Utility of microsatellites and mitochondrial DNA for species delimitation in the spruce budworm (Choristoneura fumiferana) species complex (Lepidoptera: Tortricidae)

Species identifications have been historically difficult in the Choristoneura fumiferana group, an important insect pest complex. We examined the utility of simple sequence repeats (SSRs, also referred to as microsatellites) and mitochondrial DNA (mtDNA) for delimiting and identifying eight currently recognized species sampled across North America. Four of these species formed discrete clusters using SSRs, while only two species were delimited with mtDNA. There was evidence for hybridization or incomplete lineage sorting between several species pairs. An integrative approach, using both phenotypic traits and molecular markers, allowed for the discrimination of more biologically relevant species units than did the use of molecular markers alone. As species are currently identified using putatively adaptive phenotypic traits, the differences observed between recognized species and neutral SSRs or mtDNA suggests that these species (or evolutionary significant units) have diverged via natural selection in spite of some gene flow.     

Integrating morphology and mitochondrial DNA for species delimitation within the spruce budworm (Choristoneura fumiferana) cryptic species complex (Lepidoptera: Tortricidae)

Species in cryptic complexes tend to be very difficult, if not impossible, to identify using morphological characters. One such complex is the spruce budworm (Choristoneura fumiferana Clemens, 1865) species group, an economically important group of Nearctic forest pests. Morphological, ecological, behavioural and genetic characters have been studied to try to understand the taxonomy of this group better, but diagnostic character states differ in frequency rather than being complete replacements between each species. We used mitochondrial DNA (mtDNA), together with a new morphology‐based character system that focuses on forewing colour components, to determine if one or a combination of character sources can be used for species diagnoses within the spruce budworm complex. We characterized 47 forewing morphometric measurements and sequenced a 470 bp region of cytochrome c oxidase I mtDNA for 111 ingroup individuals comprising five taxa within the complex. Larval host association and coloration or adult pheromone attraction were used as the prior method for grouping individuals. Our results showed that linear discriminant analysis of morphometric wing characters gave unique clusters for all species on the first and second canonical axes, except for a partial overlap between C. fumiferana and C. biennis, which are not sympatric in nature. In contrast, mtDNA distinguished C. fumiferana, C. pinus pinus Freeman, 1953 and a group of western species, but the three western species (C. occidentalis Freeman, 1967 , C. biennis Freeman, 1967 and C. lambertiana Busck, 1915) shared mtDNA haplotypes. On the basis of the linear discriminant analysis of the combined character set, this study supports the application of both morphology and mtDNA within a framework of integrative taxonomy as the most accurate method for species identification. Furthermore, it demonstrates the utility of quantitative colour analysis, which may be particularly helpful for groups in which colour characters are difficult to divide into discrete units due to intergrading hues.     

Effect of kaolin on fitness and behavior of Choristoneura rosaceana (Lepidoptera: Tortricidae) larvae

The mechanisms by which kaolin, a clay particle film formulation, affects the fitness and behavior of larvae of obliquebanded leafroller, Choristoneura rosaceana (Harris), were investigated. Feeding experiments tested kaolin as a physical barrier versus a physiological toxin for larvae that consumed kaolin applied either to apple (Malus spp.) leaves or mixed in artificial diet. Behavioral experiments tested the effects of kaolin applied to apple leaves on neonate dispersal and leaf rolling by third and fourth instars. When larvae fed on apple leaves sprayed with kaolin, mortality and time to pupation of larvae increased significantly, whereas pupal mass significantly decreased. When larvae consumed kaolin mixed into an artificial diet, however, the effects on mortality, pupation time, and pupal mass were negligible. There may be minor physiological effects from consumption because male time to pupation was delayed for larvae fed diets containing the highest concentration of kaolin. In behavioral experiments, neonate larvae dispersed more quickly off plants covered with kaolin than control plants, and kaolin delayed the construction of leaf shelters by third and fourth instars. We showed that the effects of kaolin on C. rosaceana larvae are primarily physical, causing changes in dispersal and rolling behaviors and as a physical barrier to feeding.     

