Culture-independent analyses of carrion beetle (Coleoptera: Silphidae) secretion bacterial communities.

IMPORTANCE: The manuscript explores the secretion bacterial community of carrion and burying beetles of the central plains of North America. A core secretion microbiome of 11 genera is identified. The host subfamily, secretion type, and collection locality significantly affects the secretion microbiome. Future culture-dependent studies from silphid secretions may identify novel antimicrobials and nontoxic compounds that can act as meat preservatives or sources for antimicrobials.

Comparative toxicity of two neonicotinoid insecticides at environmentally relevant concentrations to telecoprid dung beetles.

Dung beetles (Coleoptera: Scarabaeinae) frequently traverse agricultural matrices in search of ephemeral dung resources and spend extended periods of time burrowing in soil. Neonicotinoids are among the most heavily applied and widely detected insecticides used in conventional agriculture with formulated products designed for row crop and livestock pest suppression. Here, we determined the comparative toxicity of two neonicotinoids (imidacloprid and thiamethoxam) on dung beetles, Canthon spp., under two exposure profiles: direct topical application (acute) and sustained contact with treated-soil (chronic). Imidacloprid was significantly more toxic than thiamethoxam under each exposure scenario. Topical application LD50 values (95% CI) for imidacloprid and thiamethoxam were 19.1 (14.5-25.3) and 378.9 (200.3-716.5) ng/beetle, respectively. After the 10-day soil exposure, the measured percent mortality in the 3 and 9 µg/kg nominal imidacloprid treatments was 35 ± 7% and 39 ± 6%, respectively. Observed mortality in the 9 µg/kg imidacloprid treatment was significantly greater than the control (p = 0.04); however, the 3 µg/kg imidacloprid dose response may be biologically relevant (p = 0.07). Thiamethoxam treatments had similar mortality as the controls (p > 0.8). Environmentally relevant concentrations of imidacloprid measured in airborne particulate matter and non-target soils pose a potential risk to coprophagous scarabs.

Evaluation of A3 cytoplasmic male sterile forage sorghum lines for resistance to sugarcane aphid.

MAIN CONCLUSION: Three known sugarcane aphid-resistant pollinator parents were sterilized in A3 cytoplasmic male sterility and were confirmed in this study to be resistant to sugarcane aphid allowing for the development of sugarcane aphid-resistant forage hybrids. We utilized A3 cytoplasmic male sterility and converted known sugarcane aphid-resistant sorghum TX 2783, and newly released R. LBK1 (Reg. No. GP-865, PI 687244) and R. LBK2 (Reg. No. GP-866, PI 687245) into A3 sterility to determine if the sterile counterparts would also equally express tolerance and or antibiosis to sugarcane aphid. Free-choice flat screen trials and life-table demographic studies were utilized and compared to know susceptible/fertile entries KS 585, and TX 7000, and known resistant/fertile entries TX 2783 and DKS 37-07. The R. LBK1 fertile entry was more tolerant than the known susceptible entries KS 585 and TX 7000, but was not as resistant as the other resistant entries, sustaining a damage rating of 6.0 across two different screen trials. The sterile A3 R. LBK2 showed a greater tolerance and expressed higher levels of antibiosis during aphid reproductive studies when compared to the known resistant and fertile TX 2783. All other fertile (R. LBK2, TX2783) and the A3 male sterile counterparts (A3 R. LBK2, A3 TX2783) were very similar in expression of high levels of tolerance and exhibited statistically similar damage ratings of 3.3-4.3 when exposed to sugarcane aphids. No entry, either fertile or sterile, was as tolerant as DKS 37-07, a known resistant commercial hybrid. Other plant measurements including percent loss in chlorophyll content, difference in plant height, and number of true leaves for sugarcane aphid infested versus non-infested were very consistent and highly correlated with damage ratings. Antibiosis was also exhibited in both fertile and sterile versions of the resistant lines. There was a 2 × reduction in fecundity between the R. LBK1 fertile and its sterile A3 R. LBK1 when compared to the susceptible KS 585 and TX 7000; however, the remaining fertile and sterile entries had 3.8 × to 5.8 × decrease in fecundity when compared to the susceptible KS 585 and TX 7000. Other measurements in life-table statistics such as nymphs produced/female/d, and the intrinsic rates of increased were significantly lower for all fertile and sterile lines, showing that antibiosis significantly affected sugarcane aphid reproduction. In conclusion, the A3 cytoplasmic male sterility shows consistency for maintaining the single dominant trait SCA-resistant trait of TX 2783 for expressing both antibiosis and tolerance, and great utility in the development of sugarcane aphid-resistant forage sorghums.

