Molecular identification of two species of myiasis-causing Cuterebra by multiplex PCR and RFLP

The myiasis-causing flies Cuterebra grisea (Coquillet) and Cuterebra fontinella (Clark) (Diptera: Oestridae) are normally parasites of mice, predominantly of the genus Peromyscus. The morphological similarities of these species and the existence of intermediate morphotypes bearing characters of both species make the identification of adults problematic; furthermore the identification of larvae is apparently not possible. This study presents two molecular approaches to discriminate between these species using specific band patterns: (i) species-specific primers designed in the cytochrome oxidase II (COII) region used in multiplex polymerase chain reaction (PCR) and (ii) restriction fragment length polymorphism (RFLP) on amplified segments of cytochrome oxidase I (COI) gene. Both methods were tested on Cuterebra larvae and on adult museum specimens. The two techniques showed a clear difference between C. grisea and C. fontinella, although species-specific primers were more successful than RFLP for degraded DNA. No intraspecific variation in RFLP and species-specific amplifications were detected for the two species of Cuterebra. The results exhibit discrepancies between molecular and morphological identification, suggesting that some of the adults were misidentified.     

Butterflies of the South Pacific

ABSTRACT

Larvae of the invasive mosquito Culex quinquefasciatus Say are morphologically similar to those of the native Culex pervigilans Bergroth, yet distinguishing these species can be hampered by morphological variations in Cx. quinquefasciatus. We present detail about the extent of these variations in an urban population of Cx. quinquefasciatus in Auckland. To aid in identification of this exotic species, we provide images of key diagnostic characters and some observed exceptions to these. Details regarding variation in diagnostic characters for < 3rd instar and 3rd/4th instar larvae are given. Of the nine characters used for identification, three were highly consistent (dorsal papillae, mantle plate, pecten teeth); each observed in > 90% of larvae, although these characters were not always visible. Other characters were less reliable, for instance, the expected position of seta 1a-S in relation to the pecten teeth was observed in < 10% of larvae. Further exploration of regional morphological variation in both Cx. quinquefasciatus and Cx. pervigilans is recommended, ideally with associated molecular characterisation.     

COMPARATIVE PHYSIOLOGY AND BIOCHEMISTRY AND TAXONOMIC ASSIGNMENT OF THE CHLORELLA (CHLOROPHYCEAE) STRAINS OF THE CULTURE COLLECTION OF THE UNIVERSITY OF TEXAS AT AUSTIN1

The species of the genus Chlorella exhibit considerable biochemical and physiological differences. Therefore, it is important to select for and utilize in research or biotechnology correctly identified strains of the species having the most favorable properties for the respective project. We examined the Chlorella strains of the University of Texas collection at Austin, Texas, according to species-specific chemotaxonomic characters and assigned 58 strains to 10 well-established species (only 17 of these strains were correctly named before).     

Species-specific ability ofChlorella strains (Chlorophyceae) to form stable symbioses withHydra viridis

46 strains ofChlorella, identified by physiological and biochemical characters, were examined for their ability to form stable symbioses with aposymbioticHydra viridis. It was found to be a species-specific characteristic. Among the 15 taxa studied, onlyC. saccharophila var.ellipsoidea, C. saccharophila var.saccharophila, C. fusca var.vacuolata, C. kessleri, C. luteoviridis, andC. protothecoides formed stable symbioses withHydra viridis. Among the 11 known physiological and biochemical characters of theseChlorella species, only acid tolerance seems to be correlated with symbiosis: All symbiotic species are capable of growing at or below pH 4.0.     

Taxonomy of Electrogena antalyensis (Kazanci & Braasch, 1986) (Ephemeroptera,Heptageniidae)

Female imagines, subimagines, eggs and larvae of Electrogena antalyensis (Kazanci & Braasch) from Turkey are described for the first time. The results of a thorough analysis of larvae, based on the standard set of diagnostic characters for the identification of Electrogena species, are reported. The peculiarity of several characters places E. antalyensis in an isolated position within the genus Electrogena.     

