Evaluation of Beauveria bassiana （Hyphomycetes） isolates as potential agents for control of Dendroctonus valens

The red turpentine beetle （RTB）, Dendroctonus valens LeConte, as a destructive invasive pest, has become one of the most economically important forest pest in China. Effective control measures are desperately needed. Entomopathogenic fungi, such as Beauveria bassiana, have shown great potential for the management of some bark beetle species. In this study, 12 isolates of B. bassiana from bark beetle were examined for biological characteristics and virulence, to assess their potential as biocontrol agents for RTB. There were significant differences （at P = 0.05） in colony growth rate, conidial yield, conidial germination, tolerance to UV light and extracellular proteases activity among the tested B. bassiana isolates. Isolates, including Bbl801, Bb1906, Bb789 and Bb773, exhibited the best characteristics, because they have faster hyphal growth rate, higher spore production and faster spore germination, higher UV tolerance and protease （Prl） production. The results of a pathogenicity test ofB. bassiana on RTB larvae showed that most isolates of B. bassiana have demonstrated high efficacy and the highest virulent isolate was Bb 1801, which killed 100% of the treated insects and had a median lethal time （LT50） of 4.60 days at a concentration of 1 ×107 conidia/mL. Therefore, isolate Bb1801 has a great potential for sustainable control of RTB in the forest. The correlation between biological characteristics and virulence of the fungal isolates is discussed and the possibility of combination of entomopathogenic fungi with semiochemicals, as one of the promising strategy for RTB control, is considered.

Effects of bark beetle pheromones on the attraction of Monochamus alternatus to pine volatiles

We evaluated the attraction ofMonochamus alternatus Hope （Coleoptera： Cerambycidae）, Dryocoetes luteus Blandford and Orthotomicus erosus Wollaston （Coleoptera： Curculionidae： Scolytinae） to multiple-funnel traps baited with the pine volatiles, ethanol and （＋）-α-pinene and the bark beetle pheromones, ipsenol and ipsdienol. M. alternatus were attracted to traps baited with ethanol and （＋）-α-pinene but not those baited with ipsdienol and ipsenol. Ipsdienol and ipsenol decreased catches ofM. alternatus in traps baited with ethanol and （＋）-α-pinene. Traps baited with either binary combinations of ethanol and （＋）-α-pinene or ipsdienol and ipsenol were attractive to D. luteus and O. erosus. The addition of ipsenol and ipsdienol to traps baited with ethanol and （＋）-α-pinene synergized attraction of O. erosus but not D. luteus .

Colonization patterns of the red turpentine beetle, Dendroctonus valans （Coleoptera： Curculionidae）, in the Luliang Mountains, China

The alien red turpentine beetle （RTB）, Dendroctonus valens LeConte, is one of the most economically destructive forest pests in China, having killed more than 6 million pines in recent years. There is a need to understand the basic biology and ecology of the beetle in order to develop an effective monitoring and management strategy. In this study, the effects of hillside exposure （south- and north-facing）, host-tree locations according to relief （valley, mid-slope, and ridge-top） and tree diameters on RTB colonization were investigated in one valley （3 sites）. The results showed that （i） RTB clearly preferred colonizing pines growing on south-facing hillsides, especially in the valley; （ii） RTB preferred to colonize the pines growing at the valley rather than pines growing at mid-slope or on ridge-top; （iii） RTB preferred to colonize trees with large diameter over small and medium-sized pines; （iv） the attack density of RTBs （measured by pitch tubes/pine） was obviously higher on larger trees standing in the valley than other trees standing at other places. We conclude from RTB colonization patterns, that RTB prefers to attack large trees in the valley, which may be useful in developing a pest-management strategy.

