Pathology and ultrastructure of an intranuclear bacilliform virus (IBV) infecting brown shrimp Crangon crangon (Decapoda: Crangonidae)

The brown shrimp Crangon crangon supports an important fishery in Europe (over 25000 t, valued at 80 million euros in 2000). Through the course of histopathological screening of crustaceans from the Clyde estuary, western Scotland, for the biological effect of contaminants, we have discovered a highly prevalent (up to 100%) non-occluded intranuclear bacilliform virus (IBV) infection in the hepatopancreatic tubule epithelia and midgut epithelia of wild C. crangon. This is the first report of an IBV in this family. We have termed this virus Crangon crangon bacilliform virus (CcBV). Histological and ultrastructural observations suggest that this virus is similar to other IBVs previously described from crabs and penaeid shrimps. The nuclei of virus-infected epithelial cells contained an eosinophilic, hypertrophied viroplasm that marginalised the chromatin of the host nucleus. Infected cells were often separated from their neighbouring cells and their nuclei appeared apoptotic. In heavily infected shrimp, apoptotic cells were expelled into the lumen of the hepatopancreatic tubule or the midgut. Following this stage, some hepatopancreatic tubules became degenerate, with remnants of the basement membrane and myoepithelial lining remaining. Transmission electron microscopy of hypertrophic nuclei revealed the presence of rod-shaped and cylindrical, envelope-bound virions. These virions did not form arrays and were not embedded within occlusion bodies, but did appear to be partially occluded in an amorphous matrix that corresponded to a granular viroplasm. The ultrastructure, morphology and size of the nucleocapsid and the complete virion aligns the virus most closely to the IBVs previously reported from other decapod crustaceans. Due to the pathological manifestation of IBV infection in C. crangon, it appears likely that it can act as a population modulator, particularly at sites where infection prevalence is high, such as that observed in the Clyde estuary.     

Targeted Dispersal of the Aphid Pathogenic Fungus Erynia neoaphidis by the Aphid Predator Coccinella septempunctata

The potential of adult and larval C. septempunctata to vector the aphid-specific entomopathogenic fungus E. neoaphidis was assessed through a series of laboratory and field experiments. The ability of coccinellids to vector conidia from a colony of E. neoaphidis -infected pea aphids, Acyrthosiphon pisum, to a colony of uninfected A. pisum was demonstrated in a laboratory study. Adult coccinellids which had previously foraged on plants infested with different densities of sporulating cadavers (1, 5, 15, 30 cadavers per plant) initiated infection in a proportion of uninfected pea aphids (4, 0, 2 and 8%, respectively) when subsequently allowed to forage on A. pisum infested bean plants. Further laboratory studies demonstrated that fourth instar larvae and adult coccinellids artificially inoculated with conidia initiated infection in 11 and 13% of an A. pisum population in which they foraged, respectively. Furthermore, a proportion of A. pisum placed on bean plants which had previously been foraged on by inoculated larval and adult coccinellids also died from infection (3 and 10% of A. pisum, respectively). However, although coccinellid adults inoculated with conidia initiated infection in 19% of A. pisum, cereal aphids, S. avenae , exposed to the inoculated coccinellids did not become infected. A further laboratory study demonstrated that infection of A. pisum only occurred if inoculated coccinellids were transferred to A. pisum populations immediately post inoculation. However, a proportion of A. pisum placed on bean plants which had been foraged on by inoculated coccinellids transferred 0, 4 and 24 h post inoculation died from infection (9, 3 and 7%, respectively). A field study further demonstrated the potential of coccinellids to vector E. neoaphidis. Single spring sown field bean plants (Long Hoos Experimental Plots, IACRRothamsted Farm) were enclosed within nylon mesh bags and 25 adult A. pisum were added to each bag with one of the following treatments: no further addition (control), coccinellid adult (control), inoculated coccinellid adult, inoculated A. pisum or sporulating A. pisum cadavers. No aphids died of E. neoaphidis in the control treatments; 5, 16 and 33% of aphids were infected with E. neoaphidis on the other treatments, respectively.     

Histopathological survey of pathogens and commensals of white-clawed crayfish (Austropotamobius pallipes) in England and Wales

A histopathological survey of white-clawed crayfish (Austropotamobius pallipes) from six rivers in England and Wales was conducted between November 2007 and September 2011. A. pallipes bacilliform virus (ApBV) was present in four samples; infected crayfish showed pathological responses ranging from mild hypertrophy of infected nuclei and emargination of chromatin through to loss of architecture of the hepatopancreatic cells. Crayfish were found to be hosts to two different ciliate species, mites, nematodes, digeneans and the microsporidian Thelohania contejeani. The variation in disease presence between populations highlights the importance of conducting appropriate surveys of native crayfish prior to movement of animals to refugia sites for relocation, restocking or breeding programmes.     