Comparative morphology,biology and phylogeny of terminal‐instar larvae of the European species of Toryminae (Hym., Chalcidoidea,Torymidae) parasitoids of gall wasps (Hym. Cynipidae)

We present a phylogenetic and taxonomic study of the morphology and biology of the terminal‐instar larval stage of 19 species representing all the genera of Torymidae parasitoids of gall wasps in Europe, with the single exception of Megastigmus. The genera studied include Adontomerus Nikol'skaya, Idiomacromerus Crawford, Chalcimerus Steffan & Andriescu, Glyphomerus Förster, Pseudotorymus Masi and Torymus Dalman. We primarily used chaetotaxy and some head structures. The terminal‐instar larvae of all studied species are thoroughly described for the first time and illustrated with SEM images. We provide diagnostic characters for the family and the genera studied, and keys to genera and species for the identification of torymid larvae associated with cynipid galls. The majority of the torymid larvae studied are solitary monophagous parasitoids. Finally, to assess the potential use of larval characters in systematic studies of the family, a phylogenetic analysis of the studied taxa based on 42 larval morphological characters is proposed and compared with the current taxonomy of Torymidae. Our results suggest that body chaetotaxy, and characters of the head and mouthparts could be used for genera and species discrimination. © 2008 The Linnean Society of London, Zoological Journal of the Linnean Society, 2008, 154 , 676–721.     

DNA Taxonomy and the identification of immature insect stages: the true larva of Tauriphila argo (Hagen 1869) (Odonata: Anisoptera: Libellulidae)

For many insect taxa, larval morphology plays a decisive role in various fields like taxonomy, phylogeny or ecology. However, species identification is usually based on imaginal characters and the identification of larvae depends upon an established link to unequivocally identified imagines. This taxonomic correspondence of larvae and imagines is far from being established in many odonate species. We have employed a molecular approach to link larval and adult specimens in Tauriphila argo (Hagen, 1869). The sequenced mt SSU gene fragments of the reared female, supposedly a T. argo female, and a clearly identified male specimen of the species were identical. However, the larva of the reared female clearly differed from the described T. argo larva, previously matched to the species. From this observation, we conclude that the previously described larva of T. argo does not belong to this species because of too many phenotypic differences that far exceed the generally observed intraspecific variation.

It can be foreseen that the molecular approach will prove to be effective in identifying unknown larvae in many insect species. Additionally, the discrimination of sibling species or the linkage of allotypes and holotypes will become feasible with this approach.     

Molecular Species Identification of Newly Hatched Hawaiian Amphidromous Gobioid Larvae

This report describes a method for the determination of species identity of newly hatched larvae of five sympatric Hawaiian amphidromous gobioids (Lentipes concolor, Sicyopterus stimpsoni, Awaous guamensis, Stenogobius hawaiiensis, and Eleotris sandwichensis). Polymerase chain reaction (PCR) was used to amplify a homologous section of the cytochrome b (Cyt b) region of the mitochondrial genome (mtDNA) from adults of all five species. The resulting PCR-amplified DNA was subjected to restriction fragment length polymorphism (RFLP) analysis producing species-specific restriction patterns. PCR products from the five species were sequenced to substantiate correct amplification, restriction site locations, and fragment sizes. The sequence data were also used to construct a phylogenetic tree. Individual, newly hatched, wild-caught larvae of amphidromous gobioids of unknown species affinity were sorted into six morphotypes based on physical characteristics. These typed larvae and those from two species that spawned in captivity were subjected to the same molecular analysis as the adults. The RFLP results from adults and larvae were compared, allowing larval morphotypes to be assigned to the appropriate species. These comparisons permitted construction of an identification key to the newly hatched larvae of these species based solely on physical characteristics for use in future field studies. Received April 29, 1998; accepted September 30, 1998.     

Allozymatic diagnosis of four economically important Liriomyza species (Diptera, Agromyzidae)

Liriomyza bryoniae, L. huidobrensis, L. sativae and L. trifolii are leafminers of great economic importance; morphologically they are difficult to separate, many being inseparable at the pre-adult stages. Horizontal starch gel electrophoresis of larvae, pupae and adults was carried out in order to search for diagnostic allozymic characters. Analysis of the variation patterns at 15 genetic loci reveals that each species can be identified at all three stages of the life cycle. A biochemical key is presented.     