Desiccation resistance in pre-diapause, diapause and post-diapause larvae of Choristoneura fumiferana (Lepidoptera: Tortricidae)

Desiccation resistance was examined in pre-diapause, diapause and post-diapause larvae of the spruce budworm, Choristoneura fumiferana (Clemens), in terms of passive water evaporation under three desiccation conditions: freeze-drying, desiccant-drying at 2 degrees C and desiccant-drying at 18 degrees C. Diapausing second instar larvae and post-diapause non-feeding second instar larvae showed strongest desiccation resistance: a significant amount of water was retained after repeated drying under desiccating conditions, while pre-diapause first instar larvae and post-diapause feeding second instar larvae lost almost all their water content after one or two drying cycles. A hibernaculum covering had no effect on water evaporation. While dead larvae tended to lose significantly more water than their living counterparts, particularly among first instar larvae, such an impact was much weaker among diapausing second instar larvae. Desiccation resistance was lost when post-diapause second instar larvae were allowed access to water while the level of desiccation resistance was maintained or enhanced when the larvae did not have access to water. These results are discussed in the context of overwintering ecology of the species and possible mechanisms for the desiccation resistance are also discussed.     

Oviposition Behavior of the Eastern Spruce Budworm Choristoneura fumiferana (Clemens) (Lepidoptera: Tortricidae)

We documented 13 behaviors associated with oviposition in unfertilized and fertilized spruce budworm moths, Choristoneura fumiferana, by videotaping active moths with a macro lens. Apart from resting, the most pronounced behaviors were probing, drumming + probing, and egg laying. Probing was a bending of the abdomen and extension of the ovipositor to touch the substrate in a rocking, back and forth motion. Drumming + probing involved tapping of the substrate with the pro- and mesothocacic legs with concurrent bending of the abdomen as above. Egg laying was the actual process of egg deposition onto the needle surface of a balsam fir twig. Both the frequency and the duration of these behavioral elements varied depending on the mating status of the female. Unfertilized females exhibited a higher frequency of probing and egg laying. The duration of probing was longer in unfertilized females, while drumming + probing and egg laying were longer for fertilized females. This study is the basis for future work on the chemosensilla associated with the perception of host-plant surface chemicals by ovipositing females.     

Host-plant influence on the population ecology of the jack pine budworm, Choristoneura pinus (Lepidoptera: Tortricidae)

Abstract.

• 1 Newly-emerged, second-instar jack pine budworm (Choristoneura pinus Freeman) establish spring feeding sites preferentially in the pollen cones of their host tree, Pinus banksiana Lamb.
• 2 Laboratory studies showed that the rate of establishment and survival of jack pine budworm on pollen cones was high throughout the entire spring emergence period of the insect.
• 3 In contrast, the rate of establishment and survival of jack pine budworm on vegetative buds was very poor early in the spring. Vegetative buds were only acceptable as feeding sites to the jack pine budworm for a relatively brief period in late spring.
• 4 Field studies showed that the change in population density of jack pine budworm during the spring emergence stage, as expressed by k-values, was a function of the abundance of pollen cones in the stand. Population reduction was greatest in those stands with the fewest pollen cones.
• 5 Direct measurement of spring dispersal by jack pine budworm showed that dispersal and consequent losses to the budworm population were greatest in stands with the fewest pollen cones.
• 6 We conclude that changes in the density of jack pine budworm are strongly influenced by production of pollen cones in the host stand. Because pollen cone production is related to previous years of defoliation by the jack pine budworm, we propose that pollen cones act as a density-dependent factor governing the density of early-stage jack pine budworm.
• 7 The resulting dynamics are compared to those of other budworm species and used to explain observed regional and temporal patterns of jack pine budworm outbreaks.

     

Instar-specific phenology of Pandemis pyrusana and Choristoneura rosaceana (Lepidoptera: Tortricidae) in Washington apple orchards

Head capsule width was used to determine the instar specific phenology of the leafroller Pandemis pyrusana Kearfott and the obliquebanded leafroller, Choristoneura rosaceana (Harris) (Lepidoptera: Tortricidae), attacking apple in Washington state during 2001-2003. In total, 7012 P. pyrusana and 6122 obliquebanded leafroller larvae were measured from apple orchards from mid-March to mid-September. Degree-day accumulations from each site were paired with the head capsule data to determine the periods during which different instars were present in the field. The implications of this work for pest management and biological control of leafrollers is discussed.     

The development of spindle inclusions of Choristoneura fumiferana (Lepidoptera: Tortricidae) infected with entomopox virus

Spindle-shaped crystals from 0.1 to 0.5 μm in length develop in the cytoplasm of cells infected with entomopox virus. They become enclosed in membranes and are occluded with entomopox virions in the formation of polyhedra. Large protein masses that develop in the cytoplasm of infected cells appear to be the source of protein for both polyhedra and spindles.     

