Comparison of alpha-amylase and protease activities of a zoophytophagous and two phytozoophagous Heteroptera

To better understand the nature of facultative phytophagy in the zoophytophagous Geocoris punctipes (Say), and facultative zoophagy in phytozoophagous Lygus hesperus (Knight) and Lygus lineolaris (Palisot de Beauvois), we compared the activities of both the starch digesting enzyme alpha-amylase and of general proteases in these species. The alpha-amylases and proteases were demonstrated in L. hesperus, L. lineolaris and G. punctipes. The presence of alpha-amylase in the salivary gland complexes of G. punctipes indicates a disposition of this species toward utilization of nutrients that can be derived only from plants, either directly from ingestion of plant macromolecules or from second-hand ingestion of plant material from the digestive system of their prey. The alpha-amylase activity in G. punctipes was much less than those of phytozoophagous L. hesperus and L. lineolaris. The relative importance of amylolytic activity and proteolytic activity is also discussed.     

Partial characterization of alpha-amylase in the salivary glands of Lygus hesperus and L. lineolaris

The alpha-amylases in the salivary glands of Lygus hesperus Knight and L. lineolaris (Palisot de Beauvois) were isolated and purified by ion exchange chromatography, and by isoelectric focusing, respectively. The alpha-amylase from L. hesperus had an isoelectric point (pI) of 6.25, and a pH optimum of 6.5. The specific activity of alpha-amylases in the salivary glands of L. hesperus was 1.2 U/mg/ml. The alpha-amylase from L. lineolaris had a pI of 6.54, and a pH optimum of 6.5. The specific activity of alpha-amylase from L. lineolaris was 1.7 U/mg/ml. The activity of alpha-amylase in both species was significantly inhibited by alpha-amylase inhibitor from wheat and also by EDTA and SDS. Sodium chloride enhanced alpha-amylase activity for both species. The enzyme characteristics and relative activities are discussed in the context of differences phytophagous versus zoophagous habits in these two congeneric species.     

Injury to cotton by adult Lygus hesperus (Hemiptera: Miridae) of different gender and reproductive states

Lygus hesperus Knight (Hemiptera: Miridae) is a key pest of cotton (Gossypium spp.) in the western United States that injures floral buds (squares) and developing fruit (bolls). However, no clear relationship between Lygus population level and plant injury has been established. Age-dependent feeding activity by L. hesperus is a possible source of variation that has not been examined for its influence in studies of the impact of Lygus on cotton. Recent video-based laboratory studies indicated that feeding behaviors and trivial movement varied among L. hesperus adults of different gender and reproductive states (prereproductive; reproductive and unmated; and reproductive and mated). We compared within-plant distributions and accumulations of feeding injury to intact cotton plants corresponding to adult L. hesperus of different gender and reproductive states. Adult females, regardless of reproductive state, were observed on squares and axillary buds more often than were males. Additionally, prereproductive adults were observed on squares and axillary buds more often than were mated or unmated reproductive adults, regardless of gender. Plants that were exposed to prereproductive adults exhibited more abscised squares and more squares with injured anthers compared with plants exposed to reproductive adults. However, feeding injury did not differ by insect mating status or gender. These results are consistent with results of our previous video-based assays, and indicate adult reproductive state represents a source of variation that should be controlled in studies to evaluate Lygus-induced injury to cotton and other crop plants.     

Among-sampler variation in sweep net samples of adult Lygus hesperus (Hemiptera: Miridae) in cotton

The sweep net is a standard sampling method for adults of the western tarnished plant bug, Lygus hesperus Knight (Hemiptera: Miridae), in cotton (Gossypium spp.). However, factors that influence the relationship between true population levels and population estimates obtained using the sweep net are poorly documented. Improved understanding of these factors is needed for the development and application of refined treatment thresholds. Recent reports of significant among-sampler differences in sweep net-based population estimates of the adult tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), seem to preclude meaningful comparisons of population estimates collected by different samplers. We used a mark-release-recapture method and the standard sweep net to evaluate among-sampler differences in population estimates of L. hesperus adults. Adult lygus, marked with fingernail polish to facilitate identification and prevent flight, were released into 10-m sample rows on the evening before 10-sweep samples were collected the following morning. The experimental design was a randomized complete block with three replications of three treatments (sampler). Separate experiments were conducted in two plantings each of Pima (Gossypium barbadense L.) and Acala (Gossypium hirsutum L.) cotton. Collections of marked bugs from each study were evaluated for effects of sampler, sample date, and their interaction. Although differences in lygus collections were observed among sample dates in some tests, no differences were detected in the population estimates by different samplers. These results demonstrate that the sweep net technique can be sufficiently standardized to allow direct comparison of population estimates obtained by different samplers.     