Phenotypic and Genotypic Characterization of <Emphasis Type="Italic">Enterococcus</Emphasis> spp. of Different Origins

Sixty-four strains of Enterococcus spp. of different origin were identified using traditional phenotypic, biochemical and cultural tests, together with molecular tests. API 20 STREP tests identified the species at a preliminary level, only one strain remaining unidentified. Strains belonging to the genus Enterococcus were tested with genus-specific primers, while species-level identification was carried out with the 16S–23S rDNA intergenic region (ITS) and species-specific primers for E. faecium and E. faecalis. Those strains found to be negative with the species-specific primers were subjected to 16S rDNA partial sequencing.     

A single-step multiplex PCR assay for the detection of European Peristenus spp., parasitoids of Lygus spp.

Lygus Hahn (Hemiptera: Miridae) are serious pests of agricultural and greenhouse crops throughout North America. In Europe, bivoltine Peristenus Förster (Hymenoptera: Braconidae) species have a significant impact on Lygus populations. Release and establishment of European P. digoneutis Loan in Lygus lineolaris (Palisot de Beauvois) populations in northeastern USA has renewed interest in the intended liberation of European parasitoids for Lygus control in Canada. Accurate identification of natural enemies is the cornerstone of biological control but conventional methods for identifying Peristenus species and estimating parasitism rates rely on tedious and time-consuming dissection and rearing methods. The present study describes species-specific PCR primers for three species of Peristenus, and the use of a multiplex PCR assay to detect P. digoneutis and P. stygicus Loan eggs and larvae from Lygus rugulipennis Poppius nymphs. Results indicate that the primers amplify uniquely sized, species-specific PCR products for the three species and are capable of detecting single eggs in parasitized nymphs within 3 days post-parasitism. Using a multiplex PCR assay, the primers maintain specificity and sensitivity, and allow detection of each of the three species in a single reaction. Although molecular diagnostics have previously been used in the identification of parasitoids and estimation of parasitism rates, this is the first time a single-step multiplex PCR protocol has been described.     

Discrimination of two Japanese water pennies,Eubrianax granicollis Lewis and E. ramicornis Kiesenwetter (Coleoptera: Psephenidae), based on laboratory rearing and molecular taxonomy

Two psephenid beetles, Eubrianax granicollis Lewis and E. ramicornis Kiesenwetter, are common species on the main islands of Japan (i.e. Honshu, Shikoku and Kyushu), but diagnostic characters for larval identification are unknown. Two types of field‐collected Eubrianax larvae from Honshu and Kyushu were discriminated based on the distributions of granules on the dorsal surface. These larvae were assigned to E. granicollis and E. ramicornis by comparing them with larvae of the two species obtained via laboratory rearing. The two species were also identified unambiguously on the basis of their mitochondrial cytochrome oxidase subunit I (COI) gene sequences. Larval and pupal morphology are described based on laboratory‐reared specimens.     

The larvae of the burrowing mayfly genus Tortopus (Ephemeroptera: Polymitarcyidae)

The larval stage of Tortopus is redescribed based on three species: T. puella from North America, the only species of the genus previously known from larva, and the larvae of T. obscuripennis and T. sarae from South America described here. Generic characters of the larva include: relatively large finger-like gill near base of maxilla, inner margin of mandibular tusks with a subdistal tubercle, straight or weakly convex frontal ridge present between antennae, reduced unilamellated gill on abdominal segment I. Additionally the male imagines of both Neotropical species are described for the first time, and T. obscuripennis is recorded from Bolivia. Diagnoses, SEM photographs, and illustrations are given for the new stages described and for the identification of the three Tortopus species known as larvae.     

Molecular identification of Chironomus spp. (Diptera) for biomonitoring of aquatic ecosystems

Abstract   Chironomidae larvae often represent a major component of the benthic fauna in inland water bodies and are used frequently as bioindicators of ecosystem health. The genus Chironomus is a recognised indicator of organic enrichment and has been used extensively in the northern hemisphere and New Zealand in ecotoxicological studies. However, similar use of Chironomus in Australia is limited due to the presence of cryptic species that restrict the collection of information on species-specific responses to environmental stress. To address the problems associated with species identification, we have used PCR-RFLP of the mitochondrial COI gene to develop DNA profiles for nine common Australian Chironomus species. Species-specific haplotypes were identified using reference taxa previously identified by cytological analysis, and verified with field specimens collected from seven wetlands around Melbourne. This research provides an effective tool for species identification of this ubiquitous and often abundant genus that will provide the basis of obtaining species-specific information to inform on the health of aquatic ecosystems.     

