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.).     

Cuticular hydrocarbons on elytra of the Diaprepes root weevil Diaprepes abbreviatus (L.) (Coleoptera: Curculionidae)

Abstract 1 External gland openings and associated structures on the elytra of teneral and mature Diaprepes root weevils, Diaprepes abbreviatus (L.), were elucidated by scanning electron microscopy (SEM). 2 There were clear differences between teneral, callow adults and fully mature adults. In the field, teneral adults remain in the pupal chamber in the soil until sclerotization of the cuticle is complete or nearly so. 3 Phenotypic variation of the elytra in this species consists of varying patterns and coloration of scaled intervals between a variable number of raised ridges devoid of scales. In addition to being thinner and lighter in colour than fully mature adults, the elytra of teneral adults were devoid of waxy hydrocarbon secretions. 4 External gland openings at the base of each scale were observed on teneral elytra and mature elytra washed with methylene chloride. 5 SEM evidence to document the production of waxy filaments by these glands and partial characterization of these by gas chromatography and mass spectrometry are presented.     

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.     

Size-assortative mating, male choice and female choice in the curculionid beetle Diaprepes abbreviatus

In the beetle Diaprepes abbreviatus (L.) females are larger on average than males, as indicated by elytra length. Size-assortative matings were observed in wild populations in Florida and in laboratory mating experiments. We tested three mechanisms for this size-assortative mating: (1) mate availability; (2) mating constraints; and (3) mate choice. We found that mate choice influenced size-assortative mating by: (1) large and small males preferring to mate with large females; (2) large males successfully competing for large females, leaving small males to mate with small females; and (3) females accepting large males as mates more readily than small males. Males increased their reproductive success by mating with larger, more fecund females. They transferred protein to females during mating. Copyright 1999 The Association for the Study of Animal Behaviour.     

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.     

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.     

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.     

Xenobiotic absorption and binding by proteins in hemolymph of the weevil Diaprepes abbreviatus

A synthetic coumarin, 7-amino-3-phenyl coumarin (coumarin-10), was used to study the uptake of ingested xenobiotics into hemolymph. Larvae were forcefed coumarin-10 in peanut oil, and hemolymph was extracted and analyzed by fluorescence spectroscopy. Coumarin-10 entered hemolymph within 5 min, reaching a steady state of concentration within 1 h. Assayed 2 h after feeding, hemolymph titers of 1–5 ng/μl were proportional to log dose between 10 and 100 ng/mg body weight; hemolymph did not reach saturation. Fluorescence spectra of hemolymph in saline revealed that energy was readily transferred from hydrophobic residues of hemolymph proteins to coumarin-10. Ultracentrifugal density gradients revealed that 94% of absorbed coumarin-10 was bound to sedimenting proteins while 6% bound to lipophorin. Native polyacrylamide gel electrophoresis (N-PAGE) on minigels identified two major proteins responsible for binding. Though readily separated by native electrophoresis, these proteins were not fully separable by HPLC using a wide variety of columns. Gel permeation-HPLC of the sedimenting proteins from hemolymph revealed a single major peak of 480,000 M_r. When upper and lower electrophoretic bands were isolated by preparative N-PAGE, the upper band (band I) yielded subunits of 75,000 and 71,000 M_r, while the lower band (band II) yielded only one size subunit of 75,000 M_r on denaturing (SDS) PAGE. The fluorescent products bound by sedimenting proteins were identified by thin-layer chromatography and scanning fluorescence densitometry as coumarin-10 (80% of total) and a polar metabolite (20%). In addition, lipophorin-containing fractions contained an apolar metabolite (3% of total fluorescence). In vitro binding studies utilizing fluorescent energy transfer demonstrated saturation binding with a K_D of 1.5 μM.     

Transfer and fate of seminal fluid molecules in the beetle, Diaprepes abbreviatus: implications for the reproductive biology of a pest species

Molecules transferred from males to females via seminal fluids are important to the study of insect reproduction because they affect female physiology, reproductive behavior, and longevity. These molecules (seminal fluid molecules or SFMs) interest applied entomologists because of their potential use in insect control. SFMs are also interesting because of their relatively rapid evolution and important role in post-mating sexual selection. We studied SFMs in Diaprepes abbreviatus, a major pest of numerous plant species of economic importance. Using radiolabeled-methionine (35S), we found that D. abbreviatus males synthesized proteins de novo in their reproductive tissues after mating. Males that were fed radiolabeled methionine transferred radioactivity to females beginning within the first 10 min of mating. Male-derived substances are absorbed from the female's reproductive tract into the hemolymph and circulated throughout the body, but are found primarily in the eggs and ovaries. As a result, SFMs may be a useful means of both horizontal (to mates) and vertical transfer (to offspring) of control agents between conspecifics.     

Competition Between Entomopathogenic and Free-Living Bactivorous Nematodes in Larvae of the Weevil Diaprepes abbreviatus

