Susceptibility of four species of turfgrass-infesting scarabs (Coleoptera: Scarabaeidae) to Bacillus thuringiensis serovar japonensis strain Buibui

Bacillus thuringiensis serovar japonensis strain Buibui experimental formulations containing Cry8Cal delta-endotoxin were evaluated in the field for the control of oriental beetle, Anomala orientalis Waterhouse; Japanese beetle, Popillia japonica Newman; Asiatic garden beetle, Maladera castanea (Arrow); and European chafer, Rhizotrogus majalis (Razoumowsky) larvae infesting turfgrasses. In 17 fall applications, control of A. orientalis and P. japonica was achieved at rates as low as 100 g of toxin per ha; however, rates of 301 or 593 g/ha were consistently more effective. A. orientalis larvae were the most susceptible followed by P. japonica, R. majalis, and M. castanea. Two spring applications of 120 g of toxin per ha were not effective against oriental or Japanese beetles.     

Effect of white grub developmental stage on susceptibility to entomopathogenic nematodes

The pathogenicity of the entomopathogenic nematodes Heterorhabditis bacteriophora Poinar and Steinernema scarabaei Stock & Koppenh?fer against different developmental stages of the Japanese beetle, Popillia japonica Newman, and the oriental beetle, Anomala (=Exomala) orientalis Waterhouse, were studied under laboratory conditions. The efficacy of S. scarabaei did not differ between second and third instars in P. japonica or A. orientalis or between small (young) and large (older) third instars in A. orientalis. However, H. bacteriophora efficacy decreased from first over second to third instar and also from small third instars to large third instars in A. orientalis but did not differ significantly between P. japonica larval stages. Once A. orientalis third instars had purged their intestines in preparation for pupation, no significant mortality by S. scarabaei and H. bacteriophora was observed. In contrast, P. japonica susceptibility to both nematode species gradually decreased from stage to stage from actively feeding third instars to pupae. In two additional experiments, we found no difference in Steinernema glaseri (Steiner) susceptibility between second and third instars of A. orientalis but an increase in S. scarabaei susceptibility from the second to third instar of Asiatic garden beetle, Maladera castanea (Arrow). Our observations combined with those of previous studies with other nematode and white grub species show that nematode efficacy against white grub developmental stages varies with white grub and nematodes species, and no generalization can be made.     

Two novel insect defensins from larvae of the cupreous chafer, Anomala cuprea: purification, amino acid sequences and antibacterial activity.

A humoral immune response in larvae of the coleopteran insect, Anomala cuprea has been examined for exploring the molecular basis of host-pathogen interactions. The antibacterial activity against the Gram-positive strain, Micrococcus luteus was detected at a low level in absence of injection. The activity increased strikingly in the hemolymph of the larvae challenged with Escherichia coli, showing the fluctuating profile through a time course, which consists of the static induction phase, the production phase rising to a maximum level, and the reduction phase extending over a long duration. Two peptides were purified and characterized by reverse-phase HPLC, Edman degradation and mass spectrometry. They were isoforms, composed of similar sequences with two amino acid substitutions in 43 residues, and novel members of the insect defensins, cysteine-rich antibacterial peptides. Anomala defensins A and B showed potent activity against Gram-positive bacteria, with slight differences in activity against a few strains of tested bacteria. Anomala defensin B was active at high concentration of 40 microM against the Gram-negative strain, Xenorhabdus japonicus, a pathogen toward the host, A. cuprea larvae.     

Analysis of Tiphia parasitoids preovipositional behaviors and of their scarab host defensive responses

