Characterization of proteolytic enzymes of the midgut and excreta of the biting fly Stomoxys calcitrans

Proteolytic enzymes were characterized in the midgut and the excreta of the stable fly Stomoxys calcitrans (L) with proteins, synthetic substrates, and inhibitors. Inhibition studies suggested trypsinlike activity in sugar-fed fly midguts, whereas excreta and blood-fed fly guts exhibited other proteases. Trypsinlike activity in midguts removed 20 and 30 h after a blood meal increased from 20% to 50% of the total proteolytic enzymes present. Trypsinlike activity was inhibited with human sera, trypsin-specific inhibitors, and a protein isolated from the stable fly thorax. When human albumin and globulin fractions were incubated with trypsinlike enzymes isolated from the midgut and excreta, the albumin fraction was less inhibitory than the globulin fractions and was readily hydrolyzed by the proteolytic enzymes. These results may indicate that the proteolytic enzymes produce an abortive complex with the globulin fractions of the sera. Such a complex may explain the temporary inhibition of proteolysis by the blood meal. Soybean trypsin inhibitor fed to stable flies caused 50% inhibition in proteolytic activity in the midguts of sugar-fed stable flies and 25% inhibition in the midguts of blood-fed stable flies. Complete inhibition of proteolytic enzyme activity was achieved only in vitro. pH profiles of proteolytic enzyme activity isolated from the excreta of blood-fed stable flies indicated that several proteolytic enzymes were excreted.     

Absence of female-specific protein in the hemolymph of stable fly Stomoxys calcitrans (L.) (Diptera: Muscidae)

Changes, during the reproductive cycle, in fat body, hemolymph, and ovarian proteins of the stable fly Stomoxys calcitrans were characterized quantitatively and qualitatively using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein content of all three tissues increased after blood feeding. Fat body protein increased first, followed by hemolymph and ovarian proteins. SDS-PAGE failed to identify vitellogenin in both female hemolymph and fat body samples. No single protein or group of proteins predominated at any stage of the reproductive cycle. Comparisons between male and female stable fly hemolymph and fat body proteins failed to detect female-specific proteins. Female-specific proteins, however, were detected in the hemolymph of four other species of Diptera.     

Allozyme variation in stable flies (Diptera: Muscidae)

Polyacrylamide gel electrophoresis was used to resolve allozymes in the cosmopolitan blood-feeding stable fly,Stomoxys calcitrans (L.). Nineteen of 38 loci were polymorphic (53%). Mean heterozygosities among all loci and among only polymorphic loci were 0.096 and 0.182, respectively. These gene diversity measures are about half those among other muscid Diptera. Variation in gene frequencies was examined in 10 natural stable fly populations from Iowa and Minnesota. Gene frequencies were homogeneous at five of eight loci among six populations in 1990 and eight of eight loci among four populations in 1992. Wright'sF statistics showed no significant departure from random mating among stable flies. It was concluded that gene flow compensates for any local differentiation in stable fly populations.     

EFFECTS OF VARIATION IN TEMPERATURE. I. ON THE BIOCHEMICAL COMPOSITION OF EIGHT SPECIES OF MARINE PHYTOPLANKTON1

The influence of temperature on the biochemical composition of eight species of marine phytoplankton was investigated. Thalassiosira pseudonana Hasle and Heim-dal, Phaeodactylum tricornutum Bohlin and, Pavlova lutheri Droop (three of eight species studied) had minimum values of carbon and nitrogen quotas at intermediate temperatures resulting in a broad U-shaped response in quotas over the temperature range of 10 to 25°C. Protein per cell also had minimum values at intermediate temperatures for six species. For T. pseudonana, P. tricornutum, and P. lutheri, patterns of variation in carbon, nitrogen, and protein quotas as a function of temperature were similar. Over all species, lipid and carbohydrate per cell showed no consistent trends with temperature. Only chlorophyll a quotas and the carbon: chlorophyll a ratios (θ) showed consistent trends across all species. Chlorophyll a quotas were always lower at 10°C than at 25°C. Carbon: chlorophyll a ratios (θ) were always higher at 10°C than at 25°C. We suggest that although θ consistently increases at lower temperatures, the relationship between temperature and θ ranges from linear to exponential and is species specific. Accordingly, the interspecific variance in θ that results from species showing a range of possible responses to temperature increases as temperature declines and reaches a maximum at low temperatures. High photon flux densities appear to increase the potential interspecific variance in the carbon: chlorophyll a ratio and therefore exacerbate these trends.     

SOURCES OF INORGANIC CARBON FOR PHOTOSYNTHESIS BY THREE SPECIES OF MARINE DIATOM1

The utilization of inorganic carbon by three species of marine diatom, Skeletonema costatum (Grev.) Cleve. Ditylum brightwellii (West) Grun., and Chaetoceros calcitrans Paulsen was investigated using an inorganic carbon isotopic disequilibnum technique and inorganic carbon dose-response curves. Stable carbon isotope data of the diatoms are also presented. Observed rates of photosynthetic oxygen evolution were greater than could be accounted for by the theoretical rate of CO₂ supply from the uncatalyzed dehydration of HCO₃^? in the external medium, suggesting use of HCO₃^? as an inorganic carbon source. Data from the isotopic disequilibrium experiment demonstrate the use of both HCO₃^? and CO₂ for photosynthesis. Carbon isotope discrimination values support the use of HCO₃^? by the diatoms.     