Invasive bark beetle-associated microbes degrade a host defensive monoterpene

Conifers respond to herbivore attack with defensive chemicals, which are toxic to both insects and their associated microorganisms. Microorganisms associated with insects have been widely reported to metabolize toxic chemicals, which may help both microorganisms and host insects overcome host conifer defense. Dendroctonus valens LeConte, an introduced exotic pest from North America to China, has killed millions of healthy pines. Alpha-pinene is the most abundant defensive monoterpene in Chinese Pinus tabuliformis. Although microorganisms associated with D. valens have already been investigated, little is known about their bioactivities when encountering host defensive monoterpenes. In this study, we evaluated the influences of different concentrations ofα- pinene to D. valens and the three most frequently isolated yeasts and bacteria ofD. valens, and further assayed microorganisms＇ capabilities to degrade α-pinene. Results showed that the gallery lengths and body weight changes of bark beetles were significantly affected by 6 mg/mL and 12 mg/mL of α-pinene applied in media compared to controls. The tolerance of experimental microorganisms to α-pinene varied depending on the microbial species. Two out of three yeast strains and all three bacterial strains degraded 20%-50% of α-pinene compared to controls in 24 h in vitro. The microorganisms capable of α-pinene degradation in vitro and their tolerance to high levels ofα-pinene suggested that D. valens- associated microorganisms may help both microorganisms and the bark beetle overcome host α-pinene defense.

Comparative analysis of the Monochamus alternatus immune system

The pine sawyer beetle, Monochamus alternatus, is regarded as a notorious for- est pest in Asia, vectoring an invasive pathogenic nematode, Bursaphelenchus xylophilus, which is known to cause pine wilt disease. However, little sequence information is available for this vector beetle. This hampered the research on its immune system. Based on the transcriptome of M. alternatus, we have identified and characterized 194 immunity-related genes in M. alternatus, and compared them with homologues molecules from other species known to exhibit immune responses against invading microbes. The lower number of puta- tive immunity-related genes in M. alternatus were attributed to fewer C-type lectin, serine protease （SP） and anti-microbial peptide （AMP） genes. Phylogenetic analysis revealed that M. alternatus had a unique recognition gene, galectin3, orthologues of which were not identified in Tribolium castaneum, Drosophila melanogastor, Anopheles gambiae and Apis mellifera. This suggested a lineage-specific gene evolution for coleopteran insects. Our study provides the comprehensive sequence resources of the immunity-related genes ofM. alternatus, presenting valuable information for better understanding of the molecular mechanism of innate immunity processes in M. alternatus against B. xylophilus.

Classical biological control program for the mealybug Oracella acuta in Guangdong Province, China

The mealybug Oracella acuta, native to the southeastern US, was accidentally introduced into slash pine plantations in Guangdong Province in China in 1988. A classical biological control program was initiated in 1995, and the parasitoids Allotropa oracellae, Acerophaus coccois, and Zarhopalus debarri were imported from the US. A total of 19 972 parasitized mealybugs were shipped to China from 1996-2004, from which 15 430 wasps emerged, 12 933 of which were the three target species. Efforts to establish a mass-rearing program for the parasitoids in China failed. Five field release sites were established, and 6 020 parasitoids were released. Only 118 individuals of the three imported species were collected during establishment checks, although several wasps were collected 1-2 years after the last parasitoid release. Over 2 000 Anagyrus dactylopii, a cosmopolitan parasitoid, emerged from the parasitized mealybugs collected, a majority from the Taishan area near the site of the original introduction ofO. acuta. To date the imported parasitoids have failed to establish, and natural enemies have not noticeably reduced mealybug populations.

Fighting and aggressive sound determines larger male to win male-male competition in a bark beetle

Intrasexual selection occurs in male-male competition over access to females and usually results in the larger male winning.While much research has documented that size matters,little is known about how the larger male wins.Dendroctonus vale ns is an aggregating monogamous bark beetle in which males have large variation in body size and display intense competition over females.Behavioral observation showed two males fight each other within the gallery by pushing/shoving and stridulated more when two males encountered each other.Experiments using two different-sized males synchronously competing showed that larger males won 95%of contests.Reciprocal displacement experiments using muted and intact males of different or equal size were used to simulate male-male competition.Larger males displaced the smaller resident male in 90%of contests,while smaller males prevailed over larger residents in 6.7%of contests.With both males silenced,larger males displaced smaller males in 80%of con tests,while smaller males prevailed in 8%of con tests.Further experiments using equal-sized males showed aggressive soundemitting males displaced muted males in 67%of contests,yet intact males displaced other intact males in only 37.5%of con tests.Sound analysis showed sound pressure level is an honest signal of body size and males chose soft sounds over loud aggressive sounds in assays.Therefore,D.valens males have evolved dual behaviors,fighting and aggressive sounds associated with body size,to assess rivals to compete for a partner,gaining insights in male-male competition for this species and for other animals.