Tiphia vernalis Rohwer and Tiphia popilliavora Rohwer were introduced as biocontrol agents against Japanese beetles (Popillia japonica Newman) and oriental beetles (Anomala orientalis Waterhouse). Studies have shown that under field conditions, T. vernalis parasitize Japanese beetles more often than oriental beetles. This study was done to understand how tiphiid wasps handle the two different host species and the influence of host defensive behaviors on the oviposition process of tiphiid wasps. The preovipositional behaviors performed by Tiphia wasps included: stinging, examining, moving soil, kneading, host-feeding, and host scraping. The frequency, sequence, and total time spent on each behavior before oviposition were scored and compared between two host species. The sequence and frequency of preovipositional behaviors performed by both Tiphia wasps did not show a difference between the two host species. However, female T. vernalis spent significantly longer time trying to sting oriental beetles than Japanese beetles in order to paralyze them. The time T. popilliavora spent on prestinging behaviors did not show a difference between Japanese and oriental beetles. The defensive behaviors performed by Japanese and oriental beetle grubs included: vigorous movements, rubbing their abdomen or head against the wasp’s abdomen, and biting at the attacking wasp. The frequency and total time spent on each defensive behavior was scored and compared between two host species. Overwintered, third instar oriental beetle grubs spent significantly longer time on defensive behaviors when they were attacked by T. vernalis which likely cause wasps to spend longer time trying to sting oriental beetle grubs. The active host resistance gained through behavioral defenses could make oriental beetle grubs less susceptible to T. vernalis attack than Japanese beetle grubs, especially under field conditions. Younger grubs attacked by T. popilliavora did not exhibit these differences.     

Nucleotide sequence of gene cryIIID encoding a novel coleopteran-active crystal protein from strain BTI109P of Bacillus thuringiensis subsp. kurstaki.

The nucleotide sequence of a novel insecticidal crystal protein(Cry)-encoding gene from a Bacillus thuringiensis serotype kurstaki isolate is described. The gene is related to the known coleopteran-active cryIII genes and encodes a CryIIID that is much more active against Colorado potato beetle than other CryIII.     

Long‐term evaluation of field‐wide oriental beetle (Col., Scarabaeidae) mating disruption in blueberries using female‐mimic pheromone lures

The oriental beetle, Anomala orientalis (Waterhouse) (Col., Scarabaeidae), is the most important root‐feeding pest of blueberries and turfgrass in New Jersey, USA. Previous studies showed that mating disruption is a feasible option for oriental beetle management; however, assessing its efficiency can be challenging, and little is known on its long‐term effects. Accordingly, we conducted studies to investigate low‐dose pheromone lures equivalent to oriental beetle females (i.e. female mimics) as easy‐to‐use indicators of mating disruption success, determine the distance at which oriental beetle males respond to female‐mimic lures and assess the long‐term (3‐year) effects of mating disruption on oriental beetle populations in entire blueberry fields. Our studies showed that rubber septa baited with 0.3 μg of the oriental beetle sex pheromone (Z)‐7‐tetradecen‐2‐one attract similar numbers of males as compared with virgin females and can thus be used as a female mimic. The range of attraction of this lure was found to be also similar to virgin females and <30 m. In blueberries, mating disruption provided 87% inhibition of oriental beetle populations (trap shutdown) over a 3‐year period. Oriental beetle male captures in disrupted fields were threefold higher along the field edges than in the field interiors, indicating movement of males from nearby areas into the pheromone‐treated fields. In addition, mating disruption reduced male attraction to female‐mimic lures by 93% in all 3 years and reduced the number of larvae in sentinel potted plants in 1 of 2 years. These results show for the first time that mating disruption provides consistent long‐term field‐wide control of oriental beetle populations and that female‐mimic pheromone lures can be used as a new tool to assess oriental beetle mating disruption success.     

Effects of delayed mating on reproductive output of female oriental beetle Anomala orientalis (Coleoptera: Scarabaeidae)

1 Although management of the oriental beetle Anomala orientalis (Waterhouse) by mating disruption shows promise across a range of agricultural systems, relatively little is known about aspects of the reproductive biology of this species relevant to its management. We studied the effects of delayed mating on several aspects of the oviposition behaviour and biology of the oriental beetle using females mated in the laboratory at 4–13 days posteclosion. 2 Females exhibited a gradual decline in fecundity with increased age at mating that was largely a function of a decline in duration of the oviposition period, as well as lower female fertility: females mated at 11 days were less likely to lay any fertile eggs. However, egg fertility did not vary with female age at mating. 3 Because mating delay did not affect longevity, females mated at older ages experienced decreased oviposition periods; however, females laid more eggs per day with increased age at mating, which partially offset shorter oviposition periods. 4 A mating delay of ≥ 6 days relative to females mated within the first day of reaching sexual maturity resulted in an approximately 35–50% lower mean fecundity. 5 These results suggest that, for mating disruption to be a successful management tool for the oriental beetle, mating must be prevented rather than delayed. We compare the findings of the present study with the published research on delayed mating in moths and discuss the importance of these results in relation to management of the oriental beetle using female sex pheromones.     

