The encounter of distinct Morimus asper (Coleoptera: Cerambycidae) phylogeographic lineages on the Balkan Peninsula: conservation implications

Journal of Insect Conservation - The first step in monitoring and management of any taxa includes resolving taxonomic ambiguities which complicate the identification of its management units. One of...     

Adaptations to captive breeding of the longhorn beetle Morimus funereus (Coleoptera: Cerambycidae); application on amylase study

Captive breeding has been suggested as a method of conservation for many vertebrates, and is increasingly being proposed as a strategy for invertebrates. In this study, the growth, development and fertility of adults of the vulnerable cerambycid Morimus funereus reared in captivity are examined. Two oviposition cycles; from May to September and from January to March were studied and larvae from wild adults and from the progeny of captive adults (second generation larvae) were examined. Five to 12 instars were observed during larval development. Larval development was completed in 218 days (average) for the progeny of wild adults with an average mortality rate of 10.3% and in 226 days (average) for larvae from captive adults with mortality rate of 34.9%. First generation larval body weights were disparate during development, while second generation larvae had similar weights with no significant differences. In this study we have tested the potential of captive breaded M. funereus larvae as a model for investigation of digestive enzymes. Amylase from the midgut of larvae reared under laboratory conditions showed twofold higher specific activities with a decreased number of isoforms expressed, as compared to the enzyme from field-collected larvae. Captive breeding of M. funereus can be used in the future as a part of an effective conservation strategy for this rare insect species.     

Purification and properties of midgut alpha-amylase isolated from Morimus funereus (Coleoptera: Cerambycidae) larvae

Using soluble starch as a substrate five isoforms of alpha-amylase were identified in a crude extract of Morimus funereus larvae. The main alpha-amylase (termed AMF-3) was purified by gel filtration chromatography and anion exchange chromatography to obtain a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Its enzymatic purity was confirmed by an in-gel activity assay after SDS-PAGE. The purity of AMF-3 was increased 112-fold with a 15.4% yield. AMF-3 had apparent molecular masses of 33 and 31 kDa when analysed using SDS-PAGE and Superdex 75 FPLC gel filtration chromatography, respectively and a calculated isoelectric point of 3.2. Purified AMF-3 showed maximal activity at pH 5.2 and had an optimum activity temperature of 45 degrees C. AMF-3 retained over 90% of its maximum activity at temperatures from 45 to 60 degrees C. AMF-3 exhibited a high affinity towards soluble starch with a K(m) value of 0.43 mg/mL. Maximal AMF-3 activity was achieved in the presence of 0.1 mM CaCl(2), while at higher concentrations its activity decreased. AMF-3 activity increased with increasing NaCl concentration. AMF-3 activity was significantly inhibited by alpha-amylase wheat inhibitor. Using a number of raw starch substrates maximum AMF-3 activity was achieved with horse-radish starch, in contrast to undetectable activity towards potato starch.     

Partial purification and characterization of midgut leucyl aminopeptidase of Morimus funereus (Coleoptera: Cerambycidae) larvae

Exopeptidases of Morimus funereus larvae were partially purified and characterized. Specific leucyl aminopeptidase (LAP) activity was increased eight-fold by gel filtration of the crude midgut extract. The partially purified LAP had a molecular mass greater than 100 kDa with pH optima from 7.0-9.0 and no strict substrate specificity. M. funereus LAP preferentially hydrolyzed p-nitroanilides with hydrophobic amino acids in the active site, with a K(m) for leucine-p-nitroanilide of 0.21 mM. Zymogram analysis of an electropherogram obtained by native polyacrylamide gel electrophoresis revealed four enzymatically active proteinases using leucine-p-nitroanilide and methionine-p-nitroanilide as substrates and two enzymatically active proteinases using lysine-p-nitroanilide as a substrate. Although the optimal temperature of LAP activity was 40 degrees C, the enzyme was active over a broad temperature range from 2 to 60 degrees C. Among a number of inhibitors tested, heavy metals and 1,10-phenanthroline completely inhibited the enzyme, while methanol, ethanol and EGTA stimulated somewhat LAP activity.     

