Proliferation and copy number variation of BEL-like long terminal repeat retrotransposons within the Diabrotica virgifera virgifera genome

The proliferation of retrotransposons within a genome can contribute to increased size and affect the function of eukaryotic genes. BEL/Pao-like long-terminal repeat (LTR) retrotransposons were annotated from the highly adaptable insect species Diabrotica virgifera virgifera, the Western corn rootworm, using survey sequences from bacterial artificial chromosome (BAC) inserts and contigs derived from a low coverage next-generation genome sequence assembly. Eleven unique D. v. virgifera BEL elements were identified that contained full-length gag–pol coding sequences, whereas 88 different partial coding regions were characterized from partially assembled elements. Estimated genome copy number for full and partial BEL-like elements ranged from ~ 8 to 1582 among individual contigs using a normalized depth of coverage (DOC) among Illumina HiSeq reads (total genome copy number ~ 8821). BEL element copy number was correlated among different D. v. virgifera populations (R² = 0.9846), but individual element numbers varied ≤ 1.68-fold and the total number varied by ~ 527 copies. These data indicate that BEL element proliferation likely contributed to a large genome size, and suggest that differences in copy number are a source of genetic variability among D. v. virgifera.     

Comparative flight behavior of Diabrotica virgifera virgifera and Diabrotica barberi in the laboratory

A tethered flight system was used to characterize and compare the flight behavior of western, Diabrotica virgifera virgifera LeConte (WCR), and northern, Diabrotica barberi (Smith and Lawrence) (NCR), corn rootworms in the laboratory. Distributions of flight durations were skewed towards short flights for both species regardless of sex or age and there was high variability in flight durations and frequency in individual beetles. Flight distributions for WCR were distinctly bimodal, reflecting a dichotomy between short trivial flights and much longer sustained flights. Trivial flight performance did not vary with age but did differ between species. In comparison to WCR, NCR took shorter flights but had a higher frequency of flights over a 23-h period. Overall, WCR beetles logged substantially more flight time in the same period of time. The proportion of individual WCR undertaking sustained flight was relatively low (<24%) and, for females, declined along with sustained flight durations as a function of age. Periodicity of trivial flight did not differ betweer the species. In general, flight activity was greatest during early morning and early evening hours. No correlations were found between any feature of flight performance and body size or wing loading ratios.

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Résumé Un système de vol captif a été utilisé pour caractériser et comparer au laboratoire les comportements de vol de D. virgifera virgifera et D. barberi. La distribution des durées de vol est nettement biaisée avec la grande majorité des vols durant moins d'une minute quels que soient le sexe et l'âge. Il y a une grande variabilité interindividuelle tant en durée qu'en fréquence de vol. Les distributions des durées de vol en fonction du sexe sont bimodales chez D. virgifera virgifera, mais pas chez D. barberi, ce qui reflète la séparation entre vols brefs ordinaires et long vols prolongés. Les vols brefs ordinaires ne dépendent pas beaucoup de l'âge ou du sexe mais diffèrent suivant l'espèce. D. barberi a eu des vols plus brefs, mais en a effectué plus pendant une période de 23 heures. Au total, D. virgifera virgifera a consacré nettement plus de temps au vol pendant la même période; mais, chez cette espèce la proportion d'individus effectuant des vols soutenus était relativement faible (<24%), avec une diminution de leur nombre avec l'âge chez les femelles; le nombre le plus élevé étant chez les jeunes femelles. La périodicité des vols ordinaires n'a dépendu ni du sexe, ni de l'espèce; l'activité de vol a été en général plus importante tôt le matin et en début de soirée. Aucune corrélation n'a été trouvée entre une quelconque performance de vol et la taille du corps ou la charge alaire.

