天敌体内玉米蚜的CoⅡ标记检测

以玉米蚜Rhopalosiphum maidis Fitch及玉米田的捕食性天敌为研究对象,筛选1对特异扩增玉米蚜细胞色素氧化酶Ⅱ(COⅡ)基因序列片段的引物。种特异性检验表明,该引物只对玉米蚜基因组DNA具有扩增效果,对与其同域发生的其他种类不具扩增作用;室内以该引物检测喂食单头玉米蚜的异色瓢虫Harmonia axyridis(Pallas)成虫和大草蛉Chrysopa pallens(Rambur)成虫腹内靶标食物的衰变情况,结果表明,异色瓢虫成虫和大草蛉成虫阳性比率的半衰期分别为喂食后2·814和2·441h;对玉米田间捕食性天敌的检测结果表明,在所检测的6类群13种捕食性天敌中,大灰食蚜蝇幼虫、草蛉幼虫、异色瓢虫成虫和幼虫、龟纹瓢虫成虫和幼虫对玉米蚜的捕食作用检出率较高,大于40%。     

Rapid PCR-based test for identifying Candida albicans by using primers derived from the pH-regulated KER1 gene

A PCR-based method in combination with a simple, reliable and inexpensive DNA extraction procedure for rapid detection of Candida albicans clinical isolates is described here. The extraction protocol is based on a combination of chemical (NaOH and detergents) and physical (boiling) treatments, thus avoiding many of the problems inherent in the currently available DNA extraction protocols (basically the use of expensive and/or toxic chemical reagents), and may be useful for daily clinical routine. The PCR-based system described here uses a single pair of primers (SC1F and SC1R) deduced from the C. albicans-specific KER1 gene sequence. These primers amplify a 670-bp fragment of the KER1 gene. All the clinical C. albicans isolates generated the expected 670-bp amplicon. Other non-albicans Candida species, including the azole-resistant C. krusei and C. glabrata, and the very closely related C. dubliniensis, failed to amplify any DNA fragment. The PCR results reported here suggest that amplification with SC1F and SC1R primers is species specific and, consequently, may be useful for specifically identifying C. albicans strains.     

转Bt基因抗虫玉米对玉米蚜种群增长的影响

在人工气候箱条件下研究了玉米蚜（Rhopalosiphum maidis Fitch）取食表达cry1Ab杀虫蛋白Bt抗虫玉米的实验种群生命表.结果表明：两种不同Bt玉米杂交种DK647BTY （MON810转化事件）和NX4777（Bt11转化事件）对玉米蚜的生长、发育、繁殖和存活均无明显的不利影响,玉米蚜在DK647BTY和NX4777两种Bt玉米品种上的内禀增长率rm、周限增长率λ和种群净增殖率R0与各自对照之间没有显著差异;玉米蚜有翅蚜比率、各龄若虫的死亡率在Bt玉米和对照以及不同品种之间没有明显差异;Bt玉米对玉米蚜的寿命和繁殖历期也没有明显差异.表明表达cry1Ab杀虫蛋白的Bt玉米对玉米蚜的生长发育和繁殖没有明显影响.     

Evaluation of temperature gradient gel electrophoresis for the analysis of prey DNA within the guts of invertebrate predators

The utility of temperature gradient gel electrophoresis (TGGE) as a means of analysing the gut contents of predators was evaluated. Generalist predators consume multiple prey species and a species-specific primer approach may not always be a practical means of analysing predator responses to prey diversity in complex and biodiverse ecosystems. General invertebrate primers were used to amplify the gut contents of predators, generating banding patterns that identified component prey remains. There was no evidence of dominance of the polymerase chain reaction (PCR) by predator DNA. When applied to field samples of the carabid predator Pterostichus melanarius (Illiger) nine banding patterns were detected, including one for aphids. To further distinguish between species, group-specific primers were designed to separate species of earthworm and aphid. TGGE of the earthworm PCR products generated banding patterns that varied with haplotype in some species. Aphid and earthworm DNA could be detected in the guts of carabids for up to 24 h using TGGE. In P. melanarius, with low numbers of prey per insect gut (mean<3), interpretation of banding patterns proved to be tractable. Potential problems of interpretation of TGGE gels caused by multiple prey bands, cryptic bands, haplotype variation, taxonomic uncertainties (especially with regard to earthworms), secondary predation, scavenging and presence of parasites and parasitoids in the prey or the predators, are discussed. The results suggest that PCR, using combinations of general invertebrate and group-specific primers followed by TGGE, provides a potentially useful approach to the analysis of multiple uncharacterized prey in predators.     

