R. M. Roe

Hemolymph proteins in ticks.

In comparison to insects and Crustacea, our knowledge of the predominant hemolymph proteins in ticks is minimal. The hemolymph protein most studied in ticks has been vitellogenin (Vg). Vg is synthesized by the tick fat body after female adults obtain a blood meal, is released into the hemolymph and is absorbed by developing oocytes as vitellin (Vn). Much of what we know about Vg is from studies of Vn. In general, the carbohydrate, lipid and amino acid composition is similar to insects except that in the tick, Vg contains heme, most likely from the digestion of host hemoglobin. In the American dog tick, Dermacentor variabilis, Vg is comprised of two native proteins and seven subunits on SDS-PAGE. Vg has been characterized in five tick species but the amino acid sequence is not yet available. Another predominant hemolymph protein, apparently a carrier protein (CP), has recently been studied in two tick species. This protein is found in the hemolymph of both male and females adults, in adult tissues outside of the hemolymph in some tick species, in coxal fluid of soft ticks and in whole body homogenates from eggs, larvae and nymphs. CP from the hard tick, D. variabilis, contains cholesterol, phospholipids, monoacylglycerides, triacylglycerides, free fatty acids, carbohydrate and heme. Under identical assay conditions, the analogous protein in the soft tick, Ornithodoros parkeri, did not contain heme. CP in the American dog tick consists of two subunits, one of which has 61% identity to the biliprotein, artemocyanin, from the fairy shrimp. CP is identical to a heme-lipoprotein (HeLp) from Boophilus microplus. The exact roles of CP and HeLp have not yet been fully determined, but they apparently are important in heme sequestration and as a storage depot for protein and lipid. Macroglobulin, lectin, antimicrobial, JH binding, JH esterase, and other tick hemolymph proteins are also discussed.

Tissue distribution and characterization of predominant hemolymph carrier proteins from Dermacentor variabilis and Ornithodoros parkeri.

The tissue distribution of the predominant hemolymph protein found throughout tick development was examined in the hard tick, Dermacentor variabilis, and in the soft tick, Ornithodoros parkeri. In D. variabilis, the predominant (purified) hemolymph protein was a lipoglycoheme-carrier protein (DvCP) with a molecular weight of 200K. A protein with a similar mobility on native-PAGE was found in fat body, salivary gland, muscle and ovary from partially fed females which was most abundant in the plasma and salivary gland. DvCP from plasma, salivary gland and fat body of partially fed females consisted of two subunits on SDS-PAGE (98 and 92K). In replete females, only salivary gland exhibited protein subunits equivalent to hemolymph CP. CP in salivary gland and fat body stained positive for lipids. The concentration of CP in tissues varied between partially fed and replete females, indicating a difference in the expression and/or sequestration of CP during adult development. The predominant hemolymph carrier protein from O. parkeri (OpCP) was purified to homogeneity for the first time and is presumed to have similar functions to CP from D. variabilis. Purified OpCP exhibited a molecular weight of 668K by native-PAGE. Unlike CP from D. variabilis, OpCP was not detected in fat body or salivary gland tissues but occurred abundantly in coxal fluid. By SDS-PAGE, purified hemolymph OpCP consisted of two major subunits (114 and 93K) and a less abundant protein with an apparent molecular weight of 48K. Purified native OpCP was a lipoprotein like DvCP. A spectral analysis of purified OpCP failed to demonstrate the presence of heme like that found for CP from D. variabilis, purified by the same methods. However, plasma from O. parkeri contained heme with a lambda(max) of 410nm.

Aphid carboxylesterases: Biochemical aspects and importance in the diagnosis of insecticide resistance☆

Abstract The activity of general carboxylesterases from susceptible and malathion-resistant strans of the green peach aphid, Myzus persicae , and the tobacco aphid, M. nicotianae , was examined using 10 1-naphtholic esters. The activity showed a parabolic relationship with the number of carbon atoms in the substrate acyl moiety. The maximal activity was obtained with 1-naphthyl propionate and 1-naphthyl butyrate, respectively, for the above species. No interstrain differences in the general profile of the structure-activity relationship were observed. However, an elevated carboxylesterase activity in the resistant strains when compared to the reference-susceptible strain was observed with each of the tested substrates, and the bestowed activity correlated with the degree of malathion resistance. The zymograms and intensity of the carboxylesterase bands when resolved by wide range isoelectric focusing (pH 3.5–9.5) and stained with Fast Blue B salt using 1-naphthyl acetate, 1-naphthyl propionate, and 1-naphthyl caproate as substrates correlated favorably with the general activity. From the above results a filter paper esterase test was developed and used for the diagnosis of the level of resistance in individual aphids. This test showed an excellent agreement with the susceptibility bioassay test of malathion against the green peach aphid. However, the test did not make possible an unambiguous discrimination between susceptible and resistant tobacco aphids. The possible impact of this and other more specific tests on the management of aphid populations is discussed.

