Status of Japanese Dytiscus species (Coleoptera: Dytiscidae) based on mitochondrial DNA sequence data

We sequenced 628 base pairs of the mitochondrial cytochrome‐c oxidase subunit 1 (cox1) gene to investigate the genetic differentiation among Japanese Dytiscus diving beetles (Coleoptera: Dytiscidae). Dytiscus beetles are a charismatic part of the fauna of lakes, ponds and swamps. Three species are known from Japan: D. dauricus Gebler, 1832; D. marginalis czerskii Zeitzev * , 1953; and D. sharpi Wehncke, 1875. Adults of D. sharpi collected in Chiba and Ishikawa Prefectures were morphologically highly similar, but here we found that they differed by 20 cox1 base pairs (3.18%). Our results imply that conservation strategies especially for Japanese D. sharpi sharpi and D. sharpi validus Régimbart, 1899 might need to be adjusted to address the presence of two evolutionarily significant units.     

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.     

Phylogenetic relationships of Dysaphis pyri (Boyer de Fonscolombe) and Dysaphis reaumuri (Mordvilko) (Hemiptera, Sternorrhyncha: Aphididae): COI and EF-1α evidence

Dysaphis (Pomaphis) pyri (Boyer de Fonscolombe, 1841) and Dysaphis (Pomaphis) reaumuri (Mordvilko, 1928) are two holocyclic aphid species alternating between Pyrus (Rosaceae) and Galium (Rubiaceae). Comparative phylogenetic analysis was performed using partial mitochondrial cytochrome oxidase subunit I (COI) and nuclear elongation factor 1 alpha (EF-1??) sequences. Partial COI data indicate the possibility of the early divergence in the D. pyri?CD. reaumuri stem, which might have occurred even before the splitting of the common ancestral species of the D. reaumuri?CD. plantaginea complex. Such a conclusion seems to be compatible with the available data on host specificity, life cycles and distribution of both species. This introductory phylogenetic analysis based on partial COI and EF-1?? sequences indicates the need for reconsideration of the subgeneric structure in the genus Dysaphis.     

Mass breeding larvae of the critically endangered diving beetles Dytiscus sharpi sharpi and Dytiscus sharpi validus (Coleoptera: Dytiscidae)

For conservation purposes, and to supply critically endangered insects for laboratory use, a system for artificial breeding is crucial. However, in the case of carnivorous insects such as diving beetles, the larvae must be isolated because they are cannibalistic. We developed a method for mass breeding the larvae of two diving beetles, Dytiscus sharpi sharpi (Wehncke) and Dytiscus sharpi validus (Régimbart) (Coleoptera: Dytiscidae), which are designated critically endangered species in Japan. Ten to twenty larvae were raised in a small tank (35 cm × 25 cm × 10 cm; water depth 7 cm) with Rana ornativentris (Werner) tadpoles as prey. At low prey density, ~80 % of the larvae were cannibalized. At moderate prey density, 50–60 % were cannibalized. However, at high prey density, <3 % were cannibalized. Well-fed mass-bred adults were larger than individually bred and field-collected adults. This mass breeding method can be used for the conservation and breeding of these rare diving beetles in a manageable number of aquaria.     

Documenting neotropical diversity of phoronids with DNA barcoding of planktonic larvae

Phoronid larvae, actinotrochs, are beautiful and complicated organisms which have attracted as much, if not more, attention than their adult forms. We collected actinotrochs from the waters of the Pacific and Caribbean coasts of Panama, and used DNA barcoding of mtCOI, as well as 16S and 18S sequences, to estimate the diversity of phoronids in the region. We discovered three operational taxonomic units (OTUs) in the Bay of Panama on the Pacific coast and four OTUs in Bocas del Toro on the Caribbean coast. Not only did all OTUs differ from each other by >10% pairwise distance in COI, but they also differed from all phoronid sequences in GenBank, including the four species for which adults have been reported for the Pacific of Panama, Phoronopsis harmeri, Phoronis psammophila, Phoronis muelleri, and Phoronis hippocrepia. In each ocean region, one common OTU was more abundant and occurred more frequently than other OTUs in our samples. The other five OTUs were relatively rare, with only one to three individuals collected during the entire project. Species accumulation curves were relatively flat but suggest that at least one more species is likely to be present at each site. Actinotrochs from the seven sequenced OTUs had morphologies typical of species with non‐brooded planktotrophic development and, in some cases, may be distinguished by differences in pigmentation and the arrangement of blood masses. We found one larva with morphology typical of brooded planktotrophic larvae for which sequencing failed, bringing the total number of potential species detected to eight and representing >50% of the adult species currently recognized globally.     