     

Evolutionary directional asymmetry and shape variation in Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae): an example using hind wings

The western corn rootworm Diabrotica virgifera virgifera LeConte is a pest of maize in the USA and Europe and especially a problem in particular regions of Croatia. In the present study, patterns of variation in hind wing shape were examined. The first objective was to examine the influence of soil type on 10 populations of D. v. virgifera sampled from three regions in Croatia that differed according to edaphic factors and climate. The second objective was to investigate the potential evolutionary presence of directional asymmetry on hind wings. Geometric morphometrics was used to examine these objectives by quantifying the morphological variation within and among individuals and populations. Overall, D. v. virgifera hind wing shape changed according to major soil type classifications in Croatia. The three hind wing morphotypes found varied because of basal radial vein differences, related to landmarks 1, 3, 7, and 14. The findings of the present study show that hind wing shape in D. v. virgifera can be used to differentiate populations based on edaphic factors and may have application as a monitoring tool in the integrated management of D. v. virgifera. In an evolutionary context, the presence of directional asymmetry in the hind wings of D. v. virgifera adds to the ever growing data on the evolution of insect wings. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 110–118.     

Spatial clustering of Diabrotica virgifera virgifera and Agriotes ustulatus in small-scale maize fields without topographic relief drift

The soil‐living larvae of Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) and Agriotes ustulatus Schaller (Coleoptera: Elateridae) can cause economic damage to maize roots, Zea mays L. (Poaceae). This study investigated the spatial clustering of both pests in four small‐scale maize fields in southern Hungary, where clustering had been observed but not expected due to the lack of topographic relief drifts and soil structuring. Between 2000 and 2002, numbers of D. v. virgifera larvae and adults and of A. ustulatus larvae were determined at four randomly chosen georeferenced maize plants in each of 24 plots per field. Soil moisture, soil bulk density, and vegetational characteristics were assessed. Moran's I test for spatial autocorrelations, semivariogram analyses, and interpolated mapping revealed that D. v. virgifera larvae and adults were spatially clustered in 67 and 50% of cases, respectively. Larvae of A. ustulatus were clustered in 75% of cases. Diabrotica virgifera virgifera larval distributions were mainly determined by increasing weed density (negative correlation), in particular with high densities of Cirsium arvense (L.) (Asteraceae), as well as by increasing soil moisture (negative correlation). Adult distributions of D. v. virgifera were mainly determined by the density distribution of flowering maize. They were moreover correlated with larval distribution and with the adult distribution of the previous year. The density distributions of male adults differed from those of females. Female density was additionally correlated with higher soil moisture and Poaceae density, e.g., with Sorghum halepense (L.) Pers. No relation was found between the larvae of A. ustulatus and D. v. virgifera. Agriotes ustulatus larval distributions were mainly determined by vegetational cover (correlation with less cover). Conclusively, male and female D. v. virgifera adults, larvae of D. v. virgifera, and larvae of A. ustulatus will display different spatial clustering even within ostensibly homogeneous habitats of flat small‐scale maize fields.     

Characterization of the complete mitochondrial genome of Diaphania pyloalis (Lepidoptera: Pyralididae)

The complete mitochondrial genome (mitogenome) of Diaphania pyloalis (Lepidoptera: Pyralididae) was determined to be 15,298 bp and has the typical gene organization of mitogenomes from lepidopteran insects. It consists of 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes and an A + T-rich region. The A + T content of this mitogenome is 80.83% and the AT skew is slightly positive. All PCGs are initiated by ATN codons, except for cytochrome c oxidase subunit 1 (cox1) gene which is initiated by CGA. Only the cox2 gene has an incomplete stop codon consisting of just a T. All the tRNA genes display a typical clover-leaf structure of mitochondrial tRNA. The A + T-rich region of the mitogenome is 332 bp in length, including several common features found in lepidopteran mitogenomes. Phylogenetic analysis showed that the D. pyloalis is close to Pyralididae.     

