Impact of Manure Storage Time and Temperature on Microbial Composition and Stable Fly (Diptera: Muscidae) Development

Samples are often frozen for preservation until needed for use. It has been a common practice to store fresh dairy manure in the freezer until needed for fly development studies. However, conflicting data have suggested that freezer temperature and duration of manure may impact fly development studies, and it is likely due to the change in microbial comminutes due to the freezer conditions. In this study manure storage conditions were assessed to ascertain how temperatures impact stable fly, Stomoxys calcitrans L., survival to pupation and determine which bacterial populations impacted fly development using massively-parallel sequencing and 16S metagenomic analysis. Stable fly survival to pupation was greater in manure that was stored warm (27˚C) or frozen (-20˚C or -80˚C) for 24 days as compared to fresh manure samples. Refrigeration (4˚C) of the manure for 24 days did not affect fly development and slightly decreased the pupal weights. Over 80 bacterial families were detected by sequencing allowing for a more thorough assessment of changes in bacterial populations. Only minor shifts were observed in bacterial family composition in the manure when refrigerated or frozen for 24 days, but significant population changes were observed when the manure was incubated for 24 days at 27˚C. Since it is the temperature and incubation time that yielded the greatest pupation rate, it is hypothesized that the manure microbial community impacts the growth and development of stable flies. This study has determined suggested freezer conditions for the best storage of manure samples to maintain bacterial diversity and retain the closest bacterial populations to freshly collected manure. Although untouched, aged (20 days) manure is best to use to assess fly development, it is not always feasible in laboratory experimentations. This study demonstrates the importance of preservation techniques on manure samples, which could also confer to storage of other biological specimens that contain resident microbes.

[1]  E. Schmidtmann,et al.  Relationship between selected bacteria and the growth of immature house flies, Musca domestica, in an axenic test system. , 1992, Journal of medical entomology.

[2]  D. Adams,et al.  Effects of Stable Flies (Diptera: Muscidae) on Weight Gains of Grazing Yearling Cattle , 2001, Journal of economic entomology.

[3]  I. Mizrahi,et al.  Composition and Similarity of Bovine Rumen Microbiota across Individual Animals , 2012, PloS one.

[4]  J. Tomberlin,et al.  Development of Black Soldier Fly (Diptera: Stratiomyidae) Larvae Fed Dairy Manure , 2008, Environmental entomology.

[5]  M. Sommer,et al.  Cultivation-based multiplex phenotyping of human gut microbiota allows targeted recovery of previously uncultured bacteria , 2014, Nature Communications.

[6]  L. Ball,et al.  Differentiation of Moraxella Bovoculi sp. nov. from other Coccoid Moraxellae by the Use of Polymerase Chain Reaction and Restriction Endonuclease Analysis of Amplified DNA , 2007, Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc.

[7]  Anders F. Andersson,et al.  Transitions in bacterial communities along the 2000 km salinity gradient of the Baltic Sea , 2011, The ISME Journal.

[8]  D. Cha,et al.  Synergistic Trap Response of the False Stable Fly and Little House Fly (Diptera: Muscidae) to Acetic Acid and Ethanol, Two Principal Sugar Fermentation Volatiles , 2015, Environmental entomology.

[9]  P. Vandamme,et al.  Proposal for a New Family, Campylobacteraceae , 1991 .

[10]  P. Pullammanappallil,et al.  Microbial Ecology in Anaerobic Digestion at Agitated and Non-Agitated Conditions , 2014, PloS one.

[11]  A. Hiraishi,et al.  Members of the Family Comamonadaceae as Primary Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate)-Degrading Denitrifiers in Activated Sludge as Revealed by a Polyphasic Approach , 2002, Applied and Environmental Microbiology.

[12]  Robert G. Beiko,et al.  A Phylogenomic View of Ecological Specialization in the Lachnospiraceae, a Family of Digestive Tract-Associated Bacteria , 2014, Genome biology and evolution.

[13]  A. Klindworth,et al.  Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies , 2012, Nucleic acids research.

[14]  R. Knight,et al.  UniFrac: a New Phylogenetic Method for Comparing Microbial Communities , 2005, Applied and Environmental Microbiology.

[15]  K. Nelson,et al.  Novel Molecular Features of the Fibrolytic Intestinal Bacterium Fibrobacter intestinalis Not Shared with Fibrobacter succinogenes as Determined by Suppressive Subtractive Hybridization , 2005, Journal of bacteriology.

[16]  William Wade,et al.  Unculturable bacteria--the uncharacterized organisms that cause oral infections. , 2002, Journal of the Royal Society of Medicine.

[17]  Robert C. Edgar,et al.  BIOINFORMATICS APPLICATIONS NOTE , 2001 .

[18]  William A. Walters,et al.  QIIME allows analysis of high-throughput community sequencing data , 2010, Nature Methods.

[19]  A. Broce,et al.  Characterization of Stable Fly (Diptera: Muscidae) Larval Developmental Habitat at Round Hay Bale Feeding Sites , 2009, Journal of medical entomology.

[20]  J. Petrosino,et al.  The Gut Microbiome Modulates Colon Tumorigenesis , 2013, mBio.

[21]  D. Mark,et al.  Economic Impact of Stable Flies (Diptera: Muscidae) on Dairy and Beef Cattle Production , 2012, Journal of medical entomology.

[22]  L. Selinger,et al.  Rearing stable fly larvae (Diptera: Muscidae) on an egg yolk medium. , 1999, Journal of medical entomology.

[23]  Marti J. Anderson,et al.  A new method for non-parametric multivariate analysis of variance in ecology , 2001 .

[24]  C. Schal,et al.  Diversity and Contribution of the Intestinal Bacterial Community to the Development of Musca domestica (Diptera: Muscidae) Larvae , 2000, Journal of medical entomology.

[25]  Eoin L. Brodie,et al.  Greengenes, a Chimera-Checked 16S rRNA Gene Database and Workbench Compatible with ARB , 2006, Applied and Environmental Microbiology.

[26]  H. Drake,et al.  Clostridiaceae and Enterobacteriaceae as active fermenters in earthworm gut content , 2011, The ISME Journal.

[27]  William A. Walters,et al.  Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms , 2012, The ISME Journal.

[28]  C. Geden.  Host Location by House Fly (Diptera: Muscidae) Parasitoids in Poultry Manure at Different Moisture Levels and Host Densities , 1999 .

[29]  A. Broce,et al.  Role of bacteria in the oviposition behaviour and larval development of stable flies , 2006, Medical and veterinary entomology.

[30]  Jonathan M. Chase,et al.  Navigating the multiple meanings of β diversity: a roadmap for the practicing ecologist. , 2011, Ecology letters.

[31]  Zhongtang Yu,et al.  Status of the phylogenetic diversity census of ruminal microbiomes. , 2011, FEMS microbiology ecology.