The Microbiome of Field-Caught and Laboratory-Adapted Australian Tephritid Fruit Fly Species with Different Host Plant Use and Specialisation.

Tephritid fruit fly species display a diversity of host plant specialisation on a scale from monophagy to polyphagy. Furthermore, while some species prefer ripening fruit, a few are restricted to damaged or rotting fruit. Such a diversity of host plant use may be reflected in the microbial symbiont diversity of tephritids and their grade of dependency on their microbiomes. Here, we investigated the microbiome of six tephritid species from three genera, including species that are polyphagous pests (Bactrocera tryoni, Bactrocera neohumeralis, Bactrocera jarvisi, Ceratitis capitata) and a monophagous specialist (Bactrocera cacuminata). These were compared with the microbiome of a non-pestiferous but polyphagous tephritid species that is restricted to damaged or rotting fruit (Dirioxa pornia). The bacterial community associated with whole fruit flies was analysed by 16S ribosomal DNA (rDNA) amplicon pyrosequencing to detect potential drivers of taxonomic composition. Overall, the dominant bacterial families were Enterobacteriaceae and Acetobacteraceae (both Proteobacteria), and Streptococcaceae and Enterococcaceae (both Firmicutes). Comparisons across species and genera found different microbial composition in the three tephritid genera, but limited consistent differentiation between Bactrocera species. Within Bactrocera species, differentiation of microbial composition seemed to be influenced by the environment, possibly including their diets; beyond this, tephritid species identity or ecology also had an effect. The microbiome of D. pornia was most distinct from the other five species, which may be due to its ecologically different niche of rotting or damaged fruit, as opposed to ripening fruit favoured by the other species. Our study is the first amplicon pyrosequencing study to compare the microbiomes of tephritid species and thus delivers important information about the turnover of microbial diversity within and between fruit fly species and their potential application in pest management strategies.

Expression patterns of sex-determination genes in single male and female embryos of two Bactrocera fruit fly species during early development.

In tephritids, the sex-determination pathway follows the sex-specific splicing of transformer (tra) mRNA, and the cooperation of tra and transformer-2 (tra-2) to effect the sex-specific splicing of doublesex (dsx), the genetic double-switch responsible for male or female somatic development. The Dominant Male Determiner (M) is the primary signal that controls this pathway. M, as yet uncharacterized, is Y-chromosome linked, expressed in the zygote and directly or indirectly diminishes active TRA protein in male embryos. Here we first demonstrated the high conservation of tra, tra-2 and dsx in two Australian tephritids, Bactrocera tryoni and Bactrocera jarvisi. We then used quantitative reverse transcription PCR on single, sexed embryos to examine expression of the key sex-determination genes during early embryogenesis. Individual embryos were sexed using molecular markers located on the B. jarvisi Y-chromosome that was also introgressed into a B. tryoni line. In B. jarvisi, sex-specific expression of tra transcripts occurred between 3 to 6 h after egg laying, and the dsx isoform was established by 7 h. These milestones were delayed in B. tryoni lines. The results provide a time frame for transcriptomic analyses to identify M and its direct targets, plus information on genes that may be targeted for the development of male-only lines for pest management.

Tropical tephritid fruit fly community with high incidence of shared Wolbachia strains as platform for horizontal transmission of endosymbionts.

Wolbachia are endosymbiotic bacteria that infect 40-65% of arthropod species. They are primarily maternally inherited with occasional horizontal transmission for which limited direct ecological evidence exists. We detected Wolbachia in 8 out of 24 Australian tephritid species. Here, we have used multilocus sequence typing (MLST) to further characterize these Wolbachia strains, plus a novel quantitative polymerase chain reaction method for allele assignment in multiple infections. Based on five MLST loci and the Wolbachia surface protein gene (wsp), five Bactrocera and one Dacus species harboured two identical strains as double infections; furthermore, Bactrocera neohumeralis harboured both of these as single or double infections, and sibling species B. tryoni harboured one. Two Bactrocera species contained Wolbachia pseudogenes, potentially within the fruit fly genomes. A fruit fly parasitoid, Fopius arisanus shared identical alleles with two Wolbachia strains detected in one B. frauenfeldi individual. We report an unprecedented high incidence of four shared Wolbachia strains in eight host species from two trophic levels. This suggests frequent exposure to Wolbachia in this tropical tephritid community that shares host plant and parasitoid species, and also includes species that hybridize. Such insect communities may act as horizontal transmission platforms that contribute to the ubiquity of the otherwise maternally inherited Wolbachia.

