Polymorphic microsatellite loci for Diaprepes root weevil (Diaprepes abbreviatus L.)

Diaprepes abbreviatus (L.) is an insect pest of the US agriculture that originated from the Caribbean islands. Larvae are of economic importance in both nursery and commercial citrus plantings due to root feeding. Eight polymorphic microsatellite markers were developed from a (CA)_n‐enriched genomic library of Diaprepes root weevil. Three to eight alleles were observed for each locus during screening of 17 to 25 individuals. Observed and expected heterozygosities ranged from 0.182 to 0.864 and 0.587 to 0.835, respectively. These markers will be useful to characterize the genetic variability and track the migration patterns of populations of D. abbreviatus (L.).     

Discovery and characterization of chemical signals for citrus root weevil, Diaprepes abbreviatus

The tropical root weevil Diaprepes abbreviatus (L. 1758) (Coleoptera: Curculionidae) is a polyphagous insect from the Caribbean Islands and an invasive insect in the southern part of the United States where it is pest of citrus crops and ornamental trees. Adults feed upon foliage where aggregation, mating and oviposition take place. Here, the headspace volatiles from Citrus macrophylla Wester (Rutaceae), D. abbreviatus adults feeding on this plant, adults alone and adult feces, were collected by aeration and solid-phase microextraction (SPME) for analysis by gas chromatography-linked mass spectrometry (GC-MS). Electrophysiological responses of weevil antennal receptors to volatile headspace extracts and synthetic analogues were recorded by gas chromatography-linked electroantennographic detection (GC-EAD) and electroantennograms (EAGs). Antennal responses were recorded to the monoterpenes (R)-(−)-linalool, citronellal, nerol, citral, and geraniol; all present in the headspace of C. macrophylla. Antennal responses were also recorded to carvacrol, present in the headspace of adults and adult feces. The green leaf volatiles cis-3-hexen-1-ol and trans-2-hexen-1-ol, produced by other host plants, elicited reliable responses on the D. abbreviatus antenna. When comparing EAGs between (±)-linalool and (R)-(−)-linalool, no significant difference was found; responses to (R)-(+)-citronellal were larger than for (S)-(−)-citronellal. Among the individual compounds and blends tested using an open T-track dual choice olfactometer, only the blend of (±)-linalool, cis-3-hexen-1-ol and carvacrol (source dose 25:25:2.5 μg) elicited significant attraction of females, the same blend was repellent for males. The biologically active compounds found here likely play a role in host finding by D. abbreviatus and other interactions of the insect with its hostplant. Handling editor: Sam Cook     

Storage proteins of the larval root weevil Diaprepes abbreviatus (Coleoptera: Curculionidae): riboflavin binding and subunit isolation.

Proteins present at high concentrations in hemolymph of the larval weevil Diaprepes abbreviatus were previously shown to bind a synthetic coumarin, 7-amino-3-phenyl coumarin (coumarin-10). One of the two native proteins previously identified (protein I) is now shown to separate into two distinct bands (proteins Ia and Ib) using native gradient pore-limiting electrophoresis. The high concentration of proteins Ia, Ib, and II in larval hemolymph, their disappearance from hemolymph upon pupation, and an apparent hexameric structure shown by chemical crosslinking identify them as hexameric storage proteins (hexamerins). At least one chromatographic form of Ib isolated by anion exchange HPLC is now shown to bind riboflavin (Rb). Binding was also demonstrated by quenching of Rb fluorescence by a partially isolated mixture of the storage proteins. Lipophorin did not quench Rb fluorescence. Rb was heat-extracted from whole hemolymph and identified by its fluorescence spectra and by reverse phase HPLC with fluorescence detection. The two subunits shared by the three holoproteins have been isolated by sequential density gradient ultracentrifugation, gel permeation HPLC, and reverse phase HPLC. All three holoproteins shared the alpha subunit (M(r) 75,000), while the beta subunit (M(r) 71,000) was lacking from one of the three. Repeated passage through an anion exchange column yielded two of the three proteins (Ib and II) in homogeneous form. Chemical crosslinking with dimethylsuberimidate indicated a hexameric structure for the holoproteins. All subunits and holoproteins stained as high mannose glycoproteins when probed with biotinylated concanavalin A on PVDF membranes. The alpha subunit was high in Met, His, and Thr, and the beta subunit was high in Lys. Both were high in Pro and had approximately 16% Phe+Tyr. Sequences of the 20 N-terminal amino acid residues of each subunit showed 45-60% homology between subunits. These coleopteran proteins also showed some sequential homology but no immunological cross-reactivity with storage proteins from the lepidopterans Galleria mellonella and Heliothis virescens.     