Purification and properties of major midgut leucyl aminopeptidase of Morimus funereus (Coleoptera, Cerambycidae) larvae

The major leucyl aminopeptidase (LAP) from the midgut of Morimus funereus larvae was purified and characterised. Specific LAP activity was increased 292-fold by purification of the crude midgut extract. The purified enzyme had a pH optimum of 7.5 (optimum pH range 7.0-8.5) and preferentially hydrolysed p-nitroanilides containing hydrophobic amino acids in the active site, with the highest V(max)/K(M) ratio for leucine-p-nitroanilide (LpNA). Among a number of inhibitors tested, the most efficient were 1,10-phenanthroline having a K(i) value of 0.12 mM and cysteine with K(i) value of 0.31 mM, while EGTA stimulated LAP activity. Zn(2+), Mg(2+) and Mn(2+) all showed bi-modal effects on LAP activity (activated at low concentrations and inhibited at high concentrations). The purified LAP (after gel filtration on Superose 6 column) had molecular mass of 400 kDa with an isoelectric point of 6.2. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed one band of 67 kDa, suggesting that the enzyme is a hexamer. Six peptide sequences from protein band were obtained using ESI/MS-MS analysis. Comparison of the obtained peptide sequences with the EMBL-EBI sequence analysis toolbox and the BLASTP database showed a high degree of identity with other insect aminopeptidases.     

星天牛幼虫龄期的划分

本文测量了人工饲养的星天牛Anoplophora chinensis(Forster)幼虫的9个形态结构数据,运用频次分布、均差分析和回归分析对测量数据进行统计分析,结果表明,星天牛幼虫可分5龄,其额宽是最佳的分龄结构,其次是上颚长和上颚宽。     

Behavioral Differences of the Insect Morimus funereus (Coleoptera,Cerambycidae) Exposed to an Extremely Low Frequency Magnetic Field

Changes in the behavior of Morimus funereus individuals were investigated as early manifestations of the contact of a living system with a changed environment primarily established via the nervous system. These experiments were aimed at revealing possible behavioral differences of a laboratory population of cerambycid beetle M. funereus in an “open field” before and after exposure to an extremely low frequency magnetic field (ELF‐MF, 50 Hz, 2 mT). The experimental groups were divided into several activity categories and exposed to ELF‐MF. The results showed that the activity increased in the groups with medium and low motor activity, but decreased in highly active individuals. High individual variability was found in the experimental groups, as well as differences in motor activities between the sexes, both before and after exposure to ELF‐MF. According to preliminary results, we assume the changes of activity in both sexes after exposure to ELF‐MF. The results showed a tendency toward locomotor activity decrease, the affect being more pronounced in females. As opposed to this type of activity, stereotypic activity of males was increased after the exposure, whereas females maintained the expected tendency of decrease. However, we did not obtain statistically significant differences because of a high individual variability and a low total number of individuals in the experiment (N = 28). Only a detailed analysis of the locomotor activity at 1‐min intervals showed some statistically significant differences in behavior between the sexes.     

Surprising longhorned beetle (Coleoptera, Cerambycidae) richness along an Italian alpine valley

In this paper we report about 88 longhorned beetles (Cerambycidae) species found in 6929 hectares and distributed along an altitudinal gradient of 1500 m of an Italian alpine valley (Val Genova, central-eastern Italian Alps). The species richness, result merging data from sixty years (1947-2007) of entomological surveys, corresponds to the 32% of the Italian cerambycid fauna confirming the high richness/surface ratio, probably unique in the Alps. The effect of thirteen environmental variables was tested on the species richness, but only the elevation resulted able to affect it. The species richness decrease with altitude not gradually, but experience a strong step above 1700 m a.s.l.. The highest species richness (average values of 42 species) was recorded at the lowest and mid elevations (between 800 and 1600 m a.s.l.). The species turnover along the altitudinal gradient is low suggesting moderate habitat turnover along the valley.One of the eighty-eight observed species, Tragosoma depsarium,is classified near threatened by the IUCN. Our data suggest that the wilderness of the valley close to the suitable management of grasslands and forests, help to support high level of cerambycids diversity. This biodiversity is good indicators of health of the wood saproxylic assemblages, as well an important food source for many vertebrate predators.     