Germ-line transformation of the Queensland fruit fly, Bactrocera tryoni, using a piggyBac vector in the presence of endogenous piggyBac elements.

We report the heritable germ-line transformation of the Queensland fruit fly, Bactrocera tryoni, using a piggyBac vector marked with either the fluorescent protein DsRed or EGFP. A transformation frequency of 5-10% was obtained. Inheritance of the transgenes has remained stable over more than 15 generations despite the presence of endogenous piggyBac sequences in the B. tryoni genome. The sequence of insertion sites shows the usual canonical pattern of piggyBac integraton into TTAA target sites. An investigation of endogenous piggyBac elements in the B. tryoni genome reveals the presence of sequences almost identical to those reported recently for the B. dorsalis complex of fruit flies and two noctuid moths, suggesting a common origin of piggyBac sequences in these species. The availability of transformation protocols for B. tryoni has the potential to deliver improvements in the performance of the Sterile Insect Technique for this pest species.

Interspecific hybridization as a source of novel genetic markers for the sterile insect technique in Bactrocera tryoni (Diptera: Tephritidae).

Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) or "Qfly," is the most serious horticultural pest in Australia, with a bioclimatic range that extends from the tropical north to the temperate south. Various Australian horticultural exports depend on certification that they originated from B. tryoni-free areas. To eliminate, rather than suppress, B. tryoni in production areas, a sterile insect technique (SIT) campaign directed at B. tryoni has been in operation in southeastern Australia since 1997. Like many other SIT programs around the world, the B. tryoni SIT program relies on fluorescent dust to mark the sterile insects. However, fluorescent dust marking does not provide 100% accuracy in the identification of sterile insects, as required where the aim is to declare regions completely free of fruit fly. Here, we show that novel mitochondrial markers can be introduced into a strain of B. tryoni by interspecies hybridization between B. tryoni and a related but well-differentiated species, Bactrocera jarvisi (Tryon), followed by backcrossing of the hybrid strain with the parental B. tryoni strain. These novel markers do not affect the viability of the strain as measured by pupation and eclosion rates. A simple polymerase chain reaction-based test is described that distinguishes the marked B. tryoni from wild B. tryoni. As required in practice, the test was shown to work reliably on DNA extracted from dead flies that had remained in field traps for up to two weeks.

Effects of shaded versus unshaded wallows on behavior, performance, and physiology of the outdoor lactating sow.

The objectives of this study were to determine the effects of shading wallows during the summer months on lactating sow behavior, performance, and physiology. A total of 128 sows were used during warm weather (May to August 2001) to determine the effects of unshaded (control; n = 8) vs. shaded (SH; n = 8) wallows. Sows ranged over 6 parities and were fed a completely balanced sorghum-based diet. Behavioral data were collected by 15-min scan samples over a 24-h period/wk for a total of 16 wk. All sows were observed twice when litter age was 5 and 15 d, respectively. Respiration rates (breaths/min) were collected on 50 sows (control, n = 25; SH, n = 25) over an 8-wk period when the maximum temperature exceeded 32 degrees C. Ten milliliters of clotted blood and 20 mL of whole blood were obtained by jugular puncture from each sow on the day of weaning to determine total white blood cells, acute phase proteins, packed-cell volume, and chemotaxis and chemokinesis. Descriptive water temperature profiles were measured by using data loggers positioned at 3 levels per wallow: surface water, shallow mud, and deep mud. Behavioral, postural, location, performance, and physiological measurements did not differ (P > 0.05) among wallow treatments. Regardless of treatment, sows spent approximately 82% of their total time budget inside the farrowing hut and only approximately 7% of their total time budget in the wallow. A total of 428 piglets died, 219 in the control treatment and 209 in the SH treatment. The majority of piglets in both treatments died of crushing within the first 72 h after parturition, and most of the piglets had suckled. Shade kept the shallow water profile cooler during the hotter afternoon temperatures compared with the control wallows. In SH for both the shallow and deep mud profiles, temperatures were consistent throughout the day. In conclusion, sows spent a large percentage of their daily time budget inside the farrowing hut and spent only brief episodes in the wallow. Shading the wallow did not result in increased wallow use time or improvements in sow physiology and overall performance.