Mitochondrial nucleotide variability in invasive populations of the root weevil Diaprepes abbreviatus (Coleoptera: Curculionidae) of Florida and preliminary assessment of Diaprepes sp. from Dominica

Diaprepes abbreviatus (L.) (Coleoptera: Curculionidae) is a root weevil introduced into the United States from the Caribbean in 1964. It is associated with >300 plants, including citrus, sugarcane, and potatoes. D. abbreviatus is widespread in Florida, and it has recently been detected in limited areas of California and Texas. The purpose of this research is to evaluate the utility of 16S ribosomal (16S rRNA) and cytochrome oxidase I (COI) mitochondrial markers for the delineation of genetic populations of D. abbreviatus in Florida and for the characterization of patterns of dispersion among these populations. We also assessed these markers as genetic tools for the clarification of taxonomic uncertainties in specimens from Dominica (Lesser Antilles). We analyzed 111 weevils from six Florida populations and six specimens from Dominica. In Florida, we found three haplotypes with only one haplotype in each population. Florida haplotypes differed by one to three nucleotide substitutions, possibly the result of a recent divergence from one source population or three different introductions from closely related populations from the Caribbean. In contrast, specimens from Dominica showed a high genetic variability with three 16S haplotypes and six unique COI haplotypes, delineating two mitochondrial clades. We show that these mitochondrial markers are useful for phylogeographic studies of D. abbreviatus.     

Particle film deters oviposition by Diaprepes abbreviatus (Coleoptera: Curculionidae)

A hydrophilic formulation of the inert silicate kaolin was tested in a screenhouse for its effect on the behavior of the root weevil Diaprepes abbreviatus (L.), a pest of citrus and ornamental plants in Florida and the Caribbean. Feeding by adults on treated foliage was reduced by 68-84% compared with adults fed untreated foliage. No insecticidal activity was detected after 14 d of exposure to kaolin-treated leaves. Oviposition was completely suppressed on treated foliage. Although females oviposited >19,000 eggs during two trials on untreated foliage, no egg masses were found on foliage treated with the kaolin formulation. These data indicate potential for kaolin as a barrier to oviposition in citrus groves and may prove to be an economically viable and environmentally sound component of an integrated approach for control of D. abbreviatus and related root weevils.     

Steinernema diaprepesi n. sp. (Rhabditida: Steinernematidae), a Parasite of the Citrus Root Weevil Diaprepes abbreviatus (L) (Coleoptera: Curculionidae)

A nematode collected from Diaprepes abbreviatus is identified and described as a new species, Steinernema diaprepesi n. sp. The new species is closely related to S. feltiae, S. glaseri, and S. oregonense and can be distinguished from these species by the following characteristics: Males: Spicule averaging 79 (71-90) µm and spicule shape; D% (distance from anterior end to excretory pore/ esophagus length × 100) about 80; the ratio SW (spicule length/anal body width) about 1.8. Females: Vulva with short, double- flapped epiptygma; tail terminus usually with 5 papillae-like structures. Infective juveniles: Body averaging 1,002 (880-1,133) µm, EP (distance from anterior end to excretory pore) = 74 (66-83) µm; tail length = 83 (65-91) µm, and E% (EP/tail length × 100) = 89.6 (78-114). Lateral field pattern variable, the formula for the arrangement of ridges from head to tail is: 2, 6, 7, 8, 4, 2. The portion with eight ridges is the longest. This new species can be differentiated further from three closest species (S. feltiae, S. glaseri, and S. oregonense) by characteristic sequences of their ITS regions, including sequence lengths, ratios of similarity, composition, and differences in base characters in sequence alignment.     