球孢白僵菌对小猿叶甲的致病力测定

在室内研究了分离自小猿叶甲的一株球孢白僵菌(SCAU-BB 01D)对小猿叶甲的致病力。结果表明,该菌株能感染小猿叶甲的成虫和各龄幼虫,但对不同虫期的致病力存在差异。在105~108孢子/m l的浓度范围内,随着处理浓度的升高,各虫期小猿叶甲的感病死亡率增加,在最高浓度1×108孢子/m l,处理后成虫第14天及1~3龄幼虫第10天的累计死亡率分别为84.7%、94.0%、96.0%和81.0%。用TDM模型对成虫和各龄幼虫的致病力数据进行模拟,所建模型均顺利通过Hosm er-L em eshow拟合异质性检验,表明模型拟合良好,并由模型估计出了该菌株对小猿叶甲各虫期的致死剂量与致死时间。在处理后第10天,成虫和1~3龄幼虫的致死中浓度(LC50)分别为2.68×107、1.07×106、1.63×105孢子/m l和8.31×106孢子/m l,而第14天成虫的LC50为2.38×106孢子/m l。随着浓度的增加,各虫期所需的感病死亡时间缩短,在最高浓度1×108孢子/m l,球孢白僵菌对小猿叶甲成虫及1~3龄幼虫的致死中时(LT50)分别为9.28、4.29、4.40d和5.06 d。综合分析白僵菌对各虫期的致死剂量及致死时间可以看出,不同虫期的小猿叶甲对球孢白僵菌敏感性不同。结果表明该菌株在小猿叶甲生物防治中具有一定的潜力。     

Host density effects on efficacy of entomopathogenic nematodes against white grub (Coleoptera: Scarabaeidae) species

We tested the effect of host density on entomopathogenic nematode efficacy in 1-L pots with grass and soil. In four experiments, combinations ranged from somewhat resistant hosts (oriental beetle, Anomala orientalis, or northern masked chafer, Cyclocephala borealis, with Heterorhabditis bacteriophora) over more susceptible hosts (Japanese beetle, Popillia japonica, with Steinernema glaseri) to a highly susceptible host (P. japonica and S. scarabaei). In each experiment, four larval densities were exposed to two nematode rates over a 14-day period. A significant effect of host density on nematode efficacy occurred only in the A. orientalis–H. bacteriophora combination, but there was no clear trend in the data. This suggests that an exhaustion of available nematode populations to less lethal levels by high host numbers was counteracted by other factors such as increased chances for nematode-host contact and increased host susceptibility due to stress via reduced food resources and increased aggression between larvae.     

Unisex pheromone detectors and pheromone-binding proteins in scarab beetles

Olfaction was studied in two species of scarab beetle, Anomala octiescostata and Anomala cuprea (Coleoptera: Scarabaeidae: Rutelinae), which are temporarily isolated and use the same sex pheromone compounds, (R)-buibuilactone and (R)-japonilure. Single sensillum recordings in A. octiescostata revealed highly sensitive olfactory receptor neurons (ORNs) (threshold <1 pg) that were tuned to the detection of the green leaf volatile compound (Z)-3-hexenyl acetate. As opposed to similar ORNs in another scarab species, Phyllopertha diversa, in A. octiescostata a diazo analogue elicited much lower neuronal responses than the natural ligand. Detectors for other floral and leaf compounds were also characterized. Extremely stereoselective ORNs tuned to sex pheromone were identified in male and female antennae. Biochemical investigations showed that, in A. octiescostata and A. cuprea, the pheromone-binding proteins (PBPs) isolated from male antennae were identical to PBPs obtained from female antennae. AoctPBP and AcupPBP had seven different amino acid residues. Binding of AoctPBP to (R)-japonilure is shown. PdivOBP1, which is also known to bind to (R)-japonilure, differed from AcupPBP in only two amino acid residues, one at the N-terminus and the other near the C-terminus. The structural features of the Bombyx mori PBP are compared with the sequences of eight known scarab odorant-binding proteins.     