三种玉米蚜虫种群的生态位分析

对重庆市郊区1998～1999年春玉米上3种蚜虫种群的数量动态、生态位宽度和生态位重叠进行了系统的分析研究．结果表明,发生在春玉米上的玉米蚜(Rhopalosiphum maidis),禾谷缢管蚜(Rhopalosiphum padi)和麦长管蚜(Sitobion avenae)3种蚜虫混合种群的种群数量消长呈现出5月中下旬和6月下旬两个高峰;在生态位宽度上,Livens生态位宽度指数(Bi)分析表明,麦长管蚜表现出时间上的高度分化,玉米蚜表现出空间上的高度聚集特性,而禾谷缢管蚜的分化并不明显;在生态位重叠上,玉米蚜和禾谷缢管蚜的HturlbeIt生态位重叠指数(Lij)最高,玉米蚜和麦长管蚜次之,禾谷缢管蚜和麦长管蚜最小．在春玉米上发生的3种蚜虫中,玉米蚜具有较强的竞争优势．     

Effect of a co-occurring aphid on the susceptibility of the Russian wheat aphid to lacewing predators

Previous field experiments indicated that the presence of the bird cherry-oat aphid, Rhopalosiphum padi (L.), on perennial grasses can decrease the effectiveness of predatory lacewings, Chrysoperla plorabunda (Fitch), in reducing populations of the Russian wheat aphid, Diuraphis noxia (Mordvilko). We tested the hypothesis that R. padi deflects predation away from D. noxia because it feeds in sites that are more accessible to predators. We quantified the behavior of lacewing larvae on crested wheatgrass plants bearing either D. noxia alone or an equal mixture of D. noxia and R. padi. On non-flowering plants, R. padi typically occurred on leaf sheaths or open blades, and was encountered and captured more often than D. noxia, which usually fed within immature, rolled leaves. Overall time-budgets of lacewings did not differ between the pure-D. noxia and mixed-species treatments, but >75% of the time spent consuming aphids in the mixed-species treatment was devoted to R. padi. On flowering plants, D. noxia usually aggregated on the flag leaf below the inflorescence, whereas R. padi occurred mostly on leaf sheaths. Predators again captured R. padi more often than D. noxia, and spent more time consuming aphids in the mixed-species treatment than in the pure-D. noxia treatment. These behavioral observations support the hypothesis that non-target prey can hamper the short-term effectiveness of biological-control agents against D. noxia.     

Molecular biological detection of anaerobic gut fungi (Neocallimastigales) from landfill sites

Oligonucleotide primers were designed for the 18S rRNA genes of members of the Neocallimastigales and used in a nested PCR protocol to amplify 787-bp fragments of DNA from landfill site samples. The specificities of the primers were confirmed by phylogenetic analysis of the environmental clone sequences, and this method can therefore now be used to investigate the ecology of the obligately anaerobic fungi. To our knowledge, this is the first demonstration of the occurrence of members of the Neocallimastigales outside the mammalian gut, and their distribution across the landfill samples examined here suggests that they are actively involved in cellulose degradation.     