Identification of Orchidaceae species of Northern West of Syria based on chloroplast DNA

The plant family Orchidaceae has a great economic value (ornamental and medical uses, beside the aromatic features). Traditionally, identification of orchid species has relied heavily on morphological features. These features, however, are either not variable enough between species or too plastic to be used for identification at the species level. DNA-based markers could be the alternative strategy towards an accurate and robust identification of those species. Since the chloroplast DNA has a lower level of evolution compared to the nuclear genome, an attempt was made in this study to investigate polymorphism in the chloroplast DNA among orchid species distributed in North-West region of Syria using Cleaved Amplified Polymorphic Sequence (CAPS) technique for developing markers for the diagnosis of targeted species. CAPS analysis was carried out on 34 orchid samples that represent all species observed in the region. Universal primers were used to amplify targeted chloroplast regions. Generated PCR products were digested with various restriction enzymes. CAPS results revealed high polymorphism among species examined. This polymorphism was suffiecient for the diagnosis of all of those species apart from five species (Ophrys fuciflora (one sample), Oph. bornmuelleri, Ophrys sp., scolopax and Oph. argolica). Availability of such species-specific markers would ensure more authentic identification of orchid species compared to morphological characters and can be regarded as a valuable tool to guide in conservation programs of orchid species in Syria. CAPS data generated were converted to an identification key for orchid species studied.     

Pathology of black bass hepatic tissue infected with larvae of the tapeworm Proteocephalm ambloplitis

Intra- and interspecific differences in pigmentation between finfold larvae of the three most abundant cyprinids in Dutch eutrophic waters, bream, white bream and roach, were studied, using laboratory-raised larvae in the length range 8–11 mm. Because the internal pigmentation of the larvae has been used for identification, some attention is paid to the effects of different ways of fixation and preservation on transparency. The size and the shape of the melanophores, as described in the literature, could not be used as identification characters because of too much intraspecific variation and the effects of light conditions at the moment of fixation. Three characters proved to be significant for the identification of the larvae of the three species. Roach can be distinguished from bream and white bream by the pattern of melanophores on the belly. A second character is the pigmentation of the ventral aorta, which is only found in white bream. Lastly bream shows an irregular pattern of melanophores on the dorsal side, in contrast to roach which has a regular pattern.     

A molecular protocol using quenched FRET probes for the quarantine surveillance of Tilletia indica, the causal agent of Karnal bunt of wheat

The current surveillance protocol for Karnal bunt of wheat in most countries, including the USA, European Union (EU), and Australia, involves the tentative identification of the spores based on morphology followed by a molecular analysis. Germination of spores is required for confirmation which incurs a delay of about two weeks, which is highly unsatisfactory in a quarantine situation. A two-step PCR protocol using FRET probes for the direct detection and identification of Tilletia indica from a very few number of spores (≤10) is presented. The protocol involves amplification of the ITS1 DNA segment in the highly repeated rDNA unit from any Tilletia species, followed by FRET analysis to detect and unequivocably distinguish T. indica and the closely related T. walkeri. This rapid, highly sensitive, fluorescent molecular tool is species-specific, and could supersede the conventional microscopic diagnosis used in a quarantine surveillance protocol for Karnal bunt which is often confounded by overlapping morphological characters of closely related species.     