Effect of food source availability in the salivary gland transcriptome of the unique burying beetle Nicrophorus pustulatus (Coleoptera: Silphidae).

Nicrophorus is a genus of beetles that bury and transform small vertebrate carcasses into a brood ball coated with their oral and anal secretions to prevent decay and that will serve as a food source for their young. Nicrophorus pustulatus is an unusual species with the ability to overtake brood of other burying beetles and whose secretions, unlike other Nicrophorus species, has been reported not to exhibit antimicrobial properties. This work aims to better understand how the presence or absence of a food source influences the expression of genes involved in the feeding process of N. pustulatus. To achieve that, total RNA was extracted from pooled samples of salivary gland tissue from N. pustulatus and sequenced using an Illumina platform. The resulting reads were used to assemble a de novo transcriptome using Trinity. Duplicates with more than 95% similarity were removed to obtain a "unigene" set. Annotation of the unigene set was done using the Trinotate pipeline. Transcript abundance was determined using Kallisto and differential gene expression analysis was performed using edgeR. A total of 651 genes were found to be differentially expressed, including 390 upregulated and 261 downregulated genes in fed insects compared to starved. Several genes upregulated in fed beetles are associated with the insect immune response and detoxification processes with only one transcript encoding for the antimicrobial peptide (AMP) defensin. These results confirm that N. pustulatus does not upregulate the production of genes encoding AMPs during feeding. This study provides a snapshot of the changes in gene expression in the salivary glands of N. pustulatus following feeding while providing a well described transcriptome for the further analysis of this unique burying beetle.

Yield Response of Determinate Chipping Potato to Artificial Defoliation.

The relationship between canopy defoliation by insects and yield loss in potato (Solanum tuberosum L.) has been extensively researched. However, detailed analysis of varieties with different properties and examination of methods of defoliation are still lacking. Absence of research results has led many producers to use a very conservative economic threshold (ET) of 10% defoliation in the management of insect defoliators. A series of leaf removal experiments was conducted on a determinate chipping variety of potato at different plant stages. In 2002, the highest levels of defoliation induced upon the plant canopy were 50, 30, and 75% for tuber initiation, full bloom, and plant maturity stages, respectively. In 2003, defoliation levels were increased to 70, 80, and 90% for tuber initiation, full bloom, and plant maturity, respectively. Total yield was unaffected, and no significant changes in market sized yield, or number of marketable tubers were observed at any growth stage in either year. Because the determinate variety showed remarkable ability to recover from substantial defoliation, two different methods of defoliation: hand cut (cutting the edge of leaves with scissors while avoiding the midrib) and hole punching (hole-punching leaf tissue [15.08-mm diameter] while avoiding midrib) were tested. Although total yield was unaffected, marketable yield and small potato yield were reduced, Thus, although defoliation methods must be considered, a 10% defoliation ET is exceedingly conservative and an ET of 60% for all stages of determinate chipping potato could be adopted by growers.

Invasive Saltcedar and Drought Impact Ant Communities and Isopods in South-Central Nebraska.

The establishment and spread of non-native species often results in negative impacts on biodiversity and ecosystem function. Several species of saltcedar, Tamarix spp. L., have been recently naturalized in large portions of the United States where they have altered plant and animal communities. To test the prediction that saltcedar negatively affects invertebrates, we measured ant genera diversity and the activity density of the exotic isopod Armadillidium vulgare Latrielle (Isopoda: Oniscoidea) for 2 yr using pitfall traps located within 30 5-m2 plots with or without saltcedar at a south-central Nebraska reservoir. From 2005 to 2006, we collected 10,837 ants representing 17 genera and 4,953 A. vulgare. Per plot, the average number of ant genera was not different between saltcedar (x̅ = 3.9) and non-saltcedar areas ( x̅ = 3.9); however, saltcedar plots were compositionally different and more similar from plot to plot (i.e., they had lower beta diversity than control plots) in 2005, but not in 2006. Isopods were likewise temporally affected with higher activity density (+89%) in control plots in 2005, but higher activity density (+27%) in saltcedar plots in 2006. The observed temporal differences occurred as the drought that initially enabled the saltcedar invasion became less severe in 2006. Combined, our results suggest that invertebrate groups like ants, which are generally omnivorous, may be better equipped than more specialized taxa like detritivores to withstand habitat changes due to invasions by non-native species, especially during extreme weather events such as prolonged droughts.

