A Visit to the Zoos and Aquariums in Japan II

In the year 2013, the authors, who had already visited some Japanese aquariums and zoos in 2012 (Lange & Tai, 2012), revisited several Japanese aquariums and zoos. They were accompanied by two colleagues from Basle Zoo and the Monterey Bay Aquarium, who joined the tour.Their goals of the tour were focused on new innovative husbandry methods, on new aquarium buildings and new zoo enclosures as well as on rare Japanese endemic animals, which are never or seldom seen outside of Japan.The perfect new aquariums in Kyoto and Kitami City (Hokkaido), but also the very specialized aquariums like the Kamo Aquarium (for jellyfish) in Yamagata, the Chitose Salmon Aquarium (for salmons), the Otaru Aquarium (for sea mammals) and the Umi Kirara Kujukushima Aquarium (for the fauna of the Kujukushima island National Park) have been destinations for this trip. Besides the aquariums also the Asahiyama Zoo (new enclosures for endemic animals of Hokkaido), Kobe Oji Zoo, Kushiro Zoo (many endemic Hokkaido species), Nagasaki Bio Park, Sapporo Maruyama Zoo (new area for South East Asian animals) and the Sasebo Zoological & Botanical Garden were visited.During this tour it was obvious that neither the colleagues in Japan nor the staff in the zoos outside of Japan know sufficient enough about the conception and the husbandry methods in the different institutions in this part of the world. Therefore it is recommendable to organize such tours often for more people, thus both sides can benefit and learn from each other. This again will influence to help to keep the animals better, to attract more visitors in order to inspire them for the conservation of nature and environment.     

空心莲子草叶甲对越冬保护的响应与控害效能

空心莲子草叶甲Agasicles hygrophila是入侵杂草空心莲子草Alternan thera philoxeroide的重要天敌,冬春季低温冰冻是影响其安全越冬的关键胁迫因子。为了探明采取保护措施是否能提高越冬后的种群基数问题,在低温冰冻天气来临前,用塑料膜覆盖网室构建空心莲子草与空心莲子草叶甲保护生境(PH),分低温期(2009年1月21日至4月9日)、升温期(4月15日至5月1日)与自然生境(NH)中叶甲成虫初现期(从6月1日至29日)3个阶段,调查比较PH与NH两类生境中空心莲子草叶甲种群数量、结构、空间生态位分布特征及控害效能。结果表明:在保护生境中,低温期空心莲子草叶甲不休眠,且能安全越冬,当晴天温度较高时,成虫主要在直立茎或嫩枝上活动取食、补充营养,而在阴雨低温天气则停留在匍匐草层或土壤表层、缝隙中,少活动。随着气温回升,成虫转移到直立茎生态位上活动取食,并于4月上旬开始产卵繁殖,种群数量迅速增加,对空心莲子草的地上部分有较好的控制效果。在自然生境中,直到6月初才见少量成虫活动,此时草生长茂密,较低的空心莲子草叶甲种群难以发挥生防因子的作用。比较两种生境中6月1日与6月15日的控草效果显示,空心莲子草株高增长率分别为:PH-13.91%,NH-2.94%,NH中空心莲子草株高极显著高于PH,且单株生物量的控制率为PH(47.56%)极显著高于NH(4.89%)。在6月15日与29日的调查表明,单株叶片控制效果PH极显著优于NH,PH中的空心莲子草茎上均无叶,叶片或被取食殆尽、或被害后干枯脱落。茎被害率、茎蛀孔数与蛀茎率均为PH极显著高于NH。结果说明覆膜保护能显著提高叶甲越冬种群基数,使其提前进入繁殖期,从而能更有效地控制生境中的空心莲子草。     

Multiplex PCR assay for the detection of enterotoxic <Emphasis Type="Italic">Bacillus cereus</Emphasis> group strains and its application in food matrices