Effects of Culture Method and Formulation on the Virulence of Steinernema riobrave (Rhabditida: Steinernematidae) to Diaprepes abbreviatus (Coleoptera: Curculionidae)

The Diaprepes root weevil, Diaprepes abbreviatus, is a pest of vegetables, ornamental plants, sugarcane, and citrus in Florida and the Caribbean. The entomopathogenic nematode, Steinernema riobrave, can reduce larval populations of D. abbreviatus substantially. Efficacy of entomopathogenic nematodes, however, may be affected by culture method and formulation. Using D. abbreviatus as the host, we compared the efficacy of two commercial S. riobrave formulations, a liquid and a waterdispersible granule (WDG), with each other and with in vivo produced S. riobrave. Nematodes in the commercial formulations were produced in vitro through liquid fermentation; the in vivo nematodes were cultured in Galleria mellonella and applied in aqueous suspension. Laboratory experiments measured nematode virulence in plastic cups containing soil and seventh-eighth instar D. abbreviatus. One laboratory experiment was conducted using only fresh nematodes (less than 5 days old); another experiment included WDG nematodes that were stored for 25 days at 10 °C. Two field experiments were conducted in which nematodes were applied either to potted citrus (containing D. abbreviatus larvae) placed beneath mature citrus trees or to soil directly beneath the tree. In the latter experiment, efficacy was determined by measuring mortality of caged D. abbreviatus larvae that were buried beneath the soil surface prior to application. Mortality of D. abbreviatus treated with nematodes ranged from 80-98% and 50-75% in laboratory and field experiments, respectively. In all experiments, we did not detect any significant effects of culture method or formulation.     

Virulence of entomopathogenic nematodes to Diaprepes abbreviatus (Coleoptera: Curculionidae) in the laboratory

The Diaprepes root weevil, Diaprepes abbreviatus (L.) is the most severe weevil pest in Florida citrus. Entomopathogenic nematodes have effectively suppressed larval populations of D. abbreviatus. Our objective was to conduct a broad laboratory comparison of entomopathogenic nematodes for virulence toward larvae of D. abbreviatus. The study was conducted at three temperatures (20, 24, and 29 degrees C) and included nine entomopathogenic species and 17 strains: Heterorhabditis bacteriophora Poinar (Baine, NJl, Hb, Hbl, HP88, and Lewiston strains), H. indica Poinar, Karunakar & David (original and Homl strains), H. marelatus Liu & Berry (IN and Point Reyes strains), H. megidis Poinar, Jackson & Klein (UK21l strain), H. zealandica Poinar (NZH3 strain), Steinernema riobrave Cabanillas, Poinar & Raulston (355 strain), S. carpocapsae (Weiser) (All strain), S. feltiae (Filipjev) (SN and UK76 strains), and S. glaseri (Steiner) (NJ43 strain). At 20 degrees C, the greatest mortality was caused by S. riobrave although it was not significantly greater than H. bacteriophora (Baine), H. bacteriophora (Hb), H. bacteriophora (Hbl), and H. indica (original). At 24 and 29 degrees C, S. riobrave caused greater larval mortality than other nematodes tested. Two strains of H. indica, H. bacteriophora (Baine), and S. glaseri were next in terms of virulence at 29 degrees C. Our results suggest that S. riobrave has the greatest potential for control of D. abbreviatus.     

Prolonged mate guarding and sperm competition in the weevil Diaprepes abbreviatus (L.)

