castaneum in mill and laboratory settings:

Transcription

1 504 Sampling Tribolium confusum and Tribolium castaneum in mill and laboratory settings: differences between strains and species Karen J. Hawkin, Dean M. Stanbridge, Paul G. Fields Abstract*The efficacy of pitfall traps baited with pheromone and cereal oil in capturing Tribolium confusum Jacquelin du Val and T. castaneum (Herbst) (Coleoptera: Tenebrionidae) was low (trap catch) in mill and simulated warehouse settings. In a simulated warehouse experiment, strains of Tribolium Macleay recently taken from mills were caught 24% less often in traps than were laboratory strains, and T. confusum was caught 40% less often than T. castaneum. Both species were found together in all flour samples taken from a Canadian flour mill. A comparison of the species ratio in flour samples with that found in traps revealed that T. confusum was caught less often in traps than was T. castaneum. In flour, T. castaneum burrowed more than did T. confusum, and there were differences in burrowing behaviour between the four T. castaneum strains. Mills infested with T. confusum may have higher levels of infestation than was previously thought, indicating that further research into beetle behaviour in mills is needed. Résumé*Les pièges à fosse pour Tribolium confusum Jacquelin du Val et T. castaneum (Herbst) (Coleoptera : Tenebrionidae) appâtés avec de la phéromone et de l huile de céréales étaient de faible efficacité (insectes capturés dans les pièges) dans un moulin à farine et un entrepôt expérimental. Dans un essai d entrepôt expérimental, des souches de Tribolium Macleay prélevés pris récemment dans des moulins à farine ont été capturés dans les pièges 24 % moins souvent que les souches de laboratoire, et le T. confusum aété capturé dans les pièges 40 % moins souvent que le T. castaneum. Les deux espèces ont été trouvés ensemble dans tous les échantillons de farine prélevés dans un moulin à farine canadien. Le rapport entre les deux espèces dans les échantillons de farine a été comparé à celui dans les pièges, révélant que T. confusum a été pris moins souvent que T. castaneum dans les pièges. Dans la farine T. castaneum creusait plus que T. confusum, et il y avait des différences significatives dans le comportement de creusage dans la farine entre les quarte souches de T. castaneum.le T. castaneum a creusé plus que T. confusum,et il y avait des différences entre les quatre souches de T. castaneum. Les moulins envahis par le T. confusum pourraient en avoir plus que l on croyait auparavant, de sorte que des recherches supplémentaires sur le comportement de ces espèces s avère nécessaire. Introduction Pheromone traps can be useful for monitoring populations of pest insects. Dome traps (Mullen et al. 1992; Mullen 2011) are used to monitor populations of Tribolium confusum Jacquelin du Val and T. castaneum (Herbst) (Coleoptera: Tenbrionidae) in Canadian and American flour mills (Campbell et al. 2002, Campbell et al. 2010a, Campbell et al. 2010b; Harrison 2007). Traps are baited with an isomeric mixture of 4R,8R-()- and 4R,4S- ()-4,8-dimethyldecanal, the aggregation Received 31 December Accepted 17 May K.J. Hawkin, Department of Entomology, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2 D.M. Stanbridge, Direct Line Sales and Supplies Corporation, Suite 218, 4305 Fairview Street, Burlington, Ontario, Canada L7L 6E8 P.G. Fields, 1 Cereal Research Centre, Agriculture and Agri-Food Canada, 195 Dafoe Road, Winnipeg, Manitoba, Canada R3T 2M9 1 Corresponding author ( paul.fields@agr.gc.ca). doi: /n Can. Entomol. 143: (2011)