Volatile semiochemical impact on trapping and distribution in maize of northern and western corn rootworm beetles (Coleoptera: Chrysomelidae)

Abstract 1 Field studies evaluated plant attractants and analogues as tools to move corn rootworm beetles (Diabrotica spp.) into areas to be treated with toxic baits for population suppression via mass removal/annihilation of reproductive adults. 2 When dispensed from sticky traps in maize, 2‐phenyl‐1‐ethylamine and 2‐phenyl‐1‐ethanol captured more northern corn rootworm, Diabrotica barberi, than did 4‐methoxyphenethanol. Only 2‐phenyl‐1‐ethanol attracted the western corn rootworm, Diabrotica virgifera virgifera, but not until maize matured beyond milk stage. 3 Attraction of D. barberi to the amine, alone or blended with 2‐phenyl‐1‐ethanol, occurred before and after maize flowered but not during intervening silk or blister stages. Attraction recurred during early milk stage at or before 50% emergence of adult female D. barberi or D. v. virgifera, respectively, and before populations declined for the season. 4 Synergistic interaction of 2‐phenyl‐1‐ethylamine with 2‐phenyl‐1‐ethanol in attracting D. barberi females did not occur until maize matured to late milk stage. 5 The amine‐alcohol blend (0.44 point sources m^?2) doubled the density of D. barberi but not D. v. virgifera when applied to small plots within mostly milk‐stage or younger maize. Traps without bait within attractant‐treated plots captured more female, but not male, D. barberi than did traps in untreated control plots, hinting that females accounted for most of the observed increase in beetle density. 6 The results suggest that attractants can be used despite phenological limitations to concentrate preovipositional females within field areas and thus to complement a variety of corn rootworm control strategies.     

European populations of Diabrotica virgifera virgifera are resistant to aldrin,but not to methyl‐parathion

The western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a major pest of cultivated corn in North America and has recently begun to invade Europe. In addition to crop rotation, chemical control is an important option for D. v. virgifera management. However, resistance to chemical insecticides has evolved repeatedly in the USA. In Europe, chemical control strategies have yet to be harmonized and no surveys of insecticide resistance have been carried out. We investigated the resistance to methyl‐parathion and aldrin of samples from nine D. v. virgifera field populations originating from two European outbreaks thought to have originated from two independent introductions from North America. Diagnostic concentration bioassays revealed that all nine D. v. virgifera field populations were resistant to aldrin but susceptible to methyl‐parathion. Aldrin resistance was probably introduced independently, at least twice, from North America into Europe, as there is no evident selection pressure to account for an increase of frequency of aldrin resistance in each of the invasive outbreaks in Europe. Our results suggest that organophosphates, such as methyl‐parathion, may still provide effective control of both larval and adult D. v. virgifera in the European invasive outbreaks studied.     

Comparative assessment of the efficacy of entomopathogenic nematode species at reducing western corn rootworm larvae and root damage in maize

The western corn rootworm (Diabrotica virgifera virgifera LeConte, Coleoptera: Chrysomelidae) is an invasive maize (Zea mays L.) pest in Europe. Crop yield is significantly impacted by the feeding of all three larval instars on maize roots, making them prime targets for control measures. Therefore, the control efficacy of three entomopathogenic nematodes (EPNs), Steinernema feltiae (Filipjev), Heterorhabditis bacteriophora Poinar, and H. megidis Poinar, Jackson and Klein (Nematoda: Rhabditida), was studied in four field plot experiments in southern Hungary in 2005 and 2006. All EPN species significantly reduced D. v. virgifera independently, whether applied as a row spray with a solid stream into the soil at sowing or onto the soil along maize rows in June. When applied at maize sowing, H. bacteriophora was more effective at reducing D. v. virgifera (81%) than H. megidis (49%) and S. feltiae (36%). When applied in June, H. bacteriophora and H. megidis were more effective at reducing D. v. virgifera (around 70%) than S. feltiae (32%). All tested EPN species significantly reduced damage on maize roots independently, whether they were applied at sowing or in June. Damage, however, was not totally prevented. The use of H. bacteriophora for the development of a biological control product for inundative releases against D. v. virgifera larvae is suggested.     