温度对玉米蚜种群增长的影响

研究了温度对玉米蚜RhopalosiphummaidisFitch种群存活率、生殖率及内禀增长率的影响 ,同时测定了不同温度条件下玉米蚜种群的净增殖率、周限增长率、种群倍增时间及稳定年龄组配等种群参数。在 1 0℃和 3 5℃条件下 ,玉米蚜若蚜的死亡率较高 ,特别是 4龄若蚜 ;2 5℃最适宜玉米蚜的生长、发育和繁殖 ,其种群增长最快     

玉米田蚜虫种群的空间动态

【目的】明确我国黄淮海地区玉米蚜虫种类及其空间分布,掌握玉米蚜虫田间发生规律。【方法】采用系统调查法于2009—2010年在河北廊坊对春玉米、夏玉米上玉米蚜虫种群的发生动态进行了研究。【结果】结果表明,该地区取食为害玉米的蚜虫有5种;玉米蚜Rhopalosiphum maidis(Fitch)和禾谷缢管蚜R.padi(L.)混合发生,为玉米田蚜虫的优势种群。【结论】几种蚜虫的混合种群在玉米田间的动态分布始终呈聚集分布;二项分布k和聚集型指标m~*/x判断表明,随着玉米的生长发育,玉米蚜虫表现扩散-聚集-再扩散-再聚集的趋势。     

The influence of time and temperature on molecular gut content analysis： Adalia bipunctata fed with Rhopalosiphum padi

Gut content analysis is a useful tool when studying arthropod predator-prey interactions. We used polymerase chain reaction （PCR） technique to examine how detection of prey DNA in the gut content of predators was influenced by digestion time and temperature. Such knowledge is critical before applying PCR-based gut content analysis to field collected predators. Larvae of the two-spotted ladybeetle （Adalia bipunctata L.） were fed with the bird cherry-oat aphid （Rhopalosiphum padi L.） at either 21℃ or 14℃. After consuming one aphid, the predators were allowed to digest the prey for a range of time periods up to 24 hours. The influence of temperature on A. bipunctata feeding behavior was also recorded. From the fed larvae, total DNA was extracted and PCR reactions with R. padi specific primers were run. The number ofA. bipunctata that tested positive for R. padi DNA was negatively related to the length of digestion time. Temperature influenced larval feeding behavior but did not have a significant effect on R. padi DNA detection. After pooling the data from both temperature treatments we estimated the time point when R. padi DNA could be amplified from 50% of the fed A. bipunctata by PCR to be 4.87 hours. With such a rapid decrease in prey DNA detection success, positive PCR reactions will most likely be the result of predation events occurring shortly before capture. If a defined digestion temperature range has proven not to influence prey detection, PCR data obtained from predators collected within that particular range can be interpreted in the same way.     

Feeding mode and prey detectability half-lives in molecular gut-content analysis: an example with two predators of the Colorado potato beetle

The time during which prey remains are detectable in the gut of a predator is an important consideration in the interpretation of molecular gut-content data, because predators with longer detectability times may appear on the basis of unweighted data to be disproportionately important agents of prey population suppression. The rate of decay in detectability, typically expressed as the half-life, depends on many variables; one that has not been explicitly examined is the manner in which the predator processes prey items. The influence of differences in feeding mode and digestive physiology on the half-life of DNA for a single prey species, the Colorado potato beetle Leptinotarsa decemlineata (Say), is examined in two predators that differ dramatically in these attributes: the pink ladybeetle, Coleomegilla maculata (DeGeer), which feeds by chewing and then ingesting the macerated material into the gut for digestion; and the spined soldier bug, Podisus maculiventris (Say), which physically and enzymatically processes the prey extra-orally before ingestion and further digestion in the gut. In order to standardize the amount of DNA consumed per predator, a single L. decemlineata egg was used as the prey item; all predators were third instars. The PCR assay yields estimated prey DNA half-lives, for animals maintained under field temperatures, of 7.0 h in C. maculata and 50.9 h in P. maculiventris. The difference in the prey DNA half-lives from these two predators underscores the need to determine detectabilities from assemblages of predators differing in feeding mode and digestive physiology, in order to weight positives properly, and hence determine the predators' relative impacts on prey population suppression.     

