Inferring larval taxonomy and morphology in Maladera species (Coleoptera: Scarabaeidae: Sericini) using DNA taxonomy tools

Based on a comparative molecular study of scarab chafers we matched adult and larval instars to identify and describe unknown larvae of Sericini. Here, we use for the first time a two‐fold DNA taxonomy approach based on: (i) mitochondrial and nuclear DNA markers of a local sample (from Nepal) of adults and larvae, in combination with character and tree‐based species delimitation methods; and (ii) a global search of cytochrome c oxidase subunit I (cox1) sequences with GenBank data. In the latter analysis we used a sequence of a specimen that resulted in the first analysis conspecific with the larvae of Maladera affinis (Blanchard) as the query sequence in GenBank, and checked in a minimum evolution tree whether larva–adult matches from the local approach were altered through interference with other taxa of the worldwide database. Both approaches unambiguously identified the unknown larvae as belonging to M. affinis and Maladera cardoni (Brenske). Based on this robust framework of taxonomic identification we could associate names to the larval morphology of the third larval instar of these two Nepalese Maladera species, which are both known for their economical importance in agriculture. They are described here in detail and are compared with known related taxa, especially with Maladera castanea (Arrow).     

The evolutionary history of maternal plant-manipulation and larval feeding behaviours in attelabid weevils (Coleoptera; Curculionoidea)

Attelabid weevils manipulate specific structures of their host plants in a species-specific manner, e.g., cutting a shoot, cutting a leaf, rolling a leaf, or constructing sophisticated wrapped leaf rolls, presumably to secure the survivorship of eggs or larvae. To depict the evolutionary history of maternal plant-manipulation behaviours and larval feeding strategies of the family Attelabidae, molecular phylogenetic analyses were conducted by sequencing the nuclear 18S and 28S ribosomal DNA and the mitochondrial cytochrome oxidase subunit I genes. Our analyses indicated that the attelabid weevils form a monophyletic group, and that maternal plant-cutting behaviour originated in a common ancestor of Attelabidae, but was subsequently lost in several lineages. Monophyly of the subfamily Attelabinae was also recovered with high support, but the subfamily Rhynchitinae was not recovered as monophyletic. By employing maximum-likelihood-based ancestral state reconstructions, larval leaf-blade feeding was inferred to have evolved from boring of cut shoots/petioles. Moreover, maternal leaf-rolling behaviours likely originated independently in the Attelabinae and Byctiscini lineages, and in several Deporaini lineages. As the sophisticated behaviours constructing wrapped leaf rolls of Attelabinae originated only once and has not been lost from the lineage, these complex and innovative behaviours may have contributed to the success and diversification of the lineage.     

Application of real-time PCR for simultaneous identification and quantification of larval abalone

A paucity of direct studies of marine invertebrate larval dispersal motivated the development of a high-throughput method for identification and quantification of pinto abalone (Haliotis kamtschatkana) larvae in seawater. DNA extracted from sample retentate provided template to screen for species-specific cytochrome oxidase I (COI) mitochondrial DNA sequence via quantitative PCR (QPCR) technology. Primers and a dual-labeled probe were designed and used to identify and quantify DNA from the target species in blind tests of unknown samples alongside a standard template quantity series. Quantity estimates derived from QPCR standard curves were verified via direct enumeration of larvae using light microscopy. Multiplex reactions containing an internal positive control minimized underestimation of quantity and false negatives via partial or full PCR inhibition, respectively. Planned controlled field release and collection experiments to examine larval dispersion patterns via sampling over short and long postrelease times anticipate similar QPCR assays for other marine invertebrate species to aid investigations of larval dispersal in the marine environment.     

Genetic variation tracks ecological segregation in Pacific island black flies

Geographic isolation is widely viewed as a key component of insular radiations on islands. However, strong ecological affinities may also reinforce isolation and promote genetic divergence. The black fly fauna in the Society Islands French Polynesia is notable for the number of closely related endemic species (31), and the morphological and habitat diversity of the larvae. Here, we measure ecological and morphological differences within and between two closely related species, Simulium oviceps and Simulium dussertorum and relate these differences to genetic distance. Phylogenetic analyses of a 920 bp fragment of the cytochrome oxidase I (COI) gene revealed a well-supported, ecologically divergent S. oviceps clade (larvae found in rivers instead of cascades) that shows little morphological differentiation. For both S. oviceps and S. dussertorum, genetic distance among populations is related to larval habitat, with cascade populations showing greater isolation from each other than river populations. Our data support the hypothesis that larval ecological shifts have played a role in the radiation of this black fly fauna.     