Olfactory Responses of <Emphasis Type="Italic">Anaphes iole</Emphasis> (Hymenoptera: Mymaridae) to Volatile Signals Derived from Host Habitats

Anaphes iole Girault is a frequent parasitoid of Lygus spp. eggs in the United States, and has potential as a biological control agent against Lygus hesperus Knight in different crops. Feeding and oviposition by L. hesperus induce emission of plant volatiles, but studies to date do not address the role of plant volatiles in the host-searching behavior of A. iole. In this study, a four-arm olfactometer was used to test the responses of female parasitoids to odors emanating from cotton (Gossypium hirsutum L., Malvaceae) plants damaged by L. hesperus females, L. hesperus males, larvae of the nonhost Spodoptera exigua Hubner, or mechanically, or to odors from L. hesperus females alone. In addition, various plants damaged by L. hesperus females were evaluated in the olfactometer: cotton, alfalfa (Medicago sativa L., Fabaceae), common groundsel (Senecio vulgaris L., Asteraceae), annual ragweed (Ambrosia artemisifolia L., Asteraceae), and redroot pigweed (Amaranthus retroflexus L., Amaranthaceae). In all olfactometry bioassays, treatment odors were compared against three controls (humidified air). Results showed that A. iole females were consistently attracted to odors derived from different plant–L. hesperus complexes, while odors from plants subjected to nonhost (S. exigua) or mechanical damage and L. hesperus females alone were not attractive or only variably attractive. These findings suggest that while searching for hosts A. iole females use specific volatiles induced by L. hesperus feeding and oviposition to locate hosts inhabiting a wide variety of plants, including annual and perennial species from four plant families. It was suggested that future research should seek to identify the chemical elicitors involved in the release of plant volatiles attractive to A. iole females.     

Micro-injection of Lygus salivary gland proteins to simulate feeding damage in alfalfa and cotton flowers

Alfalfa and cotton flowers were pierced with small glass capillaries of an overall size and shape similar to that of Lygus stylets, and injected with small quantities (6 to 100 nL) of solutions that contained Lygus salivary enzymes. Crude and partially purified protein solutions from Lygus heads and isolated salivary glands showed substantial polygalacturonase (PG) activity, as has been previously reported. Following injection with both crude and partially purified protein solutions, as well as with pure fungal and bacterial PGs, flowers of both alfalfa and cotton exhibited damage similar to that caused by Lygus feeding. Injection with the same volume of a buffer control as well as a buffer control containing BSA at a comparable protein concentration (approximately 6 microg/mL) showed no symptoms. These results are consistent with a previously suggested hypothesis that the extensive tissue damage caused by Lygus feeding is primarily due to the action of the PG enzyme on the host tissue, rather than to mechanical damage caused by the insect stylet. Substantial genotypic variation for a PG inhibiting protein (PGIP) exists in alfalfa and cotton. We, therefore, suggest that breeding and selection for increased native PGIP levels, or transformation with genes encoding PGIP from other plant species, may be of value in obtaining alfalfa and cotton varieties that are more resistant to Lygus feeding damage.     

Characterization of eight polymorphic microsatellite markers in the tarnished plant bug,Lygus lineolaris (Palisot de Beauvois)

A partial genomic library of the tarnished plant bug, Lygus lineolaris, enriched for microsatellite sequences was screened to identify marker loci. Eight polymorphic loci suitable for population genetic studies were identified by screening 192 field‐collected insects. The observed number of alleles ranged from four to 21 with an average of 12.25 (SE ± 1.94) while the effective number of alleles ranged from 1.23 to 11.05 with an average of 4.49 (SE ± 1.15). No linkage disequilibria or significant deviations from Hardy–Weinberg expectations were detected at any of the loci. Seven of the eight L. lineolaris microsatellite loci were transferable to Lygus hesperus.     