Mosquito Community and West Nile Virus Risk on a National Guard Training Base in Oklahoma.

Military bases are important areas for mosquito surveillance to maintain active duty combat readiness and protect training exercises. The aim of this study was to assist Camp Gruber National Guard training facility personnel to assess their mosquito community and West Nile virus (WNV) risk using biweekly sampling of 50 sites. Between May and October 2018, 10,259 adult female mosquitoes consisting of 6 genera and 26 species were collected over 662 trap-nights using 2 trap types. The most commonly collected genus was Culex (72.2% of total), followed by Psorophora (13.3%) and Aedes (10.2%). Of note, most of the medically important species were collected in the area containing troop living quarters, including 1 WNV-positive pool of Culex tarsalis. Two specimens of Aedes aegypti were collected around a vehicle storage area. While smaller in land mass size than many other active military bases in Oklahoma, the diversity of species at Camp Gruber was comparable to collections from 4 larger bases in Oklahoma. These data demonstrate the need for regular season-long mosquito monitoring of training bases to protect the health of active duty and reserve military personnel.

Differences in Mosquito Communities in Six Cities in Oklahoma.

Vector-borne diseases in the United States have recently increased as a result of the changing nature of vectors, hosts, reservoirs, pathogens, and the ecological and environmental conditions. Current information on vector habitats and how mosquito community composition varies across space and time is vital to successful vector-borne disease management. This study characterizes mosquito communities in urban areas of Oklahoma, United States, an ecologically diverse region in the southern Great Plains. Between May and September 2016, 11,996 female mosquitoes of 34 species were collected over 798 trap nights using three different trap types in six Oklahoma cities. The most abundant species trapped were Culex pipiens L. complex (32.4%) and Aedes albopictus (Skuse) (Diptera: Culicidae) (12.0%). Significant differences among mosquito communities were detected using analysis of similarities (ANOSIM) between the early (May-July) and late (August-September) season. Canonical correlation analysis (CCA) further highlighted the cities of Altus and Idabel as relatively unique mosquito communities, mostly due to the presence of Aedes aegypti (L.) and salt-marsh species and absence of Aedes triseriatus (Say) in Altus and an abundance of Ae. albopictus in Idabel. These data underscore the importance of assessing mosquito communities in urban environments found in multiple ecoregions of Oklahoma to allow customized vector management targeting the unique assemblage of species found in each city.

New Records of Aedes aegypti In Southern Oklahoma, 2016.

Aedes aegypti is an important subtropical vector species and is predicted to have a limited year-round distribution in the southern United States. Collection of the species has not been officially verified in Oklahoma since 1940. Adult mosquitoes were collected in 42 sites across 7 different cities in Oklahoma using 3 different mosquito traps between May and September 2016. Between July and September 2016, 88 Ae. aegypti adults were collected at 18 different sites in 4 different cities across southern Oklahoma. Centers for Disease Control and Prevention mini light traps baited with CO2 attracted the highest numbers of Ae. aegypti individuals compared to Biogents (BG)-Sentinel® traps baited with Biogents (BG)-lure and octenol and Centers for Disease Control and Prevention gravid traps baited with Bermuda grass-infused water. The discovery of Ae. aegypti mosquitoes within urban/exurban areas in Oklahoma is important from an ecological as well as a public health perspective.

Causes of Rapid Carrion Beetle (Coleoptera: Silphidae) Death in Flooded Pitfall Traps, Response to Soil Flooding, Immersion Tolerance, and Swimming Behavior.

Terrestrial insects in water can often delay or escape drowning by floating and swimming. However, we observed that flooding of pitfall traps baited with rotting carrion results in high overnight mortality of captured beetles and reasoned that this risk may be enhanced by microbial respiration. By assessing carrion beetle (Coleoptera: Silphidae) response to flooding, tolerance to immersion, and swimming behavior, we offer insights to this cause of death and beetle behavioral physiology. Response of buried Nicrophorus orbicollis Say to soil flooding resulted in beetles moving to the soil surface. The lethal time to 50% mortality (LT50 (immersion); mean ± 95% CI) for Nicrophorus investigator Zetterstedt, Nicrophorus marginatus F., Necrodes surinamensis F., and Thanatophilus lapponicus Herbst was 14.8 ± 2.3, 9.0 ± 3.3, 3.2 ± 1.1, and 12.1 ± 2.5 h, respectively. Swimming behavior and survival time of N. investigator was tested using yeast:sucrose (Y:S) solutions to create a eutrophic, severely hypoxic aqueous environment. LT50 (swimming) for N. investigator was 7.5 ± 1.4, 6.0 ± 1.7, and 4.2 ± 1.2 h for the low, medium, and high Y:S solutions, respectively, and >24.0 h in control treatments. Nicrophorus investigator survived nearly twice as long when completely immersed in deoxygenated water, as might occur in flooded burrows, than when swimming on the surface. We document for the first time, the rapid induction of hypoxic coma and death for a terrestrial insect from enhanced microbial activity and CO2 production of an aqueous environment, as well as suggestions on trapping protocols related to the federally endangered Nicrophorus americanus Olivier.

