Process of infection of armored scale insects (Diaspididae) by an entomopathogenic Cosmospora sp

Several species in the fungal genus Cosmospora (synonym Nectria) (anamorph Fusarium) are specialist entomopathogens of armored scale insects (Diaspididae), known to cause periodic epizootics in host populations. Inconsistent mortality rates recorded under laboratory conditions prompted a study into the process of infection of armored scale insects by this fungus. Scale insect mortality following exposure to a Cosmospora sp. (Culture Collection Number: CC89) from New Zealand was related to insect age, with reproductively mature insects having a significantly higher infection rate than immature insects. Examination using scanning electron microscopy found no evidence that the fungus penetrated directly through the wax test (cap) of the scale insect or through the un-lifted interface between the test and the substrate on which the insect resided. However, fungal hyphae were observed growing beneath the test when the test of the reproductively mature insect lifted away from the substrate for the purpose of releasing crawlers, the mobile pre-settled juveniles. Once the hyphae of CC89 advanced under the test, germ-tubes readily penetrated the insect body through a number of natural openings (e.g. spiracles, vulva, stylet), with mycosis observed within seven days after inoculation. Direct penetration through the cuticle of the scale insect was not observed.     

一种冬虫夏草全基因组DNA的提取方法

冬虫夏草是真菌与昆虫形成的复合生物体,本研究建立了一种可同时提取冬虫夏草真菌子座和虫体全部基因组DNA的方法。该方法稳定高效,简便易行,提取纯度高,适用于冬虫夏草多重PCR、Realtime-PCR和DNA指纹图谱等分子水平的研究。     

冬虫夏草的研究进展、现存问题与研究展望

冬虫夏草Ophiocordyceps sinensis是传统的名贵中药材,具有非常重要的食用与药用价值。本文从天然冬虫夏草的地理分布与形态特征,冬虫夏草菌的分离、分类、遗传与进化、大量培养,寄主昆虫蝠蛾的分类、分布、生物学与人工饲养,冬虫夏草的化学成分与药效作用等方面综述了冬虫夏草的研究进展,分析了冬虫夏草研究现存的问题,并展望未来的研究方向。     

Cordyceps: a traditional Chinese medicine and another fungal therapeutic biofactory?

Traditional Chinese medicines (TCM) are growing in popularity. However, are they effective? Cordyceps is not studied as systematically for bioactivity as another TCM, Ganoderma. Cordyceps is fascinating per se, especially because of the pathogenic lifestyle on Lepidopteron insects. The combination of the fungus and dead insect has been used as a TCM for centuries. However, the natural fungus has been harvested to the extent that it is an endangered species. The effectiveness has been attributed to the Chinese philosophical concept of Yin and Yang and can this be compatible with scientific philosophy? A vast literature exists, some of which is scientific, although others are popular myth, and even hype. Cordyceps sinensis is the most explored species followed by Cordyceps militaris. However, taxonomic concepts were confused until a recent revision, with undefined material being used that cannot be verified. Holomorphism is relevant and contamination might account for some of the activity. The role of the insect has been ignored. Some of the analytical methodologies are poor. Data on the "old" compound cordycepin are still being published: ergosterol and related compounds are reported despite being universal to fungi. There is too much work on crude extracts rather than pure compounds with water and methanol solvents being over-represented in this respect (although methanol is an effective solvent). Excessive speculation exists as to the curative properties. However, there are some excellent pharmacological data and relating to apoptosis. For example, some preparations are active against cancers or diabetes which should be fully investigated. Polysaccharides and secondary metabolites are of particular interest. The use of genuine anamorphic forms in bioreactors is encouraged.     

