Surface ultrastructure of the puparia of the blow fly, Lucilia cuprina (Diptera: Calliphoridae), and flesh fly, Liosarcophaga dux (Diptera: Sarcophagidae)

Surface ultrastructure of the puparia of the blow fly, Lucilia cuprina, and flesh fly, Liosarcophaga dux, are presented utilizing scanning electron microscopy (SEM). Emphasis was focused on characteristic features of the puparia that could be used for differentiation from other forensically important fly species. The puparium of L. cuprina typically measures 6.2±0.2 mm in length and 2.3±0.1 mm in width. Each anterior spiracle contains a single row of 5–7 papillae. The intersegmental spines between the prothorax and mesothorax are triangular with constricted tips. A clustered bubble membrane, comprising about 30 mammillate structures, is positioned dorsolaterally on each side of the first abdominal segment in young puparia but is replaced by short, tubular respiratory horns in aged puparia. The posterior end of the puparium is broadly truncate and bears a pair of medially positioned posterior spiracles that each contains three straight spiracular slits. The puparium of L. dux is larger in comparison to L. cuprina and typically measures 9.9±0.3 mm in length and 3.8±0.2 mm in width. An anterior spiracle of this species contains 14–17 papillae. The intersegmental spines between the prothorax and mesothorax are broad and triangular. A convoluted structure of unknown function was observed at the dorsolateral edge of segments 5–11. In L. dux, the caudal segment of the puparium is slightly tapered and abruptly truncated and contains a pair of posterior spiracles that are located within a deep concavity in the terminal end. Each posterior spiracular disc appears D-shaped, with a pronounced medial projection and three vertically oriented long, narrow spiracular slits. The anatomical features presented herein may be useful for identification of fly puparia of these two species in future forensic entomological investigations.     

Survey of microhymenoptera (Hymenoptera: Chalcidoidea) parasitizing filth flies (Diptera: Muscidae, Calliphoridae) breeding in refuse and poultry farms in peninsular Malaysia

Nine species of parasitoids were found parasitizing the pupae of filth flies breeding in refuse dumps and poultry farms throughout peninsular Malaysia. Spalangia were most common, consisting of Spalangia endius Walker, S. cameroni Perkins, S. gemina Boucek, S. nigroaenea Curtis, and two undescribed species. Other parasitoids collected were Pachycrepoideus vindemmiae Rondani, Dirhinus himalayanus Westwood, and an unidentified Hymenoptera. The parasitized fly hosts included Musca domestica L., Chrysomya megacephala (F.), Fannia sp., and Ophyra sp. S. endius was the most common parasitoid attacking M. domestica and C. megacephala at refuse dumps and poultry farms D. himalayanus were found to parasitize only M. domestica pupae collected at poultry farms.     

Laboratory evaluation of novaluron as a development site treatment for controlling larval horn flies, house flies, and stable flies (Diptera: Muscidae)

A granular formulation of novaluron (Novaluron 0.2G, 0.2% [AI]), a newer benzoylphenyl urea insecticide, was evaluated for its efficacy in controlling the larval stage of horn flies, Haematobia irritans (L.); house flies, Musca domestica L.; and stable flies, Stomoxys calcitrans (L.), in cow manure. Various rates and insecticide placement locations (top, middle, and bottom of manure) were evaluated in this study and all combinations of these variables reduced adult emergence of all three species when compared with the untreated controls. The presence of deformed pupae indicated that novaluron had an insect growth regulator effect on the developing fly larvae. Top, middle, or bottom application rates of 0.125, 0.195, 0.25, and 0.375 g novaluron onto manure samples, reduced adult horn fly emergence by > 90%. Middle and bottom application rates of 0.195, 0.25, and 0.375 g novaluron reduced adult house fly emergence >93%. All rates and placement combinations resulted in >98% reduction of adult stable fly emergence. The level of control efficacy observed against these three fly species along with the ease of use of a granular formulation, make this product an ideal candidate for use in an integrated livestock pest management program.     

Thermoregulation in larval aggregations of carrion-feeding blow flies (Diptera: Calliphoridae)