Effects of a concentrated lidocaine solution on the acute phase stress response to dehorning in dairy calves.

The objective of this study was to more fully define the surgical stress response to dehorning by heat cauterization in dairy calves by measuring behavioral, hormonal, inflammatory, and immunological markers of stress and to determine whether a nerve block of the surgical site with a concentrated solution of lidocaine (5%) reduces the degree of stress. Thirty-two 10- to 12-wk-old female Holstein calves were randomly allotted to 1 of 4 treatments: 5% lidocaine followed by dehorning, 2% lidocaine followed by dehorning, saline followed by dehorning, or 5% lidocaine followed by sham dehorning. Plasma cortisol concentration was measured in blood samples collected via a jugular catheter at -0.5, 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 6, 9, 12, 24, 48, and 72 h. Various other blood constituents were measured in samples collected at -0.5, 12, 24, 48, and 72 h. Feeding, drinking, scratching, grooming, rubbing, licking, and inactivity behaviors were observed in the standing and recumbent positions using a 10-min scan sampling method analyzed on a time period and daily basis for 72 h following the dehorning procedure. The frequency of vocalization, kicking, and lying in the chute during the dehorning procedure were also assessed. The overall plasma cortisol concentrations were higher in calves subjected to dehorning than in control calves. Compared with the control group, the saline-treated calves had a higher cortisol concentration at 30 and 60 min postdehorning. Plasma cortisol concentrations were higher in all groups at 30 min postdehorning than at other sampling times. The percentage of circulating neutrophils and the neutrophil:lymphocyte ratio were increased in the saline and 2% lidocaine group. Total plasma protein, fibrinogen, and alpha1-acid glycoprotein concentrations were similar among treatments. The behavioral response to dehorning, as manifested by kicking while in the chute, was greater in the saline and 2% lidocaine group than in the control or 5% lidocaine treatment groups. In the postdehorning period, the percentage of time calves spent performing various maintenance behaviors did not differ among treatments. Thus, injection of 5% lidocaine may not provide any added comfort after the dehorning but may decrease the overall stress response during the procedure.

Social stress increases fecal shedding of Salmonella typhimurium by early weaned piglets.

"Segregated early weaning" (SEW) of pigs reduces exposure to pathogenic bacteria, but upon arrival at grower facilities pigs may be co-mingled regardless of farm of origin. The present study was designed to examine the effect of mixing (social) stress on populations of Salmonella enterica Typhimurium in SEW pigs. Piglets (7 days old; n = 28 in each of 2 replicates) were separated into 2 treatments (control and mixed groups) of 2 pens per treatment (7 piglets/pen). One (n = 1) "seeder" pig/pen was inoculated with 10(9) CFU of S. Typhimurium. Each seeder was placed with non-inoculated "contact" piglets (n = 6). A"contact" piglet was swapped each day between the "mixed" pens for 5 days; pigs in control pens were not exchanged. On day 5, the incidence of fecal Salmonella shedding was higher in the mixed contact pigs (P < 0.05). Rectal Salmonella and cecal coliform populations in mixed pigs were significantly (P < 0.05) greater than in control pigs but cecal Salmonella populations were not different. Mixed pigs were more susceptible to tissue invasiveness (i.e., Salmonella-positive tonsils and lymph nodes) than control pigs. These results indicate that social stress of weaned pigs may increase susceptibility to and/or fecal shedding of Salmonella. Food-borne Salmonella infections in the United States are estimated to cost the economy dollar 2.4 billion annually (ERS/USDA, 2001). Approximately 6-9% of human salmonellosis is associated with the consumption of pork products (Frenzen et al., 1999). Salmonella is relatively common on swine farms and has been isolated from all stages of the pork production chain (Davies et al., 1999; Fedorka-Cray et al., 1997b; Rostagno et al., 2003). Salmonella is a threat to the pork industry not only from a food-safety perspective as a public health concern, but some Salmonella serotypes can cause clinical illnesses in swine, negatively impacting production efficiency and profitability (Schwartz, 1991).

Environmental prevalence and persistence of Salmonella spp. in outdoor swine wallows.