The effect of temperature on midgut and brain protein profiles in Morimus funereus larvae (Coleoptera: Cerambycidae)

The 7-days shift of M. funereus larvae, from nature to a constant temperature of 23 degrees C led to changes in midgut and brain protein quality and quantity. The changes in midgut protein profiles are characterized by an intensified protein band Mr of 29 kD, the absence of protein Mr of 22 kD and less intense bands Mr of 8.5-2.5 kD. Electrophoretic patterns of brain proteins showed less intense Mr of 66-2.5 kD protein bands.     

Karyological notes on six beetle species from Armenia (Coleoptera: Tenebrionidae,Cerambycidae, Curculionidae)

Karyotypic details were studied in males of six beetle species from three families, viz. Tenebrionidae: Dailognatha pumila Bdy. (2n = 20, n(male) = 9 + Xy(p)), Pachyscelis musiva Ménétr. (2n = 18, n(male) = 8 + Xy(p)), Pimelia capito Kryn. (2n = 18, n(male) = 8 + Xy(p)); Cerambycidae: Agapanthia walteri Reitt. (2n = 20, n(male) = 9+Xy(p)), Agapanthia korostelevi Danilevsky (2n = 20, n(male) = 9 + Xy(p)); Curculionidae: Phyllobius caucasicus Stierl. (2n = 22, n(male) = 10 + Xy(p)). The chromosome number and sex determining system of all beetle species are described for the first time. Evolutionary trends in karyotypes of the studied beetle groups are briefly discussed.     

A new insect cell line from the longicorn beetle Plagionotus christophi (Coleoptera: Cerambycidae)

We have established a continuous cell line from the fat body tissue of the longicorn beetle Plagionotus christophi. The cells have been serially subcultured in MGM450 medium, and the line has been designated as PC-1. The cells were grown in suspension and comprise largely flattened spindle- or oval-shaped cells morphologically related to blood cells of longicorn beetles. The chromosome number ranged from 19 to 36, with a mode of 20 (diploid). The growth curve was determined at the 100th passage, and the population doubling time was calculated to be 3.79 d. Isozyme analysis of malic enzyme, phosphoglucose isomerase, and phosphoglucomutase revealed that PC-1 cells were enzymatically distinct from coleopteran XP-1, AnCu-35, dipteran AeAl-2, and lepidopteran GaMe-LF1 cell lines.     

Evidence for limited spatial spread in an exotic longhorn beetle, Tetropium fuscum (Coleoptera: Cerambycidae)

The longhorn beetle Tetropium fuscum F. (Coleoptera: Cerambycidae) has become established in Nova Scotia, Canada, where it coexists with Tetropium cinnamopterum Kirby. The two Tetropium species share a similar ecological niche and use the same volatile cues for mate attraction. Exotic T. fuscum was introduced near Halifax, Nova Scotia, in approximately 1990, but the rate of its spread 20 yr later has not been documented. We report a large-scale, 3-yr study that investigates the distribution of T. fuscum relative to its site of introduction. Traps baited with male-produced pheromone and host volatiles were used to estimate the relative abundance of the two Tetropium species. Adult T. fuscum emerged 1-2 wk earlier than T. cinnamopterum each year between 2008 and 2010. The spatial distribution of T. fuscum was characterized by a sharp decline in abundance in relation to its point of introduction, up to a threshold distance of approximately 80 km beyond which T. fuscum is rare in comparison with native T. cinnamopterum. The restricted range of T. fuscum 20 yr after its introduction may be attributed to limited dispersal of adults or reproductive failures of low-density populations. The distribution of T. fuscum seemed stable between 2008 and 2010. In 1 of 3 yr, the abundance of T. cinnamopterum increased with the distance to the site of introduction of T. fuscum, which suggests competitive interactions between the two Tetropium species.     

