Molecular methods for assessing insect parasitism

2006 ◽  
Vol 96 (1) ◽  
pp. 1-13 ◽  
Author(s):  
M.H. Greenstone

AbstractDetermining insect parasitism rates is problematic due to the small size and lack of useful distinguishing morphological characters of many parasitoid taxa. To solve this problem, entomologists have employed one of four general methods to detect parasitoid protein or nucleic acid markers: serological assay; random amplified polymorphic DNA–polymerase chain reaction “RAPD-PCR” allozyme electrophoresis; or specific PCR. Serological methods, especially with monoclonal antibodies, are unrivalled for specificity, enabling discrimination at the stage as well as species level. However, they have not found favour with many workers, possibly due to complexity and expense. RAPD–PCR has been widely used, but can only be recommended for restricted applications because of its poor reproducibility. Allozyme electrophoresis provides reproducible detection and discrimination of closely related species. Specific-PCR is highly specific and reproducible, and also has the shortest latency for detection, usually 24 h or less after parasitization. The substantial existing literature on allozyme electrophoresis and specific PCR is used to support recommendations on what are apt to be fruitful enzyme systems or genomic regions for detecting and discriminating parasitoids in untried parasitoid–host assemblages.

2008 ◽  
Vol 3 (2) ◽  
pp. 77
Author(s):  
Andi Parenrengi ◽  
Andi Tenriulo

Random Amplified Polymorphic DNA (RAPD) was employed to determine the genetic variability and population structure of grouper (Epinephelus suillus) from Makassar Strait and Bone Bay, South Sulawesi, Indonesia. Genomic DNA was isolated from preserved muscle tissue using Phenol-Chloroform technique. Among 24-screened arbitrary primers, ten primers (OPA-02, OPA-06, OPA-08, OPA-10, OPA-15, OPA-16, OPA-17, OPA-18, OPA-19 and CA-05) were selected to generate RAPD fingerprinting of grouper populations. The ten primers generated a total of 212 fragments (loci) and 120 polymorphic fragments in their size ranging from 250 to 2,500bp. The high polymorphism (60%) was obtained from Makassar population followed by Bone (59%) and Pare-Pare populations (50%). Similarity index of individuals was 0.86±0.07 for Pare-Pare, 0.80±0.11 for Makassar and 0.82±0.07 for Bone population. Fifteen fragments from ten primes were identified as species-specific markers of E. suillus. The UPGMA cluster analysis showed that the dendrogram seemed to be clustered according to their geographical location, where Pare-Pare population was genetically closer to Makassar population (D=0.20) than to Bone population (D=0.24).


2008 ◽  
Vol 140 (5) ◽  
pp. 527-538 ◽  
Author(s):  
Patricia L. Johnson ◽  
Jane L. Hayes ◽  
John Rinehart ◽  
Walter S. Sheppard ◽  
Steven E. Smith

AbstractScolytus schevyrewi Semenov, the banded elm bark beetle, and S. multistriatus Marsham, the smaller European elm bark beetle, are morphologically similar. Reliance on adult external morphological characters for identification can be problematic because of wide within-species variability and the need for good-quality specimens. The inability to identify developmental stages can also hamper early-detection programs. Using two character identification systems, genitalic (aedeagus) morphology, and DNA markers (random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR)) to distinguish S. schevyrewi from S. multistriatus, we examined specimens from geographically distinct populations of both species collected from infested host trees or semiochemical-baited funnel traps. We found that aedeagus morphology can be used to identify the two species. The use of two oligonucleotide primers in the RAPD-PCR analysis yielded distinct DNA banding patterns for the two species. Species identification using RAPD-PCR analysis was validated by a blind test and used to make species identifications of larval specimens. These tools improve the ability to differentiate between S. schevyrewi and S. multistriatus at immature and adult stages, and could be developed and used for other scolytines as well.


