Karyotypic analysis of the cotton boll weevil, Anthonomus grandis Boheman

The diploid chromosome number of the cotton boll weevil, Anthonomus grandis Boheman, is 44. Both C‐ and N‐banding techniques of mitotic cells demonstrated constitutive heterochromatin in the p arm of the eight largest chromosomes, the p arm of the X chromosome, and the centromeric region of autosomal groups A–D. Neither the y nor the group E autosomes appeared to contain constitutive heterochromatin. Supernumerary chromosomes were not found in the boll weevil. Restriction endonuclease banding of primary spermatocytes revealed a rod‐shaped Xy tetrad in which the X and y were terminally associated. The p arm of the large, submetacentric X was C‐band positive. While two of the autosomal tetrads were typically ring‐shaped in primary spermatocytes, the remaining 19 autosomal tetrads were rod‐shaped. This revised version was published online in July 2006 with corrections to the Cover Date.     

Site of pheromone biosynthesis and isolation of HMG-CoA reductase cDNA in the cotton boll weevil, Anthonomus grandis

Isolated gut tissue from male cotton boll weevil, Anthonomus grandis (Coleoptera: Curculionidae), incorporated radiolabeled acetate into components that co-eluted with monoterpenoid pheromone components on HPLC. This demonstrates that pheromone components of male A. grandis are produced de novo and strongly suggests that pheromone biosynthesis occurs in gut tissue. A central enzyme in isoprenoid biosynthesis is 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-R), and a full-length HMG-R cDNA was isolated from A. grandis. The predicted translation product was 54 and 45% identical to HMG-R from Ips paraconfusus and Drosophila melanogaster, respectively. HMG-R gene expression gradually increased with age in male A. grandis, which correlates with pheromone production. However, topical application of JH III did not significantly increase HMG-R mRNA levels.     

Functions of the spermathecal muscle of the boll weevil,Anthonomus grandis

In female boll weevils, Anthonomus grandis, spermathecal filling was not affected by severing the spermathecal muscles. Females whose spermathecal muscles were severed 2 to 4 weeks after mating laid infertile eggs and resumed virginal ovipositional behaviour indicating the importance of this muscle in supplying sperm for egg fertilization. The presence of normal active sperm within the spermatheca in no way influenced ovipositional behaviour. In females whose spermathecal muscles had been severed, 22 per cent sperm displacement occurred after a second mating compared with 66 per cent for normal females. The physical displacement of sperm was thus largely dependent on a functional spermathecal muscle.     

Isolation and characterization of farnesyl diphosphate synthase from the cotton boll weevil,Anthonomus grandis

Farnesyl diphosphate synthase (FPPS) catalyzes the consecutive condensation of two molecules of isopentenyl diphosphate with dimethylallyl diphosphate to form farnesyl diphosphate (FPP). In insects, FPP is used for the synthesis of ubiquinones, dolicols, protein prenyl groups, and juvenile hormone. A full‐length cDNA of FPPS was cloned from the cotton boll weevil, Anthonomus grandis (AgFPPS). AgFPPS cDNA consists of 1,835 nucleotides and encodes a protein of 438 amino acids. The deduced amino acid sequence has high similarity to previously isolated insect FPPSs and other known FPPSs. Recombinant AgFPPS expressed in E. coli converted labeled isopentenyl diphosphate in the presence of dimethylallyl diphosphate to FPP. Southern blot analysis indicated the presence of a single copy gene. Using molecular modeling, the three‐dimensional structure of coleopteran FPPS was determined and compared to the X‐ray crystal structure of avian FPPS. The α‐helical fold is conserved in AgFPPS and the size of the active site cavity is consistent with the enzyme being a FPPS. © 2009 Wiley Periodicals, Inc.     

