Monitoring of the saproxylic beetle Morimus asper (Sulzer, 1776) (Coleoptera: Cerambycidae) with freshly cut log piles

Quantitative information on population size, structure and dynamics are urgently needed for assessing species extinction risk and developing monitoring measures, especially for beetles belonging to the threatened guild of saproxylic organisms. Here freshly cut log piles (FCLP) were tested for the monitoring of the longhorn beetle Morimus asper (Sulzer, 1776) (Coleoptera: Cerambycidae). Novel mathematical models based on presence–absence or count data were compared with the most commonly used capture–recapture methods to investigate if less invasive and easier approaches may be suitable for large-scale monitoring of this species and other large saproxylic beetles. The use of FCLP as bait was reliable to detect the presence and abundance of M. asper, and capture events were not influenced by the phenomena of trap-happiness or trap-shyness. In order to obtain accurate presence–absence estimates of M. asper at a reasonable cost piles of at least 0.25 m³ are suggested. Models which take into account the abundance of the species without marking individuals were the best compromise between costs and accuracy of estimation. Therefore, a reliable assessment of the population size of M. asper, can be based on count data without marking individuals. Because FCLP attract adults of M. asper and other saproxylic beetles, they could act also as potential ecological traps if chipped and/or removed after the sampling season. Therefore, if FCLP are used in monitoring as a part of conservation programmes, the piles should be left until completely decayed.     

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.     

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.     

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 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.     

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.     

A method for detecting the effect of magnetic field on activity changes of neuronal populations of Morimus funereus (Coleoptera, Cerambycidae)

Modification of a new method for detecting changes in the activities of neuronal population and the nearest neuron is described. Preliminary measurements of the influence of a static magnetic field (2 mT) on neuronal population activity on eight individuals of an endangered insect species Morimus funereus are included. Five minutes exposure produced both excitatory (5/8) and inhibitory (3/8) effect on the activity of neuronal population of M. funereus antennal lobe. However, when the reversibility of induced effects was quantitatively analyzed, our results showed that they were prevailingly irreversible: (7/8) for the population, (6/8) for the nearest neuron.     

Evidence for mate‐encounter Allee effect in an invasive longhorn beetle (Coleoptera: Cerambycidae)

1. Limited empirical support is available for mate‐encounter Allee effects in invasive insects due to the logistical challenges of studying demographic trends in low‐density populations. 2. Traps baited with pheromone and spruce volatiles were used to monitor the abundance of female Tetropium fuscum F. (Coleoptera: Cerambycidae) at multiple sites in Nova Scotia in 2011 and 2012. Each female was dissected to determine the presence or absence of sperm in its spermatheca (mated or virgin female, respectively). 3. Both male and total T. fuscum abundance declined with increasing distance to the focal point of T. fuscum's invasion. Female mating probability declined with male abundance and with distance from the invasion focus, and mating probabilities were very low at the most peripheral sites. Difficulty in encountering mates may thus contribute to limiting the spread of T. fuscum. 4. The approach outlined here could be integrated into existing surveys of wood borers using traps baited with semiochemicals to improve our understanding of the role of the mate‐encounter Allee effect in invasion dynamics.     

Nested clade and phylogeographic analyses of the chub, Leuciscus cephalus (Teleostei, cyprinidae), in Greece: implications for Balkan Peninsula biogeography

Phylogenetic relationships among Greek populations of the chub, Leuciscus cephalus, were investigated using 600 bp of the cytochrome b gene. The aim of this study was to test the assumption that the main difference in ichthyological composition between both sides of the Balkan Peninsula is directly linked to differences in the dispersion mechanisms used by fish in order to extend their distribution range. Phylogenetic and nested clade analyses clearly showed that populations in Greece are significantly differentiated. Greek populations were found to descend from three lineages in three geographical provinces: Western, Central, and Eastern Greece. The chub reached Western Greece at the beginning of the Pleistocene and Eastern Greece during the mid-Pleistocene. Chub dispersion occurred mainly by river confluence due to sea level lowering and river capture in Western Greece and sea dispersal with low-salinity conditions within the Aegean Sea in Eastern Greece. However, in Central Greece, the original mtDNA lineage has presumably been lost owing to a genetic introgression following a second invasion from the Danube during the final stage of the last glaciation. This study provides new elements for a better understanding of the composition of the contemporary ichthyofauna in Greece and highlights possible evolutionary mechanisms responsible for the high endemism rate in the Western Greek biogeographic province.     

Morphing of the phylogeographic lineages of the Balkan alpine newts (Ichthyosaura alpestris, Caudata, Salamandridae): in situ morphological diversification

Numerous alpine newt (Ichthyosaura alpestris) populations from the Balkans, representing all the previously established phylogeographic lineages, were studied for variations in various morphological characteristics (body size and shape, skull qualitative traits and number of trunk vertebrae). Here, we present a decoupling of morphological and mtDNA phylogeographic substructuring in the alpine newt on the Balkan Peninsula. In sharp contrast to other European newts (Triturus spp., Lissotriton spp.), the vast majority of morphological variation in the alpine newt is concentrated at the population level indicating an in situ morphological diversification. We found that the rate of morphological change is similar to the rate of mtDNA change. We hypothesize that the alpine newts are characterized by non-adaptive morphological evolution.     

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.     