Different effects of coarse woody material on the species diversity of three saproxylic beetle families (Cerambycidae, Melandryidae, and Curculionidae)

The relationships between coarse woody material (CWM) and the diversity of three saproxylic beetle families (Cerambycidae, Melandryidae, and Curculionidae) were investigated. These three families responded differently toward the quality and quantity of CWM. The species richness of cerambycid beetles increased in forests with more CWM in the early stage of decomposition. The richness of saproxylic curculionids increased in stands with more tree species. No clear trend was evident for melandryid diversity. The CWM differed in quality and quantity among three forest types [middle-aged larch (Larix kaempferi [Lamb.] Carrière) plantation, secondary forest, and old-growth forest] and between two forest-management practices (thinning and long rotation). Forest type and management practices affected CWM and the number of tree species, and CWM and the number of tree species influenced the diversity of saproxylic families differently. Because each decomposition stage is considered to be important for saproxylic beetles at the species level, a constant supply of CWM is necessary to maintain a suitable balance of CWM through these decomposition stages in forests to maintain the diversity of saproxylic beetles.     

Impacts of chipping on surrogates for the longhorned beetle Anoplophora glabripennis (Coleoptera: Cerambycidae) in logs

As part of the eradication program for recent introductions of the longhorned beetle Anoplophora glabripennis (Motschulsky) in the United States, wood from infested trees is chipped and incinerated. Two tests were conducted to evaluate the efficiency of chipping wood from infested trees on the survival of the beetle. In the first test, plastic worms were used as surrogates for larvae of the beetle. Plastic worms of different sizes were placed in holes drilled in logs of sugar maple, Acer saccharum Marsh. In a second test, in addition to plastic worms, we used different instars and pupae of gypsy moth, Lymantria dispar (L.) (Lepidoptera: Lymantriidae); larvae of the beetle Phyllophaga annina Lewis (Coleoptera: Scarabaeidae); and larvae of an unidentified weevil (Coleoptera: Curculionidae). Although chipping did not result in an obvious damage to all plastic worms, it did kill all larvae and pupae of insects placed in holes of maple logs. The overall recovery rate (percent recovered) for the plastic worms was 96% in the first (1997) test, and 71 and 98% for 10 and 40 mm long plastic worms in the second (1998) test, respectively. Logistic regression analysis of the data from the first experiment indicates that larger worms receive more severe damage. Size of logs did not have a significant effect on the level of damage received by plastic worms. All recovered insects were severely damaged after chipping logs and we could not determine recovery rates. Results of the two tests indicate that chipping wood from infested trees without incineration of the resulting chips provides a highly effective method for destroying wood inhabiting insect pests such as A. glabripennis. The elimination of incineration saves considerable resources while effectively eliminating risks associated with movements of wood containing living wood-boring insects.     

Phenology and laboratory rearing procedures of an Asian longicorn beetle, Glenea cantor (Coleoptera: Cerambycidae: Lamiinae)