Corn rootworm survey in North Dakota and a comparison of two sticky traps

Northern, Diabrotica barberi Smith & Lawrence, and western, D. virgifera virgifera LeConte, corn rootworms are major economic pests of corn, Zea mays L., in the United States. This research was conducted to determine the geographic distribution, abundance, and species composition of Diabrotica species in North Dakota, and to compare effectiveness of unbaited green Scentry™ Multigard and unbaited yellow Pherocon^® AM/NB sticky traps for monitoring. Fifty-one corn fields were monitored using traps from July through October of the 2013, 2014, and 2015 growing seasons for rootworm beetle activity. The overall species composition was 61% D. barberi and 39% D. v. virgifera. Both species were frequently captured, and the highest densities (i.e. >10 beetles per trap per week) were found in southeastern North Dakota. Low densities (i.e. <0.1 beetles per trap per week) of D. barberi were found in areas further north, but no D. v. virgifera were captured in those fields. The two different coloured sticky traps were not significantly different across 38 sites for D. barberi and across 21 sites for D. v. virgifera. However, green Scentry™ Multigard traps captured more D. barberi beetles than yellow Pherocon^® AM/NB traps at 68% of the 38 fields. In contrast, the yellow Pherocon^® AM/NB traps captured more D. v. virgifera beetles at 57% of the 21 fields. Findings also indicated that, although D. barberi was the predominant species in surveyed fields, populations rarely reached the economic threshold. Our study observed that economic populations of corn rootworms were infrequent among the field sites trapped in North Dakota. As a result, producers should scout fields regularly for corn rootworm populations levels to make sound pest management decisions. This knowledge can enable producers to effectively protect their crop when control is economically justified, and the information can also provide input cost savings when populations do not warrant control efforts.     

Characterization of the complete mitochondrial genome of Bombyx mori strain H9 (Lepidoptera: Bombycidae)

The complete mitochondrial genome (mitogenome) of Bombyx mori strain H9 (Lepidoptera: Bombycidae) is 15,670 base pairs (bp) in length, encoding 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes and a control region. The nucleotide composition of the genome is highly A + T biased, accounting for 81.31%, with a slightly positive AT skewness (0.059). The arrangement of 13 PCGs is similar to that of other sequenced lepidopterans. All the PCGs are initiated by ATN codons, except for the cytochrome c oxidase subunit 1 (cox1) gene, which is proposed by the TTAG sequence as observed in other lepidopterans. Unlike the other PCGs, the cox1 and cytochrome c oxidase subunit 2 (cox2) genes have incomplete stop codons consisting of just a T. All tRNAs have typical structures of insect mitochondrial tRNAs, which is different from other sequenced lepidopterans. The structure of A + T-rich region is similar to that of other sequenced lepidopterans, including non-repetitive sequences, the ATAGA binding domain, a 18 bp poly-T stretch and a poly-A element upstream of transfer RNA M (trnM) gene. Phylogenetic analysis shows that the domesticated silkmoth B. mori originated from the Chinese Bombyx mandarina.     

Flight activity of Diabrotica spp. at the borders of cornfields and its relation to ovarian stage in D. barberi

Flight activity of corn rootworm beetles, the northern (NCR), Diabrotica barberi (Coleoptera: Chrysomelidae), and western (WCR), D. virgifera virgifera, was monitored using malaise traps placed at the edges of two small cornfields. Populations of beetles in the fields peaked while corn was flowering, but capture in malaise traps remained low until silks had dried. Capture of NCR and (to a lesser extent) WCR then increased and remained high through the season. Malaise traps were constructed to permit separate collections of beetles that entered on each of two opposite sides. Female NCR that were captured in sides that faced toward corn tended to contain fewer mature eggs than those in sides facing away from corn. These findings support the hypothesis that female NCR emigrate from cornfields to feed when fresh silk and corn pollen (favored foods) become unavailable, but that egg-laden NCR actively seek corn when searching for oviposition sites.