Diagnostic esterases and insecticide resistance in the tobacco aphid, Myzus nicotianae Blackman (Homoptera: Aphididae)

Abstract Insecticide resistance in the tobacco aphid, Myzus nicotianae Blackman, from different localities in the southeastern United States has been found to exist mainly in aphids with red body coloration. Frequency distribution of esterase activity in individual tobacco aphids collected from a tobacco field and a greenhouse indicated that organophosphate resistance was always linked to high carboxylesterase activity toward 1-naphthyl acetate. Two approaches were taken to speed up the detection methodology of the frequency of resistant phenotypes. First, an abbreviated bioassay test, using malathion as a reporter molecule, was developed to rapidly distinguish between OP-resistant and susceptible phenotypes of the tobacco aphid. A time threshold of 50 min following aphid exposure to 50 ppm malathion in a water suspension accurately discriminated between susceptible and resistant phenotypes. Second, assays of aphid carboxylesterases, using the acetates and propionates of 1-naphthol, 2-naphthol, and 4-nitrophenol, were performed to optimize and better understand the esterase discriminating activity between resistant and susceptible phenotypes. Only activity toward 1-naphtholic esters unambiguously discriminated between susceptible and resistant aphids. In general, resistance to malathion appeared to be esterase-mediated and some electrofocusing-detectable esterase isozymes were associated with resistance.

The effect of temperature on energy distribution during the last-larval stadium of the female house cricket, Acheta domesticus

Abstract The distribution of energy during the last stadium of the house cricket at two temperatures was the main theme of this study. Food consumption, growth, and oxygen consumption were greater in the first half of the stadium at both 25 and 35°C. An RQ > 1 indicated the conversion of carbohydrates to lipids during the first half of the instar at both temperatures. The duration of the stadium increased from 6 days at 35°C to 14 days at 25°C. The same maximal weight, protein content and lipid content were attained at both 25 and 35°C. A weight loss (mostly in stored lipids) after the midstadium peak weight was greater at the lower temperature. The absorption efficiency and the production of metabolic wastes were not affected by temperature, but the metabolic efficiency was much higher at 35 than at 25°C during the first half as well as the latter half of the stadium. Although during the first half of the stadium more energy was ingested, absorbed, and made available for growth at 25 than at 35°C, only slightly more growth occurred at 25°C. During the last half of the stadium less energy was ingested at 25 than at 35°C, and much more growth occurred at 35°C because of the even greater heat loss at 25 than at 35°C. Therefore at a lower temperature cricket larvae eat slightly more and reach the same maximal weight as at a higher temperature, but they end up smaller because they waste more energy during the extended duration of the stadium at the lower temperature.

First report of the repellency of 2-tridecanone against ticks.

2‐Tridecanone and 2‐undecanone are both found naturally in the trichomes of wild tomato plants and are important in plant resistance to herbivory. 2‐Undecanone is the U.S. Environmental Protection Agency (EPA)‐registered active ingredient in the commercially available arthropod repellent, BioUD®. The goal of this study was to examine the tick repellency of 2‐tridecanone. Two‐choice bioassays were conducted using 8% 2‐tridecanone vs. the repellent carrier (absolute ethanol) and compared with two‐choice studies using 8% 2‐undecanone vs. absolute ethanol. Unfed, host‐seeking adult (mixed sex) Amblyomma americanum (L.) (Acari: Ixodidae) and Dermacentor variabilis Say (Acari: Ixodidae) were used to evaluate repellency and time to repellent failure at room temperature. The present study shows in filter paper assays (0.63 mg test compound/cm2) that 2‐tridecanone was 87% repellent to A. americanum at 12 h after application, but had no statistically significant repellency at 15 h and 24 h, and was 72% repellent to D. variabilis at 15 h, but had no statistically significant repellency at 24 h. By contrast, 2‐undecanone was 74% and 75% repellent to A. americanum and D. variabilis, respectively, at 2 h after application, but no statistically significant repellency was noted at 2.5 h and 3 h. In two‐choice assays on cheesecloth, 2‐tridecanone at 0.25 mg/cm2 was 85% repellent to A. americanum 6 h after application, demonstrating its potential use as an arthropod repellent that can be used on clothing without the need for formulation. No statistically significant repellency was found at 9 h or 12 h. The potential use of 2‐tridecanone as a tick repellent is discussed.

Decomposition of Concealed and Exposed Porcine Remains in the North Carolina Piedmont.