Temperature dependent development of Lygus hesperus (Hemiptera: Miridae) nymphs

Lygus hesperus Knight (Hemiptera: Miridae) is a key pest of fruit and vegetable crops, forages, and cotton (Gossypium spp.) in the western United States. Accurate models describing relationships between temperature and L. hesperus development are critical to the study of seasonal L. hesperus population dynamics. Development of L. hesperus nymphs was assessed at nine constant temperatures from 10 to 37.8 degrees C. The relationships between temperature and development for each L. hesperus instar, and for the entire nymphal stage, were best described by six-parameter biophysical models indicating both low- and high-temperature inhibition of development. Development rates asymptotically approached zero with decreasing temperature in the lower thermal range, and decreased with increasing temperatures above 32.2 degrees C. Nymphs did not survive from egg hatch to adulthood at either 10 or 37.8 degrees C, and nymph mortality was > 90% at both 12.8 and 35.0 degrees C. The fifth instar exhibited the longest stadium, whereas the shortest stadia were associated with the second and third instars. Development rates of males and females did not differ, and the ratio of males to females was not different from 1:1 at any temperature. Our temperature-dependent development rate models for L. hesperus nymphs will facilitate control of insect physiological age in controlled laboratory experiments, and should be useful in planning and interpreting field studies on L. hesperus population dynamics.     

Host plants of the tarnished plant bug (Heteroptera: Miridae) in Central Texas

The tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), has taken on added importance as a pest of cotton in the Cotton Belt after successful eradication efforts for the boll weevil (Anthonomus grandis grandis Boheman). Because the Southern Blacklands region of Central Texas is in advanced stages of boll weevil eradication, blooming weeds and selected row crops were sampled during a 3-yr study to determine lygus species composition and associated temporal host plants. L. lineolaris was the sole lygus species in the region. Thirteen previously unreported host plants were identified for L. lineolaris, of which 69% supported reproduction. Rapistrum rugosum L. Allioni and Ratibida columnifera (Nuttall) Wooton and Standley were primary weed hosts during the early season (17 March to 31 May). Conyza canadensis L. Cronquist variety canadensis and Ambrosia trifida L. were primary weed hosts during the midseason (1 June to 14 August) and late-season (15 August to 30 November), respectively. Sisymbrium irio L. and Lamium amplexicaule L. sustained L. lineolaris populations during the overwintering period (1 December to 16 March). The proportion of females and numbers of nymphs found in R. rugosum, C. canadensis, A. trifida, and S. irio suggests these weeds supported reproductive adults during the early, mid-, and late season and overwintering period, respectively. Medicago sativa L. was the leading crop host for L. lineolaris; Glycine max L. Merrill did not yield L. lineolaris. Few L. lineolaris were collected in Gossypium hirsutum L. These results provide a more comprehensive assessment of host plants contributing to L. lineolaris populations in central Texas.     

Comparison of economic and plant phenology-based thresholds for management of Lygus lineolaris (Hemiptera: Miridae) in Minnesota strawberries

Field studies were conducted in southeastern Minnesota, 2000-2002, to assess damage potential and management options for adult Lygus lineolaris (Palisot de Beauvois) in June-bearing strawberries (Fragaria x ananassa). The first study was designed to assess the efficacy of a published economic threshold for L. lineolaris nymphs compared with a plant phenology-based threshold management program, targeted at L. lineolaris adults. L. lineolaris nymphs were sampled using the standard white pan beat method; adults were sampled using yellow sticky traps. In the second study, during 2001-2002, caged strawberries were artificially infested with adult L. lineolaris at specific plant growth stages (i.e., vegetative, green bud, white bud, first blossom, peak blossom, first green fruit, and first ripe fruit) to determine the most susceptible growth stages of strawberry. The phenology-based thresholds proved to be more effective in managing L. lineolaris than the current economic threshold based on nymphs. Results from the infestation timing study indicate that early-growth stages (i.e., green and white bud) are most susceptible to adult L. lineolaris feeding damage. During the early-growth stages, only L. lineolaris adults were present; infestations of nymphs occurred primarily from first blossom to green berry. Results from both studies indicate that (1) management of adult L. lineolaris during the early strawberry growth stages is recommended for maximizing marketable yield and (2) the use of plant phenology-based thresholds, when adults are present, will significantly improve insecticide spray timing, and thus minimize the number of insecticide sprays.     

