Master of Science / Department of Grain Science and Industry / Subramanyam Bhadriraju / The development, relative retention, and oviposition of the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), on six different types of starches, wheat flour, and wheat flour plus yeast were investigated in the laboratory. The particle size of starch and flours were different; the mean size of 90% of particles for starches ranged from 15 μm for high amylose corn starch to 58 μm for potato, whereas that of the flour was ≤ 133 μm. Larval length, head capsule width, and weight gain of T. castaneum larvae were measured every 3 d on starches, flour, and flour plus 5% (by wt) yeast diet for 30 d at 28oC, 65% r.h., and 14:10 (L:D) photoperiod. Larvae reared on flour and flour plus yeast developed normally and showed better survival compared to those reared on starches. Larvae on the starches failed to develop beyond second, and rarely, third instars. Adults of T. castaneum did not show any preference to flour over starches in dual-choice tests in circular arenas. On average, T. castaneum laid less than 3 eggs/female over a 15-d period on starches compared to 97 and 109 eggs/female on flour and flour plus yeast diet, respectively. These studies suggest that starches are poor substrates for larval survival and development. Starches were as attractive as flour to adults; however, starches do not appear to be a suitable medium for egg-laying. Both aggregation pheromone and volatiles did not trigger oviposition behavior. Experiments by moving adults between wheat starch and wheat flour and vice versa showed that feeding on wheat flour was necessary for egg-laying, indicating the absence of essential nutrients in wheat starch. On wheat flour, feeding for 0.5 d was necessary to lay eggs. Females that were starved failed to lay eggs, reinforcing that the nutrional status of females and not males was essential for egg-laying. A minimum of 4% of wheat gluten (wheat protein) elicited egg-laying on starches, although 4-5 times fewer eggs were laid in starch gluten compared with wheat flour alone. Supplementing wheat starch with 1% cholesterol, in addition, to gluten, did not result in an increase in egg-laying by T. castaneum females. These findings suggest that starches may have potential in managing development and reproduction of T. castaneum—a pest that is common and severe in food-processing facilities. Furthermore, starches can be used as a suitable substrate for studying the nutritional ecology of T. castaneum.

No description available.

Master of Science / Department of Grain Science and Industry / Bhadriraju Subramanyam / Hulya Dogan / Stored-product insects and stored products from feed mills and swine farms contain antibiotic and potentially virulent Enterococcus faecalis, Enterococcus faecium, Enterococcus casseliflavus, Enterococcus gallinarum, and Enterococcus hirae. Stored-product insects can serve as potential vectors of these enterococci which possess antibiotic resistance genes that can be spread by horizontal transfer to more serious human pathogens. In the present study, the species and concentration of enterococci from adults and larvae of key stored-product insects and insect diets and their antibiotic resistance profile were characterized. Adults of five species out of the 15 stored-product insects were tested positive for enterococci, and these included Callosobruchus maculatus (F.), Sitophilus granarius (L.), Stegobium paniceum (L.), Lasioderma serricorne (F.), and Sitophilus zeamais Motschulsky. Three enterococcal species (E. casseliflavus, E. faecalis, and E. faecium) were found in 53 to 97% of the 30 adults screened for each insect species, and the enterococcal concentrations ranged from 1.4 x 10³ to 3.1 x 10⁶ CFU/adult. About 10 to 100% of the mature larvae of the respective five insect species had these three enterococcal species with concentrations ranging from 0.3 x 10¹ to 1.4 x 10⁵ CFU/larvae. Only three of the eight insect diets screened had the same three enterococci species in addition to E. gallinarum and E. hirae at concentrations of 0.2 x 10¹ to 5.9 x 10³ CFU/g. The greatest enterococcal concentration was found in C. maculatus adults but not in their larvae or diet (cowpeas). In C. maculatus during a nine-day period after adult eclosion, the enterococcal concentrations increased exponentially from 0.6 x 10¹ to a maximum of 4.1 x 10⁷ CFU/adult. Enterococci were detected in the fecal material of C. maculatus during a four-day period with a maximum concentration of 3.3 x 10³ CFU/adult on the fourth day. A total of 298 enterococcal isolates from adults, larvae, and diets were represented by E. faecalis (51.7% of the total), E. faecium (19.1%), E. casseliflavus (18.8%), E. gallinarum (5.7%), and E. hirae (4.7%). Enterococci were phenotypically resistant to quinupristin (51.3% of the total), erythromycin (38.9%), tetracycline (30.1%), enrofloxacin (29.2%), doxycycline (11.5%), and tigecycline (2.7%). All isolates were susceptible to ampicillin and vancomycin.