Bacillus cereus, Bacillus thuringiensis and Bacillus anthracis are the major concerns for the food safety in terms of frequency and/or seriousness of the disease. Being members of the same group and sharing DNA homology to a larger extent, they do create problems when their specific detection/identification is attempted from different food and environmental sources. Numerous individual polymerase chain reaction (PCR) and few multiplex PCR (mPCR) methods have been employed to detect these organisms by targeting toxin genes but with lack of internal amplification control (IAC). Therefore, we attempted a mPCR with IAC for the detection of enterotoxic B. cereus group strains by selecting hbl A, nhe A and cyt K genes from B. cereus, indicative of the diarrheal potential and cry I A and pag genes, the plasmid borne phenotypic markers specific to B. thuringiensis and B. anthracis strains, respectively. Multiplex PCR assay validation was performed by simultaneous comparison with the results of single-target PCR assays and correlated to the classical conventional and biochemical identification of the organisms. The mPCR was able to detect as low as 10¹–10² organisms per ml following overnight enrichment of spiked food samples (vegetable biriyani and milk) in buffered peptone water (BPW). The presence of these organisms could also be detected by mPCR in naturally contaminated samples of rice based dishes and milk. The high throughput and cost-effective mPCR method described could provide a powerful tool for simultaneous, rapid and reliable detection of enterotoxic B. cereus group organisms.     

A visit to the zoos and aquariums in Northern Japan

For their trip to the National Parks in Hokkaido the authors also included in their schedule visits to the zoos and aquariums in Northern Japan. Their tight scheduled tour started at the Aquamarine Fukushima, which was after the large tsunami rebuilt and re-opened in July 2011. They continued with a visit to the Kamo Aquarium, which is famous for its unique and large jellyfish exhibition. On Hokkaido Island they visited the smaller zoos in Kushiro and Obihiro with a good collection of native Hokkaido animal species. Included was also the Asahiyama Zoo, which is at the moment one of the most popular zoos in Japan. It is well-known for its innovative enclosures for seals and penguins. The Sapporo Maruyama Zoo and the Chitose Salmon Aquarium were the last highlights of this tour. The themes and animal collections of these different zoos and aquariums are described. The general situation for the northern zoos and aquariums is quite different compared to the institutions in the southern parts of the country and entirely to the zoos in the large city areas like Tokyo, Osaka and Nagoya. Here in the northern zoos and aquariums many native animal species and subspecies are kept and often successfully bred.     

蝇蛹金小蜂的交配行为及雄蜂交配次数对雌蜂繁殖的影响

多数昆虫能够进行多次交配,随寄生蜂雄蜂交配次数的增加,雄蜂体内精子减少,雌蜂获得的精子数量减少,产下更多的单倍体卵,发育为雄性后代;一些单寄生性的寄生蜂雌蜂一生仅能够交配1次。描述了蝇蛹金小蜂雌雄蜂的交配行为,探讨了雄蜂交配次数对雌蜂后代产量等的影响以及雌蜂的可交配次数。结果表明,交配过程包括求偶、交尾前期、交尾和交尾后期;雄蜂已交配的次数并不能够显著影响其配偶的寿命、产卵期和后代总数量,但显著影响到其配偶的雌、雄后代数量和性比。随雄蜂交配次数的增加,与之交配的雌蜂的后代雄性百分比显著增大,雌蜂在产卵期内更早地出现较多雄性后代,体内精子不足的现象更加明显。无论已交配的蝇蛹金小蜂雌蜂在产卵期中是否出现精子不足,均不能再次完成交配。     

Changes in the reproductive biology of Biomphalara glabrata infected with different doses of Echinostoma paraensei miracidia

The egg-laying rate, number of egg masses, number of eggs/mass, number of eggs hatched/snail and egg viability of Biomphalaria glabrata exposed to different doses (5 and 50) of Echinostoma paraensei miracidia were analyzed as indicators of reproductive activity. Polystyrene plates were placed in aquariums containing the snails and every other day for four weeks after infection the plates were removed to count the number of egg masses and eggs laid. After this, the plates were numbered individually and placed in new aquariums free of snails and the egg masses were observed daily to determine the hatching rate. On average there was an increase in the parameters evaluated in the infected snails in relation to the controls (uninfected snails), except for egg viability, which was significantly lower in the groups infected with 50 miracidia. These findings indicate that when infected, this host snail is able to increase its reproductive activity, suggesting an ecological strategy to maintain the species.     