2 Hawkin et al. 505 pheromone of the two species (Suzuki et al. 1984), and food attractants in the form of cereal oil. Trap catches provide relative measures of size and rate of change of beetle populations in flour mills (Phillips 1997; Subramanyam and Hagstrum 2000; Campbell et al. 2002; Trematerra and Sciarretta 2004; Mullen 2011). However, trap catches can be influenced by many factors, including population size, nutritional and mating status, food availability, presence of other species, number of trapped insects, temperature, lighting, and type of lure (Phillips 1997; Burkholder 1985; Toews et al. 2005; Athanassiou et al. 2006; Fedina and Lewis 2007; Semeao et al. 2011). Little is known about the effect of species and strains of Tribolium Macleay on trap catch. Furthermore, the relationship between population size and number of trapped beetles is not clearly understood (Phillips et al. 2000; Subramanyam and Hagstrum 2000; Campbell et al. 2002). Currently, many food-processing plants are fumigated once or twice a year on predetermined dates, regardless of pest population size (Fields and White 2002). A better understanding of factors affecting trap catch will allow better estimation of pest population sizes, and ultimately lead to better IPM programs for Tribolium flour beetles in flour mills. Tribolium confusum and T. castaneum are economically important pests worldwide. Tribolium castaneum is found in granaries on farms and in grain elevators, whereas T. confusum rarely is, and both species are found in food-processing facilities and warehouses (Trematerra and Sciarretta 2004; Abdelghany et al. 2010). These habitat differences may be explained by behavioural differences (Rees 2004): adults of T. castaneum can fly; those of T. confusum are flightless (Rees 2004). A mixture of 4R-8R-()- and 4R-8S-()- 4,8-dimethyldecanal, at species-specific ratios, is the aggregation pheromone for both species (Suzuki and Mori 1983; Suzuki et al. 1984). Tribolium castaneum prefers fresh flour and is repelled by conditioned flour (Ghent 1966; Sokoloff 1974). In contrast, T. confusum prefers conditioned flour (Naylor 1959; Ghent 1966; Ogden 1970). In addition, there are differences in insecticide resistance, behaviour, and population growth between strains of Tribolium species (Sokoloff 1974; Rigaux et al. 2001; Sousa et al. 2009). The objective of this study was to examine the differences between strains and species of T. confusum and T. castaneum in an effort to increase the effectiveness of sampling for these beetles in flour mills. We examined the effectiveness of pheromone-trap monitoring in the laboratory, a small warehouse, and a flour mill, using mill and laboratory strains of T. confusum and T. castaneum; we tested the efficacy of pheromone traps in a flour mill; and we compared ratios of the two species in traps and flour samples in a mill. In addition, we released adults of both species into a small simulated warehouse to compare numbers of trapped beetles with actual population numbers. Because of observed differences in species ratios between mill populations and trap captures, in the laboratory we examined attractiveness to pheromone, speed of movement, and burrowing behaviour in flour for strains of both species using specimens recently taken from flour mills and established laboratory populations. Materials and methods Flour mill Flour-sample collection Flour samples were taken from roll stands, boots, and sifters in mill 1 (19 samples) and sifters in the adjoining packing plant (3 samples) during the week of 1623 November 2006 (while the baited versus unbaited monitoring trap study was underway). Roll stands contain roller mills, corrugated cylinders that grind and break up the wheat kernels and flour into progressively smaller particles. Sifters contain sieves of different sizes that sort the flour by particle size. Boots are the bottom most section of bucket elevators. Bucket elevators move wheat kernels or flour to the top of the mill (Posner and Hibbs 2005). Flour samples weighed approximately kg, and the amount sampled depended upon available flour residues. All samples were sifted using a 850 mm aperture sieve, and

3 506 Can. Entomol. Vol. 143, 2011 collected beetles were identified as T. confusum or T. castaneum. To compare species ratios in flour samples with those in traps, specimens of T. confusum and T. castaneum caught in 30 traps on four different floors of mill 1 were also collected and identified. Beetles were collected from traps daily from 18 to 23 November 2006 and from 10 traps in the packing plant on 24 November 2006, upon completion of the baited versus unbaited monitoring trap study. All traps were Dome traps baited with mixtures of 4R-8R-()- and 4R-8S-()-4,8- dimethyldecanal and a food oil (Trécé Inc., Adair, Oklahoma, United States of America). Trap efficacy A 100 cm diameter circle with release spots at 10, 30, and 60 cm along lines radiating north, south, east, and west from the centre of the circle was drawn on the fifth floor of mill 1. A single monitoring trap was placed in the middle of the circle. Depending on the treatment, the trapwas baited(pheromone and cereal oil) or unbaited (no pheromone and no oil). Recently emerged virgin male T. confusum from sexed pupae (Hinton 1942) in a laboratory strain were shipped to mill 1 for this experiment. Beetles were placed in a large Petri dish containing a sprinkle of flour from the mill and small pieces of filter paper for no more than 7 h prior to the experiment. To minimize disturbance to the beetles, each was transferred on a piece of filter paper to a randomly chosen release spot. Elapsed time (to a maximum of 10 min) was recorded from release of each beetle until it had left the circle or been caught in the trap. If a beetle went under the trap, the trap was checked after 1 min to see if the beetle had been caught. Trap touching by beetles was recorded; instances of a beetle under the trap but not caught after 1 min were counted as a touch. One repetition of this experiment consisted of 12 releases, one beetle at each of the release spots. There were seven repetitions using a baited trap with new pheromone and cereal oil. The trap was assembled on the first day of the experiment and used for the next 3 days. An additional four repetitions of the experiment were performed using a trap containing a pheromone septum that had aired in the mill for 5 days and new cereal oil. This pheromone lure was from a trap that was assembled 5 days prior to its use in this experiment, and had previously caught beetles on the fourth floor of the mill. There were also six repetitions using an unbaited trap. Ambient temperature and humidity (range, measured with a HOBO data logger (Onset Company, Bourne, Massachusetts) attached to a wall in the centre of the fifth floor) were C ( C; mean 9 SEM) and 35.0% 45.2% RH (39.1% % RH). Ten baited (cereal oil and pheromone) monitoring traps in the packing plant of mill 1 (5 each on the first and fifth floors) were used as part of the mill s IPM programs. All beetles in these traps were counted and discarded and 10 unbaited traps were placed 1 m from each of the baited traps along a wall. All traps, baited and unbaited, were left for 1 week and then the trapped beetles were counted. Laboratory and simulated warehouse The laboratory and simulated warehouse were located at the Cereal Research Centre in Winnipeg, Manitoba (approximately 49853?N, 97803?W). Insect strains All insects were maintained in the laboratory on unbleached white flour and 5% brewer s yeast at 30 o C, 65% RH. Unbleached white flour is hereinafter referred to as plain flour, and unbleached white flour 5% brewer s yeast is referred to as flour. The laboratory strain of T. castaneum was originally collected from Steinbach, Manitoba (approximately 49832?N, 96841?W), in The laboratory strain of T. confusum has been in culture since about Mill strains of T. castaneum were collected from three Canadian mills in different provinces in March (mill 2), June (mill 1), and May (mill 3) Tribolium confusum was collected from mill 1 in June 2007, and separated from T. castaneum by examining adults.