Doctor of Philosophy / Department of Grain Science and Industry / Bhadriraju Subramanyam / Alternatives to chemical grain protectants are needed that have low mammalian toxicity and high specificity to insects with no adverse environmental impacts. This project investigated the viability of using a synthetic amorphous zeolite as a potential alternative to chemical insecticides and phosphine in Australian grain. We evaluated the impacts of moisture content and application rates on dust and grain physical properties; compared registered dusts with this promising novel dust in terms of impact on grain flowability; investigated changes in bulk properties of inert-dust treated grain; and we constructed dynamic dewpoint isotherms of hard red winter wheat and amorphous silica dusts for a better understanding of moisture interactional behavior of the synthetic amorphous dust based on wheat initial moisture content. Full sorption isotherms of zeolite and wheat obtained at 25, 35, and 45⁰C clearly exhibited the hysteresis phenomenon. The intensity of hysteresis remained unchanged with increasing temperatures for Hard Red Winter wheat (HRW), whereas, the intensity of hysteresis decreased with increased temperatures during water adsorption for the porous synthetic amorphous zeolite powder. Considering the IUPAC classification of isotherms, HRW had typical type II sigmoid shape isotherm, whereas, zeolite powder had a sorption isotherm close to resembling a type IV sigmoid shape isotherm. The hysteresis loops were of type H3 for HRW, and of type H4 for zeolite powder. Irrespective of sorption direction, DLP model was the best model to estimate zeolite and HRW sorption isotherms, followed by GAB and BET models, although BET model provided almost perfect fitting to sorption data in the water activity range 0-0.5. Particle size of the amorphous dust increased with increasing moisture content. Conversely, shape parameters (circularity, aspect ratio, convexity, and solidity) generally decreased with increasing dust moisture contents. When wheat was mixed with the amorphous dust at different rates and moisture levels, the bulk density of wheat decreased, while the tapped density and the angle of repose increased, resulting in higher Hausner ratios and Carr Index values. Treating wheat with the amorphous dust caused the treated wheat to transition from an acceptable flowability to a poor flowability, based on angle of repose, Hausner ratio, and Carr index data, which do not account for the interaction of wheat with the storage vessel. Our data suggest that a range of moisture content (2-6%) and an application rate (0.5 g/kg) mitigate the adverse effects on wheat flowability. However, based on flow rate index and specific energy requirements, flowability of wheat was generally enhanced by admixing wheat with the amorphous dusts. Wheat treated with Odor-Z-Way was comparable with wheat treated with Celite or Diafil as they all exhibited low cohesion, moderate permeability, and moderate sensitivity to aeration. Wheat treated with each of the three dusts became almost unstable due to segregation, moisture uptake, lower adhesion, and coating of the blade and test vessel. A decrease in bulk density was however observed, although the decrease was smaller when wheat was admixed with Odor-Z-Way. The susceptibility to Celite, Diafil, and Odor-Z-Way varied among stored-product insect species and also among the type of substrate (wheat or concrete). Adults of the lesser grain borer, Rhyzopertha dominica, were generally least susceptible to all three amorphous silica dusts; however, a complete suppression of progeny production was possible using Celite. Adult emergence was generally not prevented by Celite, Diafil, and Odor-Z-Way which suggested a lower insecticidal efficacy of the three dusts against early developmental stages. On concrete, Odor-Z-way was particularly effective at controlling all stored-product insect species after 24 h of exposure. In view of the bulk and dynamic flow properties and the insecticidal activity, Odor-Z-Way has potential to become a grain protectant provided that segregation and the decrease in bulk density are mitigated and that the insecticidal activity is not adversely affected by the seemingly low adhesion on wheat kernels.