Molecular identification and distribution of leatherjackets (Diptera: Tipulidae) in U.K. agricultural grassland

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Molecular phylogeny of the green lacewings (Neuroptera: Chrysopidae)

Abstract  The first quantitative analysis of phylogenetic relationships of green lacewings (Chrysopidae) is presented based on DNA sequence data. A single nuclear and two mitochondrial genes are used in the analysis: carbomoylphosphate synthase (CPS) domain of carbamoyl-phosphate synthetase-aspartate transcarbamoylase-dihydroorotase (CAD) (i.e. rudimentary locus), large subunit ribosomal gene (16S) and cytochrome oxidase I (COI). This study represents the first use of the CAD gene to investigate phylogenetic relationships of the lacewings. DNA sequences for 33 chrysopid species from 18 genera, representing all subfamilies and tribes, were compared with outgroups sampled from families Hemerobiidae, Osmylidae and Polystoechotidae. Parsimony analyses of the combined data set recovered all of the previously established subfamilial and tribal groups as monophyletic clades (although relatively weakly supported) except Apochrysinae sensu lato . The enigmatic Nothancyla verreauxi Navás has historically been difficult to place in a subfamily group based on morphological characteristics; molecular data presented herein do not adequately resolve this problem.     

A phylogenetic analysis of relationships among genera of Conopidae (Diptera) based on molecular and morphological data

Members of the family Conopidae (Diptera) have been the focus of little targeted phylogenetic research. The most comprehensive test of phylogenetic support for the present subfamily classification of Conopidae is presented here using 66 specimens, including 59 species of Conopidae and seven outgroup taxa. Relationships among subfamily clades are also explored. A total of 6824 bp of DNA sequence data from five gene regions (12S ribosomal DNA, cytochrome c oxidase subunit I, cytochrome b, 28S ribosomal DNA and alanyl‐tRNA synthetase) are combined with 111 morphological characters in a combined analysis using both parsimony and Bayesian methods. Parsimony analysis recovers three shortest trees. Bayesian analysis recovers a nearly identical tree. Five monophyletic subfamilies of Conopidae are recovered. The rarely acknowledged Zodioninae is restored, including the genera Zodion and Parazodion. The genus Sicus is removed from Myopinae. Morphological synapomorphies are discussed for each subfamily and inter‐subfamily clade, including a comprehensive review of the character interpretaions of previous authors. Included are detailed comparative illustrations of male and female genitalia of representatives of all five subfamilies with new morphological interpretation.     

High-throughput molecular identification of fish eggs using multiplex suspension bead arrays

The location and abundance of fish eggs provide information concerning the timing and location of spawning activities and can provide fishery-independent estimates of spawning biomass. However, the full value of egg and larval surveys is severely restricted because many species' eggs and larvae are morphologically similar, making species-level identification difficult. Recent efforts have shown that nearly all species of fish may be identified by mitochondrial DNA (mtDNA) sequences (e.g. via 'DNA barcoding'). By taking advantage of a DNA barcode database, we have developed oligonucleotide probes for 23 marine fish species that produce pelagic eggs commonly found in California waters. Probes were coupled to fluorescent microspheres to create a suspension bead array. Biotin-labelled primers were used to amplify the mitochondrial cytochrome oxidase subunit I (COI) and 16S ribosomal rRNA genes from individual fish eggs. The amplicons were then hybridized to the bead array, and after the addition of a reporter fluorophore, samples were analysed by flow cytometry with Luminex 100 instrumentation. Probes specifically targeted eggs that are abundant and/or from morphologically indistinguishable species pairs. Results showed that the 33 different probes designed for this study accurately identified all samples when PCR was successful. Suspension bead arrays have a number of benefits over other methods of molecular identification; these arrays permit high multiplexing, simple addition of new probes, high throughput and lower cost than DNA sequencing. The increasing availability of DNA barcode data for numerous fish faunas worldwide suggests that bead arrays could be developed and widely used for fish egg, larval and tissue identifications.     

The first report of peritoneal tetrathyridiosis in squirrel monkey (Saimiri sciureus)

This report describes a case of peritoneal larval cestodiasis caused by tetrathyridia of Mesocestoides sp. in an adult female squirrel monkey. The monkey had lived in a zoological garden in Japan and had a clinical history of wasting. At necropsy, numerous whitish oval masses were found in the liver and peritoneal cavity. These masses contained larval cestodes. Morphological observation and molecular analyses of the mitochondrial 12S ribosomal RNA gene and cytochrome c oxidase subunit 1 gene sequences allowed us to identify the larva as the tetrathyridium of Mesocestoides sp. This is the first report of Mesocestoides larvae in a squirrel monkey in Japan.     