Distinguishing features of immature stages of Panchaetothripinae (Thysanoptera: Thripidae) known in New Zealand

Diagnostic morphological characters of the juvenile Panchaetothripinae in New Zealand are illustrated. Keys developed enable colonies with only immature stages to be identified without needing to rear adults. Live larvae or larvae in ethanol are distinguished by the presence of expanded tips of body setae (Parthenothrips dracaenae), the absence of setae at the abdomen tip (Hercinothrips bicinctus), setae at abdomen tip not longer than abdominal tip width (Heliothrips haemorrhoidalis) and abdominal tip setae longer than abdominal tip width (Sigmothrips aotearoana, endemic species). The presence or absence of spine-like setae on abdominal segments 9 and 10, and the number and length of setae on the wing buds, enable identification of pupae. Abdominal spine-like setae were on the prepupa and pupa of H. bicinctus and S. aotearoana, species that pupate off the plant, and are probably defensive structures. This is the first record of spine-like setae on segment 10 of terebrantian pupae.     

Identifying the ichthyoplankton of a coral reef using DNA barcodes

Marine fishes exhibit spectacular phenotypic changes during their ontogeny, and the identification of their early stages is challenging due to the paucity of diagnostic morphological characters at the species level. Meanwhile, the importance of early life stages in dispersal and connectivity has recently experienced an increasing interest in conservation programmes for coral reef fishes. This study aims at assessing the effectiveness of DNA barcoding for the automated identification of coral reef fish larvae through large‐scale ecosystemic sampling. Fish larvae were mainly collected using bongo nets and light traps around Moorea between September 2008 and August 2010 in 10 sites distributed in open waters. Fish larvae ranged from 2 to 100 mm of total length, with the most abundant individuals being <5 mm. Among the 505 individuals DNA barcoded, 373 larvae (i.e. 75%) were identified to the species level. A total of 106 species were detected, among which 11 corresponded to pelagic and bathypelagic species, while 95 corresponded to species observed at the adult stage on neighbouring reefs. This study highlights the benefits and pitfalls of using standardized molecular systems for species identification and illustrates the new possibilities enabled by DNA barcoding for future work on coral reef fish larval ecology.     

Residual mosquitoes in the northern Adriatic seacoast 50 years after the disappearance of malaria

The Northern Adriatic Sea littoral was heavily malarious; intensive land drainages, agricultural development and socioeconomic improvement were the key factors which led to malaria eradication, sped up by indoor insecticide spraying, achieved soon after World War II. Regular observations on anophelism were carried out by the Istituto Interprovinciale per la Lotta Antimalarica nelle Venezie from middle 20's until early 60's. The main vector was Anopheles sacharovi, a species which typically bred in coastal brackish swamps; other species were An. atroparvus (which was a probable secondary vector) and the usually strictly zoophilic An. maculipennis, An. melanoon, An. messeae and An. subalpinus. From 1995 to 1997 surveys were carried out in order to review the genus Anopheles in the coastal area of Friuli-Venezia Giulia and Veneto regions. A total of 11,346 females were collected from animal shelters (cow-shed, pigsties, horse stables) of 52 sites along 180 km of coast crossing 5 provinces (from North: Gorizia, Udine, Venezia, Padova and Rovigo). All specimens belonging to the An. maculipennis complex were scored for the presence of the differential characters of An. sacharovi, the only species of the complex morphologically characterized at the adult stage. The examination of morphological characters of single egg batches obtained from field collected females was the main diagnostic tool for the other species. Species identification was subject to confirmation by larval chaetotaxy analysis (number of branches of antepalmate hairs of IV and V abdominal segments) in representative samples of laboratory-reared mature larvae, while biochemical analysis (enzyme electrophoresis) on some samples of identified females was performed in the laboratory of Prof. L. Bullini and Dr. R. Cianchi of the University of Rome "La Sapienza" and partly in our laboratory. No An. sacharovi female was recorded. The examination of 6,361 single ovipositions led to the identification of three species of the An. maculipennis complex: An. atroparvus, An. maculipennis and An. messeae; An. claviger s.str. was also recorded. Larval chaetotaxy examination carried out on 1,608 larvae and the biochemical identification of 467 females confirmed the previous diagnosis based on egg characters. The relative frequency of the three species varied depending on the site: An. maculipennis was the most abundant species north of Venice; south of Venice, and particularly in the Po river delta, the most abundant species were An. atroparvus and, in some sites, An. messeae. In view of the high density recorded for An. atroparvus in some sites (corresponding to various thousands females in a single animal shelter), the vectorial capacity values may be significant and should be assessed.