Sensory and behavioural responses of the stable fly Stomoxys calcitrans to rumen volatiles

Analysis of volatiles from rumen digesta by gas chromatography linked antennogram recordings from Stomoxys calcitrans (L) (Diptera: Muscidae) antennal receptor cells revealed about 30 electrophysiologically active constituents, the most important of which is dimethyl trisulphide with a sensory threshold in the femtogram range. The behavioural responses of S. calcitrans to five chemostimulants (dimethyl trisulphide, butanoic acid, p-cresol, oct-1-en-3-ol and skatole) were tested in a wind tunnel where activation and attraction of hungry flies to rumen volatiles were recorded. Dimethyl trisulphide, butanoic acid and p-cresol were found to attract S. calcitrans. This sensitivity to rumen volatile constituents, that also occur in animal wastes used for oviposition by Stomoxys spp., as well as in flowers used by stable flies as sources of nectar is discussed in the context of the behavioural ecology of these flies.     

Nepetalactones from essential oil of Nepeta cataria represent a stable fly feeding and oviposition repellent

The stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae), is one of the most serious pests to livestock. It feeds mainly on cattle and causes significant economic losses in the cattle industry. Standard stable fly control involving insecticides and sanitation is usually costly and often has limited effectiveness. As we continue to evaluate and develop safer fly control strategies, the present study reports on the effectiveness of catnip (Nepeta cataria L.) oil and its constituent compounds, nepetalactones, as stable fly repellents. The essential oil of catnip reduced the feeding of stable flies by >96% in an in vitro bioassay system, compared with other sesquiterpene-rich plant oils (e.g. amyris and sandalwood). Catnip oil demonstrated strong repellency against stable flies relative to other chemicals for repelling biting insects, including isolongifolenone, 2-methylpiperidinyl-3-cyclohexen-1-carboxamide and (1S,2'S)-2-methylpiperidinyl-3-cyclohexen-1-carboxamide. The repellency against stable flies of the most commonly used mosquito repellent, DEET, was relatively low. In field trials, two formulations of catnip oil provided >95% protection and were effective for up to 6 h when tested on cattle. Catnip oil also acted as a strong oviposition repellent and reduced gravid stable fly oviposition by 98%.     

Mechanical transmission of Trypanosoma evansi and T. congolense by Stomoxys niger and S. taeniatus in a laboratory mouse model

Abstract .Mechanical transmission of Trypanosoma evansi (South American origin) and T. congolense of Kilifi DNA type (Kenyan origin) was studied in laboratory mice using the African stable flies Stomoxys niger niger and S. taeniatus . Altogether, 355 flies were interrupted after feeding on infected blood and then transferred immediately to an uninfected mouse to complete feeding. Microscopy and subinoculation of triturated flies into uninfected mice demonstrated the survival of T. congolense in Stomoxys for up to 210 min and T. evansi for up to 480 min. Parasites survived for much longer periods in the digestive tract than inside or on the mouthparts. Trypanosoma congolense was transmitted only by S. n. niger , and only at low rates of 3, 8 and 10% using flies of different feeding histories: fed on blood the previous day, freshly caught, and teneral. Trypanosoma evansi was transmitted by both Stomoxys species at higher rates: S. taeniatus range 13–18%; S. n. niger range 17–35%. The highest transmission rate occurred with the combination of teneral S. n. niger and T. evansi.     

Microsatellite loci in the stable fly,Stomoxys niger niger (Diptera: Muscidae) on La Réunion Island

Six polymorphic microsatellite markers have been isolated from a microsatellite‐enriched DNA library from the stable fly, Stomoxys niger niger. These loci exhibited five to 10 alleles per locus and an expected heterozygosity ranging from 0.57 to 0.81 in the studied populations from La Réunion Island (Indian Ocean). They should therefore be useful for the study of genetic diversity and population structure of S. niger. In addition, cross‐species amplification of four of these six loci in the closely allied species Stomoxys calcitrans produced interpretable results, two of which would be useful for population biology studies.     

N-acetylglucosaminyl transferases from the pupal instar of the stable fly,Stomoxys calcitrans

N-acetylglucosaminyl transferases from pupae of Stomoxys calcitrans (L.) were studied in 10,000g pellet suspensions. Characterization of these enzymes was based on formation of glycolipids (ie, Dol·PP-GlcNAc and Dol·PP-(GlcNAc)₂), oligosaccharide lipids, and giycoproteins. Studies on transferase activity during the pupal instar showed that there were two peaks of activity; the first peak was on day 0 (prepupae) and the second at 3 days after pupation. Subcellular fractionation indicated that 10,000g and 100,000g pellets contained most of the transferase activities. The transferases required divalent cations (either Mn²⁺ or Mg²⁺). The pH optimum, which varied for each of the products formed, was 7.5 for glycolipids, 7.0 for oligosaccharide lipids, and 6.5 for glycoprotein. Inclusion of dolichol monophosphate doubled the amount of Dol·PP-GlcNAc and Dol·PP·(GlcNAc)₂ formed, but had little effect on oligosaccharide lipid and glycoprotein formation. Tunicamycin was a potent inhibitor of glycolipid formation with an I₅₀ of 1.8–4.8 nM. It was confirmed that tunicamycin acts by preventing the transfer of GlcNAc-1-P from UDP-GlcNAc to Dol·P. UMP reverses glycolipid formation, yielding UDP-GlcNAc. Some characterization of the products was performed. Glycolipids were shown to be Dol·PP-GlcNAc and Dol·PP-(GlcNAc)₂. Glycoprotein was rapidly solubilized by protease and detergent treatments, whereas oligosaccharide lipids appeared to be acid-labile, pyrophosphate-containing lipids. The apparent kinetic constants for the formation of glycolipids were as follows: UDP-GlcNAc K_m = 1.55 ± 0.47 μM, V_max = 0.66 ± 0.21 pmol·min^?1·mg^?1; Dol·P K_m = 2.08 ± 0.85 μM, V_max = 0.13 ± 0.06 pmol·min^?1·mg^?1 protein.