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Résumé Les activités de vol de D. barberi (NCR) et D. virgifera virgifera (WCR) ont été contrôlées par l'utilisation de pièges malais placés en lisière de deux petits champs de maïs. Les populations de ces coléoptères sont maximales quand le maïs fleurit, mais leurs captures sont restées faibles jusqu'à ce que les barbes du maïs aient été sèches. Les captures de NCR et, dans une plus faible mesure, celles de WCR ont alors augmenté et sont restées importantes pendant toute la saison. Les pièges malais ont été conçus pour que les adultes pénétrant par les côtés opposés soient récoltés séparément. Les femelles de NCR capturées face au champ de maïs avaient tendance à contenir moins d'ovocytes mûrs que celles capturées du côté opposé. Ces observations appuient l'hypothèse que les femelles NCR émigrent des champs de maïs pour s'alimenter, quand les barbes et le pollen de maïs, aliments préférés, deviennent indisponibles, mais que les femelles NCR pleines d'ovocytes mûrs recherchent le maïs quand elles sont en quête de lieux de ponte.

     

Intercontinental effect on sexual shape dimorphism and allometric relationships in the beetle pest Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae)

Hind wing shape variation was examined in 686 adult Diabrotica virgifera virgifera collected from maize plants in Europe and the USA Corn Belt, using geometric morphometric techniques. Sexual dimorphism at an Intercontinental scale was assessed using canonical variates analysis, a multivariate statistical method used to find the shape characters that best distinguish among groups of specimens. Our results showed that each of the populations of D. v. virgifera investigated in this study showed high levels of sex based hind wing shape dimorphism. In particular a stronger and more obvious pattern of hind wing shape variation was found in the USA than in Europe. These results support previous studies on D. v. virgifera wing shape that show that female D. v. virgifera have more elongated wings than males. These differences raise the question of whether sexual dimorphism may be modulated by natural selection.     

The complete mitochondrial genome of the Ailanthus silkmoth, Samia cynthia cynthia (Lepidoptera: Saturniidae)

The complete mitochondrial genome (mitogenome) of the Ailanthus silkmoth, Samia cynthia cynthia (Lepidoptera: Saturniidae) was determined. The circular genome is 15,345 bp long, and presents a typical gene organization and order for sequenced mitogenomes of Bombycidea species. The nucleotide composition of the genome is highly A+T biased, accounting for 79.86%. The AT skew of the genome is slightly negative, indicating the occurrence of more Ts than As, as found in other Saturniidae species. All protein-coding genes (PCGs) are initiated by ATN codons, except for COI and COII, which are tentatively designated by CGA and GTG, respectively, as observed in other insects. Four of 13 PCGs, including COI, COII, ATP6, and ND3, harbor the incomplete termination codons, T or TA. With an exception for tRNA^Ser(AGN), all other tRNAs can form a typical clover-leaf structure of mitochondrial tRNA. The 359 bp A+T-rich region of S. c. cynthia contains non-repetitive sequences, but harbors several features common to the Bombycidea insects, including the motif ATAGA followed by a poly-T stretch of 19 bp, a microsatellite-like (AT)₇ element preceded by the ATTTA motif, and a poly-A element upstream tRNA^Met. The phylogenetic analyses support the morphology-based current hypothesis that Bombycidae and Saturniidae are monophyletic. Our result confirms that Saturniini and Attacini form a reciprocal monophyletic group within Saturniidae.     