铜绿丽金龟蛴螬肠道产消化酶细菌的分离与鉴定

目的分离不同生态环境中铜绿丽金龟蛴螬肠道中产消化酶细菌,明确生态环境和食物对其肠道共生细菌产酶活性的影响。方法 2016年7月,自野外林间废弃的菜园和花生田分别采集蛴螬,鉴定出铜绿丽金龟蛴螬后,采用传统分离培养法对其肠道中的共生细菌进行分离鉴定,利用平板透明圈法分别进行淀粉酶、蛋白酶、脂肪酶和纤维素酶等的分泌能力测定。结果自铜绿丽金龟蛴螬肠道中共分离出细菌24株,其中在野生铜绿丽金龟蛴螬体内分离出9株,花生田铜绿丽金龟蛴螬体内分离出15株。野生蛴螬体内分离的细菌中,产淀粉酶菌株1株,产蛋白酶菌株4株,产纤维素酶菌株1株,产脂肪酶菌株1株;花生田蛴螬体内分离的细菌中,产淀粉酶菌株1株,产蛋白酶菌株3株,产纤维素酶菌株9株,未分离到产脂肪酶的菌株。结论野生铜绿丽金龟蛴螬肠道细菌中产蛋白酶种类较多,占44.4%;花生田铜绿丽金龟蛴螬肠道细菌中产纤维素酶菌株最多,所占比例可达60.0%,反映出生境和食性与昆虫肠道共生细菌产消化酶活性的相适应性。     

A novel strain of Bacillus thuringiensis (NCIMB 40152) active against coleopteran insects

Abstract: A novel Bacillus thuringiensis strain, NCIMB 40152, was isolated from dead Tenebrio molitor L. larvae. The isolate is endowed with specific insecticide activity against the Colorado potato beetle ( Leptinotarsa decemlineata (Say), Coleoptera, Crysomelidae), but has no effect against lepidopteran or dipteran insects.
During the sporulation process it produces diamond-shaped crystals and small inclusions. Rare flat, square crystals are alsò produced. The strain can be differentiated from the other reported B. thuringiensis strains active against Coleoptera on the basis of physiological and biochemical tests, flagellar serotype and toxin electrophoretic patterns.
NCIMB 40152 toxin is easily extractable from spore/crystal preparations in neutral distilled water without detergents or reducing agents.     

Discovery and characterization of Sip1A: a novel secreted protein from Bacillus thuringiensis with activity against coleopteran larvae

Bioassay screening of Bacillus thuringiensis culture supernatants identified strain EG2158 as having larvicidal activity against Colorado potato beetle (Leptinotarsa decemlineata) larvae. Ion-exchange fractionation of the EG2158 culture supernatant resulted in the identification of a protein designated Sip1A (secreted insecticidal protein) of approximately 38 kDa having activity against Colorado potato beetle (CPB). An oligonucleotide probe based on the N-terminal sequence of the purified Sip1A protein was used to isolate the sip1A gene. The sequence of the Sip1A protein, as deduced from the sequence of the cloned sip1A gene, contained 367 residues (41,492 Da). Recombinant B. thuringiensis and Escherichia coli harboring cloned sip1A produced Sip1A protein which had insecticidal activity against larvae of CPB, southern corn rootworm (Diabrotica undecimpunctata howardi), and western corn rootworm (Diabrotica virgifera virgifera).     