Doctor of Philosophy / Department of Entomology / James F. Campbell / Phillip E. Sloderbeck / Robert "Jeff" J. Whitworth / Knowledge of factors influencing the establishment, persistence and distribution of stored-product pests aids the development of effective Integrated Pest Management (IPM) programs in food storage and processing facilities. This research focused primarily on Tribolium castaneum, which is one of the most important pests of mills. Populations of T. castaneum from different food facilities can potentially be interconnected by either their own dispersal behavior or by human transportation. Population genetic structure analyses based on microsatellites and other insertion-deletion polymorphisms (“indels”) showed that populations from different mills around the US are genetically distinct from each other, but the level of differentiation was not correlated with the geographic distance. A potential source of insect infestation within a food facility is spillage that accumulates outside or movement from bulk storage facilities on site. Results from three facilities showed that most stored-product species were captured both inside and outside buildings, but T. castaneum was rarely captured outside of the facilities. Spatial distribution of all species outside was associated with the proximity of buildings, not necessarily with areas with accumulated spillage. T. castaneum populations inside facilities are potentially exposed to frequent genetic bottlenecks resulting from structural fumigations. Changes in allele frequencies through time, based on the analysis of microsatellites and other indels in individuals collected in a mill, confirmed bottleneck effects. To understand how spatial distribution of T. castaneum within a mill could be influenced by environmental and physical factors, a range of variables were measured at each trap location. There was significant variation among trap locations regarding beetle captures and the variables measured, but increase in beetle captures correlated only with increase in temperature and spillage production. Tribolium castaneum response to visual cues could influence attraction to pheromone and kairomone olfactory cues used in traps. Results of laboratory experiments showed that adults respond to tall narrow black shapes and placing traps in front of these shapes can increase captures. This research provides new insights into factors influencing the spatial distribution of T. castaneum and could help in improving monitoring programs for this important pest of the food industry.

Doctor of Philosophy / Department of Grain Science and Industry / Subramanyam Bhadriraju / Hulya Dogan / Larvae of the Indian meal moth, Plodia interpunctella (Hübner), have the ability to invade or penetrate packaging materials and infest the food within. Energy bars with three polypropylene packaging types were challenged with eggs (for first instars), third, and fifth instars of P. interpunctella to determine package resistance to larvae at 28°C and 65% r.h. to determine which provided the greatest protection against P. interpunctella larval penetration. Third and fifth instars showed a higher propensity to infest all packaging variations. First instars showed a reduction in package pentration abiltiy compared to third and fifth instars. Methoprene is an insect growth regulator labeled in the USA for use as an aerosol spray, a residual surface treatment, and as a grain protectant, and recently has been impregnated into a polymer-based packing film to prevent insect infestations of packaged products. The objectives of these studies were 1) determine the effect of short term exposure time and temperature on four week old larvae, continual exposure on egg-to-adult emergence of beetles, and sub-lethal effect on adults of the red flour beetle, Tribolium castaneum (Herbst) and warehouse beetle, Trogoderma variabile Ballion, on the inside and outside surfaces of methoprene-treated woven packaging material at 27 and 32°C at 60% r.h.; 2) evaluate fecundity, egg hatch, and egg-to-adult emergence of T. castaneum and T. variabile, when exposed to two methoprene-impregnated polymer packaging on the inside and outside surfaces at 27 and 32°C at 60% r.h.; and 3) determine the effect of methoprene-treated foil packaging on larval emergence, penetration, and invasion ability of T. variabile and P. interpunctella at 27°C and 60% r.h. Short term exposure results indicated that adult emergence from larvae of T. castaneum and T. variabile decreased with increasing exposure time. The number of eggs laid per female of T. castaneum and T. variabile did not vary from their controls. Continual exposure demonstrated 100% supression of T. castaneum adult emergence, irrespective of exposure to outside or inside surfaces. T. variabile exposed to inside surfaces were unaffected and normal adult emergence was reduced in those exposed to outside surfaces.. The number of T. variabile eggs laid per female was not significantly different among polymer packaging types. The methoprene-treated polyethylene terephthalate to polyethylene packaging, PET-PE reduced the number of T. castaneum eggs laid per female. Both polymer packaging reduced the percent hatch of both species. No T. castaneum adults emerged on the inside surface of PET-PE and both sides of the polyethlyene to polyethylene (PE-PE). Egg-to-adult emergence of T. variabile was arrested at the pupal stage on the outside surface of PE-PE packaging. The PET-PE packaging greatly reduced the number of normal adults by 87 to 97% when exposed to inside surfaces at both temperatures. The foil packaging had no significant effect on hatch of either species. T. variabile were unable to penetrate/invade any foil packages. P. interpunctella invaded all packaging containing pinholes. Therefore, continual exposure of T. castaneum and T. variabile to methoprene impregnated packaging could be a vaiable tool to protect food packages.