4 Hawkin et al. 507 Fig. 1. Schematic diagram of the simulated warehouse, including placement of pheromone traps and release spot. Tribolium confusum and T. castaneum (500 adults per species) were placed at the release spot and the numbers caught in the traps were recorded daily over a 10-day period. Warehouse releaserecapture The simulated warehouse (Fig. 1) was cleaned, and sites potentially harbouring beetles were removed. Tests were run during the winter in 2007 and The warehouse was opened for approximately 4 days before each test, allowing temperatures to reach between 6 and 24 8C and ensuring that no beetles remained in the warehouse. Six baited traps were placed in the warehouse 4 days before the release of beetles to allow the baits to airout (new pheromone septa can have lower efficacy; Hussain et al. 1994) and then checked for beetles to verify that the warehouse was beetle-free. Floor temperature and relative humidity were measured at nine locations (Hawkin 2008) and ranged from 22.3 to C and from 23.9% to 24.1%, respectively, during the tests. Warehouse lights were left on for the duration of the experiment to simulate flour-mill working conditions. Five hundred beetles of each species were released on the middle of the warehouse floor at the same time. Mill and laboratory strains were run on different dates. Adults ranged in age from newly emerged to 3 months old. The beetles were sieved out of the flour and immediately released by overturning the sieves on the warehouse floor. After 30 min, any beetles remaining on the sieves were swept off with a paintbrush. After beetle release, all six traps were checked daily for 10 days. Beetle health was monitored by holding 20 beetles in 12 g of plain flour per vial, 3 vials per species in the warehouse. Live and dead beetles were counted at the end of the 10-day release period. The average mortality for the trials ranged from 0% to 2% for T. confusum and from 10% to 18% for T. castaneum. Arena This experiment was carried out because it was impossible to run more than two species at a time in the warehouse release-recapture experiment. To test several species and strains at the same time under the same conditions, we tested trap efficacy in small arenas containing one monitoring trap per arena but no food or harbourage sites. Six arenas were used for this experiment, each consisting of a 1.2 m 2 plywood base with 4 cm high metal sides attached to the outside of the plywood. Fluon (AGC Chemicals Americas, Inc., Bayonne, New Jersey) was applied to the metal sides to keep beetles from escaping from the arena. The arenas were placed in a grid pattern in a warehouse at the Cereal Research Center (Winnipeg, Manitoba; Fig. 1). Temperature and relative humidity inside the warehouse were measured by data loggers taped to the outside of each arena. The