The growth and development of carrion-feeding calliphorid (Diptera: Calliphoridae) larvae, or maggots, is of great interest to forensic sciences, especially for estimation of a postmortem interval (PMI). The development rate of calliphorid larvae is influenced by the temperature of their immediate environment. Heat generation in larval feeding aggregations (=maggot masses) is a well-known phenomenon, but it has not been quantitatively described. Calculated development rates that do not include internally generated temperatures will result in overestimation of PMI. Over a period of 2.5 yr, 80 pig, Sus scrofa L., carcasses were placed out at study sites in north central Florida and northwestern Indiana. Once larval aggregations started to form, multiple internal and external temperatures, and weather observations were taken daily or every few days between 1400 and 1800 hours until pupation of the larvae. Volume of each aggregation was determined by measuring surface area and average depth. Live and preserved samples of larvae were taken for species identification. The four most common species collected were Lucilia coeruleiviridis (=Phaenicia) (Macquart) (77%), Cochliomyia macellaria (F.) (8.3%), Chrysomya rufifaces (Macquart) (7.7%), and Phormnia regina (Meigen) (5.5%). Statistical analyses showed that 1) volume of a larval mass had a strong influence on its temperature, 2) internal temperatures of masses on the ground were influenced by soil temperature and mass volume, 3) internal temperatures of masses smaller than 20 cm3 were influenced by ambient air temperature and mass volume, and 4) masses larger than 20 cm3 on the carcass had strongly regulated internal temperatures determined only by the volume of the mass, with larger volumes associated with higher temperatures. Nonsignificant factors included presence of rain or clouds, shape of the aggregation, weight of the carcass, species composition of the aggregation, time since death, or season.     

Morphology of the puparia of the housefly, Musca domestica (Diptera: Muscidae) and blowfly, Chrysomya megacephala (Diptera: Calliphoridae)

Examination of the puparia of the housefly, Musca domestica L. and blowfly Chrysomya megacephala (F.), through scanning electron microscopy (SEM), revealed many differences in the profile of their morphology. Special attention was focused on puparial characteristics used to differentiate between the two fly species studied, and between other forensically important flies. Results of this study indicate that the housefly puparia are almost evenly rounded at both ends and the anterior spiracle bears six papillae. A pair of pupal respiratory horns is found laterally before the posterior boundary of the first abdominal segment, bearing numerous papillae that have a longitudinal opening along the oval convex base. The peritreme of each posterior spiracle forms a crude forward or reverse D-shape, encircling three sinuous slits. The blowfly pupariums anterior spiracle contains 8–12 papillae. The pupal respiratory horns protrude slightly and in some specimens a group of ~38 globules on the bubble-like membrane may be observed. Each of the posterior spiracles is more or less an oval- shaped peritreme, encircling three straight spiracular slits. The anatomical features presented herein allow for the differentiation of puparia of the two fly species studied and could prove useful in future forensic entomological assessments.     

Reproductive potential of stable flies (Diptera: Muscidae) fed cattle, chicken, or horse blood

Reproductive potential was assessed for stable fly cohorts fed cattle, chicken, or horse blood. Flies provided chicken blood oviposited 20% more eggs per day than did those fed cattle or horse blood. However, flies provided cattle or horse blood were fecund 50% longer. When both egg viability and number of eggs produced were considered, lifetime reproductive potential was almost twice as high for flies fed cattle or chicken blood than for flies fed horse blood. Maternal investment, which took egg production and volume into account, was higher in cohorts fed cattle blood (70 mm3) when compared with the other treatments (chicken = 54 mm3, horse = 55 mm3). This is the first report of stable flies producing viable eggs after feeding on bird blood. Results from this study in addition to field observations indicate that stable fly interactions with birds may be limited to relatively low risk scenarios.     

Dynamics of permethrin resistance in a colony of horn flies (Diptera: Muscidae)

A colony of horn fly, Haematobia irritans (L.), was established from field-collected, resistant flies. Changes in resistance levels were monitored under a regimen with and without insecticide pressure. The colony remained closed with no introduction of susceptible flies. In the absence of permethrin pressure, resistance levels dropped to 2.5 times from an initial level of 17 times at LC50 and remained at that level from generation 12 through 33. In the presence of various insecticide pressure regimens, resistance increased to 500 times at LC90 by generation 86. In a separate study, susceptible colony flies were subjected to permethrin pressure, with first resistance evident in generation 16 and increasing to greater than 100 times at LC90 in generation 88.     

Economic impact of stable flies (Diptera: Muscidae) on dairy and beef cattle production

Stable flies, Stomoxys calcitrans (L.), are among the most damaging arthropod pests of cattle worldwide. The last estimate of their economic impact on United States cattle production was published 20 yr ago and placed losses at $608 million. Subsequently, several studies of effects of stable flies on beef cattle weight gain and feed efficiency have been published, and stable flies have become increasingly recognized as pests of cattle on pasture and range. We analyzed published studies and developed yield-loss functions to relate stable fly infestation levels to cattle productivity, and then estimated the economic impact of stable flies on cattle production in the United States. Four industry sectors were considered: dairy, cow-calf, pastured stockers, and feeder cattle. In studies reporting stable fly infestation levels of individual herds, median annual per animal production losses were estimated to be 139 kg of milk for dairy cows, and 6, 26, and 9 kg body weight for preweanling calves, pastured stockers, and feeder cattle, respectively. The 200,000 stable flies emerging from an average sized winter hay feeding site reduce annual milk production of 50 dairy cows by an estimated 890 kg and weight gain of 50 preweanling calves, stockers, or feeder cattle by 58, 680, or 84 kg. In 2009 dollars, the value of these losses would be $254, $132, $1,279, or $154, respectively. Using cattle inventories and average prices for 2005-2009, and median monthly infestation levels, national losses are estimated to be $360 million for dairy cattle, $358 million for cow-calf herds, $1,268 million for pastured cattle, and $226 million for cattle on feed, for a total impact to U.S. cattle industries of $2,211 million per year. Excluded from these estimates are effects of stable flies on feed conversion efficiency, animal breeding success, and effects of infested cattle on pasture and water quality. Additional research on the effects of stable flies on high-production dairy cows and nursing beef calves is needed to increase the reliability of the estimates.     