粉棒束孢Isaria farinosa的研发进展

粉棒束孢Isaria farinosa是一种常见的昆虫病原真菌,已被用作一种生物因子防治温带农林害虫、植物病原和线虫。同时,粉棒束孢作为冬虫夏草中的定殖真菌,严重危害冬虫夏草菌Ophiocordyceps sinensis寄主蝙蝠蛾幼虫的饲养,引起了资源昆虫研究者的高度重视和控制。作为生防真菌,粉棒束孢资源的分布和筛选、分子鉴定和遗传多样性、培养、活性成分和药效、分子生物学、害虫生物防治、病害控制、安全性等是学术界的研究重点;作为冬虫夏草寄主昆虫病原菌,学界和产业界一直研究其感染特征、侵染机理和防霉剂筛选等,为这一真菌的高效安全控制提供基础。本文将综述粉棒束孢在利用和控制两大方面的研发进展。     

Entomopathogenic Nematodes for Control of Insect Pests Above and Below Ground with Comments on Commercial Production

Entomopathogenic nematodes (EPNs) have been utilized in classical, conservation, and augmentative biological control programs. The vast majority of applied research has focused on their potential as inundatively applied augmentative biological control agents. Extensive research over the past three decades has demonstrated both their successes and failures for control of insect pests of crops, ornamental plants, trees and lawn and turf. In this paper we present highlights of their development for control of insect pests above and below ground. The target insects include those from foliar, soil surface, cryptic and subterranean habitats. Advances in mass-production and formulation technology of EPNs, the discovery of numerous efficacious isolates/strains, and the desirability of reducing pesticide usage have resulted in a surge of commercial use and development of EPNs. Commercially produced EPNs are currently in use for control of scarab larvae in lawns and turf, fungus gnats in mushroom production, invasive mole crickets in lawn and turf, black vine weevil in nursery plants, and Diaprepes root weevil in citrus in addition to other pest insects. However, demonstrated successful control of several other insects, often has not lead to capture of a significant share of the pesticide market for these pests.     

Horizontal transmission of the insect symbiont Rickettsia is plant-mediated

Bacteria in the genus Rickettsia, best known as vertebrate pathogens vectored by blood-feeding arthropods, can also be found in phytophagous insects. The presence of closely related bacterial symbionts in evolutionarily distant arthropod hosts presupposes a means of horizontal transmission, but no mechanism for this transmission has been described. Using a combination of experiments with live insects, molecular analyses and microscopy, we found that Rickettsia were transferred from an insect host (the whitefly Bemisia tabaci) to a plant, moved inside the phloem, and could be acquired by other whiteflies. In one experiment, Rickettsia was transferred from the whitefly host to leaves of cotton, basil and black nightshade, where the bacteria were restricted to the phloem cells of the plant. In another experiment, Rickettsia-free adult whiteflies, physically segregated but sharing a cotton leaf with Rickettsia-plus individuals, acquired the Rickettsia at a high rate. Plants can serve as a reservoir for horizontal transmission of Rickettsia, a mechanism which may explain the occurrence of phylogenetically similar symbionts among unrelated phytophagous insect species. This plant-mediated transmission route may also exist in other insect-symbiont systems and, since symbionts may play a critical role in the ecology and evolution of their hosts, serve as an immediate and powerful tool for accelerated evolution.     

滇川桂3省暗褐网柄牛肝菌菌腔虫瘿的寄主植物与介壳虫种类调查

暗褐网柄牛肝菌Phlebopus portentosus与介壳虫形成的菌腔虫瘿是该菌营养机制研究的关键环节。本研究先后在云南、四川和广西3省区暗褐网柄牛肝菌产区的16个地点,对菌腔虫瘿的生态和生物学进行了大量的野外调查。发现根部着生菌腔虫瘿的寄主植物有31种,涉及16个科的28个属。与暗褐网柄牛肝菌形成菌腔虫瘿的介壳虫种类有12种,其中10种隶属粉蚧科Pseudococcidae、绵蚧科Monophlebidae、蚧科Coccidae各1种。在不同的寄主植物上菌腔虫瘿的寄生位置和形状会有所不同,与暗褐网柄牛肝菌菌丝形成菌腔虫瘿的寄主植物和介壳虫之间不存在专一性。上述研究结果为暗褐网柄牛肝菌的仿生栽培奠定了基础。     

Genome-wide comparative analysis of simple sequence coding repeats among 25 insect species