Experimental evidence of bark beetle adaptation to a fungal symbiont

The importance of symbiotic microbes to insects cannot be overstated; however, we have a poor understanding of the evolutionary processes that shape most insect–microbe interactions. Many bark beetle (Coleoptera: Curculionidae, Scolytinae) species are involved in what have been described as obligate mutualisms with symbiotic fungi. Beetles benefit through supplementing their nutrient‐poor diet with fungi and the fungi benefit through gaining transportation to resources. However, only a few beetle–fungal symbioses have been experimentally manipulated to test whether the relationship is obligate. Furthermore, none have tested for adaptation of beetles to their specific symbionts, one of the requirements for coevolution. We experimentally manipulated the western pine beetle–fungus symbiosis to determine whether the beetle is obligately dependent upon fungi and to test for fine‐scale adaptation of the beetle to one of its symbiotic fungi, Entomocorticium sp. B. We reared beetles from a single population with either a natal isolate of E. sp. B (isolated from the same population from which the beetles originated), a non‐natal isolate (a genetically divergent isolate from a geographically distant beetle population), or with no fungi. We found that fungi were crucial for the successful development of western pine beetles. We also found no significant difference in the effects of the natal and non‐natal isolate on beetle fitness parameters. However, brood adult beetles failed to incorporate the non‐natal fungus into their fungal transport structure (mycangium) indicating adaption by the beetle to particular genotypes of symbiotic fungi. Our results suggest that beetle–fungus mutualisms and symbiont fidelity may be maintained via an undescribed recognition mechanism of the beetles for particular symbionts that may promote particular associations through time.     

Molecular characterization,virulence and horizontal transmission of Beauveria pseudobassiana from Dendroctonus micans (Kug.) (Coleoptera: Curculionidae)

The great spruce bark beetle, Dendroctonus micans (Kugelann) (Coleoptera: Curculionidae), has been a potential threat for Turkey and the entire Eurasian spruce forests for many years. Control strategies which have been applied so far are still insufficient to prevent its damage. A previous study has shown that a Beauveria isolate (ARSEF 9271) proved to be an efficient microbial control agent against the great spruce bark beetle. In this study, this isolate was identified as B. pseudobassiana based on the partial sequence of EF1‐α and ITS sequence. A conidial suspension (1 × 10⁸/ml) of this fungus caused 100% mortality on both larvae and adults of D. micans within 5 and 6 days, respectively. Also, it caused 100% mycosis value on both larvae and adults. Mortality values of horizontal transmission experiments between larvae and adults which were contaminated with 1 × 10⁶/ml spore suspension at 25%, 50%, 75% and 100% rates were determined as 100% after 15 days at 20°C under the laboratory conditions. We also determined the decrease of the damage in spruce wood block (15 × 25 cm) when the contamination rate of the larvae was increased. Our results indicate that B. pseudobassiana ARSEF 9271 seems to be a very promising biocontrol agent against D. micans.     

Discovery of a novel Bacillus thuringiensis Cry8D protein and the unique toxicity of the Cry8D-class proteins against scarab beetles

A novel cry gene, cry8Db, highly toxic to scarab beetles such as the Japanese beetle, Popillia japonica Newman, was cloned from an isolate of Bacillus thuringiensis(Bt), BBT2-5. The cry8Db gene has 3525 bp nucleotides and codes for a protein of 1174 amino acid residues. The protein, Cry8Db, has typical Bt characteristics such as the 8-block, conserved sequences and the three-domain 3 D toxin structure as defined with Cry3Aa. When the amino acid sequence of Cry8Db was compared with that of Cry8Da whose gene was cloned and characterized in our laboratory earlier, substantial sequence diversities were found in their Domain III. The cry8Db gene was expressed in an acrystalliferous B. thuringiensis strain, BT51. BT51 expressing cry8Db formed a spherical crystal like the natural crystal of BBT2-5. The Cry8Db protein was assayed along with the other scarab active Cry8Da and Cry8Ca against the Japanese beetle. While Cry8Da and Cry8Db had toxicity against both adults and larvae of the Japanese beetle, Cry8Ca was toxic to only larvae. Cry8Ca showed no toxicity against the adult beetle up to 30 μg per 1 cm² of leaf discs on which the protein was applied. The activation process of Cry8Db by adult and larval gut juice was compared in vitro with the processes of Cry8Da and Cry8Ca. All three proteins, Cry8Db, Cry8Da and Cry8Ca, produced a toxic core of approximately 70 kDa equally indicating that the activation process does not inactivate the adult activity of Cry8Ca. We concluded that the adult activity of Cry8D proteins is encoded in Domain II. Further tests against other beetle species showed a significant difference between Cry8D’s and Cry8Ca but no difference between Cry8Da and Cry8Db. Comparison of 3D structural models of Cry8Ca, Cry8Da and Cry8Db, which were constructed by using Cry3Bb as the structural template, indicated significant structural differences, especially between Cry8Ca and Cry8D proteins, in three major surface-exposed loops of Domain II that may be involved in determining the adult beetle activity.     