Proteolytic processing of the vitellogenin precursor in the boll weevil,Anthonomus grandis

The soluble proteins of the eggs of the coleopteran insect Anthonomus grandis Boheman, the cotton boll weevil, consist almost entirely of two vitellin types with M_rs of 160,000 and 47,000. We sequenced their N-terminal ends and one internal cyanogen bromide fragment of the large vitellin and compared these sequences with the deduced amino acid sequence from the vitellogenin gene. The results suggest that both the boll weevil vitellin proteins are products of the proteolytic cleavage of a single precursor protein. The smaller 47,000 M vitellin protein is derived from the N-terminal portion of the precursor adjacent to an 18 amino acid signal peptide. The cleavage site between the large and small vitellins at amino acid 362 is adjacent to a pentapeptide sequence containing two pairs of arginine residues. Comparison of the boll weevil sequences with limited known sequences from the single 180,000 M_r honey bee protein show that the honey bee vitellin N-terminal exhibits sequence homology to the N-terminal of the 47,000 M_r boll weevil vitellin. Treatment of the vitellins with an N-glycosidase results in a decrease in molecular weight of both proteins, from 47,000 to 39,000 and from 160,000 to 145,000, indicating that about 10–15% of the molecular weight of each vitellin consists of N-linked carbohydrate. The molecular weight of the deglycosylated large vitellin is smaller than that predicted from the gene sequence, indicating possible further proteolytic processing at the C-terminal of that protein. © 1993 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Regulation of vitellogenin synthesis and uptake in the boll weevil, Anthonomus grandis

Abstract. Hormonal factors influencing reproductive development were examined in adult boll weevils, Anthonomus grandis (Boheman) (Coleoptera: Curculionidae). Long-day, high-temperature rearing conditions promote reproduction whereas short-day, low-temperature conditions do not. Implants of corpora allata (CA), brains, or brains plus retrocerebral complexes taken from long-day donors, or hormone analogue treatments were used to examine onset of vitellogenin synthesis and uptake in decapitated bodies of adult weevils reared in short-day, low-temperature conditions. Weevils decapitated within 2 days after eclosion and reared in short-day, low-temperature conditions never initiated vitellogenin production or ovarian development. Females and males decapitated on day 2 showed haemolymph vitellogenin within 5 days following treatment with Juvenile Hormone (JH) analogue or implantation of CA, but not after implantation of brain alone or implantation of muscle (sham). Uptake of vitellogenin into the oocytes did not occur unless both JH analogue and brain were given as replacement therapy. These experiments indicated that JH is necessary and sufficient to stimulate vitellogenin synthesis in this species but that a brain factor must be present for vitellogenin uptake.     

Endocrine regulation of reproduction and diapause in the boll weevil,Anthonomus grandis Boheman

A review of the literature for the hormonal control of reproduction and diapause in Coleoptera is presented. The role of juvenile hormone and juvenile hormone esterase (JHE) in the control of the different life history strategies of the boll weevil are examined. Elevated levels of hemolymph JHE were found to be positively correlated with survival throughout the winter in South Texas population of weevils. Winter weevils were examined for hemolymph vitellogenin (Vg) and their subsequent survival was monitored. The majority of weevils surviving beyond ten weeks had no hemolymph Vg. We conclude that elevated hemolymph JHE and the absence of Vg are good predictors of survivors in the South Texas population. The hormonal basis of diapause termination was examined using hormone treatment and implant therapy. We were unsuccessful in our attempt to induce post-diapause reproductive development with all of our treatments. Only weevils given access to a food source were capable of reproductive development. Our recent experimental findings in the control of Vg synthesis and uptake in the boll weevil are reviewed. Arch. Insect Biochem. Physiol. 35:455–477, 1997. © 1997 Wiley-Liss, Inc.     

Toxicity to cotton boll weevil Anthonomus grandis of a trypsin inhibitor from chickpea seeds