松果梢斑螟对虫害诱导寄主防御的抑制作用

以松果梢斑螟（Dioryctria pryeri）-油松（Pinus tabuleaformis）（2年生球果和新梢）为研究对象,探讨梢斑螟幼虫对油松球果小卷蛾（Gravitarmata margarotana）先期虫害诱导寄主防御的抑制作用,以及虫害诱导的负防御机制。结果表明,双萜松脂酸作为油松球果和新梢的主要组成和诱导性防御物质,梢斑螟虫害后球果双萜松脂酸极显著增加,10 d后降低到正常水平;而新梢虫害后,松脂酸显著增加,后随新梢基础含量而增加,10 d后虫害新梢松脂酸显著高于球果。梢斑螟幼虫以小卷蛾虫害球果、健康球果和新梢等部位为食料,均为梢斑螟5龄幼虫下唇腺葡萄糖氧化酶（Glucose oxidase,GOX）活性最高,极显著高于4龄和3龄幼虫;且同一龄期,小卷蛾虫害球果中的梢斑螟幼虫GOX活性最高,显著高于新梢和健康球果中幼虫酶活性。研究发现,虫害后萜类防御物质随幼虫GOX活性升高呈下降趋势。梢斑螟幼虫RNA和P含量比较发现,取食小卷蛾虫害球果、健康球果和新梢3种食料,均为梢斑螟3龄幼虫最高,5龄幼虫最小,差异极显著;但同一龄期,3种食料发育的幼虫,其RNA和P含量间无显著差异。这些结果说明,小卷蛾幼虫的先期危害,诱导了寄主防御,但后来的梢斑螟幼虫通过下唇腺GOX抑制了寄主的诱导防御,使其生长率与健康球果和新梢中的幼虫基本一致。     

Genome sequence of B. amyloliquefaciens type strain DSM7(T) reveals differences to plant-associated B. amyloliquefaciens FZB42

The complete genome sequence of Bacillus amyloliquefaciens type strain DSM7^T is presented. A comparative analysis between the genome sequences of the plant associated strain FZB42 (Chen et al., 2007) with the genome of B. amyloliquefaciens DSM7^T revealed obvious differences in the variable part of the genomes, whilst the core genomes were found to be very similar. The strains FZB42 and DSM7^T have in common 3345 genes (CDS) in their core genomes; whilst 547 and 344 CDS were found to be unique in DSM7^T and FZB42, respectively. The core genome shared by both strains exhibited 97.89% identity on amino acid level. The number of genes representing the core genome of the strains FZB42, DSM7^T, and Bacillus subtilis DSM10^T was calculated as being 3098 and their identity was 92.25%. The 3,980,199 bp genome of DSM7^T contains numerous genomic islands (GI) detected by different methods. Many of them were located in vicinity of tRNA, glnA, and glmS gene copies. In contrast to FZB42, but similar to B. subtilis DSM10^T, the GI were enriched in prophage sequences and often harbored transposases, integrases and recombinases. Compared to FZB42, B. amyloliquefaciens DSM7^T possessed a reduced potential to non-ribosomally synthesize secondary metabolites with antibacterial and/or antifungal action. B. amyloliquefaciens DSM7^T did not produce the polyketides difficidin and macrolactin and was impaired in its ability to produce lipopeptides other than surfactin. Differences established within the variable part of the genomes, justify our proposal to discriminate the plant-associated ecotype represented by FZB42 from the group of type strain related B. amyloliquefaciens soil bacteria.     