The EU protected taxon <Emphasis Type="Italic">Morimus funereus</Emphasis> Mulsant, 1862 (Coleoptera: Cerambycidae) and its western Palaearctic allies: systematics and conservation outcomes

Morimus funereus is a large longhorn beetle included in the European Habitats Directive and in previous releases of the IUCN red list. It represents a flagship species of old-growth forest saproxylic communities in E and SE Europe. The morphologically based taxonomy of W Palaearctic Morimus is rather unstable due to high phenetic intrapopulational and geographic variability and different authors have attempted to recognise one to five different taxa of specific/subspecific rank. No previous molecular data are available for the genus Morimus. Here, for the first time, a molecular approach based on COI and ITS2 gene sequences was applied in European and Anatolian Morimus specimens. The genetic variability among Euro-Anatolian Morimus populations and the geographical structure suggest that they can not be ascribed to the currently accepted five W Palaearctic Morimus species and may actually represent a single, genetically and morphologically variable biological species (M. asper), highlighting the necessity of an extended taxonomical revision. In light of these results, a phylogeographical hypothesis of postglacial colonisation of the central Mediterranean area has been developed and the consequences of this new taxonomic arrangement regarding conservation strategies for “Morimus funereus” and allied taxa in Europe and Turkey are discussed.     

Mating behavior ofPhytoecia rufiventris Gautier (Coleoptera: Cerambycidae)

Sexual behavior between males and females, as well as between males, is described and discussed for the cerambycid beetlePhytoecia rufiventris. The beetles' taxis toward plants taller than average height brings the sexes together from a distance. A male may mount another individual (male or female) and attempt copulation without sex discrimination. The male can discern the sex of another individual only when the terminal part of his abdomen touches the ventral surface of the fifth visible sternite of the latter. No evidence of a sex pheromone is found in this species. Within 1.5–5.5 cm the substrateborne vibrations produced by a moving individual may be the important factor which elicits males to approach a moving individual and attempt copulation. If a female is receptive when a male touches her, he can copulate with her without any courtship display. However, if the female runs away and appears unreceptive, the male will perform courtship displays. Copulation is usually terminated by males. Homosexual behavior between males is discussed.     

中国泥色天牛属Uraecha Thomson 分类研究 (鞘翅目：天牛科：沟胫天牛亚科)

记述泥色天牛属 Uraecha的主要特征及地理分布 ,并报道 1新种 :龙州泥色天牛 Uraecha longzhouensis,sp.nov.,编制了我国已知 9种的检索表。新种的模式标本保存在西南农业大学植物保护系昆虫标本馆。     

TheRorippa pyrenaica group (Brassicaceae) in the Balkan Peninsula

TheRorippa pyrenaica group is represented in the area of Balkan Peninsula byR. pyrenaica (diploid),R. lippizensis (diploid), andR. thracica (diploid and tetraploid). They differ morphologically from all other EuropeanRorippa species by their pitted reticulate seed surface.Rorippa pyrenaica is characterized by ovoid elipsoidal siliculae, up to three times shorter than pedicels.Rorippa thracica andR. lippizensis possess linear siliquae, equal or longer than pedicels, they differ in length of fruits and length of petals. For all three species full synonymy, geographical distribution, ecological characteristics and karyotype data are provided.     

Effect of entomopathogenic nematodes on Plectrodera scalator (Fabricius) (Coleoptera: Cerambycidae)

Entomopathogenic nematodes were screened for efficacy against the cottonwood borer, Plectrodera scalator (Fabricius). Steinernema feltiae SN and S. carpocapsae All killed 58 and 50% of larvae, respectively, in filter paper bioassays but less than 10% in diet cup bioassays. S. glaseri NJ, S. riobrave TX, and H. indica MG-13 killed less than 10% of larvae in both assays. H. marelata IN was ineffective in the diet cup bioassay and killed 12.9% of larvae in a filter paper bioassay. The nematode isolates we tested are not suitable for use as biological control agents against P. scalator.     

松褐天牛的交配行为

采用室内试验和野外观察相结合的方法,对松材线虫病的主要媒介昆虫松褐天牛Monochamus alternatus Hope的交配行为进行了研究。结果表明： 松褐天牛一次完整的交配包括相遇抱对、插入输精和配后保护3个阶段,在交配过程中雄虫有多次插入输精现象发生。室内试验中共观察到松褐天牛的交配123次,松褐天牛一次完整的交配过程平均需时63.49 min,其中输精前的抱对时间平均为1.68 min,交配过程中每次输精插入时间平均为57.60 s,配后保护时间为15.18 min。松褐天牛在开始交配的4天内平均交配5.15次,不同雄性个体所获得的交配机会差异很大。松褐天牛的交配行为表现出强烈的雄性竞争现象,雄虫能根据雌虫或自身的交配经历调整交配投入,当雌虫或者雄虫是初次交配时,总输精时间和插入输精的次数显著大于与有交配经历的雌虫或雄虫交配时的输精时间和插入输精次数。田间松褐天牛的交配行为与室内观察结果基本一致。     

栗山天牛成虫的活动节律

研究了栗山天牛Massicus raddei（Blessig）成虫的日活动节律,结果表明,栗山天牛成虫的取食、交配、产卵、爬行和飞翔等行为的昼夜节律均表现为单峰型.其取食的高峰期是在晚上18时至20时;交配高峰期为17时至22时;产卵和爬行的高峰期均为19时至22时;飞翔的高峰期为19时至21时.提出了昆虫活跃值的概念...