Plant Disease ◽  
2001 ◽  
Vol 85 (1) ◽  
pp. 97-97 ◽  
Author(s):  
A. Pane ◽  
M. G. Li Destri Nicosia ◽  
S. O. Cacciola

Feijoa (Feijoa sellowiana) is native to South America. In the early 20th Century it was introduced into Sicily (southern Italy), where it is grown as an ornamental plant and for its fruits. In 1985 a Phytophthora brown rot of feijoa fruits was reported in the province of Syracuse (eastern Sicily) (2). Several species of Phytophthora, including P. citricola, P. citrophthora, and P. nicotianae, were recovered from soil samples taken from trees with infected fruits. These species were experimentally inoculated on detached feijoa fruits and all incited symptoms of brown rot. However, only P. citricola was isolated from naturally infected fruits. In early autumn 1999, an outbreak of Phytophthora brown rot of feijoa fruits was observed in the Syracuse province, in the same site where the disease had been first recorded. P. citricola (95% of the isolates) and P. citrophthora (5% of the isolates) were recovered from symptomatic fruits. The species were identified on the basis of morphological and cultural characters according to Erwin and Ribeiro (1). The P. citricola isolates formed colonies with a distinctive chrysanthemum pattern on potato-dextrose agar (PDA), had an optimum temperature for radial growth of 25°C, and were homothallic with paragynous antheridia and spherical oogonia (mean diameter of oogonia= 20 μm). Sporangia, which were produced only in water or saline solution, were semi-papillate (often with two apices) and variable in shape. The P. citrophthora isolates formed petaloid colonies on PDA, had an optimum temperature of 25°C, and produced noncaducous, papillate (frequently bipapillate), ovoid to limoniform sporangia. They did not produce gametangia. The identification of both species was confirmed by the electrophoresis of mycelial proteins on polyacrylamide slab gel. The electrophoretic patterns of total proteins and four isozymes (alkaline phosphatase, esterase, malate dehydrogenase, and superoxide dismutase) of the P. citricola and P. citrophthora isolates from feijoa were identical to those of reference isolates of these two species from various other hosts. Conversely, they were clearly distinct from the electrophoretic patterns of reference isolates of P. cactorum, P. capsici, P. nicotianae, and P. palmivora. The random amplified polymorphic DNA patterns of the P. citrophthora isolates from feijoa obtained by polymerase chain reaction (RAPD-PCR) were compared with those of reference isolates of other species of Phytophthora and those of P. citrophthora isolates obtained from citrus trees with symptoms of trunk gummosis and root rot, grown in the immediate vicinity of feijoa trees. DNA was extracted and analyzed following previously described procedures, using 16 decamer primers (3). The RAPD-PCR patterns of the P. citrophthora isolates from feijoa were identical to those of the isolates from citrus but were distinct from those of reference isolates of the other species of Phytophthora, suggesting that inoculum of P. citrophthora may have originated from infected citrus trees. P. citricola is known as a causal agent of fruit brown rot of feijoa and guava (Psidium guajava), a closely related species (1). Conversely, this is the first report of natural infections of P. citrophthora on feijoa fruits. References: (1) D. C. Erwin and O. K. Ribeiro, 1996. Phytophthora Diseases Worldwide. The American Phytopathological Society. St. Paul, MN. (2) G. Magnano di San Lio and R. Tuttobene. Inf. Fitopatol. 85:43, 1985. (3) Q. Migheli et al. Eur. J. Plant Pathol. 104:49, 1998.


Author(s):  
C. André ◽  
M. Lindegarth ◽  
P.R. Jonsson ◽  
P. Sundberg

The polymerase chain reaction (PCR) was used to produce species-specific DNA markers (RAPDs) from two sibling cockle species and five other co-occurring intertidal bivalves. Amplification reactions with one single primer readily distinguished larvae and adults of Cerastoderma edule from larvae and adults of C. lamarcki, and from adults of Mya arenaria, Macoma balthica, Scrobicularia plana, Venerupis pulastra and Mytilus edulis. Random amplified polymorphic DNA (RAPD) is suggested as a simple and quick method to determine species identity in taxa that are difficult to identify on the basis of morphological characters alone, such as marine bivalve larvae.


Parasitology ◽  
1995 ◽  
Vol 111 (1) ◽  
pp. 119-125 ◽  
Author(s):  
J. Liu ◽  
R. E. Berry

SUMMARYWe combined polymerase chain reaction (PCR) amplification of DNA sequences and important morphological characters as a technique to differentiate nematode isolates in the genus Steinernema. Five decamer oligonucleotide primers were used to generate random amplified polymorphic DNA (RAPD) fragments from 11 nematode isolates. The primers generated 8–12 fragments, ranging from 220 to 1300 bp in size. Reproducible amplified DNA fragments of 11 isolates showed obviously inter- or intra-specific polymorphisms, enabling us to differentiate easily the nematode species and isolates. Combining RAPD–PCR fragments with the examination of morphological characters of infective juveniles and 1st-generation males, we identified isolate OH1S, collected from Newport, Oregon, as S.feltiae; isolate OS21, collected from Grants Pass, Oregon, belonged to a previously undescribed species. Our study may provide a rapid and reliable method for the identification of Steinernema nematodes.