Correlation of oxygen consumption, cytochrome c oxidase, and cytochrome c oxidase subunit I gene expression in the termination of larval diapause in the bamboo borer, Omphisa fuscidentalis

The moth Omphisa fuscidentalis (Lepidoptera, Pyralidae) is a univoltine insect with a larval diapause period lasting up to 9 months. We studied changes in O(2) consumption in conjunction with cytochrome c oxidase activity and cytochrome c oxidase subunit I (cox1) gene expression. O(2) consumption changed within a day, showing a supradian rhythm with a ca.12-h cycle at 25 degrees C. During the first two-thirds of the diapause period, from October to March, O(2) consumption was constant until January and then increased by March. Topical application of methoprene, a juvenile hormone analog (JHA), to diapausing larvae terminated the diapause and was associated with an increase in O(2) consumption rate at diapause termination. In JHA-treated larvae, cytochrome c oxidase activity in fat bodies was high at the beginning of the prepupal period and highest at pupation. cox1 expression in fat bodies displayed a transient peak 8 days after JHA application and peaked in the prepupal period. Taken together, our results show that the break of diapause by JHA is associated with the activation of cox1, bringing about an increase in cytochrome c oxidase activity, followed by an increase in O(2) consumption rate.     

New family of allomorphic jellyfishes, Drymonematidae (Scyphozoa, Discomedusae), emphasizes evolution in the functional morphology and trophic ecology of gelatinous zooplankton

Molecular analyses have revealed many cryptic species in the oceans, often permitting small morphological differences to be recognized as diagnosing species, but less commonly leading to consideration of cryptic ecology. Here, based on analyses of three nuclear DNA sequence markers (ribosomal 18S, 28S, and internal transcribed spacer 1 [ITS1]), two mitochondrial DNA markers (cytochrome c oxidase subunit I [COI] and ribosomal 16S), and 55 morphological features, we revise the classification of the enigmatic jellyfish genus Drymonema. We describe a new scyphozoan family, Drymonematidae, elevating the previous subfamily Drymonemidae to accommodate three species: the type species D. dalmatinum from the Mediterranean region, for which we identify a neotype; the western South Atlantic species D. gorgo; and a new species, D. larsoni from the western Atlantic and Caribbean, which also is described here. This revision emphasizes the remarkable morphological disparity of Drymonematidae from all other scyphomedusae, including allometric growth of the bell margin distal of the rhopalia, an annular zone of tentacles on the subumbrella, and ontogenetic loss of gastric filaments. Anatomical innovations are likely functionally related to predatory specialization on large gelatinous zooplankton, most notably the phylogenetically younger moon jellyfish Aurelia, indicating evolution of the feeding niche in Drymonematidae. This family-level revision contributes to the growing body of evidence that scyphomedusae are far more taxonomically rich, their biogeography is a more detailed mosaic, and their phenotypes are more nuanced than traditionally thought. Ecological and evolutionary responses to environmental change, past or future, are likely to be commensurately diverse.     

Species composition and seasonal occurrence of Phyllophaga (Coleoptera: Scarabaeidae) infesting intensely managed Bermudagrass in Oklahoma

Larvae of Phyllophaga spp. (Coleoptera: Scarabaeidae) are important turfgrass pests in many regions of the United States. However, not all of the species associated with turfgrass are known, including species most likely to be of economic concern in Oklahoma turfgrasses, especially Bermuda grass. This study documented the species composition and seasonal occurrence of Phyllophaga associated with high maintenance Bermuda grass turf in Oklahoma over a 2-yr period. In 2005 and 2006, adult Phyllophaga spp. were collected with blacklight traps from selected golf courses throughout Oklahoma Phyllophaga larvae were obtained from Bermuda grass stands at selected sod production facilities adjacent to or near the light traps. We collected 20 species of Phyllophaga beetles in light traps, and nine species of Phyllophaga larvae from turfgrass. Peak flight periods for most species occurred in May and June, but some were captured as early as mid-April and others as late as September. The cytochrome c oxidase I (COI) gene from adults and larvae was amplified using polymerase chain reaction, sequenced, and then used to compare larval DNA against DNA from identified adults. These results confirmed the validity of using COI sequences to identify species of some Phyllophaga larvae. The identifications will aid in optimizing the timing of insecticide applications against Phyllophaga white grubs as discussed.     