Phenology of Lygus lineolaris (Hemiptera: Miridae) in Minnesota June-bearing strawberries: comparison of sampling methods and habitats

Field studies were conducted in southeastern Minnesota from 2000 to 2002 to determine the phenology of Lygus lineolaris in various habitats and to compare yellow and white sticky traps as a sampling method for adult L. lineolaris. Strawberry fields were sampled for L. lineolaris adults using yellow sticky traps, and nymphs were sampled using the standard white pan beat method. Adult L. lineolaris abundance in alfalfa, an adjacent fence-row, and a wooded habitat were also compared. The nonlinear relationship between cumulative trap catch and cumulative degree-days was modeled with a two-parameter cumulative Weibull function to predict early-season adult capture using yellow sticky traps. Adult L. lineolaris were detected in bearing-year strawberries at the onset of vegetative growth in all years. Yellow sticky traps caught significantly higher densities of adult L. lineolaris than white sticky traps. The Weibull model predicted 50% capture at 10 DD (>12.4 degrees C), which corresponds to the vegetative strawberry growth stage. L. lineolaris nymphs were not detected until the blossom stage. Alfalfa harbored significantly higher densities of L. lineolaris than other habitats during early-season sampling (i.e., March-June). Late-season sampling (July-September) revealed significantly higher densities in bearing-year strawberries. These results suggest that monitoring at the onset of vegetative growth, using yellow sticky traps, will be an efficient method for detecting early L. lineolaris adult activity.     

Effect of host instar on measuring parasitism of Lygus spp. (Hemiptera: Miridae) nymphs by Peristenus spp. (Hymenoptera: Braconidae)

The accurate measurement of insect mortality by parasites is critical in biological control research, both in baseline studies to determine the absence or inadequacy of native parasites and in subsequent efforts to measure the effectiveness of introduced endoparasitic species. Although rearing has been most frequently used to measure parasitism, dissection has been shown to be more accurate in several cases. Selection of the host instar, whether for rearing or dissection, was also found to be important in this study. In two species [Lygus lineolaris (Palisot) and L. hesperus Knight], parasitism by Peristenus digoneutis Loan and P. howardi Shaw, respectively, was highest in instars 3 and 4. Parasitism was underestimated in instars 1 and 2 (because of reduced exposure time) and in instar 5 (because of parasites killing the hosts in instar 4).     

Molecular cloning and partial characterization of a trypsin-like protein in salivary glands of Lygus hesperus (Hemiptera: Miridae)

Trypsin-like enzymes from the salivary gland complex (SGC) of Lygus hesperus Knight were partially purified by preparative isoelectric focusing (IEF). Enzyme active against Nalpha-benzoyl-L-arginine-p-nitroanilide (BApNA) focused at approximately pH 10 during IEF. This alkaline fraction gave a single activity band when analyzed with casein zymograms. The serine proteinase inhibitors, phenylmethylsulfonyl fluoride (PMSF) and lima bean trypsin inhibitor, completely inhibited or suppressed the caseinolytic activity in the crude salivary gland extract as well as the IEF-purified sample. Chicken egg white trypsin inhibitor also inhibited the IEF-purified sample but was not effective against a major caseinolytic band in the crude salivary gland extract. These data indicated the presence of serine proteinases in the SGC of L. hesperus. Cloning and sequencing of a trypsin-like precursor cDNA provided additional direct evidence for serine proteinases in L. hesperus. The encoded trypsin-like protein included amino acid sequence motifs, which are conserved with five homologous serine proteinases from other insects. Typical features of the putative trypsin-like protein from L. hesperus included residues in the serine proteinase active site (His(89), Asp(139), Ser(229)), conserved cysteine residues for disulfide bridges, residues (Asp(223), Gly(252), Gly(262)) that determine trypsin specificity, and both zymogen signal and activation peptides.     