Geographic distribution of stomoxyine flies (Diptera: Muscidae) and diurnal activity of Stomoxys calcitrans in Thailand

Stomoxyine flies (Stomoxys spp.) were collected in 10 localities of Thailand using the Vavoua traps. These localities represented four major ecological settings, as follows: small local dairy farms, large industrial dairy farms, a national park, and one elephant conservation area. Three species of stable flies were identified in the following proportions: Stomoxys calcitrans (91.5%), Stomoxys indicus (7.9%), and Stomoxys sitiens (0.6%). The number of flies collected differed significantly among collection sites (chi2 = 360.15, df=3, P < 0.05). The greatest number of stomoxyine flies was captured in dairy farms. Seasonal and daily activity of S. calcitrans was observed during a 1-yr period at two selected locations (Dairy Farming Promotion Organization of Thailand and Khao Kheow Open Zoo). S. calcitrans was more abundant during the rainy season (March-September), but was not associated with the total rainfall (r2 = 0.0002, P > 0.05). Peak of daily flight activity of males S. calcitrans was at 1000 and 1600 h, whereas females showed an increase of activity all along the day until 1600 h. A better understanding of stomoxyine fly behavior related to patterns of daily activity will facilitate and improve the efficiency of fly control measures in private and government sectors.     

Seasonal prevalence and transmission of salivary gland hypertrophy virus of house flies (Diptera: Muscidae)

A survey (2005-2006) of house fly, Musca dormestica L. (Diptera: Muscidae) populations on four Florida dairy farms demonstrated the presence of flies with acute symptoms of infection with salivary gland hypertrophy (SGH) virus on all farms. Disease incidence varied among farms (farm averages, 0.5-10.1%) throughout the year, and it showed a strong positive correlation with fly density. Infections were most common among flies that were collected in a feed barn on one of the farms, especially among flies feeding on wet brewers grains (maximum 34% SGH). No infections were observed among adult flies reared from larvae collected on the farms, nor among adults reared from larvae that had fed on macerated salivary glands from infected flies. Infected female flies produced either no or small numbers of progeny, none of which displayed SGH when they emerged as adults. Healthy flies became infected after they fed on solid food (a mixture of powdered milk, egg, and sugar) that had been contaminated by infected flies (42%) or after they were held in cages that had previously housed infected flies (38.6%). Healthy flies also became infected after they fed on samples of brewers grains (6.8%) or calf feed (2%) that were collected from areas of high fly visitation on the farms. Infection rates of field-collected flies increased from 6 to 40% when they fed exclusively on air-dried cloth strips soaked in a suspension of powdered egg and whole milk. Rates of virus deposition by infected flies on food were estimated by quantitative polymerase chain reaction at approximately 100 million virus copies per fly per hour. Electron microscopy revealed the presence on enveloped virus particles in the lumen of salivary glands and on the external mouthparts of infected flies.     

Surface characteristics, morphology, and ultrastructure of human adherent lymphokine-activated killer cells

Human adherent lymphokine-activated killer (A-LAK) cells represent a population of highly cytotoxic, interleukin-2 (IL-2)-activated peripheral blood lymphocytes that have large granular lymphocyte (LGL) morphology and display natural killer (NK) cell-associated surface markers (CD3-CD56+). The ultrastructure of A-LAK cells also is consistent with that of highly activated NK cells. After their initial isolation, continued culture of A-LAK cells in the presence of IL-2 resulted in cyclic shifts between adherent and non-adherent phases with about 90% of the cells floating and non-adherent. All of these A-LAK cells were spheroidal with numerous villi and pseudopodia. In the adherent phase, A-LAK cells were motile, moving along the solid surface at a speed of about 1 micron per minute, and polar, with a ruffled leading edge at one end of the cell and a terminal tuft of villi at the opposite end. Transmission electron microscopy of these cells also demonstrated a high degree of internal polarity, with the nucleus at the leading edge of the cell and richly granulated cytoplasm at the terminal end. Extensive cytoskeletal structures, multivesicular bodies, and an active Golgi complex characterized these cells. A-LAK cells in the adherent phase were found to express numerous point contacts (podosomes) and larger adherence structures containing polymerized actin, which appear to be important for interactions of these cells with the substrate. Increased expression of adhesion molecules in association with surface structures mediating adherence is also responsible for effective binding of A-LAK cells to solid surfaces.