Clenea cantor (F.) (Coleoptera: Cerambycidae: Lamiinae) is an Asian longicorn beetle whose larvae bore under the bark of living trees of at least seven plant families and enter the wood for pupation. Here, we determined the phenology of this beetle on its natural host, kapok [Bombax ceiba L. = Cossampinus malabaricus (DC.) Merr], in an uncontrolled insectary at ambient environmental conditions, and we compared the efficiency of four larval rearing procedures at 25 +/- 2 degrees C, 75 +/- 5% RH, and a photoperiod of 14:10 (L:D) h in the laboratory. It had five generations, including an overwintering generation, a year in southern China, with overlaps between generations and no diapause. Adults were present in early March-early December; eggs in early April-early December; larvae almost year-round, and pupae in mid-February-mid-April as well as mid-May-late November. All larvae of the overwintering generation and the larvae of the fourth generation that hatched in and after mid-November overwintered. The longevity of females and males was 71.94 +/- 1.21 d and 46.87 +/- 1.11 d, respectively, and mean fecundity was 106.65 +/- 3.61 eggs. Four larval rearing procedures using kapok twigs were tested: (1) 10 neonate larvae were left in the original host twig, (2) 10 neonate larvae were removed from the original bark and transferred to a new host twig, 3) 10 neonate larvae embedded in the original bark were transferred to a new host twig, and 4) a single neonate larva embedded in the original bark was transferred to a new host twig. We observed the survival of 300 neonate larvae for each rearing procedure. Approximately 81-85% of neonate larvae successfully developed to adult stage when neonate larvae were transferred together with the original bark to new host twigs; when neonate larvae were transferred without the original bark, only approximately 38% of the inoculants became adults, and when larvae were left in original twigs, approximately 52% of them reached adult stage. Resulting adults from different rearing methods and collected from the field had similar body weight and sex ratio. In terms of time, labor, and the number of resulting adults, procedure 3 is the most effective method for maintaining a laboratory colony.     

Incorporation of mitochondrial fragments in the nuclear genome (Numts) of the longhorned beetle Monochamus galloprovincialis (Coleoptera, Cerambycidae)

A mitochondrial DNA (mtDNA) study, based on 43 European populations (33 of them sampled in France) of Monochamus galloprovincialis , vector of the pinewood nematode, and 14 populations of its sister species Monochamus sutor was realized. Sequencing of 792 bp of the cytochrome oxidase I (COI) and 521 bp of the COII genes revealed numerous ambiguities on multiple nucleotide sites for half of M. galloprovincialis specimens studied (44.8%). Hypotheses of heteroplasmy and pseudogenes ( Numts ) were examined. The mtDNA isolation by alkaline lysis and cloning (for three successfully used individuals) both support the hypothesis that the ambiguous sequences amplified were not of mtDNA nature and validate the presence of Numts in the nuclear genome of M. galloprovincialis . Multiple copies of mtDNA-like sequences were found paralogous to COI, tRNA leucin and COII regions. Phenetic analysis placed different recently diverged mtDNA-like sequences as a close relative of mtDNA sequences, and grouped 10 closely related mtDNA-like sequences as a more basal clade, closer to ancestral states and to M. sutor . This result supports that this nuclear family of pseudogenes arose independently of the other events and may represent mitochondrial haplotypes sampled from M. galloprovincialis ancestral populations. This is the first time that Numts are proved for a longhorned beetle, whereas no Numts were found within its sister species M. sutor. The incorporation mechanism of Numts in unknown for M. galloprovincialis , however, excess of ambiguous sites corresponding to synonymous mutations placed on third codon position as well as the absence of Numts in M. sutor , conducted to the hypothesis of a recent transfer of these Numts in the nuclear genome of M. galloprovincialis .     

Biogeography of the phoracanthine beetles (Coleoptera: Cerambycidae)

Six biogeographic subregions of the phoracanthine beetles are proposed on the basis of 136 valid species in all eleven genera of this Australasian group of insects: the Kosciuskan, Western, Eyrean, Torresian, Timorian and New Guinean. Both phenetic and cladistic approaches are employed in the biogeographic study of these beetles. Phenetically, the Kosciuskan and Torresian fauna are the most similar and the least faunal similarity occurs between the New Guinean subregion and all others. Cladistically, two distinct peripheral faunas are divided: the southern Western+Kosciuskan and the northern New Guinean+Torresian+Timorian. The Eyrean fauna in the central Australian continent is more distantly related to the remainder than the latter are to each other. Biogeographic history of the phoracanthine beetles is discussed extensively.     