5 508 Can. Entomol. Vol. 143, 2011 distance between arenas was m. The arenas were vacuumed between repetitions. The beetles used in the experiments were reared by placing 100 adults in 200 g flour, and adults used were 46 weeks old. For each repetition, 200 unsexed adults of each of the six types of beetles were released in the lefthand corner of each arena. A monitoring trap was placed in the centre of each arena prior to release of the insects; a new trap and pheromone lure were used for each repetition. Baited traps contained a pheromone septum and three or four drops of cereal oil; unbaited traps had no oil or pheromone. New traps were used for each repetition and the three unbaited replications were done first in order to avoid contamination. Beetles were left in the arenas for 24 h in the dark and then beetles in the trap, under the base of the trap, and elsewhere in the arena were counted. The arenas were vacuumed after each trial. Average temperature and relative humidity during the tests ranged from 15.1 to C and from 29.1% to 55.5%, respectively (Hawkin 2008). There were three replicates, each done on a different date. All experiments were completed in a 2-week period. Movement rates The rate of movement of adults of T. confusum and T. castaneum and two strains (mill 1 and laboratory) was measured using techniques similar to Rigaux et al. s (2001), with 17 cm diameter rings (acrylonitrilebutadiene-styrene (ABS) plastic) placed over black paper. The sides of the rings were painted with Fluon to prevent escape of beetles. Mill 1 beetles were the progeny of beetles taken from the mill. Beetles were sexed as pupae and mated as adults, and the marked mates (DayGlo Color Corporation, Cleveland, Ohio, United States of America (Hawkin 2008)) were separated from the unmarked test insects. Experiments were run in the dark. There were 1315 beetles of each sex per treatment. Ten beetles were run at once, each in a separate ring. Beetle order was random. Average temperature and humidity during the experiment were C and 37.4% %, respectively. We observed beetles with an infrared camera (WV-CD820, Panasonic Inc., Tokyo, Japan), after letting them acclimate for at least 1 h. A remotecontrol system was used to move the camera and minimize disturbance to beetles. Rate of movement was measured by placing a sheet of acetate over a TV screen, playing the recording and constantly marking the path of the beetle for 1 min, and estimating path length using a ruler placed beside the rings. Pheromone-pitfall-trap bioassay We used a paired pitfall-trap bioassay modified from Boake and Wade (1984) and Phillips et al. (1993) to test the attractiveness of pheromone in the absence of trap-design effects. Species, strains, and rearing method were as in the arena experiment. Pupae were sexed and males and females held in separate jars with flour. Tested adults were at least 1 week old. Response to synthetic aggregation pheromone (Trécé Inc., Adair, Oklahoma, United States of America) was tested against a hexane control. Rubber septa were loaded with 1 ml of pheromone in 19 ml of hexane, or 19 ml of hexane alone, using a syringe, and allowed to dry at room temperature for 24 h. A glass plate (16 cm 16 cm) with two 0.8 cm diameter holes 8 cm apart was sanded to provide a roughened surface upon which beetles could walk. The sides of the holes were coated with Fluon to increase the efficiency of beetle capture. The glass plate was placed level in a wood casing above two glass vials that held the rubber septa and collected beetles. A flat-bottomed glass bowl (6.5 cm high, 12.5 cm diameter, with a 1 cm diameter hole in the centre) was placed over the glass plate to prevent beetle escape. An inverted glass funnel was placed on the glass plate over a 4.25 cm diameter filter paper. Ten beetles were dropped onto the filter paper through the stem of the inverted funnel, which held them in place until the funnel was raised after 30 min. The number of beetles in the vials was recorded after 10 min. All glass was washed with 95% ethanol between runs. There were six replicates for each sex, species, and strain combination. The experiments were run in the dark at C, 60% 9 10% RH.

6 Hawkin et al. 509 Burrowing This experiment tested for differences in burrowing behaviour between the six strains of the two Tribolium species. Beetles for this experiment were collected from one of mills 1 3 less than 7 months prior to the experiment or were taken from existing laboratory strains. All mill beetles and laboratory T. confusum were reared for almost 3 months prior to the experiment in groups of 100 adults in 200 g flour. Six polyvinyl chloride tubes, 100 cm 10 cm, were used to measure burrowing distributions. There were horizontal slots every 10 cm of height and metal separators were fashioned to fit into these slots. Tape was used to cover the slots during the experiment. Plain flour (1800 g) was placed in each of six tubes, resulting in a flour depth of approximately 36 cm. The tubes were placed vertically in an incubator for at least 10 h to allow the temperature to stabilize at C. Two hundred beetles of each of the six strains were placed on the surface of the flour (one strain per tube) and allowed to burrow for 24 h in the dark. Tubes were removed from the incubator and the metal separators were inserted into the 10, 20, and 30 cm horizontal slots, resulting in three 10 cm deep and one 6 cm deep sections of flour in each tube. The flour was sifted, section by section, using a 850 mm aperture sieve and the beetles in each section were counted. Statistical analysis Differences in the ratio of T. confusum to T. castaneum from various locations, obtained by means of various sampling methods, were tested using subdivided contingency tables and the x 2 test with Holm s correction. Differences between species and strains in their rate of movement, mass, and number of beetles caught in traps or found under traps were analyzed using three-way, two-way, and one-way ANOVAs. As necessary, data were transformed using log(x1)-transformation to normalize means and equalize variances. Significant differences between treatments were determined with Tukey s multiple-range test (P 0.05). We employed a general linear model using a multinomial model (in our case different depths) to determine whether the distribution of the depths of burrowing differed between species and strains. This analysis uses the entire distribution to compare groups. We used specific contrasts to compare particular groups and a x 2 test for significant differences. All statistics were run using SigmaStat (Systat Software Inc. 2006), except for the general linear model, for which we used SAS (SAS Institute Inc. 2004). Results Flour mill Flour-sample collection Flour samples from boots located on the ground floor of mill 1 contained approximately 50% T. confusum and 50% T. castaneum, whereas flour samples from the roll stands on the fourth floor and sifters in the packing plants contained over 90% T. confusum (Table 1). Beetles in flour samples from the packing plant were almost entirely T. confusum (95%), whereas beetles trapped in pheromone traps were mostly T. castaneum (81%). Of beetles from mill 1 flour samples, 45%95% were T. confusum, whereas of beetles from mill 1 pheromone traps, 45% were T. confusum (Table 1). All 22 flour samples collected contained both species. Trap efficacy When a trap with new pheromone and cereal oil was placed in the middle of the circle, only 2 of 84 released beetles were captured; these 2 beetles were released either 10 or 60 cm away from the trap. No beetles were captured in unbaited traps or traps using 5-day-old pheromone and new cereal oil (Table 2). Pheromone did not affect the number of insects that touched the trap (x , P 0.48). Of traps placed throughout the mill for 6 days, baited traps caught more ( ) beetles than did unbaited traps (0 9 0 beetles; paired t test, t 3.1, P 0.01, n 10).