A New Species and Genus Distribution Record from China: Neoscytalidium novaehollandiae

One isolate from bark beetle galleries was identified and characterized using morphological and molecular methodology, and described and illustrated based on these data. These results identified a new species distribution record in China for Neoscytalidium novaehollandiae. Species morphology and micrographs are provided in this paper. The specimens are stored in Northeast Forestry University.     

杉木天然次生林金龟子多样性及季节动态变化

1　引　言杉木 (Ｃｕｎｎｉｎｇｈａｍｉａｌａｎｃｅｏｌａｔａ(Ｌａｍｂ .)Ｈｏｏｋ .)在我国南方各省有着悠久的栽培历史 ,它生长快 ,材质好 ,在我国林业生产和国民经济建设中占有重要地位。但长期生产实践表明 ,杉木人工林重复连栽会引起地力衰退及生产力下降。影响杉木人工林稳定性的因素很多 ,其中包括地下害虫蛴螬。但有关方面的研究很少 ,只保留在个体生物学和生态学水平上 ,并且许多是室内工作 ,并未把金龟子种群在杉木天然次生林生态系统中分布、发生及季节动态作为一个整体加以研究。本研究工作通过对杉木天然次生林生态系统中金…     

金龟子在句容丘陵地区葡萄园的种类和发生规律

采用灯下诱集和田间调查的方法,研究了句容丘陵地区葡萄园发生的金龟子的种类、发生数量和发生规律。结果表明,在句容丘陵地区葡萄园发生的金龟子有双叉犀金龟、华北大黑腮金龟、暗黑腮金龟、铜绿异丽金龟、深铜绿异丽金龟、四纹丽金龟、斑喙丽金龟、毛黄腮金龟等8种。发生量较大,对葡萄危害较重的有铜绿异丽金龟和双叉犀金龟,并比较了这两种金龟子的发生规律和危害高峰期。文中还讨论了金龟子对葡萄危害和防治方法。     

海南椰心叶甲病原菌金龟子绿僵菌的分离、鉴定及其生防潜力

椰心叶甲[Brontispa longissima（Gestro）]是椰子的重要害虫,近年来,该虫在海南岛发生普遍,椰子受害严重。由于椰心叶甲受到自然界中某些致病微生物的侵袭,在受害的椰子树心叶上常可发现椰心叶甲僵虫,并发现大部分僵虫表面长出了霉菌,本研究的目的在于从椰心叶甲僵虫表面的霉菌中分离出绿僵菌,并对分离菌株进行鉴定和致病性测定。从僵虫表面刮下孢子或菌丝体,置于绿僵菌选择性培养基（DOA）上培养,挑出真菌菌落,经纯化后,进行生物学特性、菌落生长速率及产孢量的测定,并从PPDA、OMA、VSA和PDA中筛选菌落生长及产孢最适培养基,同时对所分离的菌株进行对椰心叶甲的致病性测定。结果表明,所有分离菌株均鉴定为金龟子绿僵菌[Metarhizium anisopliae（Metschnikoff）],PPDA是菌落生长及产孢的最适培养基,大多数菌株对椰心叶甲有较强的致病力。选取强毒菌株MA4在田间进行防治效果的初步测定,结果表明,该菌株能显著降低椰心叶甲成虫的虫口密度。这些金龟子绿僵菌菌株是首次从海南的椰心叶甲僵虫中分离到的昆虫病原真菌,该菌对海南的椰心叶甲具有很好的生防潜能。