<p>Hermetic storage systems have gained global popularity for their ability to minimize stored product losses by depleting oxygen. However, relying solely on insects to deplete oxygen in hermetic storage, when this process takes longer, can result in (further) damage to stored commodities. This study was conducted to investigate: (i) the potential of four different germinating seeds (soybean, rice, cowpea, and corn) in scavenging oxygen within hermetic storage systems; (ii) the impact of container volume and the number of germinating seeds on oxygen depletion; and (iii) the effects of germinating seeds on insect mortality and grain quality. Among the crops tested, cowpea, during their fourth, fifth, and sixth germination stages (T4, T5, and T6), depleted oxygen below 5% within 12 hours. The fourth stage of cowpea (T4) was identified as a potential oxygen scavenger due to its shorter germination time and ease of handling. Moreover, increasing the number of germinating seeds resulted in a faster initial rate of oxygen depletion in all-sized jars. Doubling both the volume of the jars and the number of germinating seeds had a similar rate of oxygen depletion. Additionally, an equation was derived to predict the required number of germinating seeds based on data from different numbers of seed and container volume combinations. Relative humidity levels increased to approximately 90% when empty jars were used but remained consistent at 40% when the jars were filled with grains. Furthermore, using 10, 20, and 30 germinating cowpea seeds with stored grains and insects, oxygen levels were reduced below 5% at different time intervals. Complete adult mortality of <em>C. maculatus</em> was achieved within 3-5 days of exposure, depending upon the number of germinating seeds. 20 and 30 seeds achieved complete mortality within 72 hours, while 10 seeds required 120 hours. As the number of germinating seeds increased, egg counts decreased, and moisture content significantly increased in the treatment involving 30 seeds. Furthermore, no adult emerged after 96 and 120 hours of exposure to normoxia for the 30 and 20 seed treatments, respectively. However, in the 10 seeds treatment, a small percentage of adults (0.29%) did emerge even after 120 hours of exposure.</p>

No description available.