Physical contact, volatiles, and acoustic signals contribute to monogamy in an invasive aggregating bark beetle

The behavioral strategies and mechanisms by which some insects maintain monogamous mating systems are not well understood.We investigated the mating system of the bark beetle Dendroctonus valens,and identified several contributing mechanisms.Field and laboratory observations suggest the adults commonly form permanent bonds during host colonization.Moreover,it showed mated females that remained paired with males produced more offspring than mated females that were alone in galleries.In bioassays,a second female commonly entered a gallery constructed by a prior female.Videos show she commonly reached the location of the first female,but they did not engage in actual fighting.Rather,the second female typically departs to form her own gallery.Acoustic signaling likewise does not appear to influence female-female encounters,based on controlled muting experiments.Instead the intruder appears to perceive the residents presence by physical contact.Both acoustic signals and volatiles released by females during gallery constructing were shown to attract males.After a male joined a female in a gallery,the male-produced aggressive sounds,which were shown by playback to deter other males from entering the gallery.Unlike female-female interactions,resident males use their head and rear to push intruders out of galleries.Additionally,volatiles released by males during feeding repelled intruding males,discouraging them from entering the gallery.Males also construct plugs that block the entrance,which may prevent subsequent males and predators from entering the gallery.Thus,D.valens has evolved multifaceted mechanisms contributing to single pairings that confer benefits to both sexes.

Effects of fertilization and herbicides on growth of young loblolly pine and infestations of Nantucket pine tip moth（Lepidoptera： Tortricidae）

A 2-year-old pine plantation was selected to receive treatments of fertilizers and herbicides to evaluate effects on Nantucket pine tip moth infestations and the tree growth parameters of height, diameter and volume increment. Nitrogen and phosphorus fertilizers, and hexazinone and sulfometuron methyl herbicides were used in creating six treatments： （i） control; （ii） phosphorus; （iii） nitrogen and phosphorus; （iv） phosphorus and herbicide; （v） nitrogen, phosphorus and herbicide; and （vi） herbicide. Treatments were applied in 1987 and 1988. In 1987, trees treated with nitrogen, phosphorus and herbicide had significantly greater height, diameter and volume growth than trees not receiving fertilizer treatments, but did not have significantly higher tip moth infestations than control trees. Treatments receiving phosphorus only had much lower tip moth infestation rates than other treatments except nitrogen and phosphorus. In 1988, tip moth infestations were uniformly low, with no differences in treatment effects observed.

Effect of associated fungi on the immunocompetence of red turpentine beetle larvae, Dendroctonus valens （Coleoptera： Curculionidae： Scolytinae）

Abstract Dendroctonus-fungus symbioses are often considered as the ideal model sys- tems to study the development and maintenance ofectosymbioses, and diverse interactions, including antagonism, commensalism and mutualism, have been documented between these organisms. The red turpentine beetle, Dendroctonus valens LeConte （Coleoptera： Curculionidae： Scolytinae） is a pine-killing invasive beetle in northern China. Fungi species Ophiostoma minus, Leptographium sinoprocerum, L. terebrantis and L. procerum were associated with this bark beetle. Antagonistic interactions between D. valens and its as- sociated fungi, such as O. minus and L. sinoprocerum, have been demonstrated, but the underlying causes of this phenomenon are unknown. Here, we first found the two tested fungi species retarded the net weight gain of D. valens larvae after completing 3-day feeding on their media. Furthermore, we provide direct evidence indicating the effect of associated fungi on the immunocompetence olD. valens larvae to explain the documented antagonism. Our results showed that the activity of phenoloxidase and total phenoloxi- dase in D. valens larvae were significantly upregulated by two strains of associated fungi, O. minus and L. sinoprocerum as compared with the controls. The phenoloxidase ratio increased significantly in the larvae which had fed for 3 days on media inoculated with O. minus. Because insect immtme defenses are costly to be deployed, these results could be explored as one of the underlying mechanisms of the documented antagonism.