Morphological and molecular characterization of Anopheles (Anopheles) maculipennis Meigen, type species of the genus and nominotypical member of the Maculipennis Complex

Abstract. Adults and larvae of Anopheles maculipennis Meigen were collected in Greece in May and June 2001. Larvae and the progeny of wild‐caught females were individually reared to obtain samples of all life stages for integrated morphological and molecular study. Specimens were identified on the basis of egg morphology and correlation of their ITS2 rDNA sequences with those in GenBank. The adult, pupal, larval (fourth‐instar) and egg stages are described, and all stages except the egg are illustrated. Partial sequence data are provided for the mitochondrial cytochrome c oxidase I (COI) gene and complete sequences for the nuclear ITS2 region. Additionally, the bionomics and distribution of the species are reviewed, and its taxonomy and systematics are discussed. This study provides the first fully integrated morphological and molecular assessment of An. maculipennis, the nominotypical member of the Maculipennis Complex, and the type species of genus Anopheles, and serves as a foundation for further studies of the complex and subfamily Anophelinae.     

Molecular characterization of the mitochondrial cytochrome oxidase I gene of Oestridae species causing obligate myiasis

A 688-bp region of the mitochondrial cytochrome oxidase I gene was sequenced from larvae of 18 species of Oestridae causing obligate myiasis. Larvae belonged to the four Oestridae subfamilies (Cuterebrinae, Gasterophilinae, Hypodermatinae and Oestrinae), which are commonly found throughout the world. Analysis of both nucleotide and amino acid data was performed. Nucleotide sequences included 385 conserved sites and 303 variable sites; mean nucleotide variation between all species was 18.1% and variation within each subfamily ranged from 5.3% to 13.34%. Intraspecific pairwise divergences ranged from 0.14% to 1.59%, and interspecific variation ranged from 0.7% to 27%. Of the 229 amino acids, 76 were variable (60 of which were phylogenetically informative), with some highly conserved residues identified within each subfamily. Phylogenetic analysis showed a strong divergence among the four subfamilies, concordant with classical taxonomy based on morphological and biological features. This study provides the first molecular data set for myiasis-causing Oestridae species, providing an essential database for the molecular identification of these parasites and the assessment of phylogenetic relationships within family Oestridae.     

Differentiation of the dragonfly genus Davidius (Odonata: Gomphidae) in Japan inferred from mitochondrial and nuclear gene genealogies

To infer the differentiation of Japanese Davidius dragonflies, we investigated the genealogies of the mitochondrial cytochrome oxidase subunit I gene (COI) and the nuclear ribosomal RNA gene region encompassing 18S, ITS1, 5.8S, and ITS2 sequences for three species endemic to Japan--Davidius nanus, D. fujiama, and D. moiwanus--as well as D. lunatus from the Korean Peninsula. According to the mitochondrial and nuclear gene genealogies, D. nanus and D. moiwanus are closely related and are sister to the continental species D. lunatus, whereas D. fujiama differentiated from an ancestor of the other three species. Although the mitochondrial DNA data did not resolve the relationships between D. nanus and three D. moiwanus subspecies, the nuclear DNA data indicate the monophyly of D. moiwanus and its subspecies. The nuclear gene genealogy suggests that isolated wetlands used by larval D. moiwanus derive from the ancestral riverine habitats of D. nanus and other Davidius species. The COI sequence divergence among local populations was much greater in D. moiwanus than in D. nanus, which may be the result of differences in the dispersal ranges associated with the habitat types of these species.     

Is assessment of oviposition sites using conspecific larval cues a general mechanism in aphidophagous ladybirds (Coccinellidae)?

Aphidophagous ladybirds of the Coccinellinae subfamily are deterred from oviposition in the presence of chemical cues deposited by conspecific larvae, therefore avoiding the detrimental effects of competition and cannibalism to their offspring. However, it is still unknown whether aphidophagous species from other Coccinellidae subfamilies similarly behave. Here, we investigate this question for species of the Scymninae subfamily. A GC‐MS analysis of Scymnus interruptus (Goeze) larval tracks shows that larvae deposit a cocktail of hydrocarbons containing at least five branched‐chain alkanes. Furthermore, our experiments on the oviposition behaviour of S. interruptus and S. nubilus (Mulsant) in the presence of conspecific larval tracks and of conspecific larval wax covering Scymninae larvae show that females do not refrain from ovipositing in the presence of these larval cues. We recommend that more attention is paid to the role of Scymnus spp. in the regulation of aphids because their oviposition strategy might strengthen aphid suppression in agrosystems.