中国绿小卷蛾属Eucoenogenes分类研究(鳞翅目:卷蛾科:新小卷蛾亚科)

本文报道了中国绿小卷蛾属6种,包括2新种和1新记录种：长绿小卷蛾Eucoenogenes elongata,sp．nov．,武夷绿小卷蛾Eucoenogenes wuyiensis,sp．nov．和叉绿小卷蛾Eucoenogenes cyanopsis(Meyrick)。文中给出中国已知6个种的检索表。模式标本保存在南开大学生物系。1．长绿小卷蛾Eucoenogenes elongata,新种(图1～2)翅展13．5mm。正模：♂,云南巍山县巍宝山,海拔2200m,2001-Ⅶ-20,李后魂,玻片号ZAH03725。本种与同属其它种的区别在于：雄性爪形突下垂,抱器瓣狭长,左侧颈部具5根粗刺,右侧具四根。2．武夷绿小卷蛾Eucoenogenes wuyiensis,新种(图3～4)翅展13．0mm。正模：♂,福建武夷山仙峰岭,海拔1000m,2004-Ⅴ-26,于海丽采,玻片号ZAH04215副模：1♂,同正模。本种与其余种明显不同,区别在于：雄性爪形突细长,尾突侧生,三角形,抱器瓣颈部具一根粗刺。     

Male larval nutrition in Choristoneura rosaceana (Lepidoptera: Tortricidae): an important factor in reproductive success

This study examines how Choristoneura rosaceana male quality, as determined by larval diet, age and mating history, affects the reproductive success of both sexes. While the size of the spermatophore produced at first mating increased linearly with male age, the frequency of mating was significantly higher for middle-aged males (2–4 days old) than younger (0–2 days old) or older (6–8 days old) individuals, when both sexes were fed on artificial diet. However, the duration of copulation was longer in couples with older than younger males. The observed age-related changes in spermatophore size had no significant effect on female longevity, fecundity or fertility, suggesting no direct relationship between male investment and spermatophore size under these experimental conditions. Different larval food sources (artificial diet, maple and hazelnut) did not affect the proportion of 2-day-old virgin males that mated; however, the proportion that remated was significantly higher for males reared on high-quality food (maple and artificial diet) than those on hazelnut, a poorer food source. There was a 5-fold decline in spermatophore size between the first and second matings on all diets, but female reproductive output was reduced by only 25%. In contrast, while the first spermatophore produced by males on hazelnut was 1.5 times smaller than those produced on maple and artificial diet, the fecundity of their mates was 40% less than those mated with high-quality virgin males. These results provide additional support to the idea that spermatophore size is not a valuable indicator of male quality. Most tethered females placed in the field during the first flight period mated with virgin males (based on the size of the spermatophore), suggesting that female choice exists in this species. These results are discussed in relation to the incidence of polyandry in naturally occurring populations of Choristoneura and the potential use of size and/or chemical cues by females to assess male quality.     

Comparative Genome Sequence Analysis of Choristoneura occidentalis Freeman and C. rosaceana Harris (Lepidoptera: Tortricidae) Alphabaculoviruses

The complete genome sequences of Choristoneura occidentalis and C. rosaceana nucleopolyhedroviruses (ChocNPV and ChroNPV, respectively) (Baculoviridae: Alphabaculovirus) were determined and compared with each other and with those of other baculoviruses, including the genome of the closely related C. fumiferana NPV (CfMNPV). The ChocNPV genome was 128,446 bp in length (1147 bp smaller than that of CfMNPV), had a G+C content of 50.1%, and contained 148 open reading frames (ORFs). In comparison, the ChroNPV genome was 129,052 bp in length, had a G+C content of 48.6% and contained 149 ORFs. ChocNPV and ChroNPV shared 144 ORFs in common, and had a 77% sequence identity with each other and 96.5% and 77.8% sequence identity, respectively, with CfMNPV. Five homologous regions (hrs), with sequence similarities to those of CfMNPV, were identified in ChocNPV, whereas the ChroNPV genome contained three hrs featuring up to 14 repeats. Both genomes encoded three inhibitors of apoptosis (IAP-1, IAP-2, and IAP-3), as reported for CfMNPV, and the ChocNPV IAP-3 gene represented the most divergent functional region of this genome relative to CfMNPV. Two ORFs were unique to ChocNPV, and four were unique to ChroNPV. ChroNPV ORF chronpv38 is a eukaryotic initiation factor 5 (eIF-5) homolog that has also been identified in the C. occidentalis granulovirus (ChocGV) and is believed to be the product of horizontal gene transfer from the host. Based on levels of sequence identity and phylogenetic analysis, both ChocNPV and ChroNPV fall within group I alphabaculoviruses, where ChocNPV appears to be more closely related to CfMNPV than does ChroNPV. Our analyses suggest that it may be appropriate to consider ChocNPV and CfMNPV as variants of the same virus species.     