7 510 Can. Entomol. Vol. 143, 2011 Table 1. Percentages of adult Tribolium confusum in flour samples or pheromone traps (remaining beetles were Tribolium castaneum) collected from mill 1 on 1824 November Table 2. Numbers of adult beetles (Tribolium confusum and T. castaneum) that were caught in or touched a trap in a flour mill. Treatment Sampling method Location Age of pheromone and cereal oil* Laboratory and simulated warehouse Warehouse releaserecapture More T. castaneum than T. confusum were caught overall during both trials (Table 3). When mill beetles were released into the warehouse (Fig. 1), no beetles of either species were caught on the first day of trial 1 and on the first 3 days of trial 2. No T. confusum were caught during trial 1 (Fig. 2). Both species were caught in trial 2, but T. confusum was caught only on days 7, 8, and 10 of sampling (Fig. 2). When laboratory beetles were released into the warehouse, both species were caught in every trap in both trials. In trial 1, no T. confusum were caught on the first day of sampling. In trial 2, no T. confusum were caught on days 1 and 3 of sampling (Fig. 2). Arena experiment Baited traps caught fewer beetles than did unbaited traps (three-way ANOVA, F 1,28 Description No. of samples Release distance from centre of trap (cm) Tribolium confusum (%)* Flour Mill Roll stands 7 91a 1220 Mill Boots 12 55c 685 Packing plant Sifters 3 95b 555 Trap Mill 4 floors 30 45c 20 Packing plant 2 floors 10 9d 23 No. caught in trap Total no. of beetles *Values followed by a different letter are significantly different (subdivided contingency tables, x 2 test, P 0.05, using Holm s correction). No. of touches Pheromone and cereal oil New pheromone (84) day-old pheromone (48) No pheromone, no cereal oil N/A (72) *Numbers in parentheses show the number of beetles in the treatment. No. of beetles not responding 19.0, P B 0.001). This was mainly due to the higher number of laboratory and mill 1 T. confusum and laboratory T. castaneum in unbaited traps than in baited traps (Table 3). To simplify analysis, we analyzed the data from baited and unbaited traps separately. Tribolium castaneum was caught more often than T. confusum in baited traps (two-way ANOVA, F 1, , P B 0.001), laboratory beetles were caught more often than mill beetles (F 1,14 4.8, P 0.05), and there was no interaction between species and strain (F 1, , P 0.48). Laboratory T. castaneum were caught more often than any other group, T. confusum from mill 1 were caught least, and the others fell in the middle (one-way ANOVA, F 5,12 8.3, P 0.001; Table 3). There were no differences in the number of beetles under the trap between strains (two-way ANOVA, F 1,14 2.2, P 0.16) or species (F 1,14 3.7, P 0.08). More mill 3 T. castaneum than laboratory

8 Hawkin et al. 511 Table 3. Numbers of adult beetles caught in traps or found under traps after 24 h in dark conditions. Arena test $ With bait Without bait Species Origin Warehouse test (with bait, beetles in traps)* Beetles in traps Beetles under traps Beetles in traps Beetles under traps Tribolium confusum Laboratory 115b 291bc 090b 27910b 292b Mill 1 6d 191c ab 1697b 090b Tribolium castaneum Laboratory 220a 35916a 191b 7799a 392ab Mill 1 75c 391bc 993ab 291b 492ab Mill 2 * 1796ab 292ab 1295b 16913ab Mill 3 * 992abc 20913a 995b 27911a *Two hundred beetles were released into each of six 1.2 m 2 arenas, each with a trap in the middle, one species/strain per arena. Traps contained pheromone and cereal oil (with bait) or no pheromone and no oil (without bait). There were two replicates, 500 T. castaneum and 500 T. confusum were released at the same time, mill and laboratory strains were tested on different dates; differences between species/strains were tested using subdivided contingency tables, x 2 test, P 0.05, using Holm s correction. $ Values are given as the mean 9 SEM. Values in each column followed by different letters are significantly different (Tukey s multiple-range test, P 0.05, n 3). T. confusum and T. castaneum were under the trap (one-way ANOVA, F 5,12 5.0, P 0.011; Table 3). There was no difference in numbers between species in unbaited traps (two-way ANOVA, F 1, , P 0.59), and laboratory beetles were caught in the traps more often than mill beetles (F 1, , P 0.001). There was a significant interaction between species and strain (F 1,14 6.1, P 0.03) (probably because there was no difference between laboratory and mill strains for T. confusum), but there were differences for T. castaneum. One-way ANOVA showed differences between the groups (F 5, , P B 0.001). Laboratory T. castaneum were caught most frequently, with no differences between the other groups (Table 3). For unbaited traps, T. castaneum were found under the trap more often than were T. confusum (two-way ANOVA, F 1,14 7.0, P 0.02), with no difference between strains (F 1, , P 0.71) or interaction between species and strain (F 1,14 2.2, P 0.16). There were differences between groups (one-way ANOVA, F 5,12 4.8, P 0.01). ANOVA, F 1, , P 0.20); sex did not affect movement rates (F 1, , P 0.43), but T. confusum moved faster than T. castaneum (F 1, , P 0.001; Table 4). No significant interactions between sex, species, and strain were found (P 0.05). Significant differences in mass were found: T. confusum weighed more than T. castaneum (three-way ANOVA, F 1, , P B 0.001), laboratory strains weighed more than mill strains (F 1, , P 0.003), and males weighed more than females (F 1, , P B 0.001), with significant interactions between species and strain (F 1, , P B 0.001) and between species and sex (F 1, , P B 0.001). Female laboratory T. confusum were heaviest, female mill T. confusum and male laboratory T. confusum were second heaviest, and there was no significant differences in mass between the other beetle groups (Table 4). There was a significant positive correlation between movement rate and mass (r , n 111; mass mg; rate of movement cm/min; slope, intercept, and r 2 value were significantly different from 0, P B 0.01). Movement rates There was no difference in movement rates between mill and laboratory strains (three-way Pheromone pitfall trap bioassay There was no difference between males and females for a given species and strain