Doctor of Philosophy / Department of Grain Science and Industry / Subramanyam Bhadriraju / Ludek Zurek / Feed samples and live stored-product insects from feed mills and swine farms were collected and cultured for Enterococcus spp. The mean concentration of enterococci in insect and feed were 2.7 ± 0.5 × 101 cfu/insect and 6.3 ± 0.7 × 103 cfu/g respectively. A total of 362 isolates of enterococci collected from 89 feed samples and 228 stored-product insects were identified to the species level using PCR. These isolates were represented by Enterococcus casseliflavus (53.0%), E. gallinarum (20.4%), E. faecium (16.2%), E. hirae (5.2%), and E. faecalis (5.0%). Enterococci were phenotypically resistant to tetracycline (48.0%), erythromycin (14.3%), streptomycin (16.8%), kanamycin (12.1%), ciprofloxacin (11.0%), ampicillin (3.3%), and chloramphenicol (1.1%). All isolates were susceptible to vancomycin and gentamicin. Tetracycline resistance was encoded by tetM (50.0%), tetO (15.1%), tetK (0.5%), tetS (0.2%) and other unknown tetracycline determinants. Enterococci carried virulence genes including gelatinase (gelE; 21.5%), an enterococcus surface protein (esp; 1.9%), and cytolysin (cylA; 2.2%). An aggregation substance (asa1) gene was detected in 61.0% of E. faecalis isolates. Fifty perncet of E. faecalis isolates were phenotipically tested positive for aggregation substances. Enterococci with cylA genes were hemolytic (52.0%) and with gelE genes were gelatinolytic (18.5%). The ermB gene, encoding erythromycin resistance was detected in 8.8% of the total isolates. The Tn916/1545 family of conjugative transposons was detected in 10.7% of the isolates. Laboratory experiments showed that adults of the red flour beetle, Tribolium castaneum (Herbst), fed on poultry and cattle feeds inoculated with E. faecalis OG1RF:pCF10, were able to successfully acquire enterococci and contaminate sterile poultry and cattle feeds. To assess the potential of horizontal gene transfer, conjugation assays were carried out with E. faecalis using a donor (wild strains) and recipient (E. faecalis OG1SSP) in ratio of 1:10. Only one isolate (1 out of 18 E. faecalis) could transfer tetM to a recipient using broth mating. However, filter mating assay, followed by PCR confirmation revealed that 89.0% (16 out of 18 E. faecalis) of isolates could transfer tetM to E. faecalis. Transfer ratios of transconjugant per recipients ranged from 2.6 × 10-4 to 1 × 10-9. In summary, feed (52.0%) and stored-product insects (41.6%) collected from feed mills and swine farms carried antibiotic-resistant and potentially virulent enterococci. Our study showed that T. castaneum, a pest commonly associated with feed, served as a potential vector for enterococci in the feed environment. Conjugation assays followed by PCR confirmed presence of the tetM gene on a mobile genetic element(s) such as Tn916 and may be horizontally transferred to other Enterococcus species and to other bacteria of clinical significance.

Doctor of Philosophy / Department of Entomology / James F. Campbell / Thomas W. Phillips / Movement is a fundamental feature of animals that impacts processes across multiple scales in space and time. Due to the heterogeneous and fragmented nature of habitats that make up landscapes, movement is not expected to be random in all instances, and an increase in fitness is an expected consequence for those that can optimize movement to find valuable and scarce recourses. I studied the movement of Rhyzopertha dominica (Coleoptera: Bostrichidae), one of the most important pests of stored grain worldwide, within and between resource patches. At a fine spatial scale, I identified factors that contribute to overall and upward movement in the grain mass. Three-week-old insects tented to stay closer to the surface than one or two-week-old insects. Females tended to be more active and to explore more than males. I also found that males tended to stay closer to the surface than females and that might be related to the ability to attract females from outside the patch since there was no significant difference regarding female’s attraction within the grain patch. Interaction with feeding sites or other individuals of the same sex creates positive feedback and a more clumped spatial pattern of feeding and foraging behavior. On the other hand, interaction with individuals of different sex creates negative feedback and a more random or overdispersed pattern. At a broad spatial scale, I studied the long-term consequence of R. dominica movement on the development of population structure within the U.S. To evaluate population structure, I used reduced representation of the genome followed by direct sequencing of beetles collected from different locations across the U.S where wheat or rice is produced and stored. Ecoregions were more important in explaining structure of R. dominica populations than crop type. I also found significant isolation by distance; however, model selection primarily elected grain production and movement variables to explain population differentiation and diversity. Understanding animal movement is essential to establishing relationships between distribution and surrounding landscape, and this knowledge can improve conservation and management strategies.