Nectar feeding by the Cape rock elephant-shrew Elephantulus edwardii (Macroscelidea) — A primarily insectivorous mammal pollinates the parasite Hyobanche atropurpurea (Orobanchaceae)

Cape rock elephant-shrews, Elephantulus edwardii (Macroscelidea) were found to be pollinators of Hyobanche atropurpurea (Orobanchaceae), a member of a holoparasitic plant genus, lacking up to now any detailed pollinator observations. The elephant-shrews were live-trapped near the plants in the Western Cape of South Africa. One animal was released in a terrarium, containing the plants. It licked nectar with its long and slender tongue while being dusted with pollen and touching the stigmas of the flowers with its nose and the fur above the snout. H. atropurpurea pollen was found in swabs of the fur around noses and snouts as well as in the faeces of captured animals. The occurrence of pollen in the faeces of the elephant-shrews is likely a result of grooming their fur as they visited the flowers without eating or damaging them. This is one of the first records of pollinating, nectar-drinking elephant-shrews, contributing to the very scarce knowledge about the behaviour of those primarily insectivorous animals, as well as small mammal pollination.     

美洲黑杨凋落叶分解初期对小白菜生长的影响

采用盆栽试验,研究了美洲黑杨(Populus deltoides)凋落叶分解初期对受体植物小白菜(Brassica chinensis)生长和生理的影响。试验设置0、30、60和90 g/盆4个凋落叶施用水平(分别记作CK、L30、L60和L90)。同时,为检验凋落叶施入是否对土壤通气透水性产生明显影响进而影响受体植物的生长,用蒸煮后的凋落叶设置平行空白试验,即30、60、90 g/盆3个蒸著后的凋落叶处理(分别记作Z30、Z60和Z90)。将各处理的凋落叶分别与7 kg土壤混合,播种小白菜。在播种后50、80 d测定小白菜株高和生理指标。结果表明:1)高量(L90)凋落叶下小白菜的高生长和鲜重于50 d时被显著抑制,80 d时长势恢复正常;2)80 d时各处理净光合速率(Pn)与CK水平相当,色素含量略低于CK;3)50、80 d时,低(L30)、中(L60)量处理的超氧化物歧化酶(SOD)活性无明显变化,高量处理下SOD活性升高;4)各处理丙二醛(MDA)含量在50、80 d时与CK均无显著差异。总的来看,杨树各凋落叶量处理对小白菜的影响表现为:低、中量促进,高量抑制,而经蒸煮后的凋落叶处理间差异不显著。表明,低、中量杨树凋落叶在土壤中分解对小白菜生长及生理代谢的影响主要表现为促进作用,而施入高量凋落叶的初期,化感抑制作用明显。     

Rubrimonas shengliensis sp. nov. and Polymorphum gilvum gen. nov., sp. nov., novel members of Alphaproteobacteria from crude oil contaminated saline soil

Four bacterial strains were isolated from a crude oil contaminated saline soil in Shengli Oilfield, China. Strains SL014B-28A2^T and SL014B-80A1 were most closely related to Rubrimonas cliftonensis OCh 317^T, while strains SL003B-26A1^T and SL003B-26A2 were most closely related to but readily different from the species in the Pannonibacter-Labrenzia-Roseibium-Stappia cluster. The major fatty acids were C_18:1ω7c, C_16:0, C_18:0 and 11-Methyl C_18:1ω7c, and C_18:1ω7c, 11-Methyl C_18:1ω7c and C_18:0, respectively, for these two groups of isolates. Q-10 was the predominant ubiquinone. The G + C contents of genomic DNA of the four isolates were 67.9, 69.7, 65.6 and 65.6 mol%. Based on the polyphasic taxonomic characteristics, strains SL014B-28A2^T and SL014B-80A1 represented a novel species of the genus Rubrimonas, for which the name Rubrimonas shengliensis sp. nov. is proposed, with strain SL014B-28A2^T (=LMG 26072^T = CGMCC 1.9170^T) as the type strain. Isolates SL003B-26A1^T and SL003B-26A2 represented a novel genus and species of the family Rhodobacteraceae, for which the name Polymorphum gilvum gen. nov., sp. nov. is proposed, with strain SL003B-26A1^T (=LMG 25793^T = CGMCC 1.9160^T) as the type strain.     