Forms of Melanoplus bowditchi (Orthoptera: Acrididae) collected from different host plants are indistinguishable genetically and in aedeagal morphology.

The sagebrush grasshopper, Melanoplus bowditchi Scudder (Orthoptera: Acrididae), is a phytophilous species that is widely distributed in the western United States on sagebrush species. The geographical distribution of M. bowditchi is very similar to the range of its host plants and its feeding association varies in relation to sagebrush distribution. Melanoplus bowditchi bowditchi Scudder and M. bowditchi canus Hebard were described based on their feeding association with different sagebrush species, sand sagebrush and silver sagebrush, respectively. Recently, M. bowditchi have been observed feeding on other plant species in western Nebraska. We collected adult M. bowditchi feeding on four plant species, sand sagebrush, Artemisia filifolia, big sagebrush, A. tridentata, fringed sagebrush, A. frigidus, and winterfat, Krascheninnikovia lanata. We compared the specimens collected from the four plant species for their morphological and genetic differences. We observed no consistent differences among the aedeagal parameres or basal rings among the grasshoppers collected from different host plants. Amplified Fragment Length Polymorphism markers were used to test the genetic relationships among the grasshoppers. Analysis of Molecular Variance and distance-based Unweighted Pair Group Method with Arithmetic mean dendrogram failed to reveal significant differences. Although the forms showed behavioral and minor color and size differences, the genetic data suggest all forms under study likely interbreed, which indicates they are a single species instead of four species or subspecies. These results indicate that host plant use may influence melanopline phenotype and suggest the need of further genetic analysis of subspecies recognized based on morphology, distribution, and ecology.

A comparison of dung beetle (Coleoptera: Scarabaeidae) attraction to native and exotic mammal dung.

Although the preference of dung beetles (Coleoptera: Scarabaeidae) for specific types and conditions of dung has been given substantial attention, little has been done to investigate the potential effects of exotic mammal introduction for game farms or rewilding projects. We used pitfall traps baited with various native and exotic herbivore, carnivore, and omnivore dung to evaluate dung beetle preference in the Great Plains of North America. Additionally, we analyzed of the nutrient quality of each dung type. In total, 9,089 dung beetles from 15 species were captured in 2 yr of sampling. We found significant differences (P < 0.05) in mean dung beetle capture among omnivore, herbivore, and carnivore dung, as well as differences in individual species preference for dung type. Omnivore dung was the most attractive with chimpanzee and human dung having the highest mean capture (291.1 ± 27.6 and 287.5 ± 28.5 respectively). Carrion also was highly attractive with a mean of 231.9 ± 20.6 beetles per trap (N = 8). Our results suggest definitive local preference of carrion in Phanaeus vindex Macleay and Onthophagus hecate (Panzer), while the congener, O. pennsylvanicus (Harold), was rarely captured in carrion and highly preferred omnivore dung. Preference for a specific bait type does not appear to be correlated with dung quality, mammalian diet, or origin of mammal. Results suggest niche segregation by dung type among dung beetle species.

Effect of investigator disturbance in experimental forensic entomology: carcass biomass loss and temperature.

