Investigation on Color Pattern Diversity of Overwintering Harmonia axyridis in Southern Suburb of Jiamusi

Five sampling points for Harmonia axyridis （Pallas） were set in Sifeng Mountain scenic spot and four school districts of Jiamusi University, and diversity of color pattern of overwintering H. axyridis was surveyed from October to December in 2010. Survey results found that there were 16 color patterns of H. axyridis in southern suburb of Jiamusi, 11 species showed yellow background, while the other 5 species were black background, accounting for 68.75% and 31.25% of all the species, respectively. The majority of H. axyridis with yellow background had 18 or 19 spots. Compared with the sampling data in 2006 and 2008, the propor- tions of the color patterns had no significant difference in different years, but it slightly varied with the changes of environment.

Identification and tissue distribution of odorant binding protein genes in Harmonia axyridis (Coleoptera:Coccinellidae)

The olfactory system of insects is crucial in modulating behaviors such as host seeking,mating,and oviposition.Odorantbinding proteins(OBPs)are involved in semiochemical recognition.OBPs recognize and bind odorants and transport them to odorant receptors located in olfactory neurons.Harmonia axyridis(Coleoptera:Coccinellidae)is a widely used predacious biological control agent for many agricultural and forestry pests.This study identified 19 OBPs in H.axyridis based on the antennal and whole-body transcriptomes of adults and obtained all the full-length open reading frames,including 11‘Classic’OBPs,7‘Minus-C’OBPs and 1‘Plus-C’OBP.They encoded 125 to 241 amino acid proteins with molecular weights ranging from 13.75 to 27.75 kDa and isoelectric points ranging from 4.15 to 8.80.Phylogenetic analyses were used to study the relationships between H.axyridis OBPs and OBPs from other species of Coleoptera.Quantitative real-time PCR(qPCR)analysis showed that HaxyOBP2,3,5,8,10,12,13,14,and 15 were highly expressed in antennae of both adult females and males.Moreover,HaxyOBP2,3,5,12,and 15 were more abundantly expressed in antennae than other body parts,while HaxyOBP13 and HaxyOBP14 were expressed predominantly,and at similar levels,in the head and antennae.The other OBP genes were highly expressed in non-olfactory tissues including the thorax,abdomen,legs,and wings.These results provide valuable information for further study of H.axyridis olfaction,which may ultimately enhance its use as a biocontrol agent.

Survival and development of immature Harmonia axyridis (Pallas) feeding on Chaitophorus populeti (Panzer) propagated on transgenic Populus alba × P. glandulosa

Laboratory feeding experiments with the poplar aphid, Chaitophorus populeti （Panzer）, feeding on transgenic poplar （P. alba × P. glandulosa） varieties C13-5 and C013-5, were carried out to study the effect of transgenic poplar on the ladybird Harmonia axyridis （Pallas）. The mortality and development time of the immature stages, the eclosion rate and body mass of H. axyridis were measured. The results indicated that C. populeti feeding on different varieties of transgenic plants had no statistically significant ef- fect on the mortality ofH. axyridis larvae. The development time of larval and pupal stages were not significantly different between the two transgenic poplars and a non-transgenic poplar. Furthermore, the body mass and eclosion rate did not show any difference between the H. axyridis feeding on aphids reared on transgenic plants and those from non-transgenic plants. It is suggested that transgenic plants have no deleterious effect on the predatory ladybird.

Fast-GC Quantification of Harmonine,the Major Defense Alkaloid of the Multicolored Asian Ladybird Harmonia axyridis(Pallas)

These last few years the multicolored Asian ladybird, Harmonia axyridis (Pallas), previously introduced in Europe and North America as a biological control agent, has swiftly spread out on those territories and turns out to be a pest in several ways. In order to improve understanding of several key points of the biology of this ladybird, a novel fast-GC method of quantification of harmonine, the main defense alkaloid of H. axyridis, has been designed and validated using the accuracy profile concept for concentrations ranging from 13 to 264 μg/individual. This method allows quantifying harmonine in a single insect with a maximal error risk of 20%. For ladybirds collected in spring, mean harmonine concentration observed was 106.6 μg/individual with a very high standard deviation of 80.2 μg/individual, which can easily be explained by the strong asymmetry of the results distribution. Males and females seem equally chemically protected as the harmonine concentration did not significantly differ between sexes. This method is currently used to improve our comprehension of several key points of H. axyridis biology including aggregation behavior and aposematism.