Lygus hesperus （Heteroptera： Miridae） tolerates high concentrations of dietary nickel

Nickel hyperaccumulator plants contain unusually elevated levels of Ni （〉 1 000 μg Ni/g）. Some insect herbivores, including Lygus hesperus （Western tarnished plant bug）, have been observed feeding on the California Ni hyperaccumulator Streptanthus polygaloides. This bug may be able to utilize S. polygaloides as a host either through its feeding behavior or by physiological tolerance of Ni. This experiment determined the Ni tolerance of L hesperus by offering insects artificial diet amended with 0, 0.4, 1, 2, 4.5, 10, 20 and 40 mmol Ni/L and recording survival. Survival varied due to Ni concentration, with diets containing 10 mmol Ni/L and greater resulting in significantly lower survival compared to the control （0 mmol Ni/L） treatment. Insects tolerated diet containing as much as 4.5 mmol Ni/L, a relatively elevated Ni concentration. I conclude that L hesperus can feed on S. polygaloides because it is Ni-tolerant, probably due to physiological mechanisms that provide it with resistance to plant chemical defenses including elemental defenses such as hyperaccumulated Ni.     

The piercing-sucking herbivores Lygus hesperus and Nezara viridula induce volatile emissions in plants

Plant volatiles induced by herbivory are often used as olfactory cues by foraging herbivores and their natural enemies, and thus have potential for control of agricultural pests. Compared to chewing insects and mites, little is known about plant volatile production following herbivory by insects with piercing-sucking mouthparts. Here, we studied factors (insect life stage, gender, the role of salivary glands, and type of bioassay used for volatile induction) that influence the induction of plant volatiles by two agriculturally important hemipterans, Lygus hesperus and Nezara viridula. Feeding on intact cotton by virgin females of L. hesperus induced 2.6-fold greater volatile response compared to that induced by mated females, possibly due to increased feeding activity by virgin females. This plant volatile response was associated with elicitors present in the insect's salivary glands as well as to the degree of mechanical injury. Feeding injury by N. viridula females also increased volatile emissions in intact maize by approximately 2-fold compared to control plants. Maize seedlings injured by N. viridula emitted higher amounts of the monoterpene linalool, the sesquiterpenes (E)-beta-caryophyllene, alpha-trans-bergamotene, and (E,E)-beta-farnesene, and the homoterpene (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene, but not amounts of green leaf volatiles, compared to uninjured plants. Emissions from intact maize injured by adult males were lower than those emitted by adult females of the same age and did not differ from those emitted by uninjured plants. Similarly, feeding by virgin female N. viridula followed by excision led to 64% higher quantities of volatiles compared to untreated plants. Volatile emission in excised plants, however, was considerably greater than in intact plants, suggesting that careful consideration must be given to bioassay design in studies of herbivore-induced plant volatiles. Salivary gland extracts of N. viridula led to sesquiterpene emissions approximately 2.5-fold higher than for controls, although no significant differences were observed for green leaf volatiles, monoterpenes, and homoterpenes. These results indicate that L. hesperus and female N. viridula feeding induce volatile production in plants, and that volatile production is affected by gender and life stage of the bug. Although oviposition and mechanical injury by stylets may increase release of volatiles, elicitors from salivary glands of L. hesperus and N. viridula also seem to play a role in the emission of plant volatiles.     

Partial characterization of trypsin-like protease and molecular cloning of a trypsin-like precursor cDNA in salivary glands of Lygus lineolaris

Based on substrate specificity, an alkaline pH optimum, sensitivity to selected proteinase inhibitors, and molecular analysis, we provide evidence for the presence of a trypsin-like serine proteinase in the salivary gland complex (SGC) of the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) (Heteroptera: Miridae). The predominant activity in extracts of the SGC against N(2)-benzoyl-L-arginine-p-nitroanilide (L-BApNA) was at pH 10, but a minor peak of activity also occurred at pH 5. The major BApNAase activity focused at 10.4 during preparative isoelectric focusing and was eluted with an apparent molecular weight of 23,000 from a calibrated gel filtration column. The BApNAase fraction gave a single major band when analyzed on a casein zymogram. The activity was completely suppressed by the serine protease inhibitors, phenylmethylsulfonyl fluoride (PMSF) and lima bean trypsin inhibitor. A cDNA coding for a trypsin-like protein in the salivary glands of L. lineolaris was cloned and sequenced. The 971bp cDNA contained an 873-nucleotide open reading frame encoding a 291-amino acid trypsin precursor. The encoded protein included amino acid sequence motifs that are conserved with four homologous serine proteases from other insects. Typical features of the putative trypsin-like protein from L. lineolaris included the serine protease active site (His(89), Asp(139), Ser(229)), conserved cysteine residues for disulfide bridges, the residues (Asp(223), Gly(252), Gly(262)) that determine trypsin specificity, and both zymogen signal and activation peptides. Cloning and sequencing of a trypsin-like precursor cDNA provided additional direct evidence for trypsin like enzymes in the salivary glands of L. lineolaris.     