Often carrion decomposition studies are conducted using a single carcass or a few carcasses sampled repeatedly through time to reveal trends in succession community composition. Measurements of biomass and other abiotic parameters (e.g., temperature) are often collected on the same carcasses but are rarely a focal point of the studies. This study investigated the effects that repeated sampling during experiments have on the decomposition of carrion, measured as both gross biomass (carcass plus fauna) and net biomass (carcass only), on carcasses disturbed on every visit (with weighing only or also with the collection of fauna) and on carcasses disturbed only once. Each trial lasted at least 21 days, with samples taken in triplicate. Rat carcasses used in this study were placed in the field on the same day and either weighed on every visit or ignored until a given day. Internal and ambient air temperatures were recorded on each carcass at the time of sampling and on undisturbed carcasses using temperature loggers. The presence of succession fauna did not result in significant biomass loss on most days; however, there were individual days early in decomposition (days 3 through 6) when the succession fauna comprised a large portion of the gross biomass. With the exception of biomass loss by the emigration of maggots on days 4 and 5, neither repeated weighing of the carcasses nor repeated weighing and faunal sampling of the carcasses statistically affected the rate of biomass loss. Internal temperatures of carcasses sampled repeatedly were frequently 2-5°C lower than those that had not been disturbed, and ambient temperatures differed significantly depending on the location of measurement device. Results indicate that methods used historically for biomass loss determination in experimental forensic entomology studies are adequate, but further refinements to experimental methodology are desirable.

Degree-day requirements for eight economically important grasshoppers (Orthoptera: Acrididae) in Nebraska using field data.

The timing of application for the management of rangeland grasshoppers (Orthoptera: Acrididae) is critical, especially as insecticides become more specialized and the use of Insect Growth Regulators becomes more widespread. The general seasonal occurrence of adults of many grasshopper species has been well documented; however, their appearance varies widely between years. We analyzed sweep samples collected over the western two thirds of Nebraska from a 3-yr period and noted the occurrence of adults by region for eight species of rangeland grasshoppers. We analyzed occurrence based on degree-day accumulations for the region and developed estimates of degree-day requirements for these species. Because these grasshopper species are common rangeland pests, degree-day requirements to reach adulthood should improve the effectiveness of grasshopper treatment programs over a large geographic area.

Effects of invasive eastern redcedar on capture rates of Nicrophorus americanus and other Silphidae.

Because of fire suppression and improper land management, native eastern redcedar, Juniperus virginiana L., is invading many grassland areas. Among these areas are the Loess Canyons of southeastern Lincoln County, NE, which support a large population of the federally endangered American burying beetle, Nicrophorus americanus Olivier. In the past 30 yr, cedar encroachment has covered >30% of this landscape and is increasing at a rate of 2% per year. The effects of cedar trees on remaining populations of American burying beetle are currently unknown. In this study, we sampled two sites in Lincoln County, NE, by placing paired baited pitfall traps (N = 16) in cedar-dominated and open grassland habitats during June and August 2004 and 2005. We collected significantly more Silphidae, including American burying beetle in open habitats. All American burying beetle were marked and released. The recapture rate of these beetles was two times higher in cedar sites than in open sites. Among the Silphidae, only Nicrophorus orbicollis Say was collected significantly more often in cedar areas. The results of this study indicate that cedars reduce numbers of most silphid species, likely by limiting their ability to forage for carrion, a required resource for feeding and reproduction. This is the first study to directly examine the impact of cedar on Silphidae, and our results have important implications in the Great Plains and beyond. However, future studies must examine the impacts of cedar management, including mechanical control or prescribed fire, on American burying beetle and other silphid species.

Differences among antimicrobial properties of carrion beetle secretions reflect phylogeny and ecology.

Carrion beetles (Coleoptera: Silphidae) consist of two subfamilies in North America. Members of the Silphinae arrive at carcasses during the mid-stage of decay and their larvae feed on developing maggots, while members of the Nicrophorinae bury and tend carcasses upon which their developing larvae feed. The Nicrophorinae maintain the condition of the carcass by applying oral and anal secretions that reduce carcass decay apparently through bacterial inhibition, although quantification has not been made. We hypothesized that enzymes in the oral and anal secretions of the subfamily Nicrophorinae would inhibit bacterial growth, while secretions from the subfamily Silphinae would not. The secretions were assayed for inhibitory effects with a Microtox Analyzer that monitors the decrease in bioluminescence from the bacterium Vibrio fischerii. We found a significant difference of bioluminescence in the control compared to secretions of 8 out of 10 tested Nicrophorinae (with oral secretions being most active), while only anal secretions from Necrodes surinimensis of the Siphinae significantly reduced bacterial survival. These data follow the known phylogenic relationship in which Necrodes is the closest genus to the Nicrophorinae. The two species of Nicrophorinae, which did not show significant reductions in bacterial growth, differ ecologically from the others. Thus, the presence of antimicrobial compounds in most Nicrophorinae secretions, but not in most other Silphinae, represents an adaptation to preserve the buried carcass.