Black yeast habitat choices and species spectrum on high altitude creosote-treated railway ties

Polyextremotolerant black yeast-like fungi thrive in moderately hostile environments where they are concomitantly subjected to several types of stress, such as toxicity, scarce nutrient availability, and high or low temperature extremes. Their ability to assimilate alkylbenzenes (toxic environmental pollutants) enhances their growth in harsh conditions, including on railway ties. Samples were collected using cotton swabs, premoistened with physiological saline, from 658 oak and concrete railway ties at six train stations in Turkey at altitudes ranging between 1026 and 1427 m. The samples were inoculated on malt extract agar supplemented with chloramphenicol, and incubated at 26 °C for 4 weeks. Twenty-four samples (3.6 %), 17 from oak and 7 from concrete (5.6 % vs. 2 %; P = 0.02), tested positive for fungi. Exophiala crusticola was found to be the most common species (n = 13), followed by Exophiala phaeomuriformis (n = 7) and Exophiala heteromorpha (n = 4). These results suggest that hydrocarbons, particularly creosote-treated oak woods, support the growth of black yeasts, some of which are opportunists in humans.     

Effects of infection with Nosema pyrausta on survival and development of offspring of laboratory selected Bt-resistant and Bt-susceptible European corn borers

Infection with Nosema pyrausta Paillot lengthens developmental period of Bt-susceptible Ostrinia nubilalis (Hübner) to a similar extent as feeding on Cry1Ab-incorporated diet in Cry1Ab-resistant O. nubilalis, and these two factors combined lengthen developmental period further than either alone. Resistant O. nubilalis mating with infected susceptible, or infected resistant partners would produce partially- and fully-resistant offspring, respectively, infected with N. pyrausta. To investigate the impacts on the progeny of such matings, test crosses were set up to produce partially- and fully Cry1Ab-resistant O. nubilalis offspring transovarially infected and not infected with N. pyrausta, which were exposed to Cry1Ab toxin at doses of 0, 3, or 30 ng/cm² for 7 days. Transovarial infection with N. pyrausta significantly decreased 7 day survival of partially and fully-resistant O. nubilalis feeding on 30 ng/cm² Cry1Ab. In addition, N. pyrausta infection delayed larval development (as measured by weight) of partially- and fully-resistant O. nubilalis feeding on 3 and 30 ng/cm² Cry1Ab. Impacts of natural enemies on target pests may have the potential to impact evolution of resistance. N pyrausta-infected O. nubilalis are more strongly affected by feeding on Bt, and would be less likely to survive to adulthood to pass on resistance to the next generation. This indigenous microsporidium may work to delay evolution of resistance in O. nubilalis by lowering their ability to survive on Bt.     

重金属Zn2+胁迫下麦长管蚜的取食行为

为了探索重金属锌长期胁迫对麦长管蚜(Sitobion avenae)取食行为的变化影响,在模拟自然的实验室条件下,用不同浓度Zn~(2+)溶液浇灌土壤,通过土壤-小麦-蚜虫体系连续处理麦长管蚜15代,用EPG(刺探电位技术)对第1、5、10、15代成蚜的取食行为进行了监测。结果表明,第1代和第5代时,200 mg/kg的Zn~(2+)处理后np波和C波的总持续时间和数量显著低于对照,800mg/kg的Zn~(2+)使其显著增加。到第15代,高剂量的Zn~(2+)处理后np波和C波的总持续时间和数量均显著高于对照。涉及分泌唾液的E1波持续时间及涉及被动取食营养的E2波出现次数并未受到低剂量Zn~(2+)的显著影响,但高剂量的Zn~(2+)处理后单独E1波、伴随稳定E2的E1波总持续时间及E2波的数量均显著降低。麦长管蚜的取食行为会受到重金属锌的影响并且会在高剂量Zn~(2+)的胁迫条件下产生积累效应,而低剂量的Zn~(2+)则促进麦长管蚜对小麦的取食行为。针对重金属而言,此效应发生改变的关键浓度为400 mg/kg,蚜虫取食行为发生改变的关键世代为第5代和第10代。     