Swine can harbor Salmonella in their gastrointestinal tracts. It has been estimated that up to 48% of the U.S. swine herd may carry Salmonella. Housing sows in farrowing stalls has become controversial due to animal welfare-based criticisms. An alternative production system is to keep sows outdoors on pasture with access to individual farrowing huts. This study was designed to determine the effects of two production systems on indicator bacteria and Salmonella of sows housed indoors in farrowing stalls (n = 52) compared to sows housed outdoors (n = 52) in English style huts. Each farrowing radial contained one wallow, from which mud (n = 290) and water (n = 290) samples were collected weekly. All samples were analyzed for generic E. coli, coliforms and Salmonella. No differences (p > 0.05) were detected in Salmonella, generic E. coli and coliform populations between indoor farrowing stalls and outdoor farrowing huts. However, all 8 outdoor wallows contained Salmonella spp. at some point during the study (n = 49 Salmonella isolates). Salmonella genotypes persisted within some wallows for >5 months, and genetically indistinguishable Salmonella isolates were found in multiple wallows. Salmonella isolated from outdoor sow feces were genetically indistinguishable by PFGE from Salmonella isolated from wallows (n = 33) throughout the study, indicating that pathogenic bacteria were cycling between swine and their environment. In conclusion, the role of wallows in disseminating Salmonella within an outdoor swine herd appears to be significant.

Effect of water sprinkling on incidence of zoonotic pathogens in feedlot cattle.

Heat stress and dusty conditions are common challenges for cattle during the summer, and a typical method of alleviating these problems involves sprinkling cattle and pens with water. The effect of sprinkling water on the incidence of zoonotic pathogens has not been previously studied. Four pens of heifers (n = 41) were cooled using sprinklers, and four pens (n = 43) served as controls. Heifers were crossbred Charolais, with white and red hair coats. Sprinkling was initiated when cattle were on full concentrate feed (July). Fecal samples, hide swipes, and BW were collected on d 0, 28, 63, 95, and 98. Average daily gain, DMI, and G:F were calculated, and carcass traits were collected 36 h after processing. Performance data were analyzed as a randomized complete block design, and zoonotic pathogen data were analyzed using chi2 analysis. Sprinkling tended (P = 0.054) to increase the incidence of fecal Salmonella spp. populations on d 98, but simultaneously tended to decrease (P = 0.058) the Escherichia coli O157:H7 incidence on hides on d 98. The most prevalent Salmonella serovars in this study were Kentucky, Muenster, Meleagridis, and Cerro. Performance measures and carcass traits did not differ between treatments (P > 0.10). Under our conditions, sprinkling cattle with water did not affect the incidence of zoonotic pathogens in feces or on hides.

Hepatic corticosteroid-binding globulin (CBG) messenger RNA expression and plasma CBG concentrations in young pigs in response to heat and social stress.

Plasma cortisol, porcine corticosteroid-binding globulin (pCBG), hepatic CBG expression, and other physiological and behavioral measures of stress were studied in pigs in response to elevated temperature in conjunction with establishing a social hierarchy. Twenty-four crossbred pigs were weaned at 25 d of age (three or six pigs from six sows) and housed in littermate groups at 23 +/- 2 degrees C. At 57 d of age (d 0), animals were weighed and placed under general anesthesia for collection of blood (10 mL) and liver (approximately 100 mg) samples. On d 1, three unacquainted pigs of similar BW (23 +/- 1 kg) from different litters were allotted to each of eight nursery pens within two environmentally controlled rooms (12 pigs per room). From d 1 to 7, one room was maintained at 23 +/- 2 degrees C (CON) and the other at 33 +/- 2 degrees C (HEAT). Both rooms were kept at 23 +/- 2 degrees C from d 8 to 14. Animals were videotaped for 72 h beginning on d 1 and 8 to document behavioral changes in response to room temperature. The social hierarchy of pigs within each pen was based on fight activity recorded on d 1 to 3. Blood and liver tissue were collected again on d 7 and 14. The ADG for HEAT pigs increased (P < 0.05) over d 8 to 14 compared with d 1 to 7. In contrast to CON pigs, HEAT pigs displayed increased (P < 0.01) drinking but decreased feeding and lying in contact with other pigs from d 1 to 3, and similar drinking and feeding but increased (P < 0.01) lying with contact behaviors from d 8 to 10. With the exception of subordinate pigs exhibiting less (P < 0.05) frequent standing/walking behavior than the dominant or intermediate pigs on d 1 to 3, frequency of behaviors for both recorded time periods did not differ among pigs due to social status, regardless of treatment. The concentration of plasma haptoglobin in HEAT pigs on d 7 compared with d 0 increased (467 vs. 763 mg/L; P < 0.05), whereas cortisol and pCBG decreased (274 vs. 235 nmol/L and 11.4 vs. 9.9 mg/L, respectively; P < 0.05) as a result of treatment. The free cortisol index (total cortisol/pCBG) was greater (P < 0.05) in HEAT pigs on d 14 than on d 0 or 7. Hepatic CBG mRNA level was not affected by treatment. On d 14, HEAT pigs had plasma cortisol, pCBG, and haptoglobin concentrations similar to those of CON pigs. These results indicate that measured behavioral and physiological responses were not related to social status, and decreased circulating levels of cortisol and pCBG in pigs following a 7-d exposure to elevated temperature may not be determined by hepatic CBG mRNA expression.