Comparative analyses of the complete mitochondrial genomes of the two ruminant hookworms Bunostomum trigonocephalum and Bunostomum phlebotomum

Bunostomum trigonocephalum and Bunostomum phlebotomum are blood-feeding hookworms of sheep and cattle, causing considerable economic losses to the live stock industries. Studying genetic variability within and among hookworm populations is critical to addressing epidemiological and ecological questions. Mitochondrial (mt) DNA is known to provide useful markers for investigations of population genetics of hookworms, but mt genome sequence data are scant. In the present study, the complete mitochondrial DNA (mtDNA) sequences of the sheep and goat hookworm B. trigonocephalum were determined for the first time, and the mt genome of B. phlebotomum from yak in China was also sequenced for comparative analyses of their gene contents and genome organizations. The lengths of mt DNA sequences of B. trigonocephalum sheep isolate, B.trigonocephalum goat isolate and B. phlebotomum China yak isolate were 13,764 bp, 13,771 bp and 13,803 bp in size, respectively. The identity of the mt genomes was 99.7% between B. trigonocephalum sheep isolate and B. trigonocephalum goat isolate. The identity of B. phlebotomum China yak isolate mt genomes was 85.3% with B. trigonocephalum sheep isolate, and 85.2% with B. trigonocephalum goat isolate. All the mt genes of the two hookworms were transcribed in the same direction and gene arrangements were consistent with those of the GA3 type, including 12 protein-coding genes, 2 rRNA genes and 22 tRNA genes, but lacking ATP synthetase subunit 8 gene. The mt genomes of B. trigonocephalum and B. phlebotomum were similar to prefer bases A and T, the contents of A + T are 76.5% (sheep isolate), 76.4% (goat isolate) and 76.9% (China yak isolate), respectively. Phylogenetic relationships reconstructed using concatenated amino acid sequences of 12 protein-coding genes with three methods (maximum likelihood, Bayesian inference and neighbor joining) revealed that the B. trigonocephalum and B. phlebotomum represent distinct but closely-related species. These data provide novel and useful genetic markers for studying the systematics, and population genetics of the two ruminant hookworms.     

The complete mitogenome of Apocheima cinerarius (Lepidoptera: Geometridae: Ennominae) and comparison with that of other lepidopteran insects

The complete mitochondrial genome (mitogenome) of a female flightless geometrid moth Apocheima cinerarius was found to be 15,722 bp in length, containing 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and a control region. The A + T content of the complete mitogenome is 80.83%. The AT skew value ([A − T] / [A + T]) is 0.027. The 13 PCGs of the mitogenome start with typical ATN codons, except for cox1 with the start codon CGA. All the tRNA genes have typical cloverleaf secondary structures, except for trnSer(AGN). The secondary structures of rrnL and rrnS were predicted. Six structural domains including conserved regions (IV, V) and variable regions (I, II, III, VI) were identified in the secondary structure of rrnL. The secondary structure of rrnS consists of 3 structural domains. The control region of A. cinerarius begins with conserved motifs of “ATAGA” + 19-bp poly T. It also contains a microsatellite-like (TA)₂₆, a stem-and-loop structure, and a poly-A stretch. Phylogenetic analysis showed that Geometroidea is more closely related to Bombycoidea than to Noctuoidea. A. cinerarius is more closely related to Biston panterinaria than to Phthonandria atrilineata, which is in accordance with the conventional morphology-based classification.     

Entomopathogenic fungi in cornfields and their potential to manage larval western corn rootworm Diabrotica virgifera virgifera

Entomopathogenic ascomycete fungi are ubiquitous in soil and on phylloplanes, and are important natural enemies of many soil-borne arthropods including larval western corn rootworm, Diabrotica virgifera virgifera, which is a major pest of corn. We measured the prevalence of Beauveria bassiana and Metarhizium anisopliae sensu lato in ten cornfields in Iowa, USA by baiting with larval insects. B. bassiana and M. anisopliae s.l. were present in 60% ± 6.3% and 55% ± 6.4% of soil samples, respectively. Subsequent laboratory bioassays found that some M. anisopliae s.l. strains collected from cornfields killed a greater proportion of D.v. virgifera larvae than a standard commercial strain.     