The hypothesis that prolonged copulatory mate guarding coexistswith last male sperm precedence was tested for the sugarcanerootstalk borer weevil, Diaprepes abbreviatus (L.) (Coleoptera:Curculionidae). Male D. abbreviatus showed a long copulatoryguarding. Both males and females were less likely to rematewhen prolonged guarding occurred compared with terminating copulationearly. Guarding was generally terminated by the struggling behaviorof the female. Mating experiments using normal and sterile (X-rayirradiated) males revealed a similar value of last-male spermprecedence for both irradiated and normal males. The P₂ valuesof normal and sterile males were similar when all ovipositedeggs were counted over 30 days. These data made it possibleto calculate the expected gain to a male from prolonged guardingcompared with leaving a female early and seeking out an additionalmate. We show that guarding has the higher fitness. Eggs weredeposited in clutches in which normal fertilized eggs were groupedtogether and were attached to a group of sterile eggs. This,together with identifying the form of the cul-de-sac typespermatheca,allowed us to suggest a unique repositioning process, whichhas not been described elsewhere, as the likely mechanism bywhich last-male sperm precedence was achieved.     

Status of biological control by egg parasitoids of Diaprepes abbreviatus (Coleoptera: Curculionidae) in citrus in Florida and Puerto Rico

Eggs of Diaprepes abbreviatus (L.) were routinely monitored in citrus groves at ten locations in Florida during 1997 and 1998 to study egg parasitism. One citrus location was studied in Puerto Rico. No native parasitoids were recovered from 1,337 D. abbreviatus egg masses studied in Florida citrus. In contrast, an average of 35.5% (range 12.5 to 68.8%) parasitism of egg masses was reported in Puerto Rico. The parasitoids Aprostocetus gala, Horismenus spp, and Quadrastichus haitiensis were recovered from the eggs of D. abbreviatusfstudied in Puerto Rico. The Horismenus parasitoids were suspected hyperparasitoids. Releases of the parasitoid Ceratogramma etiennei from Guadeloupe were initiated during 1998 at each of the Florida research sites. By the end of 1998, C. etiennei had been recovered from D. abbreviatus eggs at two of nine locations in Florida citrus. The parasitoid was recovered from 1 of 34 egg masses at one of these locations during the month of September and from 3 of 34 egg masses at the other location during the month of November. Whether or not C. etiennei establishes itself at one or more locations in Florida remains to be seen.     

Gut chitin synthase and sterols from larvae of Diaprepes abbreviatus (Coleoptera: Curculionidae)

Gut chitin synthase was characterized and the sterols and ecdysteroids in the sugarcane rootstalk borer weevil, Diaprepes abbreviatus, were identified. An in vitro cell-free chitin synthase assay was developed using larval gut tissues from D. abbreviatus. Subcellular fractionation experiments showed that the majority of chitin synthase activity was located in 10,000g pellets. The gut chitin synthase requires Mg²⁺ to be fully active: 7–8-fold increases in activity were obtained with 10 mM Mg²⁺ present in reaction mixture. Calcium also stimulated activity (4–5-fold with 10 mM Ca²⁺), while Cu⁺² completely inhibited at 1 mM. Other monovalent and divalent cations had little or no effect on activity. The pH and temperature optima were 7 and 25°C, respectively. Gut chitin synthesis was activated ca. 50% by trypsin treatments. GlcNAc stimulated chitin synthase activity, but Glc, GlcN and glycerin did not. Polyoxin D, UDP, and ADP inhibited the chitin synthase reaction with I₅₀'s of 75 μM, 2.3 mM, and 3.6 mM, respectively. Nikkomycin Z was a potent inhibitor of chitin synthase (91% inhibition at 10 μM). Tunicamycin and diflubenzuron had no effect on the enzyme. The apparent Km and V_max for the gut chitin synthase were, respectively, 122.5 ± 7.4 μM and 426 ± 19.7 pmol/h/mg protein utilizing UDP-GlcNAc as the substrate. Sterol analyses indicated that cholesterol was the major dietary and larval sterol. HPLC/RIA data indicated that 20-hydroxyecdysone was the major molting hormone.     