We present a detailed genome-scale comparative analysis of simple sequence repeats within protein coding regions among 25 insect genomes. The repetitive sequences in the coding regions primarily represented single codon repeats and codon pair repeats. The CAG triplet is highly repetitive in the coding regions of insect genomes. It is frequently paired with the synonymous codon CAA to code for polyglutamine repeats. The codon pairs that are least repetitive code for polyalanine repeats. The frequency of hexanucleotide and dinucleotide motifs of codon pair repeats is significantly (p<0.001) different in the Drosophila species compared to the non-Drosophila species. However, the frequency of synonymous and non-synonymous codon pair repeats varies in a correlated manner (r(2)=0.79) among all the species. Results further show that perfect and imperfect repeats have significant association with the trinucleotide and hexanucleotide coding repeats in most of these insects. However, only select species show significant association between the numbers of perfect/imperfect hexamers and repeat coding for single amino acid/amino acid pair runs. Our data further suggests that genes containing simple sequence coding repeats may be under negative selection as they tend to be poorly conserved across species. The sequences of coding repeats of orthologous genes vary according to the known phylogeny among the species. In conclusion, the study shows that simple sequence coding repeats are important features of genome diversity among insects.     

Phylogeography and evolution of a fungal–insect association on the Tibetan Plateau

Parasitoidism refers to a major form of interspecies interactions where parasitoids sterilize and/or kill their hosts typically before hosts reach reproductive age. However, relatively little is known about the evolutionary dynamics of parasitoidism. Here, we investigate the spatial patterns of genetic variation of Chinese cordyceps, including both the parasitoidal fungus Ophiocordyceps sinensis and its host insects. We sampled broadly from alpine regions on the Tibetan Plateau and obtained sequences on seven fungal and three insect DNA fragments from each of the 125 samples. Seven and five divergent lineages/cryptic species were identified within the fungus and host insects, respectively. Our analyses suggested that O. sinensis and host insects originated at similar geographic regions in southern Tibet/Yunnan, followed by range expansion to their current distributions. Cophylogenetic analyses revealed a complex evolutionary relationship between O. sinensis and its host insects. Significant congruence was found between host and parasite phylogenies and the time estimates of divergence were similar, raising the possibility of the occurrence of cospeciation events, but the incongruences suggested that host shifts were also prevalent. Interestingly, one fungal genotype was broadly distributed, consistent with recent gene flow. In contrast, the high‐frequency insect genotypes showed limited geographic distributions. The dominant genotypes from both the fungus and the insect hosts may represent ideal materials from which to develop artificial cultivation of this important Chinese traditional medicine. Our results demonstrate that both historical and contemporary events have played important roles in the phylogeography and evolution of the O. sinensis–ghost moth parasitoidism on the Tibetan Plateau.     

Perspectives on the Behavior of Entomopathogenic Nematodes from Dispersal to Reproduction: Traits Contributing to Nematode Fitness and Biocontrol Efficacy

The entomopathogenic nematodes (EPN) Heterorhabditis and Steinernema are widely used for the biological control of insect pests and are gaining importance as model organisms for studying parasitism and symbiosis. In this paper recent advances in the understanding of EPN behavior are reviewed. The “foraging strategy” paradigm (distinction between species with ambush and cruise strategies) as applied to EPN is being challenged and alternative paradigms proposed. Infection decisions are based on condition of the potential host, and it is becoming clear that already-infected and even long-dead hosts may be invaded, as well as healthy live hosts. The state of the infective juvenile (IJ) also influences infection, and evidence for a phased increase in infectivity of EPN species is mounting. The possibility of social behavior - adaptive interactions between IJs outside the host - is discussed. EPNs’ symbiotic bacteria (Photorhabdus and Xenorhabdus) are important for killing the host and rendering it suitable for nematode reproduction, but may reduce survival of IJs, resulting in a trade-off between survival and reproduction. The symbiont also contributes to defence of the cadaver by affecting food-choice decisions of insect and avian scavengers. I review EPN reproductive behavior (including sperm competition, copulation and evidence for attractive and organizational effects of pheromones), and consider the role of endotokia matricida as parental behavior exploited by the symbiont for transmission.     