Testing semiochemicals from aphid, plant and conspecific： attraction of Harmonia axyridis

Harmonia axyridis Pallas （Coleoptera： Coccinellidae） is an invasive specie affecting the dynamics and composition of several guilds. Nowadays, no biological control method is available to reduce the populations of this harmful coccinellid. Attractants and semiochemicals seem to be the best alternative but only few studies have tested the impact of semiochemicals on this Asian lady beetle. In this work, through wind- tunnel experiments, semiochemicals from aphids （Z,E-nepetalactone, [E]-β-farnesene, a-pinene and β-pinene）, from coccinellids （[-]-β-earyophyllene） and from the nettle Urtica dioica L. were evaluated as potential attractants. The nettle volatile compounds （[Z]-3- hexenol and [E]-2-hexenal） were extracted using a Clevenger Apparatus and identified by headspace gas chromatography-mass spectroscopy. In the wind-tunnel experiments, the main components of the aphid alarm pheromone as well as a component of the aphid sexual pheromone strongly attracted both sexes of the Asian lady beetle while （-）-β- caryophyllene only attracted few individuals and had no impact on the males. The nettle extract as well as the （Z）-3-hexenol oriented both males and females to the odor source. The （E）-2-hexenal was shown to have no effect on females even if this green leaf volatile attracted males. Because Z,E-nepetalactone was identified as the most efficient attractant in the wind-tunnel experiments, this volatile was also tested in a potato field where H. axyridis has been showed to respond to this semiochemical. This study highlighted that Z,E-nepetalactone orientated the Asian lady beetle H. axyridis under natural condi- tions, indicating that this volatile compound could certainly help for an efficient biological control approach against this invasive specie.

Honeydew volatile emission acts as a kairomonal message for the Asian lady beetle Harmonia axyridis （Coleoptera： Coccinellidae）

The Asian lady beetle Harmonia axyridis Pallas is considered as an invasive species in most territories where it has been introduced. Because aphid honeydew acts as an attraetant for many aphid predators and parasitoids, the objectives of this work were to collect and identify the volatile compounds released from the aphid excretory product to evaluate how these semiochemicals could affect the H. axyridis foraging behavior. Twelve volatile chemicals were identified from the Megoura viciae Buckton honeydew including four alcohols, three ketones, three aldehydes, a pyrazine and a monoterpene. The volatiles 3-methyl-l-butanol and 3-methyl-butanal were highlighted as the two most abundant semiochemicals released from the M. viciae honeydew. Vieia faba L. plants treated with crude honeydew attracted more than 80% of the tested individuals with 40% of attracted beetles located on the plant. Four volatile compounds （3-hydroxy-2-butanone, 3-methyl-butanal, 3-methyl-1-butanol and limonene） were also highlighted to attract more than 75% of the coccinellids toward the odor source and to locate about 35% of them on the plants. Limonene was the most efficient attractant since 89% of the H. axyridis responded to this odor. The use of the identified semiochemicals as well as the composition of an artificial honeydew could certainly be helpful to control the dispersal of the Asian lady beetle H. axyridis.