Comparison of complete mitochondrial genomes of pine wilt nematode Bursaphelenchus xylophilus and Bursaphelenchus mucronatus (Nematoda: Aphelenchoidea) and development of a molecular tool for species identification

We determined the complete mitochondrial genome sequences for Bursaphelenchus mucronatus, one species of pinewood nematode. The genome is a circular-DNA molecule of 14,583 bp (195 bp smaller than its congener Bursaphelenchus xylophilus) and contains 12 protein-coding genes (lacking atp8), 22 tRNA genes, and 2 rRNA genes encoded in the same direction, consistent with most other nematodes. Based on sequence comparison of mtDNA genomes, we developed a PCR-based molecular assay to differentiate B. xylophilus (highly pathogenic) and B. mucronatus (relatively less virulent) using species-specific primers. The molecular identification system employs multiplex-PCR and is very effective and reliable for discriminating these Bursaphelenchus species, which are economically important, but difficult to distinguish based on morphology. The comparison of the mitochondrial genomes and molecular identification system of the two species of Bursaphelenchus spp. should provide a rich source of genetic information to support the effective control and management (quarantine) of the pine wilt disease caused by pinewood nematodes.     

The complete mitochondrial genome of Sasakia funebris (Leech) (Lepidoptera: Nymphalidae) and comparison with other Apaturinae insects

Sasakia funebris, a member of the lepidopteran family, Nymphalidae (superfamily Papilionoidea) is a rare species and is found only in some areas of South China. In this study, the 15,233 bp long complete mitochondrial genome of S. funebris was determined, and harbors the gene arrangement identical to all other sequenced lepidopteran insects. The nucleotide composition of the genome is highly A + T biased, accounting for 81.2%. All protein-coding genes (PCGs) start with typical ATN codons, except for COI which begins with the CGA codon. All tRNAs have a typical clover-leaf secondary structure, except for tRNA^Ser(AGN), the dihydrouridine (DHU) arm of which forms a simple loop. The S. funebris A + T-rich region of 370 bp contains several features common to the Lepidoptera insects, including the motif ATAGA followed by a 19 bp poly-T stretch, and two tandem repeats consisting of 18 bp repeat units and 14 bp repeat units. The phylogenetic analyses of Apaturinae based on mitogenome sequences showed: (S. funebris + Sasakia charonda) + (Apatura metis + Apatura ilia). This result is consistent with the morphological classification.     

A multifaceted hemolymph defense against predation in Diabrotica virgifera virgifera larvae

The physical and chemical aspects of Diabrotica virgifera virgifera larval hemolymph were quantitatively assessed against two predatory beetle species in the laboratory. Adult Poecilus cupreus and Harpalus pensylvanicus (Coleoptera: Carabidae) were fed pupae, second or third instar D. v. virgifera or a palatable surrogate prey, i.e., Calliphora vicina or Sarcophaga bullata larvae (Diptera: Calliphoridae, Sarcophagidae, respectively) of equivalent size. The ethanol-soluble fraction of third instar D. v. virgifera hemolymph was extracted and suspended in a 0.24 M sucrose solution and offered to H. pensylvanicus (using a sucrose only control for comparison). The mean duration until first consumption was recorded for each predator, as was the amount of time spent eating, cleaning, resting, or walking for 2 min post-attack (or 5 min for the sugar assay). Maggots and D. virgifera larvae and pupae were attacked equally by both predators. But upon attack, D. v. virgifera larval hemolymph coagulated onto the mouthparts of the predators, which they began vigorously cleaning. Predators ate the sucrose solution for significantly longer than hemolymph + sucrose solution, indicating the presence of deterrent chemicals in the hemolymph. This research suggests that D. v. virgifera larvae are defended from predation by sticky and repellent hemolymph. We hypothesize that this defense partially explains the widespread success of D. v. virgifera as an invasive pest.