Dynamics of carbon dioxide release from insects infected with entomopathogenic nematodes

The quality of an insect as a host to an entomopathogenic nematode infective juvenile depends in part on whether or not the insect is already infected and on the stage of that infection. Previous research has shown that nematode response to hosts can change after infection and that, for uninfected hosts, CO(2) can be an important cue used by infective stage juveniles during attraction. We hypothesized that CO(2) production from an insect changes after it is infected, and that these changes could influence nematode infection decisions. Changes in CO(2) released by two insect species (Galleria mellonella and Tenebrio molitor) after infection by one of four nematode species (Steinernema carpocapsae, Steinernema feltiae, Steinernema glaseri, or Steinernema riobrave) were measured. Measurements were taken every 2h from time of initial exposure to nematodes up to 224 h after infection. Dead (freeze-killed) and live uninfected insects were used as controls. Infected G. mellonella showed two distinct peaks of CO(2) production: one between 20 and 30 h and the other between 70 and 115 h after exposure to the nematodes. Peaks were up to two times higher than levels produced by uninfected insects. Infected T. molitor showed only one peak between 25 and 50h. We found differences in peak height and timing among nematode and insect species combinations. The influence of these changes in CO(2) production on IJ attraction and infection behavior remains to be determined.     

冬虫夏草无性型研究概况*

文献调查结果表明与冬虫夏草有关的无性型菌种已报道有22个学名,涉及13个属。针对这些名称上存在的命名问题本文展开了讨论,并评述了不同名称与冬虫夏草的关系及其有关菌种的研究进展。在已报道的分离菌种中,作为新种正式描述的名称有8个,其中6个为合格发表,其余2个未能满足名称合格发表的条件;还有4个名称在文献中作为新种提到,但没有合格发表的出处,也有一例只提到名称而无其他要素;另有5个名称则只有属名,尚未鉴定到种的水平。中国被毛孢为多个独立的研究证明为冬虫夏草的无性型,还有3个无性型名称已被证明不可能是冬虫夏草的无性型。其他无性型名称与冬虫夏草的关系有待进一步验证。文中对与冬虫夏草有关的菌种定名、无性型分离和验证方法、复无性型现象,以及有关学名问题作了讨论。     

Identification of MicroRNAs in Helicoverpa armigera and Spodoptera litura Based on Deep Sequencing and Homology Analysis

The current identification of microRNAs (miRNAs) in insects is largely dependent on genome sequences. However, the lack of available genome sequences inhibits the identification of miRNAs in various insect species. In this study, we used a miRNA database of the silkworm Bombyx mori as a reference to identify miRNAs in Helicoverpa armigera and Spodoptera litura using deep sequencing and homology analysis. Because all three species belong to the Lepidoptera, the experiment produced reliable results. Our study identified 97 and 91 conserved miRNAs in H. armigera and S. litura, respectively. Using the genome of B. mori and BAC sequences of H. armigera as references, 1 novel miRNA and 8 novel miRNA candidates were identified in H. armigera, and 4 novel miRNA candidates were identified in S. litura. An evolutionary analysis revealed that most of the identified miRNAs were insect-specific, and more than 20 miRNAs were Lepidoptera-specific. The investigation of the expression patterns of miR-2a, miR-34, miR-2796-3p and miR-11 revealed their potential roles in insect development. miRNA target prediction revealed that conserved miRNA target sites exist in various genes in the 3 species. Conserved miRNA target sites for the Hsp90 gene among the 3 species were validated in the mammalian 293T cell line using a dual-luciferase reporter assay. Our study provides a new approach with which to identify miRNAs in insects lacking genome information and contributes to the functional analysis of insect miRNAs.     