Cotton (Gossypium hirsutum L.) is an important agricultural commodity, which is attacked by several pests such as the cotton boll weevil Anthonomus grandis. Adult A. grandis feed on fruits and leaf petioles, reducing drastically the crop production. The predominance of boll weevil digestive serine proteinases has motivated inhibitor screenings in order to discover new ones with the capability to reduce the digestion process. The present study describes a novel proteinase inhibitor from chickpea seeds (Cicer arietinum L.) and its effects against A. grandis. This inhibitor, named CaTI, was purified by using affinity Red-Sepharose Cl-6B chromatography, followed by reversed-phase HPLC (Vydac C18-TP). SDS-PAGE and MALDI-TOF analyses, showed a unique monomeric protein with a mass of 12,877 Da. Purified CaTI showed significant inhibitory activity against larval cotton boll weevil serine proteinases (78%) and against bovine pancreatic trypsin (73%), when analyzed by fluorimetric assays. Although the molecular mass of CaTI corresponded to alpha-amylase/trypsin bifunctional inhibitors masses, no inhibitory activity against insect and mammalian alpha-amylases was observed. In order to observe CaTI in vivo effects, an inhibitor rich fraction was added to an artificial diet at different concentrations. At 1.5% (w/w), CaTI caused severe development delay, several deformities and a mortality rate of approximately 45%. These results suggested that CaTI could be useful in the production of transgenic cotton plants with enhanced resistance toward cotton boll weevil.     

Cuticle proteins of the boll weevil,Anthonomus grandis,abdomen: Structural similarities and glycosylation

Cuticle proteins are thought to be important in defining the structural and functional differences occurring in insect cuticle. In order to explain and better understand the structural similarities among the cuticle proteins of the cotton boll weevil, Anthonomus grandis Boheman, described in a previous study (Stiles and Leopold, 1990, Insect Biochem.20, 113–125) three series of monoclonal antibody producing hybridoma cell lines were produced. Larval, pupal or adult cuticle proteins were used as antigens. While some of the monoclonal antibodies were specific for one or two cuticle proteins from a single developmental stage, the majority showed multiple cuticle protein binding patterns on Western blots. To determine whether this cross-reaction was due to common oligosaccharide chains bound to the proteins, lectins were used to probe Western blots. Many of the cuticle proteins were found to be glycosylated. The majority of the Con A reactive carbohydrate could be removed from the protein by N-glycosidase F digestion (specific for N-asparagine linked carbohydrate). N-glycosidase F digestion did not reduce the multiple cross-reactions of the monoclonal antibodies, nor did periodate oxidation of the CP. The carbohydrate remaining after enzyme digestion is presumably O-linked to serine/threonine.     

Oviposition behaviour of Bracon mellitor, a parasitoid of the boll weevil (Anthonomus grandis)

ABSTRACT. The parasitoid, Bracon mellitor , will probe with its ovipositor plant structures containing its host. The identification of methyl parahydroxy-benzoate (methyl parasept), an antimicrobial additive to the semi-synthetic diet used for rearing the host, as a releaser of this behaviour prompted an investigation of the parasitoid's response to this chemical. Its absence from field hosts suggested the response was learned. Female parasitoids reared in host-infested cotton plant buds from the fields or in methyl parasept-free hosts did not respond to methyl parasept alone until exposed to hosts contaminated with it. This is taken to imply associative learning by the females reared from the methyl parasept-free hosts. A few responses to methyl parasept were elicited from inexperienced females reared from methyl parasept-contaminated hosts, but their responsiveness increased after they had been exposed to methyl parasept-contaminated hosts as oviposition stimuli. These results point to the caution that must be exercised in the isolation and identification of kairomones.     

The effects of dietary nitrogen on reproductive development in the female boll weevil, Anthonomus grandis

ABSTRACT. The role of dietary nitrogen in reproductive development was investigated in the female boll weevil, Anthonomus grandis (Boh.)(Coleoptera: Curculionidae). Artificial diets were employed containing 1.7%, 2.6% and 4.0% N. Each diet was labelled with a ¹⁴C-amino acid mixture and standardized relative to cpm and N per mg of diet dry weight. Egg production, diet consumption, nitrogen consumption and nitrogen utilization were measured by recovering the cpm in the faeces, ovaries plus eggs and the carcass.
Females given the 4% N diet initiated oviposition on day 4 with maximum egg production of 11 eggs/day/female occurring on day 6. Females on the 2.6% N diet also initiated oviposition on day 4, but the time necessary to reach maximum egg production was extended. Egg production by these females eventually equalled the total production of the group on the 4% N diet. This was achieved by consuming more diet per female and dedicating a higher percentage of the absorbed diet to egg formation. With the 1.7% N diet, females showed both a delay and a decrease in total egg production because of reduced feeding and a reduced commitment of consumed nutrients to oöcyte maturation.
Switching dietary nitrogen concentrations after the initiation of oviposition resulted in changes in consumption and reproductive allocation within 48 h.     

