 mM) cells with bradykinin, however, did result in increased cAMP levels, presumably due to PGI2 formation and subsequent activation of adenylate cyclase. In addition to phosphodiesterase inhibition, IBMX inhibited PGI2 formation (72% at 1 mM) in a dose-dependent manner so that, at higher doses of IBMX, cAMP levels returned to baseline. Thus, prostacyclin synthesis inhibition by IBMX could not be attributed to elevated cAMP. In other experiments, IBMX (1 mM) was found to directly inhibit arachidonic acid release (32%) and arachidonic acid metabolism (65%) in endothelial cells and to inhibit arachidonic acid conversion to PGE2 by sheep seminal vesicle microsomes (65%). These data suggest that IBMX directly inhibits both phospholipase and cyclooxygenase activities. These experiments do not support the contention that cAMP regulates these enzymes in cultured aortic endothelial cells.

Ethanol and other host-derived volatiles as attractants to beetles that bore into hardwoods.

Ethanol, methanol, acetone, and acetaldehyde-chemicals identified in the inner bark of living trees-were used to bait vane traps placed in crowns of oak trees in Connecticut. Ethanol-baited traps caught more cerambycid, scolytid, and clerid beetles than unbaited traps. Buprestidae were not attracted to ethanol. Acetaldehyde and acetone were not attractive to any family. A mixture of ethanol, methanol, and acetaldehyde was no more attractive than ethanol alone. The vane traps were very effective at catching Cerambycidae and Scolytidae, but ineffective compared to sticky panels at catching Buprestidae.

Sesquiterpene progenitor, germacrene A: an alarm pheromone in aphids.

Germacrene A, the elusive biogenetic "parent" of many sesquiterpenes, has been isolated from the spotted alfalfa aphid and identified as a new intrageneric aphid alarm pheromone.