9 512 Can. Entomol. Vol. 143, 2011 Fig. 2. Numbers of beetles caught in pheromone traps over a 10-day period when 500 Tribolium confusum and 500 T. castaneum from mill strains (A, B) or laboratory strains (C, D) were released into a simulated warehouse. (A) Trial 1, 414 February (B) Trial 2, 1424 December (C) Trial 1, 717 March (D) Trial 2, 616 January combination (Fisher s exact test, P 0.41). When the data from male and female beetles were combined, there was no difference between species or strains (x , df 5, P 0.171). More than 88% of beetles selected the pheromone vial over the control vial. Burrowing Most of the comparisons between the distributions of burrowing depths of the different species and strain combinations showed a difference (P B 0.05; Table 5). Tribolium confusum burrowed less than T. castaneum (Fig. 3, Table 5). For both species, distributions of burrowing depths of laboratory strains differed from those of mill strains. There were significant differences in distribution between the four strains tested for T. castaneum (Fig. 3), except between the strains from mills 2 and 3 (P 0.44; Table 5). Discussion Traps baited with pheromone and cereal oil capture Tribolium species in flour mills (Campbell et al. 2002, Campbell et al. 2010a, Campbell et al. 2010b). However, our study showed that these traps have low efficacy in mills; fewer than 2% of T. confusum released within 60 cm from a pheromone trap were caught. One reason for the low efficacy could be that the trap was placed in the middle of the floor, not along a wall or a corner, structural features that are often followed by insects (Mullen 2011). The effect of monitoring released beetles compared with resident beetles may also be a factor. Fresh lures in traps can repell individuals of T. castaneum (Hussain et al. 1994). In this study, fresh lures

10 Hawkin et al. 513 Table 4. Movement rates and masses of Tribolium confusum and T. castaneum from laboratory cultures and recently collected from a flour mill and reared for 1 month in the laboratory. Species Origin Rate of movement (cm/min)* Mass (mg)* n Tribolium confusum Laboratory a a 27 Mill ab b 30 Tribolium castaneum Laboratory b b 27 Mill b b 27 *Values are given as the mean 9 SEM; within a column, values followed by a different letter are significantly different (Tukey s multiple-range test, P 0.05). Table 5. Contrasts of Tribolium beetle distributions of beetles (Tribolium confusum and T. castaneum) measured after 24 h in a column of flour, using a generalized linear model procedure. Beetle group Comparison beetle group x 2 P Laboratory T. castaneum Laboratory T. confusum B Mill 1 T. castaneum 58.8 B Mill 2 T. castaneum 22.3 B Mill 3 T. castaneum Laboratory T. confusum Mill 1 T. confusum Mill 1 T. castaneum Mill 1 T. confusum B Mill 2 T. castaneum B Mill 3 T. castaneum B Mill 2 T. castaneum Mill 3 T. castaneum and 5-day-old lures did not differ appreciably in trapping efficiency. It is possible that 5 days did not allow for sufficient ageing of lures, but this is unlikely because traps reach peak effectiveness at 1 week (Hussain et al. 1994) and Dome traps had caught beetles on the floor of the mill prior to the experiment. Trap catches were higher for T. castaneum than for T. confusum, so further testing with T. castaneum would probably show efficacy greater than 2%. Ideally, traps could be used to estimate the total population in a flour mill or warehouse. In our simplified warehouse, we released approximately six T. castaneum or T. confusum per square metre, and the daily trap-capture rate over the 10-day trapping period was relatively constant at 02.4 beetles/trap-day. However, even in this simplified warehouse, there was significant variation among dates, strains, and species. In our tests, insects were released onto the floor; to be caught in traps in a commercial setting, the adults would need to leave the food deposits where they developed deep inside processing equipment or the building structure (Campbell and Hagstrum 2002; Hawkin 2008; Romero et al. 2010). Processing plants such as flour mills are complex structures with many floors and different habitats with variable food availability, temperature, and humidity. This is very different from the simplified warehouse in which we tested traps. Therefore, more research is required to determine whether traps can be used reliably to estimate total beetle populations in commercial facilities. The monitoring traps used in this study seemed to be less attractive than sex-pheromonebaited traps used for monitoring Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae) and Lasioderma serricorne (F.) (Coleoptera: Anobiidae). In simulated warehouse experiments, up to 30% of L. serricorne were captured in pitfall traps on the floor over 2 days (Arbogast et al. 2003) and approximately 20% of P. interpunctella were captured in sticky traps on the wall over 3 days (Arbogast et al. 2005). In our experiments fewer than 1% of T. confusum and fewer than