Factors contributing to resistance and susceptibility of cotton and other hosts to the boll weevil, Anthonomus grandis

A large portion of the world's germ plasm of cotton has been screened at the USDA Boll Weevil Research Laboratory, State College, Mississippi during the period 1962–68 utilizing developed techniques.An oviposition suppression factor causing 25–40% reduction in number of eggs laid by the weevil has been found in Gossypium barbadense and successfully moved into upland cotton, G. hirsutum.Two years field data with frego cottons indicate that the frego character contributes a significant degree of non-preference for egg laying by the boll weevil under field conditions. The frego character is currently being combined with selected lines of G. hirsutum carrying the oviposition suppression factor.A seedling screening technique utilizing field cages and greenhouse sand flats has been shown to have significant value in screening for individual resistant plants within a commercial variety. A number of selections with 25% or more resistance to oviposition are under extensive investigation.A number of biologically active materials have been detected in the cotton plant and other hosts of the boll weevil which significantly influences feeding stimulation, feeding deterrency, repellency and attractancy of the weevil to its hosts.A highly active square abscission factor has been detected and extracted successfully from 2nd and 3rd instar larvae. The material injected into cotton squares causes 95% abscission of squares within 48–60 hours.

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Zusammenfassung Ein großer Teil des Weltsortiments der Baumwolle wurde in den Jahren 1962–68 im USA-Baumwollkäfer-Institut der Staatshochschule von Mississippi mit modernen Methoden getestet.Ein die Eiablage unterdrückender Faktor, der die Anzahl der abgelegten Eier um 25–40% herabsetzt, wurde bei Gossypium barbadense gefunden und erfolgreich in Hochland-Baumwolle, G. hirsutum, eingekreuzt.Zweijährige Erfahrungen mit frego-Baumwolle weisen darauf hin, daß das frego-Merkmal unter Feldbedingungen in einem signifikanten Ausmaße zur Nonpräferenz für die eierablegenden Käfer beiträgt. Das frego-Merkmal wird laufend mit selektierten Linien von G. hirsutum kombiniert, die den Faktor für Eiablage-Unterdrückung tragen.Eine Sämlings-Prüfungstechnik, die Feldkäfige und Gewächshaus-Sandbetee benutzt, hatte nachweislich signifikanten Wert für die Auslese resistenter Einzelpflanzen innerhalb einer Handelssorte. Eine Anzahl Selektionslinien mit 25 oder mehr Prozent Eiablage-Resistenz wird intensiv geprüft.Eine Anzahl biologisch aktiven Materials wurde bei Baumwolle und anderen Wirtspflanzen endeckt, das Fraßstimulation, Fraßabschreckung, Repellenz und Attraktanz der Käfer in bezug auf ihre Wirte signifikant beeinflußt.Ein hochaktiver Kapsel-Abwurf-Faktor wurde entdeckt und erfolgreich aus Larven des 2. und 3. Stadiums extrahiert. In Baumwollkapseln injiziert, verursachte dieses Material innerhalb 48–60 Stunden Abwurf von 95% der Kapseln.

     

Effects of soybean Kunitz trypsin inhibitor on the cotton boll weevil (Anthonomus grandis)

The cotton boll weevil, Anthonomus grandis, is an economically important pest of cotton in tropical and subtropical areas of several countries in the Americas, causing severe losses due to their damage in cotton floral buds. Enzymatic assays using gut extracts from larval and adult boll weevil have demonstrated the presence of digestive serine proteinase-like activities. Furthermore, in vitro assays showed that soybean Kunitz trypsin inhibitor (SKTI) was able to inhibit these enzymes. Previously, in vivo effects of black-eyed pea trypsin chymotrypsin inhibitor (BTCI) have been demonstrated towards the boll weevil pest. Here, when neonate larvae were reared on an artificial diet containing SKTI at three different concentrations, a reduction of larval weight of up to 64% was observed for highest SKTI concentration 500 microM. The presence of SKTI caused an increase in mortality and severe deformities of larvae, pupae and adult insects. This work therefore represents the first observation of a Kunitz trypsin inhibitor active in vivo and in vitro against A. grandis. Bioassays suggested that SKTI could be used as a tool in engineering crop plants, which might exhibit increased resistance against cotton boll weevil.     