不同猎物饲喂对南方小花蝽捕食量和喜好性的影响

为探讨南方小花蝽对不同猎物的捕食喜好性,室内用西花蓟马、蚕豆蚜、二斑叶螨、混合饲料(同时饲喂3种猎物)分别饲喂南方小花蝽驯化两代,研究了4种饲喂处理的南方小花蝽初孵若虫、5龄若虫和雌成虫对西花蓟马、蚕豆蚜和二斑叶螨的捕食量和喜好性。结果显示不同猎物饲喂处理驯化的南方小花蝽1龄若虫对同一种猎物的捕食量和喜好性均不存在显著差异。南方小花蝽5龄若虫和雌成虫对某种猎物的捕食量因前期取食的猎物种类不同而有显著差异。南方小花蝽5龄若虫和雌成虫均表现出对西花蓟马2龄若虫的正喜好性。蚕豆蚜饲喂处理的5龄若虫和雌成虫对蚕豆蚜表现出正喜好性,除二斑叶螨饲喂处理外其余3种处理的南方小花蝽5龄若虫和雌成虫均表现出对二斑叶螨的负喜好性。以上结果表明4种饲喂驯化处理的南方小花蝽1龄若虫的喜好性不受前期取食猎物的影响,但5龄若虫和雌成虫对前期取食过的猎物的喜好性增强,存在一定的学习行为。     

Simultaneous detection of pathogenic <Emphasis Type="Italic">B. cereus,S. aureus</Emphasis> and <Emphasis Type="Italic">L. monocytogenes</Emphasis> by multiplex PCR

Three important foodborne pathogens, Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus are of major concern for food safety in terms of frequency and seriousness of the disease. The occurrence these three important pathogens and their coexistence in food matrices are predominant. Moreover, symptoms associated with B. cereus and S. aureus food poisoning not only closely resembles each other but can also be overlapping with other foodborne infections. In this context, detection of these three pathogens simultaneously in food samples by a single multiplex PCR (mPCR) would have advantages in terms of rapidity and cost saving, when compared with single organism specific PCRs. mPCR has been standardized by targeting three major diarrheal enterotoxin genes hbl A, cyt K and nhe A of B. cereus, virulence associated nuc and Ent B genes of S. aureus and virulence associated hly and iap genes of L. monocytogenes along with internal amplification control (IAC). The results showed that mPCR accurately identified all the three organisms individually or in combination without non-specificity. The mPCR was able to detect as low as 10 to 100 organisms per ml of growth following overnight enrichment of spiked food samples (vegetable biriyani and milk) and their presence in naturally contaminated samples also. The high throughput and cost effective multiplex PCR method developed in this study could provide a powerful tool for simultaneous, rapid and reliable detection of B. cereus, S. aureus and L. monocytogenes in food samples.     

北长山岛森林乔木层碳储量及其影响因子

人工林是庙岛群岛典型的陆地生态系统的组成部分,对维护海岛地区生态环境具有重要作用。采用现有生物量相对生长方程和样地调查数据相结合的方法,以庙岛群岛中北长山岛为研究区,对海岛黑松纯林与黑松×刺槐混交林两种林型的碳储量进行了估算,并分析了土壤质地及其理化性质对海岛乔木层碳储量的影响。结果表明:黑松乔木层平均碳储量为84.00 t/hm²,接近于世界平均水平(86.00 t/hm²);黑松×刺槐混交林乔木层平均碳储量为29.60 t/hm²,高于山东省乔木层的平均碳储量(27.62 t/hm²)。应用因子分析法研究影响乔木层碳储量的主要因子,结果表明:土壤质地、pH值、含水量及含盐量是影响海岛乔木碳储量重要的影响因子。北长山岛土壤全氮、总磷、土壤有机质、碳氮比等其他理化性质对乔木层碳储量影响不是非常明显。     

Repellent activity of catmint, Nepeta cataria, and iridoid nepetalactone isomers against Afro-tropical mosquitoes, ixodid ticks and red poultry mites