11 514 Can. Entomol. Vol. 143, 2011 Fig. 3. Distributions of mill and laboratory strains of Tribolium confusum and T. castaneum in a column of flour after 24 h (n 4, except for mill 3 T. castaneum, for which n 3). 7% of T. castaneum were captured in 2 days. The Tribolium aggregation pheromone utilizes food volatiles attractive to both sexes, whereas the P. interpunctella and L. serricorne pheromones are attractive only to males. These traps are attractive enough to control populations of P. interpunctella and L. serricorne through mass-trapping (Pierce 1994; Phillips et al. 2000). Nothing similar has been attempted with Tribolium. In these mill, simulated-warehouse, and arena tests, T. confusum were caught in pheromone traps less often than T. castaneum, and mill strains were caught less often than laboratory strains. Three possible reasons for these differences relate to the pheromone/food

12 Hawkin et al. 515 bait used, rate of beetle movement, and beetletrap interactions. The isomeric mixture of 4R-8R-()- and 4R-8S-()-4,8-dimethyldecanal in the synthetic pheromone may have been less attractive to T. confusum. Attraction of T. confusum to the 4R, 8S-() isomer is one-tenth that of T. castaneum (Suzuki and Mori 1983; Suzuki et al. 1984). We found no difference in the attraction of either species to pheromone in a pitfall-trap bioassay. However, Boake and Wade (1984) found that different strains of T. castaneum react differently to natural and synthetic pheromone in two-choice pitfall-trap bioassays. Female T. castaneum from four different strains, all reared in the laboratory for at least 7 years, differed in their sensitivity to synthetic aggregation pheromone, with some beetles preferring higher concentrations than others (Boake and Wade 1984). We tested only a single concentration. Perhaps by testing the pheromone at several doses, we could have revealed differences in attractiveness to the pheromone. Also T. confusum may be less attracted than T. castaneum to the cereal oil used in the trap. There were differences in movement rates, and we expected that faster moving insects would be captured more often. However, this was not the case. Specimens of T. castaneum moved more slowly than did T. confusum but were captured more often in traps. There were no significant differences in movement rates between laboratory and mill strains, but there were differences in trap catches in the warehouse and arena tests. We did not test for beetle-trap interactions; however, more T. castaneum than T. confusum were caught in unbaited traps. Further tests are required to examine whether there are differences in how the different species and strains approach traps, climb up the trap ramps, and are captured. In addition to the differences in trap capture, there were significant differences in burrowing behaviour between species and strains. Tribolium castaneum burrowed more than did T. confusum, as was seen in previous studies (Ghent 1966). Also there were differences between the four strains of T. castaneum. This could have been due to differences between mills and between mills and the laboratory. In the laboratory, beetles were typically taken from filter paper protruding above the culture; this could select, over many generations, for strains that burrow less. Similarly, mill fumigation or contactinsecticide treatments may select for beetles that burrow deeply and survive treatments. Mixed-species laboratory cultures of T. castaneum and T. confusum do not co-exist indefinitely; one species is eventually eliminated (Park 1948, Leslie et al. 1968). In contrast, we found a mix of T. confusum and T. castaneum in every flour sample taken from mill 1. Trematerra and Sciarretta (2004) and Abdelghany et al. (2010) also found both beetle species in the same facility, although in the former study, populations of T. confusum and T. castaneum occurred in different parts of the mill. Lack of competition may encourage co-existence of the two species in commercial facilities. Beetle populations in mills are regularly severely reduced by fumigation and sanitation and generally occur at lower densities than they do in laboratory colonies. For example, Park (1948) reared beetles in the laboratory at much higher densities (hundreds of beetles in 8 g of flour) than are seen in mills. The results reported here have significant implications for trap monitoring in mills. Since a smaller proportion of T. confusum are caught in traps, mills infested with T. confusum may have higher levels of infestation than is indicated by trap catches. Also, much of our knowledge of pheromone attractiveness and trap monitoring comes from laboratory-reared beetles, which, as indicated by our work, do not behave like mill beetles. Because mill beetles burrow more, they may be even less prone to being captured in mills. Beetles in mills may also be caught less than the mill strains seen here because they move more slowly and would be less likely to come in contact with traps. More research is needed on the differences in trap efficacy between species and strains, as we tested only one location. Further research on the chemical components of T. confusum and T. castaneum aggregation pheromones would be useful, as would research on the effects of adding other food