Rates of Juvenile Hormone biosynthesis and degradation during reproductive development and diapause in the boll weevil, Anthonomus grandis

Abstract. The role of Juvenile Hormone (JH) during reproductive development and diapause was investigated in the boll weevil, Anthonomus grandis Boheman (Coleoptera: Curculionidae). JH sythesized by corpora allata (CA) in vitro of A.grandis was identified as JH-UJ by high-performance liquid chromatography and by conversion to the methoxyhydrin. Optimal conditions for the use of a short-term assay in vitro were established to examine profiles of CA activity. In addition, rates of JH degradation by JH-specific esterase were determined. Patterns of CA and JH-esterase activity during reproductive development and the diapause state were established with laboratory-reared reproductive weevils and diapausing weevils collected as larvae and pupae in the field after the cotton-growing season. The results indicate that JH production is elevated in reproductive females whereas males and winter field-collected females show no CA activity. Vitellogenin concentrations in haemolymph and rates of oviposition were studied in relation to CA activity and JH degradation. An attempt to induce diapause in the laboratory failed.     

Molecular cloning of a cysteine proteinase cDNA from the cotton boll weevil Anthonomus grandis (Coleoptera: Curculionidae)

The cotton boll weevil (Anthonomus grandis) causes severe cotton crop losses in North and South America. This report describes the presence of cysteine proteinase activity in the cotton boll weevil. Cysteine proteinase inhibitors from different sources were assayed against total A. grandis proteinases but, unexpectedly, no inhibitor tested was particularly effective. In order to screen for active inhibitors against the boll weevil, a cysteine proteinase cDNA (Agcys1) was isolated from A. grandis larvae using degenerate primers and rapid amplification of cDNA ends (RACE) techniques. Sequence analysis showed significant homologies with other insect cysteine proteinases. Northern blot analysis indicated that the mRNA encoding the proteinase was transcribed mainly in the gut of larvae. No mRNA was detected in neonatal larvae, pupae, or in the gut of the adult insect, suggesting that Agcys1 is an important cysteine proteinase for larvae digestion. The isolated gene will facilitate the search for highly active inhibitors towards boll weevil larvae that may provide a new opportunity to control this important insect pest.     

Isolation and characterization of polymorphic microsatellite loci in the boll weevil,Anthonomus grandis Boheman (Coleoptera: Curculionidae)

The boll weevil (Anthonomus grandis Boheman) is a major insect pest of cotton in North America. Dispersal activity poses a threat to ongoing eradication efforts in the US, but little is known about the frequency of long‐distance migration. Nuclear molecular markers are needed to assess gene flow in relation to geographical distance. A biotin‐enrichment strategy was employed to develop microsatellite markers for the boll weevil. Of 23 loci isolated, 14 were polymorphic with three to 10 alleles per locus. Twelve of the polymorphic loci showed Mendelian inheritance and are likely to be useful in population genetics studies.     

Cholesterol oxidase: an oöstatic and larvicidal agent active against the cotton boll weevil,Anthonomus grandis

The enzyme cholesterol oxidase (E.C. 1.1.3.6), purified from Streptomyces culture filtrate was previously found to have oral insecticidal activity on neonate larvae of the boll weevil (Anthonomus grandis grandis Boheman) from a laboratory population. In the present study, second instar larvae were also controlled by the enzyme at diet concentrations similar to those which control neonates (12 day LC₅₀ = 2.4 μg.ml^?1 in diet). Larvae from field-collected adults were similarly susceptible to cholesterol oxidase in the diet. When ingested by adult females during the mating/pre-oviposition period, cholesterol oxidase greatly reduced subsequent oviposition (83% reduction in eggs laid as compared to controls) and larval survival (97% reduction from controls). Dissection of treated adult females revealed poorly developed ovaries and few developing oöcytes. These studies were conducted to further evaluate the utility of cholesterol oxidase in a program to establish boll weevil-resistant transgenic cotton.