Environmental contamination with Metarhizium anisopliae from fungal bands for control of the Asian longhorned beetle, Anoplophora glabripennis (Coleoptera: Cerambycidae)

Our objective was to determine whether conidia from non-woven fiber bands impregnated with Metarhizium anisopliae F 52 could be spread to other parts of the environment, and whether Anoplophora glabripennis could become infected with conidia dispersed from fungal bands. In the field, bands containing M. anisopliae were hung at 3 m height on 15 trees. Bark samples were taken 10, 20, and 30 cm above the bands and 10, 30, and 60 cm below the bands 2, 5, and 9 days after band placement to quantify conidial densities. There were more conidia in samples taken below bands compared with samples taken above bands. A significant positive correlation was found between rainfall and the occurrence of conidia on any of the bark samples. A laboratory study was conducted to determine whether conidia from M. anisopliae fungal bands could be spread to other parts of the environment by A. glabripennis adults and whether A. glabripennis could become infected by conidia dispersed from fungal bands. One or five adult A. glabripennis were used to contaminate artificial environments with conidia. All adults subsequently exposed to contaminated environments were killed by fungal infection. Beetles exposed to environments that had been contaminated by five beetles died in fewer days compared with environments contaminated by one beetle. Beetles in both density treatments died in fewer days than beetles exposed to environments without M. anisopliae conidia. Our results indicate that environmental contamination with conidia from fungal bands can occur, and that adult A. glabripennis can acquire infection from a contaminated environment.     

Resource distribution and its effect on the mating system of a longhorned beetle,Perarthrus linsleyi (Coleoptera: Cerambycidae)

Summary Adults of Perarthrus linsleyi feed on flowers of creosote bush, Larrea tridentata. Mating also occurs on the flowers and foliage of this plant. This food resource is widely and evenly distributed in space, and is usually abundant. The spatial distribution of the beetles bore no relationship to the spatial distribution of flowers among creosote bushes, nor were female beetles distributed predictably with respect to flower distribution. Males moved widely over the area under study, and moved much more frequently than females. Males actively searched for females, and mounted and began copulation attempts without preliminary courtship. Males did not engage in aggressive defense of females, creosote flowers, or creosote bushes. This species exhibits a scramble competition mating system. The spatial distribution of the food resource is a primary factor in the evolution of the mating system of Perarthrus linsleyi.     

Further contributions to the longhorn beetle (Coleoptera,Cerambycidae) fauna of New Brunswick and Nova Scotia,Canada

Sixteen species of Cerambycidae are newly recorded for New Brunswick, Canada; Arhopalus obsoletus (Randall), Atimia confusa confusa (Say), Callidium frigidum Casey, Phymatodes amoenus (Say), Phymatodes testaceus (Linnaeus), Neoclytus mucronatus mucronatus (Fabricius), Xylotrechus aceris Fisher, Xylotrechus sagittatus sagittatus (Germar), Tylonotus bimaculatus Haldeman, Lepturges angulatus (LeConte), Lepturges symmetricus (Haldeman), Urgleptes querci (Fitch), Oplosia nubila (LeConte), Eupogonius subarmatus (LeConte), Monochamus carolinensis (Olivier), and Pogonocherus parvulus LeConte. Urgleptes signatus (LeConte) and Urgleptes querci are newly recorded from Nova Scotia. All but two specimens were collected in 12-funnel Lindgren traps. Xylotrechus aceris, Tylonotus bimaculatus, Lepturges angulatus, Lepturges symmetricus, Urgleptes signatus (NS), and Pogonocherus parvulus were detected exclusively in traps deployed in the forest canopy, and most individuals of Oplosia nubila and Monochamus carolinensis were captured in canopy traps. Arhopalus obsoletus, Atimia confusa confusa, Callidium frigidum, Phymatodes testaceus, and Xylotrechus sagittatus sagittatus were captured almost exclusively in traps near (1 m above) the forest floor. These results highlight the importance of sampling both the understory and upper canopy when using traps for surveying diversity of Cerambycidae.