Field and laboratory experiments were conducted to determine the degree to which free-living, bactivorous nematodes (FLBN) are able to competitively displace entomopathogenic nematodes (EPN) from insect cadavers. Two hundred larvae of the insect Diaprepes abbreviatus were buried at regular intervals during 2 years in experimental plots that were untreated or treated twice annually with Steinernema riobrave. Larvae were recovered after 7 days, and nematodes emerging from cadavers during the next 30 days were identified. The monthly prevalence of FLBN was directly related to that of S. riobrave (r = 0.38; P = 0.001) but was not related to the prevalence of the endemic EPN, S. diaprepesi, Heterorhabditis zealandica, H. indica, or H. bacteriophora (r = 0.02; P = 0.80). In a second experiment, treatment of small field plots with S. riobrave increased the prevalence of insect cadavers in which only FLBN were detected compared to untreated controls (30% vs. 14%; P = 0.052), and increased numbers of FLBN per buried insect by more than 10-fold. In the laboratory, sand microcosms containing one D. abbreviatus larva were treated with (i) the FLBN, Pellioditis sp.; (ii) S. riobrave; (iii) S. riobrave + Pellioditis; or (iv) neither nematode. Insect mortality was higher in the presence of both nematodes (57%) than when S. riobrave was alone (42%) (P = 0.01). An average of 59.2 Pellioditis sp. g^-1 insect body weight emerged in the presence of S. riobrave, whereas 6.2 nematodes g^-1 insect were recovered in the absence of the EPN (P = 0.01). Pellioditis sp. reduced the number of S. riobrave per cadaver by 84%; (P = 0.03), and per available insect by 82% (P = 0.001), compared to S. riobrave alone. Population size of S. diaprepesi was not affected by Pellioditis sp. in experiments of the same design. Faster development (P = 0.05) and nutrient appropriation within the insect cadaver by S. diaprepesi compared to S. riobrave may increase the fitness of the former species to compete with Pellioditis sp. The results of these studies demonstrate the potential of FLBN to regulate population densities of EPN and to dampen estimates of EPN-induced mortality of insect pests in the field.     

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.     

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.     

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.     

Citrus rootstock responses to herbivory by larvae of the sugarcane rootstock borer weevil (Diaprepes abbreviatus)

The responses of roots to feeding by larvae of a citrus root weevil (Diaprepes abbreviatus) were investigated in Citrus grandis (L.) Osb. x Poncirus trifoliata (2N) (L.) Raf.; C. grandis x P. trifoliata (4N); P. trifoliata x C. grandis (Flying Dragon x Nakon); C. paradisi Macf. x P. trifoliata (Swingle citrumelo); C. aurantifolia (Christm.) Swingle (Citrus macrophylla); C. reticulata Blanco (Cleopatra mandarin); C. sinensis (L.) Osb. x P. trifoliata (Carrizo citrange); C. aurantium (L.) (sour orange). Chitinase, chitosanase. β-1,3-glucanase, peroxidase and lysozyme activities were measured and significant differences were observed for some of the cultivars between infested and uninfested rootstocks. Generally, increased activities were observed for chitinases and decreased activities were observed for the other enzymes measured. Numerous significant differences in hydrolase and peroxidase activities were observed between cultivars. Immunological detection revealed that new protein bands occurred in root protein extracts for six of the eight cultivars infested with larvae when an antibody to a class I potato leaf chitinase was used. Antibodies generated against two citrus chitinases of M_r 24 000 (basic chitinase cv. Valencia (C. sinensis) callus, BCVC) and M_r 28 000 (basic chitinase/lysozyme cv. Valencia callus, BCLVC) indicated that chitinases in Carrizo were induced in infested roots when the BCVC antibody was employed. These findings justify calling these proteins pathogenesis-related proteins. The chitinase that BCLVC was prepared from exhibited high lysozyme activities, and the results of western blots showed the presence of proteins at M_r 24 000 and 27 000 which are presumed to be lysozymes. Similar tests using antibodies against β-1, 3-glucanases and peroxidases indicated a diminution of protein bands that cross-reacted with infested root protein extracts compared with what occurred in controls. All of the root extracts were tested against chitosans with various percentages of acetylation; activities were linearly dependent on the amount of chitosan acetylation; i.e. the larger the amount of acetylation, the greater the activity. Significant differences in hydrolase activities were observed between infested and uninfested roots for the rootstocks using the variously acetylated substrates. All of the root protein extracts were capable of degrading peritrophic membranes removed from larvae of D. abbreviatus. This suggests that citrus chitinases may play a role in disrupting the peritrophic membrane such that ingested substances that pose a hazard to the insect may penetrate the membrane more easily.     

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.     

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.     

Impact of Surfactants on Control of the Root Weevil Diaprepes abbreviatus Larvae with Steinernema riobravis

A filter paper bioassay was developed for testing the efficacy of nematodes and other agents for control of Diaprepes abbreviatus larvae. Surfactants, with and without Steinernema riobravis, were screened first in the filter paper bioassay and then in a potted citrus seedling study. The results of the two assays were in agreement on the relative merits of the compounds tested. Surfactants increased larval mortality at 4 days for the filter paper bioassay and 1 week for the potted plant bioassay. At 8 days for the filter paper bioassay and 2 weeks for the potted plant bioassay, nematodes alone were equal to treatments of nematodes plus surfactants.     

Comparison of Two Steinernematid Species for Control of the Root Weevil Diaprepes abbreviatus

Steinernema carpocapsae Weiser All strain was compared to Steinernema riobravis Cabanillas, Poinar, and Raulston for control of the root weevil, Diaprepes abbreviatus (L.), in the laboratory and in potted citrus. In the laboratory bioassay, D. abbreviatus larvae were exposed to 30, 60, and 120 nematodes/cm³ in sand. Insect mortality 1 week after application was greater (P ≤ 0.05) for S. riobravis than for S. carpocapsae in the laboratory bioassay. In the greenhouse bioassay, D. abbreviatus larvae were exposed to 3 and 9 nematodes per cm³ of soil in potted citrus. Again, at each rate, mortality was greater (P ≤ 0.05) in pots treated with S. riobravis than in pots treated with S. carpocapsae. The results of this study suggest that S. riobravis is a better biological control agent against D. abbreviatus larvae in potted plants than S. carpocapsae.