PCR-based assay for the detection of Curtobacterium flaccumfaciens pv. flaccumfaciens in bean seeds

AIMS: To develop a PCR-based protocol for the rapid, sensitive and specific detection of Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff) in bean seeds. METHODS AND RESULTS: A pair of PCR primers (CffFOR2-CffREV4), targeting the sequence of a cloned DNA fragment of 550 bp amplified in Repetitive-sequence-based-PCR (Rep-PCR) experiments, were designed and shown to specifically amplify a 306-bp DNA fragment using Cff DNA as template. Moreover, this PCR protocol was demonstrated to successfully detect Cff in naturally infected bean seeds in 36 h. CONCLUSIONS: A specific, highly sensitive and rapid PCR assay for the detection of Cff was achieved. SIGNIFICANCE AND IMPACT OF THE STUDY:Cff is a seed-borne bacterium on the EPPO A2 quarantine list; this procedure may be useful for routine diagnosis of Cff, overcoming the problems of conventional techniques.     

Molecular Biological Detection of Anaerobic Gut Fungi (Neocallimastigales) from Landfill Sites

Oligonucleotide primers were designed for the 18S rRNA genes of members of the Neocallimastigales and used in a nested PCR protocol to amplify 787-bp fragments of DNA from landfill site samples. The specificities of the primers were confirmed by phylogenetic analysis of the environmental clone sequences, and this method can therefore now be used to investigate the ecology of the obligately anaerobic fungi. To our knowledge, this is the first demonstration of the occurrence of members of the Neocallimastigales outside the mammalian gut, and their distribution across the landfill samples examined here suggests that they are actively involved in cellulose degradation.     

Application of polymerase chain reaction with specific and arbitrary primers to identification and differentiation of Leishmania parasites

Abstract Two oligonucleotide primers Lsmc1 and Lsmv1 derived from the conserved and the variable region of a major class kinetoplast DNA (kDNA) minicircle (pLURkE3) of Leishmania strain UR6 were used for the polymerase chain reaction (PCR) in order to amplify a 461-bp fragment from the kDNAs of different Leishmania species. These primers amplify the specific fragment from the kDNAs of cutaneous species only. The cutaneous species can further be distinguished by randomly amplified polymorphic DNA (RAPD) analysis of the kDNAs of these organisms using arbitrarily chosen oligonucleotides. The arbitrary primers also generate polymorphic DNA fingerprints at the genomic level with different L. donovani isolates. The results indicate that the PCR and arbitrarily primed PCR (AP-PCR) may be extremely useful approaches for identifying and distinguishing Leishmania parasites.     

A DNA marker to identify predation of Plutella xylostella (Lep., Plutellidae) by Nabis kinbergii (Hem., Nabidae) and Lycosa sp. (Aranaea, Lycosidae)

Abstract:  Predators are important biotic factors in the population dynamics of the diamondback moth, Plutella xylostella . A specific DNA marker was developed to detect P. xylostella in the gut contents of two polyphagous predators, Nabis kinbergii and Lycosa sp. A distinct 275-bp product was amplified by polymerase chain reaction (PCR) from the internal transcribed spacer (ITS-1) of the ribosomal gene of P. xylostella , but not from 11 other arthropod species collected from Brassica fields in South Australia. Fortuitously, the primer set could also amplify DNA products from two species and three varieties of Brassica plants, with the fragment size about 600 bp. When N. kinbergii was analysed after feeding a single fourth instar P. xylostella , 67% of individuals were positive with the 275-bp PCR product up to 16 h after feeding. Likewise, the PCR product was detected in 80% individuals of Lycosa sp. up to 72 h after feeding on a single fourth instar P. xylostella larva. Initial tests of samples collected from the field showed that the predation incidences for both N. kinbergii and Lycosa sp. determined by the 275-bp fragment corresponded to the density of P. xylostella in the field.     