Abstract We examined the decomposition and subsequent insect colonization of small pig carrion (Sus scrofa (L.)) placed in concealed and open environments during spring, summer, and fall in Raleigh, North Carolina, as a model for juvenile human remains. Remains were concealed in simulated attics in three manners, ranging from minimal to well-concealed. Concealment had a significant effect on the insect community colonizing the remains across all three seasons; the beetles Necrobia rufipes (DeGeer) (Cleridae) and Dermestes maculatus (DeGeer) (Coleoptera: Dermestidae) were the only species indicative of remains located indoors, whereas numerous fly (Diptera: Calliphoridae, Muscidae, Sepsidae, and Piophilidae) and beetle (Coleoptera: Silphidae, Staphylinidae, and Histeridae) species and an ant species (Hymenoptera: Formicidae, Prenolepis sp.) were indicative of remains located outdoors. Season also significantly affected the insect species, particularly the blow flies (Diptera: Calliphoridae) colonizing remains: Lucilia illustris (Meigen) was indicative of the spring, Cochliomyia macellaria (F.) and Chrysomya megacephala (F.) were indicative of the summer, and Calliphora vicina Robineau-Desvoidy and Calliphora vomitoria (L.) were indicative of the fall. In addition, across all seasons, colonization was delayed by 35–768 h, depending on the degree of concealment. These differences among the insect communities across seasons and concealment treatments, and the effects of concealment on colonization indicate that such information is important and should to be considered when analyzing entomological evidence for criminal investigations.

Toxicity of the crystalline polypeptides of Bacillus thuringiensis subsp. israelensis in Japanese quail

Abstract The solubilized parasporal crystalline proteins (SPCP) of Bacillus thuringiensis subsp. israelensis when administered to Japanese quail by intraabdominal (IA) injection were toxic with an LD 50 at 24 hr of 22.8 mg/kg. No deaths were noted by intranasal, subcutaneous, and intravenous administration. SPCP were highly hemolytic against quail red blood cells but this SPCP-induced hemolysis was inhibited by preincubation of SPCP with quail serum. Electrocardiographic analyses of IA-injected quail demonstrated cardiac dysfunction consistent with bradycardia. Hypothermia was also positively correlated with heart rate reduction. SPCP IA injection reduced serum lipid levels and alkaline phosphatase, and increased serum glucose, creatine phosphokinase, and lactate dehydrogenase. Induction of a septic shock-like condition may be the toxic mode of action of IA-injected SPCP in Japanese quail.

Comparison of synganglion neuropeptides, neuropeptide receptors and neurotransmitter receptors and their gene expression in response to feeding in Ixodes scapularis (Ixodidae) vs. Ornithodoros turicata (Argasidae)

Illumina GAII high‐throughput sequencing was used to compare expressed genes for female synganglion neuropeptides, neuropeptide receptors and neurotransmitter receptors of the soft tick Ornithodoros turicata with the hard tick Ixodes scapularis. Gene ontology molecular level three mapping revealed no significant differences amongst the same categories represented in O. turicata and I. scapularis. Transcripts predicting 22 neuropeptides or their receptors in the O. turicata synganglion were similar to annotations for 23 neuropeptides or receptors previously identified from I scapularis, with minor exceptions. A transcript predicting ecdysis triggering hormone receptor was identified in O. turicata; transcripts encoding for proprotein convertase and glycoprotein B were identified in both species. Transcripts predicting the same neurotransmitter receptors were found in the synganglion of both species. Gene expression of the transcripts showed numerous differences in response to feeding. Major differences were observed in expression of genes believed important in regulating slow vs. rapid feeding, blood water elimination, cuticle synthesis plasticity and in signalling reproductive activity. Although the glutamate receptor was strongly upregulated in both species, the gamma aminobutyric acid receptor, which inhibits glutamate, was upregulated significantly only in I. scapularis. These differences are consistent with the slow vs. rapid action of the pharyngeal pump in the two species.

Conjugation of chlorodinitrobenzene with reduced glutathione in the absence and presence of glutathione transferase from larvae of the southern armyworm, Spodoptera eridania

Abstract The reaction of 1-chloro-2,4-dinitrobenzene (CDNB) with reduced glutathione in the absence and presence of glutathione transferases from larvae of the southern armyworm, Spodoptera eridania , produced the conjugate 2,4-dinitrophenyl S -glutathione with identical uv absorption spectra. The extinction coefficient was 10.63 m M −1 cm −1 at 340 nm (λ max ). Kinetics of spontaneous conjugation in 0.1 M Tris-HCl buffer (pH 8.0) at 30°C indicated that a reversible complex between CDNB and glutathione preceded the formation of the stable conjugate. The kinetic parameters associated with these two steps were 2.9 × 10 −2 M and 0.22 min −1 for the dissociation equilibrium constant ( K d ) and the first order rate constant of conjugation ( k c ), respectively. Glutathione transferase activity was present almost exclusively in the cytosolic fractions of the gut, head capsule, integument, and fat body of the last instar larva of the southern armyworm. Among the above tissue preparations, the head capsule and gut showed the highest activity per unit protein. Hemolymph glutathione transferase activity was approximately double the activity of the head and gut. The transferase activity from the hemolymph and gut exhibited identical K m values for CDNB and glutathione. This, in addition to a high correlation between the activity of the hemolymph and gut in individual larvae, argues that the two activities are due to the same enzyme(s) and are regulated by similar factors.