Juvenile hormone regulates photoperiod-mediated male reproductive diapause via the methoprene-tolerant gene in the ladybeetle Harmonia axyridis

Juvenile hormone (JH) absence induces photoperiod-mediated reproductive diapause, which is characterized by reproductive cessation. Although the role of methoprene-tolerant (Met)-mediated JH signaling in photoperiod-mediated female reproduction has been well documented, its role in male reproduction remains unclear. In this study, we investigated the role of JH in regulating photoperiod-mediated development of the male internal reproductive system (IRS) in the predatory ladybeetle Harmonia axyridis (Pallas). In a previous study, we found that adult male H. axyridis reared under either a short-day (SD) or long-day (LD) photoperiod had obvious differences in IRS development, but we were unable to identify the regulators of male reproductive diapause. In this study, we found that beetles reared under an SD photoperiod had significantly lower JH titer and a relatively undeveloped male IRS compared with those reared under an LD photoperiod. Additionally, application of the JH analog (JHA) methoprene promoted IRS development. Furthermore, Met knockdown strongly blocked JH signaling in males reared under the LD photoperiod, thereby slowing IRS development. Moreover, exogenous JHA did not reverse the suppressed development of the male IRS caused by Met knockdown. These results indicate that photoperiod regulates male IRS development in H. axyridis through a conserved Met-dependent JH signaling pathway.

Understanding nutritive need in Harmonia axyridis larvae:Insights from nutritional geometry

The multicolored Asian lady beetle,Harmonia axyridis(Pallas)(Coleoptera:Coccinellidae),is an important natural enemy in agricultural ecosystems.In spite of being a carnivore consuming protein-rich preys,the lady beetles often consume carbohydrate-rich food like nectar or honeydew.However,most studies on nutrition regulation of carnivores mainly focus on protein and lipid,two major macronutrients in preys.In this study,nutrition regulation of protein and carbohydrate has been investigated in the 4th instar larvae of H.axyridis using Geometric Framework.We provided the insects two pairs of foods,one a protein-biased one and the second carbohydrate-biased,to determine the intake target.We then confined them to nutritionally imbalanced foods to examine how they regulated food intake to achieve maximal performance.The larvae performed well on the 2 foods that containing the closest P:C ratios to the intake target,but,surprisingly,the lipid content was much lower than that in the choice experiment.The lady beetles seemed to maintain the optimal lipid content by consuming carbohydrate-rich food.Moreover,consuming the carbohydrate-rich food was less metabolically expensive than the protein-rich food.Therefore,switching behavior between plant and animal foods actually reflects their nutritive needs.These findings extended our understanding of predator forage behavior and its influence on food web in ecosystems,and shed light on the role of agri-environment schemes in meeting the nutritional need of predators in field.

Aspartate-β-alanine-NBAD pathway regulates pupal melanin pigmentation plasticity of ladybird Harmonia axyridis

Phenotypic plasticity is observed in many animal species and it is effective for them to cope with many types of environmental threats.The multicolored Asian ladybird Harmonia axyridis shows a cuticular pigmentation plasticity that can be rapidly induced by temperature changes,and in the form of changeable melanin spot patterns to adjust heat-absorbing.Here,H.axyridis with thermal stimulation were selected for determining the molecular regulations behind it.First,we confirmed the melanin level changes of H.axyridis pupa could be induced by temperature,and then screened the efficient time window for thermal sensing of H.axyridis pre-pupa;it is suggested that the late stage of pre-pupa(late stage of 4th instar larva)is the critical period to sense thermal signals and adjust its pupal melanin spot area size to adapt to upcoming thermal conditions.The Ha-ADC(aspartate decarboxylase)and Ha-ebony(NBAD synthase)of aspartate-β-alanine-NBAD pathway were then proved in regulation of cuticular melanization for pupa through RNA interference experiments;knockdown of these two genes enlarged the melanin spot size.Finally,we designed a random injection of Ha-ADC at different pre-pupal stages,to further study the regulation window during this process.Combined with all evidence observed,we suggested the spot size determination can be regulated very close to the time point of pupation,and genes of the aspartate-β-alanine-NBAD pathway play an important role at the molecular level.In brief,H.axyridis exhibits a flexible active physiological regulation through transcriptional modification to thermal changes.