Herbivory effects of Argopistes tsekooni, a chrysomelid beetle, on container-grown Chinese privet, Ligustrum sinense

The impact ofArgopistes tsekooni Chen （Coleoptera： Chrysomelidae）, a herbivore, on Chinese privet, Ligustrum sinense Lout. （Scrophulariales： Oleaceae）, an invasive shrub in the United States, was studied in China. Five densities of adults were inoc- ulated into 3-year-old potted Chinese privet plants in cages under field conditions for 1 month. Plants exposed to high densities of adults were severely damaged and the aboveground portions of some were killed, while the survival rates of adult A. tsekooni were comparatively high. The amount of new growth of the main stem, the number of new leaves, and oven-dried biomass were significantly reduced by the combined feeding of larvae and adults. Above-ground plant mortality was 100% when plants were exposed to 24 and 30 adults/plant. In this study A. tsekooni had a significant negative impact on Chinese privet growing in pots, which suggests that it may be a promising candidate for biological control of Chinese privet in the field in North America.

Effects of methamidophos and deltamethrin on in vitro protein phosphorylation in Monochamus alternatus

Monochamus alternatus Hope （Coleoptera： Cerambycidae） is not only a serious pest insect to pine trees but also the main vector of pine wood nemadote Bursaphelenchus xylophilus, which causes pine wilt disease. To explore the insecticidal mechanism of insecticides to M. alternatus, we chose methamidophos and deltamethrin as the representatives of two groups of insecticides （organophosphates and pyrethroids）, which are widely used for pest control in China and investigated their effects on phosphorylation of proteins from the insect. Phosphorylation of proteins from the insect fat body and head was determined by in vitro 32P-labelling. In the fat body, deltamethrin obviously reduced basal phosphorylation levels of proteins at 111, 95, 77, and 44 kDa, but enhanced the basal phosphorylation level of a protein at 138 kDa. However, in the presence of calmodulin but not cyclic adenosine monophosphate （cAMP）, deltamethrin increased phosphorylation of the protein at 111 kDa. In the head, deltamethrin inhibited basal phosphorylation levels of proteins at 113, 98, and 51 kDa, but potentiated phosphorylation of a protein at 167 kDa activated by cAMP. Methamidophos inhibited phosphorylation of a protein at 44 kDa in the fat body. Although methamidophos did not impact basal phosphorylation levels of any proteins in the head, it inhibited calcium/calmodulin （Ca^2＋CaM）-stimulated phosphorylation of a protein at 51 kDa. Together, our data indicate that methamidophos and deltamethrin altered phosphorylation levels of various proteins in the head and fat body of the pine insect and these two kinds of insecticides acted on the proteins that can be phosphorylated in the tissues respectively, which is possibly related to their toxicity.

Biology and damage traits of emerald ash borer planipennis Fairmaire）in China

Emerald ash borer （Agrilus planipennis Fairmaire） （Coleoptera： Buprestidae） is a major stem borer of ash （Fraxinus spp.）. It is univoltine in Tianjin, while it is semivoltine in Heilongjiang Province, and both univoltine and semivoltine in Changchun, Jilin Province, where the majority is univoltine. The longevity of emerald ash borer adults is 17.2 ± 4.6 days （n = 45）, eggs 9.0 5：1.1 days （n = 103）, univoltine larvae 308 days, semivoltine larvae 673 days, and pupae 61.2 ± 1.6 days （n = 45）. It takes about 100 days from the time larvae bore into the phloem to when they complete the pupal cell. In a 10-year-old velvet ash （Fraxinus velutina Tort.） plantation in Tianjin, emerald ash borer preferred to oviposit on the regions of boles from 50-150 cm above ground, accounting for 76.7% of the total girdling. Girdling on the south side of the tree boles accounted for 43.40% of the total girdling. The emerald ash borer population density is higher at the edge of the plantation compared with the center.