Dytiscus latissimus and D. circumcinctus (Coleoptera,Dytiscidae) larvae as predators on three case-making caddis larvae

The predacious behaviour of Dytiscus circumcinctus and D. latissimus larvae was studied experimentally. When offered different prey simultaneously, D. latissimus larvae preferred cased caddis larvae relative to mayfly nymphs and isopods, whereas in D. circumcinctus the preference order was reversed. Notonectid nymphs and tadpoles were consumed in higher numbers by D. circumcinctus than by D. latissimus larvae. D. circumcinctus larvae and instar III larvae of D. latissimus most frequently captured caddis larvae through the case wall, whereas the instar I and II larvae of D. latissimus normally attacked the thorax of the exposed larva from above the front opening of the case. Limnephilus borealis, L. nigriceps and L. rhombicus caddis larvae differed in case structure, and they were all successfully captured by D. latissimus and D. circumcinctus instar II and III larvae. Neither capture success nor ingestion efficiency varied significantly between the two Dytiscus species or between different prey species. Instar II and III D. circumcinctus larvae had shorter reaction times than those of D. latissimus. The larger L. borealis and L. rhombicus larvae were preferred by the two last Dytiscus larval instars, and the handling time of these two prey was longer than that of L. nigriceps larvae.     

Molecular identification of dipteran pests (Diptera: Sciaroidea) from shiitake mushroom

On shiitake farms, mycophagous maggots can cause serious damage by preventing formation of the fruiting body. Recently, these pests have significantly reduced shiitake production in Korea. However, larvae and female adults cannot be identified due to their lack of morphological characteristics. Therefore, farmers and applied entomologists are unable to determine which species is the primary cause of the shiitake damage. In this study, mycophagous flies (colonized larvae) were collected from damaged shiitake farms and subsequently identified by matching identified males with the cytochrome c oxidase subunit I (COI) sequences from the larvae. Divergences of the COI sequences among the species discriminated the clusters clearly, and the mycophagous pests were identified as Camptomyia corticalis and C. heterobia. Interestingly, these two species coexisted under the bark of shiitake oak bed logs.     

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.     

Low temperature shock and chill-coma consequences for the red flour beetle (Tribolium castaneum) and the rice weevil (Sitophilus oryzae)

Insects face several (environmental) abiotic stressors, including low temperature, which cause the failure of neuromuscular function. Such exposure leads insects toa reversible comatose state termed chill-coma, but the consequences of this state for the organism biology were little explored. Here, the consequences of the chill-coma phase were investigated in two of the main stored product pest species – the red flour beetle Tribolium castaneum (larvae and adults) and the rice weevil Sitophilus oryzae (adults). For this purpose, a series of low-temperature shocks were used to estimate the chill-coma recovery time (CCRT), survival, nutrition and weight gain/growth of T. castaneum (larvae and adults) and S. oryzae, as well as the development of T. castaneum life stages. The relatively long CCRT was characteristic of beetle larvae, at different low-temperature shocks, and CCRT increased with decreasing temperatures and increasing exposure intervals for both pest species. The survival was little affected by the low-temperature shocks applied, but such shocks affected insect feeding and growth. Tribolium castaneum larvae was more sensitive than adults of both insect species. Moreover, the relative consumption and weight gain of S. oryzae adults were lower than those of T. castaneum adults and mainly larvae, while feeding deterrence was not affected by low temperature shocks, unlike food conversion efficiency. Low-temperature shocks, even under short duration at some temperatures, significantly delayed development. The lower the temperature and the higher the exposure period, the more delayed the development. Thus, the physiological costs of chill-coma are translated into life-history consequences, with potential implications for the management of this insect pest species in stored products and even more so on red flour beetles and rice weevils.     

Selective breeding and characterization of a black mealworm strain of Tenebrio molitor Linnaeus (Coleoptera: Tenebrionidae)

Larvae of Tenebrio molitor Linnaeus are edible insects and are approved as a food ingredient in Korea. They are typically yellow; however, rare black larvae have been found in breeding boxes at insect farms. It is not clear whether black larvae represent a different species that invaded and hybridized with the yellow larvae of T. molitor or whether T. molitor shows intraspecific color variation. In this study, we characterized and identified black larvae for applications in industrial fields as well as accurate breeding and management. First, in a comparative analysis, we did not detect differences in the morphological characteristics of yellow and black larvae and adults, with the exception of larval body color. For accurate species identification, molecular analyses (p-distances and neighbor-joining) were performed based on partial COI sequences of 33 yellow and seven black larvae. Genetic divergence between yellow and black larvae ranged from 0.0% to 2.1%, revealing intraspecific variation. A neighbor-joining analysis strongly supported the classification of the two morphs as a single species. Black larvae were separated from yellow larvae and maintained by selective breeding. As a result, black larvae were completely fixed in the F2 generation (F1 = 96% and F2 = 100%). Yellow and black larvae showed no significant differences in developmental characteristics and fecundity. These findings improve our understanding of diversity within an important edible insect species and contribute to quality assurance in the food industry based on clear species identification.     