Abundance of Diaprepes abbreviatus (L.) (Coleoptera: Curculionidae) neonates falling to the soil under tree canopies in Florida citrus groves

The purpose of these experiments was to estimate the number and distribution of Diaprepes abbreviatus (L.) neonate larvae dropping from the canopy of infested citrus trees. The number of neonates was monitored in the field using passive funnel traps in two simultaneous experiments and a separate experiment for an additional year. In one experiment, traps were placed from trunk to dripline in the cardinal directions under each of five trees (132 traps total). In a second experiment, eight traps were placed under each tree in the cardinal directions, one trap 30 cm from the trunk and one trap 30 cm from the dripline/direction for 25 trees (200 traps total). Larvae were collected weekly for 50 wk in conical tubes containing ethylene glycol as a preservative. Traps closer to the tree trunk captured more larvae than traps nearer the dripline. The area under the tree canopy was positively correlated with the total estimated number of larvae captured per tree. The estimated number of total larvae/tree over the course of our experiments ranged from 955 to 7,290. The highest number of neonate larvae observed in 1 wk was 67 +/- 6/m2. There was an inverse relationship between the number of traps beneath a tree and the number of trees that needed to be sampled to estimate mean population density with a given precision. However, there was a direct relationship between number of traps/tree and the total number of traps needed for a given precision. This passive technique could be used to quantify the destructive larval stage and to assess D. abbreviatus management strategies.     

Toxicity and repellency of Tephrosia candida to larval and adult Diaprepes root weevil (Coleoptera: Curculionidae)

Leaves of the tropical legume Tephrosia candida DC deterred feeding by adults of the Diaprepes root weevil, Diaprepes abbreviatus (L.), compared with leaves of Citrus macrophylla Wester, a common citrus rootstock, or T. vogelii Hook. f. When larvae were placed in pots containing plants of the three species for 28 d in a growth chamber, larval survival and weight gain were significantly reduced in pots containing plants of T. candida compared with larvae in pots with C. macrophylla or T. vogelii. Diet incorporation of lyophilized roots of T. candida into an artificial diet increasingly inhibited the growth of larvae and increased larval mortality with increased concentration of roots, whereas roots from C. macrophylla or T. vogelii had no effect compared with the diet-only control. T. candida, but not T. vogelii, contains at least one constituent that acts as an antifeedant toward adult D. abbreviatus and as a toxicant toward larvae. No antifeedant effect of roots of T. candida toward larvae was observed in no-choice pot tests or in a diet incorporation bioassay. In pots, larval feeding damage to roots of T. candida was evident. In the diet incorporation assay, 97% of larvae survived 29 d on a diet of cellulose powder (a nutritionally inert filler) despite losing weight. We conclude that decreased survival and weight gain of larvae-fed fresh or lyophilized roots of T. candida were the result of ingestion of a toxicant and not deterrence from feeding.     

Thermal requirements of Fidiobia dominica (Hymenoptera: Platygastridae) and Haeckeliania sperata (Hymenoptera: Trichogrammatidae), two exotic egg parasitoids of Diaprepes abbreviatus (Coleoptera: Curculionidae)

Diaprepes abbreviatus is an exotic root weevil occurring in southern US. It is a highly polyphagous species which can complete its entire life cycle on citrus and several woody ornamental plants. The lack of native egg parasitoids for this weevil in citrus orchards has triggered efforts to evaluate candidate egg parasitoids from the Caribbean Region into Florida. The egg parasitoids Fidiobia dominica and Haeckeliania sperata are two exotic natural enemies of D. abbreviatus recently introduced in the US in a classical biological control program. The thermal requirements of both parasitoids were studied in the laboratory. The upper development threshold (UDT) of F. dominica was 30.0°C, its maximal development rate (MDR) occurred at 27.6°C, its lower development threshold (LDT) was 9.6°C and its thermal constant (K) for development from egg to adult was 293.1 DD. For H. sperata, UDT was 35.0°C, MDR occurred at 31.0°C, LDT was around 15°C and K was 188.1 DD. Based on these results, both species would be able to complete 17 to 18 generations annually in southern Florida. However, host availability during critical periods could severely impair the ability of these egg parasitoids to establish and successfully control D. abbreviatus in areas where winter temperatures fluctuate around 12°C, the LDT for this pest.     