2018 ◽  
Author(s):  
Sean P. Gordon ◽  
Joshua J. Levy ◽  
John P. Vogel

AbstractExisting methods for assigning sequences to individual species from pooled DNA samples rely on differences in genome properties like GC content or sequences from related species. These approaches do not work for closely related species where gross features are indistinguishable and related genomes are lacking. We describe a method and associated software package that uses rapidly evolving repetitive DNA to circumvent these limitations. By using short, repetitive, DNA sequences as species-specific signals we separated closely related genomes without any prior knowledge. This approach is ideal for separating the subgenomes of polyploid species with unsequenced or unknown progenitor genomes.


1998 ◽  
Vol 36 (7) ◽  
pp. 2057-2062 ◽  
Author(s):  
Mary E. Brandt ◽  
Arvind A. Padhye ◽  
Leonard W. Mayer ◽  
Brian P. Holloway

We developed a method for the identification of Aspergillus fumigatus fungal isolates by using random amplified polymorphic DNA (RAPD) PCR (RAPD-PCR) cloning and the TaqMan LS50B fluorogenic detection system (Perkin-Elmer Corp., Applied Biosystems, Foster City, Calif.). DNA from seven clinically important Aspergillusspecies was screened by RAPD-PCR to identify section- or species-specific amplicons. With the OPZ19 RAPD primer a 1,264-bp product was amplified from all A. fumigatus strains initially examined but not from other species. A partial DNA sequence of this product was used to design a specific primer pair, which generated a single 864-bp fragment with DNA from 90 of 100 A. fumigatus isolates when a “touchdown” (65→55°C) annealing protocol was used. The TaqMan system, a fluorogenic assay which uses the 5′→3′ endonuclease activity of Taq DNA polymerase, detected this 864-bp product with DNA from 89 of these 90 A. fumigatus strains; 1 DNA sample generated an indeterminate result. With DNA from three morphologically typical A. fumigatus isolates, six white (“albino”) A. fumigatus isolates, and five of six Neosartoryaspecies (non-A. fumigatus members of the sectionFumigati), the 864-bp product was amplified differentially at an annealing temperature of 56°C but not with the touchdown annealing format. No amplicon was detected with DNA from 56 isolates of heterologous Aspergillus, Penicillium, andPaecilomyces species or from Neosartorya fennelliae; TaqMan assay results were either negative (51 isolates) or indeterminate (5 isolates) for all isolates. This RAPD-PCR and TaqMan assay offers promise as a nucleic acid-based system that can be used for the identification of filamentous fungal isolates and that requires no postamplification sample manipulations.


Plant Disease ◽  
2005 ◽  
Vol 89 (2) ◽  
pp. 134-145 ◽  
Author(s):  
Rodney G. Roberts

In 2001, numerous Ya Li pear fruit from China with a disease apparently caused by an Alternaria sp. were intercepted at U.S. ports, raising concern because the disease was unknown in the United States. To identify the etiologic agent of the disease, single-spore cultures were established from intercepted fruit and were characterized by pathogenicity to wounded fruit, morphology, and sporulation pattern on three agar media, random amplified polymorphic DNA fragment pattern analysis, and species-specific polymerase chain reaction (PCR). These data indicated that the disease likely was caused by more than one species of Alternaria, and that none of the isolates from intercepted Ya Li fruit was A. alternata or A. gaisen. A species-specific PCR assay for A. gaisen demonstrated that no isolate from Ya Li was A. gaisen. A PCR assay for the AMT gene demonstrated that no isolate from Ya Li was A. ‘mali’. Because of these data and the absence of a description to accommodate at least one of the taxa involved, A. yaliinficiens R. G. Roberts sp. nov. is described, and the disease caused by this and other species of Alternaria on Ya Li pear from China is referred to as “chocolate spot of Ya Li pear” to distinguish it from black spot of Japanese pear.


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