13 516 Can. Entomol. Vol. 143, 2011 volatiles to these monitoring traps to make them generally more attractive to beetles. Because effective population sampling is a critical part of any IPM program, and behavioural differences can affect trap efficacy, further research into the behaviour of beetles in mills is needed. Acknowledgements We thank Tannis Mayert and Alicia Leroux for technical assistance. Partial funding for this project was provided by the Manitoba government (Manitoba Graduate Scholarship) and Steritech Inc. Flour-milling staff were exceptionally helpful; without their assistance none of the mill-related work reported here would have been possible. References Abdelghany, A.Y., Awadalla, S.S., Abdel-Baky, N.F., El-Syrafi, H.A., and Fields, P.G Stored-product insects in botanical warehouses. Journal of Stored Products Research, 46: doi: /j.jspr Arbogast, R.T., Kendra, P.E., and Chini, S.R Lasioderma serricorne (Coleoptera: Anobiidae): spatial relationship between trap catch and distance from an infested product. Florida Entomologist, 86: doi: / (2003)086[0437:LSCASR]2.0.CO;2. Arbogast, R.T., Chini, S.R., and McGovern, J.E Plodia interpunctella (Lepidoptera: Pyralidae): spatial relationship between trap catch and distance from a source of emerging adults. Journal of Economic Entomology, 98: doi: / Athanassiou, C.G., Kavallieratos, N.G., and Trematerra, P Responses of Sitophilus oryzae (Coleoptera: Curculionidae) and Tribolium confusum (Coleoptera: Tenebrionidae) to traps baited with pheromones and food volatiles. European Journal of Entomology, 103: Boake, C.R.B., and Wade, M.J Populations of the red flour beetle Tribolium castaneum (Coleoptera: Tenebrionidae) differ in their sensitivity to aggregation pheromones. Environmental Entomology, 13: Burkholder, W.E Pheromones for monitoring and control of stored-product insects. Annual Review of Entomology, 30: doi: /annurev.en Campbell, J.F., and Hagstrum, D.W Patch exploitation by Tribolium castaneum: movement patterns, distribution, and oviposition. Journal of Stored Products Research 38: doi: /S X(00) Campbell, J.F., Mullen, M.A., and Dowdy, A.K Monitoring stored-product pests in food processing plants with pheromone trapping, contour mapping, and mark-recapture. Journal of Economic Entomology, 95: doi: / Campbell, J.F., Toews, M.D., Arthur, F.H., and Arbogast, R.T. 2010a. Long-term monitoring of Tribolium castaneum in two flour mills: seasonal patterns and impact of fumigation. Journal of Economic Entomology, 103: doi: /ec Campbell, J.F., Toews, M.D., Arthur, F.H., and Arbogast, R.T. 2010b. Long-term monitoring of Tribolium castaneum populations in two flour mills: rebound after fumigation. Journal of Economic Entomology, 103: doi: /ec Fedina, T.Y., and Lewis, S.M Effect of Tribolium castaneum (Coleoptera: Tenebrionidae) nutritional environment, sex, and mating status on response to commercial pheromone traps. Journal of Economic Entomology, 100: doi: / (2007)100[1924 :EOTCCT]2.0.CO;2. Fields, P.G., and White, N.D.G Alternatives to methyl bromide treatments for stored-product and quarantine insects. Annual Review of Entomology, 47: doi: /annurev.en to Ghent, A.W Studies of behavior of the Tribolium flour beetles. II. Distributions in depth of T. castaneum and T. confusum in fractionable shell vials. Ecology, 47: doi: / Harrison, G Comparative evaluation of intensive pest management, heat treatments and fumigants as alternatives to methyl bromide for control of stored product pests in Canadian flour mills. Report to Agriculture and Agri-Food Canada, Ottawa, Ontario Hawkin, K.J Monitoring populations of the flour beetles Tribolium castaneum Jacquelin du Val and Tribolium confusum (Herbst) in flour mills and in laboratory settings. M.Sc. thesis, University of Manitoba, Winnipeg, Manitoba. Hinton, H.E Secondary sexual characters of Tribolium. Nature (London), 49: 500. doi: /149500b0. Hussain, A., Phillips, T.W., Mayhew, T.J., and AliNiazee, M.T Responses of Tribolium castaneum to different pheromone lures and traps in the laboratory. In Proceedings of the 6th International Working Conference on Stored-Product Protection, 1723 April 1994, Canberra, Australia. Edited by E. Highley, E.J. Wright, H.J. Banks, and B.R. Champ. CAB International, Wallingford, United Kingdom. pp

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