Sampling methods as abundance indices for adult Diaprepes abbreviatus (Coleoptera: Curculionidae) in citrus

Beat sampling and two type of traps, cup traps and Tedders traps, were evaluated as sampling methods to detect and estimate population densities of adult Diaprepes abbreviatus L. weevils newly colonizing young citrus trees. The study was conducted over a 65-wk period across a 0.25-ha area of 80 citrus trees [Citrus sinensis (L.) Osbeck] (1.2-1.5 m tall). Beat samples were taken weekly to determine the number of trees infested and number of new adult weevils per tree. Sixteen of the 80 trees studied were each monitored weekly using one of the following trapping methods: cup traps in trees, cup traps on a stake in the ground within the tree drip line, cup traps on a stake in the ground outside of the drip line, Tedders traps on the ground within the drip line, and Tedders traps on the ground outside of the drip line. Weevils collected each week from trees and traps were removed from the study site. Based on the coefficients associated with Taylor's power law, the optimum numbers of trees to sample for an SEM equal to 25% of the mean estimate decreased from 50 trees at a mean of 0.5 new weevils per tree to 30 trees at a mean of 0.8 new weevils per tree. A significant relationship was found between the weekly mean number of new weevils per tree and the proportion of trees infested, a binomial relationship that could be further explored in the search for a sampling program for adult D. abbreviatus. Regression analyses indicated that three of the trapping methods served at least as weak indices of the presence and abundance of new weevils: cup traps in trees, Tedders traps inside the dripline and Tedders traps outside the dripline. Cup traps in trees and Tedders traps inside the dripline captured the most weevils and most frequently detected weevils. Although relatively inefficient as abundance indices of populations of new weevils, these two trapping methods appeared to have some value with respect to signaling when weevils first appeared in trees during the spring.     

Larval growth of Diaprepes abbreviatus (Coleoptera: Curculionidae) and resulting root injury to three citrus varieties in two soil types

Larval growth and intraspecific competition of Diaprepes abbreviatus (L.) larvae and consequent root injury in container-grown citrus in the greenhouse were evaluated. Roots of Carrizo citrange, Citrus sinesis L. Osbeck x Poncirus trifoliata (L.) Raf.; Cleopatra mandarin, C. reticulata Blanco, and Swingle citrumelo, C. paradisi Macf. x P. trifoliata (L.) Raf. rootstock seedlings grown in Candler fine sand and potting soil were colonized with different populations of D. abbreviatus larvae. Larvae were exposed to the seedlings for 79 d. Larval growth and development increased steadily for approximately 70 d on all rootstock-soil combinations, at which time most larvae were instars 6-8. Most feeding injury occurred to roots when larvae were between instars 3 and 6. Larval weight reached a plateau at approximately 70 d, but often declined between 70 and 79 d. When larvae were small, injury to seedlings developed slowly, primarily on fibrous roots, then feeding increased rapidly, often resulting in total consumption of both fibrous root and bark tissue. Although not statistically significant, root injury developed slightly slower on Swingle citrumelo compared with Carrizo and Cleopatra rootstocks, but damage was comparable by 79 d. Little or no difference in consumptive benefit to the larvae was found between the rootstocks. Based on larval weight days, little feeding injury occurred during the first 21 d, but increased rapidly between 21 and 60 d. Soil type affected the rate of larval growth and development, with potting soil contributing to greater growth rates. Detritus in potting soil provided little or no nutritional resource, suggesting that the effect of potting soil on larval development was primarily physical. In addition, fewer inoculated larvae per seedling exhibited greater weight gains than higher infestation densities, suggesting that intraspecific competition for nutritional resources influenced larval development.     

Effect of root feeding by Diaprepes abbreviatus (Coleoptera: Curculionidae) larvae on leaf gas exchange and growth of three ornamental tree species