Involvement of glucose transporter 4 in ovarian development and reproductive maturation of Harmonia axyridis(Coleoptera:Coccinellidae)

Glucose is vital to embryogenesis,as are glucose transporters.Glucose transporter 4(Glut4)is one of the glucose transporters,which is involved in rapid uptake of glucose by various cells and promotes glucose homeostasis.Although energy metabolism in insect reproduction is well known,the molecular mechanism of Glut4 in insect reproduction is poorly understood.We suspect that Glut4 is involved in maintaining glucose concentrations in the ovaries and affecting vitellogenesis,which is critical for subsequent oocyte maturation and insect fertility.Harmonia axyridis(Pallas)is a model organism for genetic research and a natural enemy of insect pests.We studied the influence of the Glut4 gene on the reproduction and development of H.axyridis using RNA interference technology.Reverse transcription quantitative polymerase chain reaction analysis revealed that HaGlut4 was most highly expressed in adults.Knockdown of the HaGlut4 gene reduced the transcript levels of HaGlut4,and the weight and number of eggs produced significantly decreased.In addition,the transcript levels of vitellogenin receptor and vitellogenin in the fat bodies and the ovaries of H.axyridis decreased after the interference of Glut4,and decreased the triglyceride,fatty acid,total amino acid and adenosine triphosphate content of H.axyridis.This resulted in severe blockage of ovary development and reduction of yolk formation;there was no development of ovarioles in the developing oocytes.These changes indicate that a lack of HaGlut4 can impair ovarian development and oocyte maturation and result in decreased fecundity.

Abdominal-B regulates structure and development of the Harmonia axyridis cremaster

The ladybird Harmonia axyridis is an insect that exhibits pupal attachment to plants,which facilitates development and environmental adaptation.The cremaster is highly specialized for this behavior.However,the underlying molecular regulation of the cremaster remains unclear;therefore,we performed experiments to investigate the transcriptional regulation of cremaster development.First,we examined the morphological structure of the cremaster to reveal its function in pupal attachment of H.axyridis.Next,we analyzed the Hox gene Ha-Abd-B using RNA interference(RNAi)to determine its function in regulating cremaster formation;Ha-Abd-B up-regulation promoted effective pupal attachment,whereas successful RNAi caused severe down-regulation of this gene,and pupae were unable to attach.Furthermore,successful RNAi and subsequent Ha-Abd-B down-regulation caused phenotypic changes in cremaster structure,including its complete disappearance from some individuals.Finally,we observed unique development of the cremaster and dynamic expression of Ha-Abd-B during pre-pupal development;consequently,we hypothesized that there was specific pre-pupal development of the cremaster.Overall,based on these results,the specialized cremasteric structure located on the posterior side of H.axyridis was determined to be a key organ for pupal attachment.Cremaster identification in H.axyridis is regulated by Ha-Abd-B and exhibits preferential development.Pupal attachment of H.axyridis reveals an environmental adaptation of this species;thus,this study and future molecular studies will help determine the role of Hox genes in regulation of insect attachment and further our understanding of the multiple functions of Hox genes.

Do microsporidia function as ＂biological weapon＂ for Harmonia axyridis under natural conditions？

Invasive alien species, such as the multicoloured Asian ladybird Harmonia axyridis, are often regarded as major drivers ofbiodiversity loss. Therefore understanding which characteristics or mechanisms contribute to their invasive success is important. Here the role of symbiotic microsporidia in the hemolymph of H. axyridis was investigated in the context of intraguild predation between wild-caught H. axyridis and the native ladybird species Coccinella septempunctata. The microsporidia were recently discussed to contribute to the unpalatability of Harmonia for other coccinellids during intraguild predation and to function as ＂biological weapons＂. In the present study, visual detection of microsporidia in hemolymph samples revealed that 73.5 % of H. axyridis were infected. Intraguild predation experiments between larvae of the two species showed a significant competitive advantage for H. axyridis, even against larger larvae of C. septempunctata. Adult C. septempunctata always killed and fed on H. axyridis larvae. However only 11.4 % （4 of 47） of C. septempunctata that fed on infected H. axyridis died within 4 months. In contrast to previous studies this suggests that microsporidia or harmonine, the chemical defense compound of H. axyridis, do not lead to death of C. septempunctata preying on larvae ofH. axyridis. Instead our results support the idea that competitive advantage during intraguild predation greatly facilitates the success of H. axyridis and that this may help this highly invasive species to outcompete native species. The impact of microsporidia on Harmonia itself as well as on interspecific interactions require further studies.