The period gene in two species of tephritid fruit fly differentiated by mating behaviour.

The period gene is important for the generation and maintenance of biological rhythms. It served as an ideal candidate for the investigation of the mating time isolation between two sibling Queensland fruit fly species, Bactrocera tryoni and Bactrocera neohumeralis. We have isolated the homologues of the period gene in the two species, and show that their putative amino acid sequences are identical. No length polymorphism was detected in the Thr-Gly repeat region. per mRNA expression, assayed in light-dark diurnal conditions, displayed circadian oscillation in both the head and abdomen of B. tryoni and B. neohumeralis, with the same cycling phase. An alternatively spliced intron was identified in the 3' untranslated region. The effect of temperature on the splicing and mRNA expression was examined.

The effects of split marketing on the physiology, behavior, and performance of finishing swine.

One hundred twenty 8-wk-old barrows (20.3 +/- 2.0 kg BW) were used to examine the effect of split marketing on selected behavioral, physiological and performance parameters. Pigs were assigned by weight in a randomized complete block design to one of three treatments: SM (split-marketed), six pigs/pen (1.83 m2/pig); C (control), six pigs/pen (1.83 m2/pig); or MC (modified control), three pigs/pen (3.66 m2/pig). The heaviest half of SM animals were removed 1 wk prior to marketing penmates. Control and MC animals remained in their respective groups until marketing. Animals were videotaped during the first 72 h of the study (INITIAL), 72 h prior to (PRE), and following the removal (POST) of pigs in the SM treatment to quantify maintenance behaviors and to identify socially dominant, intermediate, and submissive pigs. A blood sample was collected from each animal upon completion of INITIAL, PRE, and POST time periods to determine neutrophil:lymphocyte ratio and plasma haptoglobin, cortisol, and corticosteroid-binding globulin (CBG) levels. Animals were weighed and feed disappearance was calculated biweekly. Tenth-rib backfat and area of the longissimus muscle at marketing were ultrasonically evaluated on all animals. Regardless of treatment, animals were more (P < 0.01) active (eating, standing/walking, fighting) at INITIAL than at PRE or POST times. Frequency and duration of fights per pen were less (P < 0.01) in MC than in C or SM pigs for all periods observed. Neutrophil:lymphocyte ratio, plasma haptoglobin, and CBG levels were greater (P < 0.01) during the INITIAL period than during the PRE or POST periods but did not differ between treatments. No treatment or time differences were detected in plasma cortisol levels. The MC pigs exhibited greater (P < 0.01) ADFI with poorer feed efficiency compared to C or SM pigs up to split marketing. During the POST period, both MC and SM pigs had greater (P < 0.01) ADFI with poorer (P < 0.01) feed efficiency than C pigs. The ADG was not different among animals as a result of treatment. There were no treatment differences for any of the carcass measurements. Significant differences in performance between the treatment groups could not be attributed to any physiological or behavioral measures reported here.

Shade and water misting effects on behavior, physiology, performance, and carcass traits of heat-stressed feedlot cattle.