三江源区冬虫夏草资源适宜性空间分布

冬虫夏草(Ophiocordyceps sinensis)是生长在青藏高原高寒地区的一种珍稀物种资源,具有极高的药用和经济价值,然而冬虫夏草资源的数量特征与空间格局尚不明确。以三江源区为例,结合冬虫夏草的生境条件,选取适宜冬虫夏草生长的生态环境指标,利用地理信息系统(GIS)中的空间分析方法对冬虫夏草资源的适宜性空间分布进行了研究,并用野外实地采样和入户调研数据进行验证。结果表明:三江源区冬虫夏草资源主要分布在东南部和中南部,分布面积约为18.45×10~4km~2,占该区总面积的50.74%。达日县、甘德县、玉树县、称多县、玛沁县、班玛县、久治县这7个县冬虫夏草适宜性面积占到了该县50%以上的面积,是三江源区冬虫夏草资源的主产区。基于GIS空间分析的结果与实地调查结果相符,说明利用空间分析方法划分冬虫夏草资源的适宜性分布区域是可行的,可为系统的全面调查冬虫夏草资源的分布及保护与合理开发利用冬虫夏草资源提供科学依据。     

Polyphenoloxidase activity in coffee leaves and its role in resistance against the coffee leaf miner and coffee leaf rust

In plants, PPO has been related to defense mechanism against pathogens and insects and this role was investigated in coffee trees regarding resistance against a leaf miner and coffee leaf rust disease. PPO activity was evaluated in different genotypes and in relation to methyl-jasmonate (Meja) treatment and mechanical damage. Evaluations were also performed using compatible and incompatible interactions of coffee with the fungus Hemileia vastatrix (causal agent of the leaf orange rust disease) and the insect Leucoptera coffeella (coffee leaf miner). The constitutive level of PPO activity observed for the 15 genotypes ranged from 3.8 to 88 units of activity/mg protein. However, no direct relationship was found with resistance of coffee to the fungus or insect. Chlorogenic acid (5-caffeoylquinic acid), the best substrate for coffee leaf PPO, was not related to resistance, suggesting that oxidation of other phenolics by PPO might play a role, as indicated by HPLC profiles. Mechanical damage, Meja treatment, H. vastatrix fungus inoculation and L. coffeella infestation caused different responses in PPO activity. These results suggest that coffee resistance may be related to the oxidative potential of the tissue regarding the phenolic composition rather than simply to a higher PPO activity.     

Alternaria alternata, a new pathotype pathogenic to aphids

A fungus, identified as Alternaria alternata, was isolated from dying or dead aphids and proved to be pathogenic. It was isolated from different parts of Greece from aphid specimens on cultivated plants, ornamentals and weeds. In the laboratory, disease development started with the germination of spores on the insect integument and the subsequent growth of mycelium. The fungus formed apical and intercalary, globose or lobate appressoria which were firmly attached onto the host exoskeleton and facilitated entrance of the mycelium into the insect body. Under favorable conditions of temperature (15–35 °C) and relative humidity (100%), infected aphids died in 2–4 days. A characteristic brown discoloration accompanied the death of the insects. Both mycelial growth and sporulation were profuse on dead specimens. The pathogen infected all 26 aphid species tested but was unable to infect other insects (Drosophila melanogaster and Ceratitis capitata) or aphid host plants. There were significant differences in mortality rate among aphid species only during the first two days after inoculation. It is suggested that A. alternata may be a good candidate to be exploited for the biological control of aphids.     

Several deep-sea mussels and their associated symbionts are able to live both on wood and on whale falls

Bathymodiolin mussels occur at hydrothermal vents and cold seeps, where they thrive thanks to symbiotic associations with chemotrophic bacteria. Closely related genera Idas and Adipicola are associated with organic falls, ecosystems that have been suggested as potential evolutionary 'stepping stones' in the colonization of deeper and more sulphide-rich environments. Such a scenario should result from specializations to given environments from species with larger ecological niches. This study provides molecular-based evidence for the existence of two mussel species found both on sunken wood and bones. Each species specifically harbours one bacterial phylotype corresponding to thioautotrophic bacteria related to other bathymodiolin symbionts. Phylogenetic patterns between hosts and symbionts are partially congruent. However, active endocytosis and occurrences of minor symbiont lineages within species which are not their usual host suggest an environmental or horizontal rather than strictly vertical transmission of symbionts. Although the bacteria are close relatives, their localization is intracellular in one mussel species and extracellular in the other, suggesting that habitat choice is independent of the symbiont localization. The variation of bacterial densities in host tissues is related to the substrate on which specimens were sampled and could explain the abilities of host species to adapt to various substrates.