一种炭基有机肥对“大豆-大豆蚜-异色瓢虫”系统的影响

有机肥能否通过强化作物营养促成自身代谢物的合成,增加植物的抗虫特性,影响植株上取食的植食性昆虫及其捕食性天敌个体的生理生化指标和种群指标,从而为植物保护的免疫学方法提供理论基础。本研究以“大豆-蚜虫-瓢虫”为研究对象,分别构建有机肥和化肥(无蚜无瓢、有蚜无瓢、有蚜有瓢) 6个处理;测定各处理组中大豆植株株高、地上地下生物量、大豆蚜种群数量动态、瓢虫发育历期、蛹质量,以及各处理中大豆、蚜虫、瓢虫的超氧化物歧化酶(SOD)/过氧化氢酶(CAT)/谷胱甘肽S-转移酶(GST)活性水平等指标。主要结果如下:(1)在“有蚜有瓢”最接近田间情况的大豆植株上施用有机肥会导致大豆植株显著“矮化”(-12.3%,P=0.021 1)。(2)有机肥在第7天时显著减少苗期大豆蚜种群数量(-25.9%,P=0.024 9,P=0.057 5),在第5个观测点减少有翅蚜数量(-40.7%)。(3)有机肥升高瓢虫蛹质量(2.4%),缩短瓢虫幼虫(-2.5%)和蛹的发育历期(-13.4%)。(4)大豆抗逆酶活性受到有机肥显著影响,SOD活性下降20.7%,CAT活性升高14.8%,GST活性升高27.8%。有机肥能够在有蚜有瓢接近田间实际的条件下优化作物性状,对同时受植物寄主及瓢虫天敌调节的植食性昆虫表现为中和的种群受抑制;有翅蚜减少,传毒潜力下降;对瓢虫天敌能够提供更好的营养条件,表现为蛹质量增加,低龄幼虫发育加快等变化。因此,有机肥对植物害虫免疫力的增强有积极影响。

两种瓢虫对茶蚜的防治效果研究

为探索茶蚜(Toxoptera aurantii Boyer)的较优生物防控技术,采用田间释放七星瓢虫(Coccinella septempunctata Linnaeus)和异色瓢虫(Harmonia axyridis Pallas)卵块的方法,探究2种捕食性瓢虫对茶蚜的田间防治效果与田间定殖能力。结果表明,在释放瓢虫卵卡15 d后,七星瓢虫和异色瓢虫防治效果分别为73.48%和74.35%,而后逐渐降低,但均维持在60%以上,2种瓢虫对茶蚜的防治效果无显著差异。七星瓢虫卵卡的田间孵化率为54.13%,异色瓢虫卵卡的田间孵化率为58.87%,在瓢虫卵卡释放30 d内均发现有瓢虫存留,且异色瓢虫各虫态存留数均略多于七星瓢虫,但除异色瓢虫的蛹存留数显著高于七星瓢虫外,其余虫态的存留数二者均无显著差异。

异色瓢虫成虫对豌豆修尾蚜的捕食作用

初步研究了异色瓢虫(Harmonia axyridis Pallas)的雌、雄成虫对豌豆修尾蚜(Megoura japonica Matsumura)的捕食作用,采用Holling-Ⅱ型方程功能反应模型对异色瓢虫雌、雄成虫捕食豌豆修尾蚜的效果进行拟合。结果表明,异色瓢虫雌、雄成虫的功能反应均符合Holling-Ⅱ模型。而在一定空间和相同比例猎物条件下,种内干扰会对异色瓢虫成虫捕食豌豆修尾蚜产生影响,此试验中干扰系数为1.09。