Intraguild predation of the aphid parasitoid Aphelinus certus by Coccinella septempunctata and Harmonia axyridis

Coincidental intraguild predation is expected to be less disruptive to biological control than omnivorous intraguild predation, and strong intraguild predation is not expected to occur in natural systems. Coincidental intraguild predation in a foodweb involving introduced pest and natural enemy species was examined to determine whether intraguild predation would be disruptive of biological control services in soybean agroecosystems. Introduced natural enemies are important regulators of soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), populations in North America. Seven-spotted lady beetles, Coccinella septempunctata L., and multicolored Asian lady beetles, Harmonia axyridis Pallas (Coleoptera: Coccinellidae), are key predators of soybean aphid in North America while the chalcidoid wasp, Aphelinus certus Yasnosh (Hymenoptera: Aphelinidae), is the most common parasitoid of soybean aphid in Ontario, Canada. Predation of parasitized soybean aphids at two stages (newly parasitized aphids and mummified aphids) by adults and third instar larvae of both C. septempunctata and H. axyridis was examined in laboratory experiments. In choice experiments, all stages of lady beetles preferred non-parasitized aphids over mummified aphids. In cage experiments, third instar larvae and male and female adults of both lady beetles did not discriminate between newly parasitized and non-parasitized aphids. The influence of coincidental intraguild predation on the efficacy of parasitoids as biological control agents, and implications for soybean aphid management decisions based on natural enemies, are discussed.     

Identifying Fishes through DNA Barcodes and Microarrays

Background

International fish trade reached an import value of 62.8 billion Euro in 2006, of which 44.6% are covered by the European Union. Species identification is a key problem throughout the life cycle of fishes: from eggs and larvae to adults in fisheries research and control, as well as processed fish products in consumer protection.

Methodology/Principal Findings

This study aims to evaluate the applicability of the three mitochondrial genes 16S rRNA (16S), cytochrome b (cyt b), and cytochrome oxidase subunit I (COI) for the identification of 50 European marine fish species by combining techniques of “DNA barcoding” and microarrays. In a DNA barcoding approach, neighbour Joining (NJ) phylogenetic trees of 369 16S, 212 cyt b, and 447 COI sequences indicated that cyt b and COI are suitable for unambiguous identification, whereas 16S failed to discriminate closely related flatfish and gurnard species. In course of probe design for DNA microarray development, each of the markers yielded a high number of potentially species-specific probes in silico, although many of them were rejected based on microarray hybridisation experiments. None of the markers provided probes to discriminate the sibling flatfish and gurnard species. However, since 16S-probes were less negatively influenced by the “position of label” effect and showed the lowest rejection rate and the highest mean signal intensity, 16S is more suitable for DNA microarray probe design than cty b and COI. The large portion of rejected COI-probes after hybridisation experiments (>90%) renders the DNA barcoding marker as rather unsuitable for this high-throughput technology.

Conclusions/Significance

Based on these data, a DNA microarray containing 64 functional oligonucleotide probes for the identification of 30 out of the 50 fish species investigated was developed. It represents the next step towards an automated and easy-to-handle method to identify fish, ichthyoplankton, and fish products.     

Larvae and pupae of two North American darkling beetles (Coleoptera,Tenebrionidae, Stenochiinae), Glyptotus cribratus LeConte and Cibdelis blaschkei Mannerheim,with notes on ecological and behavioural similarities

This study describes and illustrates the larvae and pupae of two North American darkling beetles (Coleoptera: Tenebrionidae) in the subfamily Stenochiinae, Glyptotus cribratus LeConte from the southeastern United States, and Cibdelis blaschkei Mannerheim from California. Both species inhabit forested regions where adults and larvae occur in soft rotten dry wood of dead branches on living trees or in sections recently fallen from them. Species identity was confirmed by rearing of adults and pupae and the discovery of both in pupal cells with associated exuvia. Specimen label data and notes on habitats are provided. Antipredator defense structures and behaviour are noted for larvae and pupae of both species.     