One hundred twelve crossbred feedlot heifers were used in two experiments to assess the impact of heat stress and its relief by shade and(or) water misting on behavior, physiology, performance, and carcass traits. Treatments were 1) no shading or misting (CONT); 2) only misting (MIST); 3) only shading (SHADE); and 4) shading plus misting (SHMI). Head in the feed bunk, head in or above the waterer, walking, standing, and lying behaviors were observed. Rectal temperature, respiration rate, and carcass traits were measured, as well as DMI, ADG, and feed:gain. Dietary NEm and NEg concentrations were calculated from performance data. In Exp. 1, (32 heifers; average BW 288 kg) the CONT heifers spent more time lying down than all others (P < 0.01). In addition, CONT heifers spent less time (P < 0.01) standing than SHADE and MIST heifers. Misting decreased (P < 0.01) rectal temperature and MIST as well as SHADE lowered (P < 0.05) respiration rates. In Exp. 2 (80 heifers; average BW = 336 kg), lying and walking behaviors did not differ among treatments, but shade increased (P < 0.01) standing behavior in heifers. The MIST cattle performed less (P < 0.05) head-above-water behavior than unmisted cattle. Rectal temperatures did not differ among treatments, but respiration rate was lower in shaded than in unshaded heifers (P < 0.05). Shaded compared with unshaded heifers had greater DMI (9.46 vs 8.80 +/- 0.14 kg/d, P < 0.01) and ADG (1.6 vs 1.41 +/- 0.1 kg/d, P < 0.01). Heifers provided with shade reached their target BW 20 d earlier than the unshaded heifers and differed in final BW (547 vs 520 +/- 6 kg, P < 0.01). Feed:gain and calculated NEg and NEm concentrations did not differ among treatments, and carcass traits were generally similar among treatments. In conclusion, cattle without shade had a physiological and behavioral stress response to heat that negatively affected productivity. Providing shade for beef cattle was a suitable solution to decrease heat stress and to lower the negative effects of heat on performance, whereas misting was largely ineffective.

Photoperiod and heat stress influence on lactating sow performance and photoperiod effects on nursery pig performance.

Three experiments were conducted to evaluate sow and piglet productivity under extended photoperiod. In Exp. 1, 98 crossbred, lactating sows were housed in one of four treatments: thermoneutral air temperature (23.6 degrees C) in either (h of light:dark) 1:23 or 16:8 photoperiods, or heat stress (30.4 degrees C) in either 1:23 or 16:8 photoperiods. Heat stress reduced (P less than .05) sow feed intake, piglet mortality and piglet weaning weight and increased (P less than .01) sow lactation weight loss. Number of pigs weaned per litter was increased (P less than .01) when sows were heat-stressed. Extended photoperiod reduced (P greater than .05) time for sows to rebreed postweaning by .4 d. The interaction between air temperature and photoperiod was significant only for sow lactation weight loss. Heat stress increased sow lactation weight loss, but this effect was more severe in the 1:23 than in the 16:8 photoperiod. Experiments 2 and 3 examined the effects of 1:23 or 16:8 photoperiods on nursery pig performance when pigs were weaned from sows experiencing 1:23 (Exp. 3) or 16:8 (Exp. 2) photoperiods. In both nursery studies, photoperiod did not influence (P greater than .10) postweaning pig mortality, feed intake, weight gain or gain:feed ratio. In conclusion, extended photoperiod reduced days to return to estrus and reduced sow lactation weight loss, especially during heat stress. No benefits in preweaning or postweaning piglet weight or survival were observed by use of extended photoperiod.

Reduction of pig agonistic behavior by androstenone.

One hundred twenty-four prepuberal crossbred pigs were used in a series of behavioral bioassays to determine the minimum dose of androstenone (5 alpha-androst-16-en-3-one) that would reduce the level of agonistic behavior among dyads of newly regrouped pigs. Randomly selected females and castrated males were used in 21-h videotaped observation periods. In Exp. 1, isopropyl alcohol was tested against no aerosol to determine if the vehicle (isopropyl alcohol) influenced agonistic behavior. Level of submissive and aggressive behaviors were similar (P greater than .10) between treatments. In Exp. 2, vehicle or androstenone in vehicle was sprayed on the snout and head of both pigs at the start of the encounter. Four bioassays were performed with four levels (.05, .5, 5 and 50 micrograms/pig) of androstenone dissolved in isopropyl alcohol. Sprayed isopropyl alcohol served as a control. At concentrations of .5 and 5 micrograms/pig, androstenone reduced aggressive behavior (P less than .05). Androstenone had no consistent effect on submissive behavior. In Exp. 3, androstenone was sprayed on pigs at the start of the encounter and again at 30, 60 and 90 min after pairs of pigs were mixed. Repeated application of this androgen resulted in levels of agonistic behavior similar to those recorded when nothing was applied (P greater than .10). A single application of as little as .5 micrograms androstenone per pig reduced aggressive behavior among prepuberal pigs and, therefore, may be a way of reducing fighting among newly regrouped prepuberal pigs.