释放异色瓢虫对甜瓜蚜虫的防控效果

为探究在设施栽培条件下,释放异色瓢虫(Harmonia axyridis)防治甜瓜蚜虫(Aphis gossypii Glover)的综合效果,按不同益害比释放异色瓢虫,评价其在设施条件下对甜瓜蚜虫的防控效果以及天敌和害虫的消长动态。结果表明:异色瓢虫对于甜瓜蚜虫具有良好的防控效果,特别是长期防控效果优于化学农药,益害比1∶5与益害比1∶10的投放密度对蚜虫的防控效果好,两者比较差异未达显著水平,在释放后28 d时防效仍在70%以上。但考虑到生产成本,益害比1∶10为甜瓜上异色瓢虫释放的最佳密度;前期异色瓢虫定殖量随着蚜虫数量增加而升高,14 d时天敌投放处理的瓢虫定殖量达到峰值,故应结合病虫害测报,在蚜虫发生高峰期前尽早释放。

Increased nitrogen fertilization inhibits the biocontrol activity promoted by the intercropping partner plant

The examination of the compatibility between agricultural practices and biocontrol activities is crucial for establishing an efficient,eco-friendly,and sustainable pest management program.In this study,we examined the population dynamics of two specialist aphids,the English grain aphid(Sitobion avenae)on potted wheat and the pea aphid(Acyrthosiphon pisum)on potted alfalfa,as well as the biocontrol activity of a generalist predator,the harlequin ladybird beetle(Harmonia axyridis).We investigated their responses to the presence of the intercropping partner plant species(alfalfa and wheat,respectively)through plant volatiles or visual cues at three nitrogen fertilizer levels in a greenhouse.In the absence of the predator,the English grain aphid population growth rate increased significantly with increasing nitrogen levels,whereas the pea aphid population increased significantly more slowly in response to high nitrogen levels.The English grain aphid and pea aphid population dynamics were unaffected by the presence of the intercropping partner.However,the presence of the intercropping partner enhanced the control of both aphid populations by the harlequin ladybird beetle.Increasing nitrogen fertilizer levels decreased the predation rates,which were otherwise increased by the intercropping partner.The beneficial effects of the intercropping partner were eventually non-existent at the highest nitrogen level tested.These results imply that the interaction between the presence of intercropping partner and the nitrogen fertilizer application affects the biocontrol activity of the natural enemies of insect pests.Thus,the compatibility between agricultural intensification and biocontrol strategies in integrated pest management programs need to be investigated.

Role of social and individual experience in interaction of the meadow ant Formica pratensis （Hymenoptera： Formicidae） with ladybird imagines and hoverfly larvae

The ability to recognize aphidophages is one of the key points in the protection ants provide aphids against their natural enemies. Behavior of honeydew collectors from nature （＂field,＂ control） and laboratory reared ＂naive＂ ants of Formica pratensis Retzius, which had never met either ＂mature＂ workers or aphids and aphidophages, was observed during their pairwise interactions with ladybird imagines and hoverfly larvae. The majority of the ＂naive＂ ants perceived ladybirds as an enemy at their first encounter attacking them immediately without any prior antennation. Ants seem to have a certain innate ＂enemy image＂ that lets them react very quickly to protect aphids. Hoverfly larvae were rarely attacked by both ＂field＂ and ＂naive＂ ants （〉 15%）. During tests with ladybirds ants from nature attacked them and also demonstrated the most aggressive reactions （series of bites and ＂death grip＂） less frequently than the ＂naive＂ ants. The percentage of ants avoiding aphidophages after a contact with their chemical defense （reflex bleeding and glue-like saliva） was significantly higher in the control group. Whereas the ＂naive＂ ants did not learn to avoid danger, foragers from nature usually tried to avoid negative experience and used tactics of ＂short bites.＂ Overall, experience has been proved to be unimportant for displaying key behavioral reactions underlying ant-ladybird interaction. However, accumulation of experience has been assumed to play an important role in the formation of behavioral strategy that allows honeydew collectors to drive aphidophages away with lower energy costs and avoid or minimize negative consequences of aphidophages＇ chemical defense.