Evaluation of green lacewings, Chrysoperla plorabunda (Fitch) (Neurop., Chrysopidae), for augmentative release against Toxoptera citricida (Hom., Aphididae) in citrus

Larvae of the lacewing Chrysoperla plorabunda (Fitch) were evaluated in laboratory and field tests for potential to control the brown citrus aphid, Toxoptera citricida (Kirkaldy) in inundative releases. Larvae surviving to pupation consumed an average of 1676, 1297, 392, 165 and 130 1st–4th instar T. citricida nymphs and apterous adults, respectively, and the mean developmental time was 27.0, 23.0, 16.5, 13.8 and 18.3 days, respectively. The average pupation rate was 37.5%, but only 6.3% of pupae yielded adults. Developmental time and survival to pupation varied with the life stage of aphids fed to larvae, 4th instars yielding the highest survival and shortest developmental time. Development of C. plorabunda was compared on diets of T. citricida and Aphis spiraecola . Only 6.3% of larvae completed development on T. citricida , whereas 37.5% yielded adults on A. spiraecola . Larvae consumed more of the brown T. citricida than the green A. spiraecola in a choice test on a white background, but no preference was evident when aphids were offered on a black background. Two separate field trials were performed in citrus groves with T. citricida infestations in which an average of 275 and 116 C. plorabunda larvae/tree were released, respectively. The rate of T. citricida colony maturation (= survival to alate production) was very low in the first trial, and relatively high in the second trial, but did not differ between control and release trees in either trial. The most apparent cause of aphid colony mortality was predation by the coccinellids Cycloneda sanguinea L. and Harmonia axyridis Pallas. Despite acceptance of T. citricida in the laboratory, very few C. plorabunda larvae were observed feeding on aphid colonies on release trees.     

Effects of Brassica oleracea waxblooms on predation and attachment by Hippodamia convergens

Four mutations that reduce waxbloom in Brassica oleracea L. were examined for their effects on predation, mobility, and adhesion to the plant surface by the general predator Hippodamia convergens (Guérin-Menéville) (Coleoptera: Coccinellidae). The mutations reduce waxbloom to different degrees, but all produce a glossy phenotype. Plants tested were inbred lines, near isogenic lines, or segregating F₂ populations, depending on the mutation. In an experiment on caged leaves, predation of Plutella xylostella L. larvae by H. convergens adult females was significantly greater on glossy types as compared with normal-wax or wild-type counterparts. Although the trend was the same for each mutation, individual comparisons between glossy and normal-wax lines or segregants were only significant for two of them, those producing mutant alleles gl_a and gl_d. Individual H. convergens were observed to spend more time walking on leaf edges and less time walking on leaf surfaces of normal-wax plants than glossy plants. Hippodamia convergens also obtained better adhesion to the surfaces of glossy plants than to normal-wax plants when tested using a centrifugal device. Two of the mutations produced similarly strong effects on predation, behaviour, and adhesion by H. convergens. These two are the same previously determined by us to provide the strongest similar effects on another generalist predator, Chrysoperla plorabunda (Fitch). The results indicate that waxbloom variation in nature could affect herbivore populations through its effects on generalist predators.     

Impact of abiotic factors on predator-prey interactions: DNA-based gut content analysis in a microcosm experiment

The effects of predators on prey populations can be modified by a number of abiotic factors. Here, we investigated the combined and separate effects of rain and ground-dwelling predators on aphid populations in a microcosm experiment lasting for 21 days, using PCR to analyse the gut content of the predators. Rain significantly dislodged aphids from shoots and ears by 57% and 25%, respectively. The gut content analysis showed that more predators consumed aphids in the rain treatment than without rain, indicating higher availability of aphids to ground-dwelling predators after rain. However, no synergistic effects of rain and ground-dwelling predators on aphid population development could be demonstrated. Rain alone significantly decreased aphid populations by 27%, suggesting that this is a significant mortality factor. Predators alone had no significant effect on aphid numbers, but the gut content analyses showed aphid consumption also in the no-rain treatments, indicating that aphids were available to the predators on the soil surface even without rain. Our results suggest that weather conditions such as rain can modify predator-prey interactions in the field. Employing PCR-based predator gut content analyses proved to be useful as trophic links could be directly verified.