The repellent activity of the essential oil of the catmint plant, Nepeta cataria (Lamiaceae), and the main iridoid compounds (4aS,7S,7aR) and (4aS,7S,7aS)-nepetalactone, was assessed against (i) major Afro-tropical pathogen vector mosquitoes, i.e. the malaria mosquito, Anopheles gambiae s.s. and the Southern house mosquito, Culex quinquefasciatus, using a World Health Organisation (WHO)-approved topical application bioassay (ii) the brown ear tick, Rhipicephalus appendiculatus, using a climbing repellency assay, and (iii) the red poultry mite, Dermanyssus gallinae, using field trapping experiments. Gas chromatography (GC) and coupled GC-mass spectrometry (GC-MS) analysis of two N. cataria chemotypes (A and B) used in the repellency assays showed that (4aS,7S,7aR) and (4aS,7S,7aS)-nepetalactone were present in different proportions, with one of the oils (from chemotype A) being dominated by the (4aS,7S,7aR) isomer (91.95% by GC), and the other oil (from chemotype B) containing the two (4aS,7S,7aR) and (4aS,7S,7aS) isomers in 16.98% and 69.83% (by GC), respectively. The sesquiterpene hydrocarbon (E)-(1R,9S)-caryophyllene was identified as the only other major component in the oils (8.05% and 13.19% by GC, respectively). Using the topical application bioassay, the oils showed high repellent activity (chemotype A RD₅₀ = 0.081 mg cm⁻² and chemotype B RD₅₀ = 0.091 mg cm⁻²) for An. gambiae comparable with the synthetic repellent DEET (RD₅₀ = 0.12 mg cm⁻²), whilst for Cx. quinquefasciatus, lower repellent activity was recorded (chemotype A RD₅₀ = 0.34 mg cm⁻² and chemotype B RD₅₀ = 0.074 mg cm⁻²). Further repellency testing against An. gambiae using the purified (4aS,7S,7aR) and (4aS,7S,7aS)-nepetalactone isomers revealed overall lower repellent activity, compared to the chemotype A and B oils. Testing of binary mixtures of the (4aS,7S,7aR) and (4aS,7S,7aS) isomers across a range of ratios, but all at the same overall dose (0.1 mg), revealed not only a synergistic effect between the two, but also a surprising ratio-dependent effect, with lower activity for the pure isomers and equivalent or near-equivalent mixtures, but higher activity for non-equivalent ratios. Furthermore, a binary mixture of (4aS,7S,7aR) and (4aS,7S,7aS) isomers, in a ratio equivalent to that found in chemotype B oil, was less repellent than the oil itself, when tested at two doses equivalent to 0.1 and 0.01 mg chemotype B oil. The three-component blend including (E)-(1R,9S)-caryophyllene at the level found in chemotype B oil had the same activity as chemotype B oil. In a tick climbing repellency assay using R. appendiculatus, the oils showed high repellent activity comparable with data for other repellent essential oils (chemotype A RD₅₀ = 0.005 mg and chemotype B RD₅₀ = 0.0012 mg). In field trapping assays with D. gallinae, addition of the chemotype A and B oils, and a combination of the two, to traps pre-conditioned with D. gallinae, all resulted in a significant reduction of D. gallinae trap capture. In summary, these data suggest that although the nepetalactone isomers have the potential to be used in human and livestock protection against major pathogen vectors, intact, i.e. unfractionated, Nepeta spp. oils offer potentially greater protection, due to the presence of both nepetalactone isomers and other components such as (E)-(1R,9S)-caryophyllene.     

Captive breeding and larval morphology of Tylototriton shanjing Nussbaum, Brodie & Yang, 1995, with an updated key of the genus Tylototriton (Amphibia: Salamandridae)

Tylototriton shanjing Nussbaum, Brodie & Yang, 1995 was described as a distinct species hidden before under the widely distributed T. verrucosus Anderson, 1871. Therefore, papers published before 1995, including those on reproductive biology, could not distinguish between these two close relatives. Consequently, data on the reproductive biology of T. shanjing are scarce. Here, we report on the first captive breeding of T. shanjing in the Aquarium of the Cologne Zoo, and we document the ontogenetic developmental stages and describe the larva for the first time. Moreover, we give a literature review for both T. verrucosus and T. shanjing and compare our data with published information referring undoubtedly to the latter species. To avoid future misidentifications and to facilitate the composition of breeding groups, we present an identification key for the East Asian salamandrid genera in general, and for the species of Tylototriton in particular.