Immunocompetence of the red turpentine beetle, Dendroctonus valens LeConte (Coleoptera: Curculionidae, Scolytinae): Variation between developmental stages and sexes in populations in China

Immune defense imposes fitness costs as well as benefits, so organisms should optimize, not maximize, their immune function through their life cycle. We investigated this issue in the red turpentine beetle, Dendroctonus valens LeConte (Coleoptera: Curculionidae, Scolytinae), which is a pine-killing invasive beetle in China, though it is usually considered as a secondary pest in its native range of North America. We hypothesized that pathogen pressure may affect these beetles differently throughout their life history. We measured the insect's immunocompetence throughout life, determining encapsulation ability and phenoloxidase activity in larval stages, pupae and adults. Pupae had the highest encapsulation ability, but encapsulation was not different between final instar larvae and adults. Phenoloxidase (PO) activity was highest in final instar larvae and pupae, followed by the second instar larvae and adults. Total phenoloxidase activity increased significantly from the second instar larval stage to pupae, and then decreased in adults. Although the second instar larvae had the lowest phenoloxidase activity, more than 90% of total PO existed in the hemolymph in the form of the active enzyme, as compared with pupae, in which over 60% of PO occurred as a proenzyme. Both active PO and total PO were much higher in females than in males, though no significant differences were detected between the encapsulation ability of male and female adults. This result suggests the existence of a sexual dimorphism of immunocompetence in D. valens adults. Variations in immunocompetence across developmental stages suggest that D. valens adopts diverse investment strategies in immunocompetence during different stages. Potential reasons for variation in immunocompetence among developmental stages and between the sexes of D. valens are discussed.     

Phylogeny and Diagnostic Markers of Six Tetranychus Species (Acarina: Tetranychidae) in Korea Based on the Mitochondrial Cytochrome Oxidase Subunit I

To understand the evolutionary relationship among six Tetranychus species in Korea, 598 nucleotides of cytochrome oxidase subunit I (COI) were sequenced and analyzed. The sequences were extremely rich in A+T (77% on average). The nucleotide differences in pairwise comparison among species ranged from 6.7% to 14.9%. Nucleotide substitutions based on all pairwise comparison showed 55% of transversions. The largest genetic distance was found between species T. viennensis and T. piercei. The inferred phylogenetic tree indicates that T. urticae, T. cinnabarinus and T. kanzawai are clustered together and showing sister-group relationship with T. truncatus at the highest bootstrap support. T. viennensis showed the largest genetic distance from all other species. T. piercei, a species recently found in Korea, was also distinct from the group composed of four species and also from T. viennensis. The restriction sites of Alu I, Dde I and Sau 3A were searched in the sequences of COI. The restriction fragment length polymorphisms (RFLPs) in COI genes and intraspecific variabilities of three restriction sites in diverse colonies of each species collected from different locations and various host plants were shown.     

Changes in tarsal morphology and attachment ability to rough surfaces during ontogenesis in the beetle Gastrophysa viridula (Coleoptera,Chrysomelidae)

Insects live in a three-dimensional space, and need to be able to attach to different types of surfaces in a variety of environmental and behavioral contexts. Adult leaf beetles possess great attachment ability due to their hairy attachment pads. In contrast, their larvae depend on smooth pads to attach to the same host plant. We tested friction forces generated by larvae and adults of dock leaf beetles Gastrophysa viridula on different rough surfaces, and found that adults generate much higher attachment to various substrates than larvae, but are more susceptible to completely losing attachment ability on surfaces with “critical” roughness. Furthermore, sex-specific setal morphology has the effect that attachment forces of male adults are generally higher than those of females when adjusted for body weight. The results are discussed in the context of development, ecology, and changing behavioral strategies of successive life stages.     

Genetic diversity of genus Vespa including an invaded species of V. velutina (Hymenoptera: Vespidae) in Korea inferred from DNA barcoding data

With about 5000 known species, the Vespidae is a large family belongs to order Hymenoptera. The genus Vespa with 22 species is one of the four genera of the subfamily Vespinae. In Korea, 10 species and subspecies are recognized. Because of their social behavior, their treat to human health and their impact in apiculture, the reliable and sometimes automated identification of these insects to species level are important. To test the efficacy of DNA barcoding method for identification of species of the genus Vespa in Korea, 30 samples of eight Korean species of genus Vespa were collected and mitochondrial DNAs of 658 bp fragment cytochrome oxidase subunit 1 (CO1) region were sequenced. A Bayesian Inference based on COI gene of the Korean Vespa species was constructed. The phylogenetic tree shoed that identification of all specimens is possible based on COI gene and we found strong relation between the sequences of the collected species from different localities in South Korea which clustered together with 100% support with sequences of the same species in GenBank. The results demonstrate that DNA barcoding is a useful technique for rapid and accurate species recognition in Korean Vespa species. The DNA barcode part of COI for V. binghami is provided for the first time that can help for identification of this species through DNA barcoding. Also, the genetic diversity among Korean Vespa velutina was zero suggests that the invasion might have occurred in a single event with small number of founders.