Diaprepes abbreviatus L. (Coleoptera: Curculionidae), feeds on a variety of ornamental plants grown in southern Florida. Studies were conducted to evaluate the effects of root feeding by D. abbreviatus larvae on leaf gas exchange and growth of three ornamental tree species commonly grown in southern Florida that are known hosts of this weevil: green buttonwood, Conocarpus erectus L.; live oak, Quercus virginiana Mill.; and pygmy date palm, Phoenix roebelenii O'Brien. These hosts were grown in containers and infested with weevil larvae. Net CO2 assimilation, transpiration, and stomatal conductance of CO, were measured monthly. Leaf, stem, and root fresh and dry weights of each species also were determined. In one of two tests, larval root feeding significantly reduced net CO2 assimilation, transpiration, and stomatal conductance of CO2 of infested green buttonwood trees. Leaf gas exchange of live oak was not affected by larval infestation. In addition to testing cumulative effects of multiple infestations of larvae, the effects of incremental infestations on leaf gas exchange and fresh and dry weights also were tested for each plant species. Net CO2 assimilation, transpiration, stomatal conductance of CO2, and dry weights of green buttonwood were reduced as a result of larval root feeding, whereas there was no effect of incremental larval infestations on leaf gas exchange of live oak or pygmy date palm within the experimental time frame. There was no effect of incremental larval infestations on dry weights of live oak, but leaf, stem, and dry root weight of pygmy date palm were lower for infested plants than for noninfested plants. Overall, green buttonwood was more susceptible to larval root feeding damage than either live oak or pygmy date palm.     

Cloning and characterization of a basic cysteine-like protease (cathepsin L1) expressed in the gut of larval Diaprepes abbreviatus L. (Coleoptera: Curculionidae)

Diaprepes abbreviatus is an important pest that causes extensive damage to citrus in the USA. Analysis of an expressed sequence tag (EST) library from the digestive tract of larvae and adult D. abbreviatus identified cathepsins as major putative digestive enzymes. One class, sharing amino acid sequence identity with cathepsin L’s, was the most abundant in the EST dataset representing 14.4% and 3.6% of the total sequences in feeding larvae and adults, respectively. The predominant cathepsin (Da-CTSL1) among this class was further studied. Three dimensional modeling of the protein sequence showed that the mature Da-CTSL1 protein folds into an expected cathepsin L structure producing a substrate binding pocket with appropriate positioning of conserved amino acid residues. A full-length cDNA was obtained and the proCTSL1 encoding sequence was expressed in Rosetta™ Escherichia coli cells engineered to express tRNAs specific for eukaryotic codon usage. The Da-CTSL1 was expressed as a fusion protein with GST and His₆ tags and purified in the presence of 1% Triton X-100 by Ni-NTA affinity and size exclusion chromatography. Recombinant mature Da-CTSL1 (23 KDa) exhibits optimal activity at pH 8, rather than at acidic pH that was shown of all previously characterized cathepsins L. Substrate specificity supports the hypothesis that Da-CTSL1 is a unique basic cathepsin L and protease inhibitor studies also suggest unique activity, unlike other characterized acidic cathepsin Ls. This paper describes for the first time a prokaryotic expression system for the production of a functional eukaryotic cathepsin L1 from larval gut of D. abbreviatus.     

Reduced oviposition by Diaprepes abbreviatus (Coleoptera: Curculionidae) and growth enhancement of citrus by Surround particle film

Regularly applied sprays of a particle film, Surround WP, greatly enhanced the growth of citrus trees on a poorly drained Winder soil at Fort Pierce, FL. After 3 yr of applications every 3 or 4 wk, Surround-treated trees had at least 5 times the mass, 6 times the canopy volume, and approximately 4 times the cross-sectional area of the tree stems at the graft union compared with untreated trees. The larger Surround-treated trees attracted a higher number of adult weevil Diaprepes abbreviatus (L.) and to a lesser extent citrus root weevil, Pachnaeus litus (Germar), per tree, but there was an equivalent number of egg masses per tree compared with the control trees. The number of egg masses per female weevil oviposited on Surround-treated trees was significantly less than either the control trees or trees treated biannually with an entomopathogenic nematode, BioVector. The number of larvae per tree recovered from the roots of excavated trees was greater from trees treated with Surround once every 3 wk compared with control trees. The data suggest that Surround particle film greatly enhanced the growth of citrus trees grown in a poorly drained soil. The reduction in oviposition by D. abbreviatus was insufficient to significantly reduce the number of root weevil larvae per tree feeding on the roots. However, the more vigorous trees resulting from Surround applications may be more resistant or tolerant to root weevil feeding.