Field evaluation of efficacy and persistence of an insect vacuum device against the tarnished plant bug (Hemiptera: Miridae) in a day-neutral strawberry field

The efficacy of a vacuuming device to reduce Lygus lineolaris (Palisot de Beauvois) populations was evaluated over 3 yr in a day-neutral strawberry field. Immediately after treatments, a significant reduction of population estimates (by tapping of flower clusters) was observed for adults (75% of the time) and nymphs (50% of the time). Under the same conditions, control (i.e., vacuum turbine off) treatments significantly reduced adult population estimates 25% of the time. Lygus lineolaris tapping samples taken from strawberry plants adjacent to the treated zone did not show significant variations over sampling time, suggesting that no escape behavior occurred. This was supported by adult catches on sticky traps located in zones adjacent to the treated one. Our results suggest that vacuuming had inconsistent effect on tarnished plant bug populations and that L. lineolaris mobility plays a marginal role in the use of this control method.     

Development of a Bacillus thuringiensis-based assay on Lygus hesperus

Lygus hesperus Knight (Hemiptera: Miridae) is an economically important insect pest controlled primarily by chemical pesticides. Bacillus thuringiensis Berliner is a gram-positive bacterium, which upon sporulation produces a parasporal inclusion body, the crystal. The latter, in some strains, exhibits specific insecticidal activities against lepidopteran, coleopteran, or dipteran pests. The aim of the present work was to develop a Bacillus thuringiensis-based assay on L. hesperus. Several factors involved in the expression of the B. thuringiensis insecticidal activity were assayed for their effects on L. hesperus mortality. We show that the choice of the L. hesperus diet type, the L. hesperus developmental stages, the B. thuringiensis crystal alkaline solubilization buffers, the dialysis buffers, the dialyzed solubilization buffers, and the proteolytic activation can all significantly affect the L. hesperus viability. This work provides essential information in pinpointing key steps before the establishment of a thorough screening program for B. thuringiensis strains expressing antihemipteran activity.     

Development and population growth of Lygus hesperus on selected weed hosts, artificial diet and cotton in the laboratory

Laboratory studies were conducted to obtain basic biological information of Lygus hesperus Knight (Hemiptera: Miridae) on alfalfa (Medicago sativa L.), pigweed (Amaranthus palmeri S. Wats.), Russian thistle (Salsola iberica Sennen and Pau), green bean (Phaseolus vulgaris L.), artificial diet, and cotton (Gossypium hirsutum L.) square and cotton boll at 27 degrees C. The nymphal developmental duration was 28.8 d on cotton bolls, nearly twice longer, compared with approximately 15 d in other hosts. The nymphal survivorship was significantly lowest on the cotton boll (18%), whereas the survivorship in other hosts ranged from 33% (cotton square) to 93% (green bean). The average total nymphal survivorship was approximately 60% and the most nymphal mortalities occurred on the first three instars. The life-table parameters were obtained only in cotton square, artificial diet, green bean, and alfalfa. The average adult longevity in artificial diet was the shortest (18.7 d),with the longest longevity observed (36.1 d) on green beans. The intrinsic rates of population increase (r(m)) were much higher in artificial diet (0.0771) and green beans (0.0671) than in alfalfa (0.0327) and cotton square (0.0317). Although statistically significant, the difference in r(m) values between alfalfa and cotton square was very small, indicating the similarity in reproductive suitability of cotton square and alfalfain a no-choice test. Thus, even though cotton is not a preferred host, when blooming alfalfa and roadside weeds are mowed in the Texas High Plains during June-July, it is the most likely time that Lygus bugs may move from alfalfa and other host plants into adjacent cotton fields and become effective pest of actively fruiting cotton.