Phylogenetics,historical biogeography and molecular species delimitation of Gnaptorina Reitter (Coleoptera: Tenebrionidae: Blaptini)

With 38 described species or subspecies, Gnaptorina Reitter is the second‐most species‐rich genus in the darkling beetle subtribe Gnaptorinina (Tenebrionidae: Tenebrioninae). In this study, we reconstructed a phylogeny of the genus based on one nuclear and three mitochondrial genes and used this phylogeny to explore the historical biography and diversification of Gnaptorina species. We implemented multiple molecular species delimitation approaches to reassess the status of Gnaptorina species and taxonomic subdivisions of the genus. Dating and historical biogeography analyses suggest an early Eocene origin of the genus, with the southeastern regions of the Tibetan Plateau most likely as areas of origin. Based on these results, we propose a new classification for Gnaptorina with three major clades identified. Consequently, the monotypic subgenus Boreoptorina is newly synonymized with the more species‐rich subgenus Hesperoptorina, and G. dongdashanensis Shi is transferred from Hesperoptorina to the subgenus Gnaptorina. In addition, G. minxiana Medvedev, formerly treated as a subspecies of G. potanini Reitter, is elevated to species. Results of molecular species delimitation analyses are largely congruent and confirm the status of most morphological species.     

Systematics, biogeography and diversification of the Indo-Australian genus Delias Hübner (Lepidoptera: Pieridae): phylogenetic evidence supports an 'out-of-Australia' origin

Abstract Two alternative hypotheses for the origin of butterflies in the Australian Region, that elements dispersed relatively recently from the Oriental Region into Australia (northern dispersal hypothesis) or descended from ancient stocks in Gondwana (southern vicariance hypothesis), were tested using methods of cladistic vicariance biogeography for the Delias group, a diverse and widespread clade in the Indo‐Australian Region. A phylogenetic hypothesis of the twenty‐four species‐groups recognized currently in Delias and its sister genus Leuciacria is inferred from molecular characters generated from the nuclear gene elongation factor‐1 alpha (EF‐1α) and the mitochondrial genes cytochrome oxidase subunits I and II (COI/COII) and NADH dehydrogenase 5 (ND5). Phylogenetic analyses based on maximum parsimony, maximum likelihood and Bayesian inference of the combined dataset (3888 bp, 1014 parsimony informative characters) confirmed the monophyly of Delias and recovered eight major lineages within the genus, informally designated the singhapura, belladonna, hyparete, chrysomelaena, eichhorni, cuningputi, belisama and nigrina clades. Species‐group relationships within these clades are, in general, concordant with current systematic arrangements based on morphology. The major discrepancies concern the placement of the aganippe, belisama and chrysomelaena groups, as well as several species‐groups endemic to mainland New Guinea. Two species (D. harpalyce (Donovan), D. messalina Arora) of uncertain group status are currently misplaced based on strong evidence of paraphyly, and are accordingly transferred to the nigrina and kummeri groups, respectively. Based on this phylogeny, a revised systematic classification is presented at the species‐group level. An historical biogeographical analysis of the Delias group revealed that the most parsimonious reconstruction is an origin in the Australian Region, with at least seven dispersal events across Wallacea to the Oriental Region. The eight major clades of Delias appear to have diverged rapidly following complete separation of the Australian plate from Gondwana and its collision with the Asian plate in the late Oligocene. Further diversification and dispersal of Delias in the Miocene–Pliocene are associated with major geological and climatic changes that occurred in Australia–New Guinea during the late Tertiary. The ‘out‐of‐Australia’ hypothesis for the Delias group supports an origin of the Aporiina in southern Gondwana (southern vicariance hypothesis), which proposes that the ancestor of Delias + Leuciacria differentiated vicariantly on the Australian plate.     

A biological assessment of Glypta variegata (Hymenoptera: Ichneumonidae) as a parasitoid of Choristoneura rosaceana (Lepidoptera: Tortricidae)

In laboratory trials, Glypta variegata, a common endoparasitoid of leafrollers in North America, successfully parasitized first through third instar Choristoneura rosaceana and first to fourth instar Pandemis limitata. Significantly more second instar C. rosaceana were parasitized when temperatures fluctuated between 30.3 and 12.0°C (16 L:8 D) than when temperatures fluctuated between 17.5 and 4.0°C (12 L:12 D), similar to fall conditions in southern British Columbia, Canada. Under the warmer simulated summer conditions, an average female wasp began to parasitize C. rosaceana 8 days post-emergence (range: 4-16 days), lived 31 days (range: 24-37 days) and successfully parasitized 28 larvae (range: 1-89). When parasitized, C. rosaceana larvae consumed less food in their last two instars than did unparasitized female larvae.