昆虫病原真菌长孢蜡蚧菌TF-2菌株对豌豆蚜的侵染过程和致病力

【目的】豌豆蚜Acyrthosiphon pisum是世界性的重要农业害虫,给豆类作物造成巨大的经济损失。本研究在前期筛选已获得豌豆蚜致病菌长孢蜡蚧菌Lecanicillium longisporum TF-2菌株的基础上,检测了该致病菌分生孢子对豌豆蚜的毒力效用及侵染方式,以期为利用昆虫病原真菌防治豌豆蚜提供理论基础。【方法】采用离体叶片饲养法检测在不同浓度长孢蜡蚧菌TF-2菌株孢子悬浮液(1.0×10³～1.0×10⁷孢子/mL)中浸渍后对豌豆蚜成虫的致病力;应用扫描电镜和体视显微镜观察豌豆蚜成虫接种TF-2菌株(1.0×10⁷孢子/mL)后长孢蜡蚧菌TF-2菌株侵染症状和侵染过程。【结果】不同浓度长孢蜡蚧菌TF-2菌株分生孢子浸染豌豆蚜成虫致病力随分生孢子浓度升高而逐渐增强,侵染后6 d对豌豆蚜成虫的半致死浓度(LC₅₀)为3.26×10⁴孢子/mL,最高浓度分生孢子(1.0×10⁷孢子/mL)对豌豆蚜成虫的半致死时间(LT₅₀...     

Application of DL-beta-aminobutyric acid (BABA) as a root drench to legumes inhibits the growth and reproduction of the pea aphid Acyrthosiphon pisum (Hemiptera: Aphididae)

DL-beta-aminobutyric acid (BABA) is a non-protein amino acid that is an effective inducer of resistance against a variety of plant pathogens. However, examples of BABA-induced resistance against insect herbivores have not been reported. We applied BABA as a soil drench to legumes and monitored its effects on the pea aphid Acyrthosiphon pisum (Harris). On tic bean (Vicia faba var. minor), BABA increased aphid mortality, caused a reduction in the mean relative growth rate of individual insects and lessened the intrinsic rate of population increase (rm). BABA also caused significant reductions in the growth rate of A. pisum on pea (Pisum sativa), broad bean (Vicia faba var. major), runner bean (Phaseolus coccineus), red clover (Trifolium pratense) and alfalfa (Medicago sativa). No direct toxic effects of BABA against A. pisum were found, and no phytotoxic effects that may have caused a reduction in aphid performance were detected. Possible mechanisms behind this BABA-induced inhibition of aphid performance are discussed.     

Diversity and geographic distribution of secondary endosymbiotic bacteria in natural populations of the pea aphid,Acyrthosiphon pisum

In addition to the essential intracellular symbiotic bacterium Buchnera, several facultative endosymbiotic bacteria called collectively secondary symbionts (S-symbionts) have been identified from the pea aphid Acyrthosiphon pisum. We conducted an extensive and systematic survey of S-symbionts in Japanese local populations of A. pisum using a specific PCR detection technique. Five S-symbionts of A. pisum, PASS, PAUS, PABS, Rickettsia and Spiroplasma, and two facultative endosymbionts universally found in various insects, Wolbachia and Arsenophonus, were targeted. Of 119 isofemale strains originating from 81 localities, 66.4% of the strains possessed either of four S-symbionts: PASS (38.7%); PAUS (16.0%); Rickettsia (8.4%); and Spiroplasma (3.4%), while 33.6% of the strains contained only Buchnera. PABS, Wolbachia and Arsenophonus were not detected from the Japanese strains of A. pisum. In order to understand intra- and interpopulational diversity of S-symbiont microbiota in detail, 858 insects collected from 43 localities were examined for infection with the four S-symbionts. It was demonstrated that different S-symbionts coexist commonly in the same local populations, but double infections with two S-symbionts were rarely detected. Notably, the S-symbionts exhibited characteristic geographical distribution patterns: PASS at high frequencies all over Japan; PAUS at high frequencies mainly in the northeastern part of Japan; and Rickettsia and Spiroplasma at low frequencies sporadically in the southwestern part of Japan. These results indicate that the geographical distribution and infection frequency of the S-symbionts, in particular PAUS, might be affected by environmental and/or historical factors. Statistical analyses suggested that the distribution of PAUS infection might be related to host plant species, temperature and precipitation.     

Spiroplasma symbiont of the pea aphid, Acyrthosiphon pisum (Insecta: Homoptera)

From a laboratory strain of the pea aphid, Acyrthosiphon pisum, we discovered a previously unknown facultative endosymbiotic bacterium. Molecular phylogenetic analysis based on 16S ribosomal DNA revealed that the bacterium is a member of the genus Spiroplasma. The Spiroplasma organism showed stable vertical transmission through successive generations of the host. Injection of hemolymph from infected insects into uninfected insects established a stable infection in the recipients. The Spiroplasma symbiont exhibited negative effects on growth, reproduction, and longevity of the host, particularly in older adults. Of 58 clonal strains of A. pisum established from natural populations in central Japan, 4 strains possessed the Spiroplasma organism.     

Complex interactions between a plant pathogen and insect parasitoid via the shared vector-host: consequences for host plant infection

Plant viruses modify the development of their aphid vectors by inducing physiological changes in the shared host plant. The performance of hymenopterous parasitoids exploiting these aphids can also be modified by the presence of the plant pathogen. We used laboratory and glasshouse microcosms containing beans (Vicia faba) as the host plant to examine the interactions between a plant virus (pea enation mosaic virus; PEMV) and a hymenopterous parasitoid (Aphidius ervi) that share the aphid vector/host Acyrthosiphon pisum. Neither PEMV-infection of V. faba, nor the carriage of PEMV virions by A. pisum, affected the growth or morphology of the aphid, or the oviposition behaviour and development of A. ervi. The presence of developing Aphidius ervi larvae within Acyrthosiphon pisum did not affect the ability of the aphids to transmit PEMV. However, by reducing their longevity, parasitism ultimately decreased the time viruliferous aphids were able to inoculate plants. In terms of virus dispersal, parasitized aphids exhibited more movement around experimental arenas than unparasitized controls, causing a slight increase in the proportion of beans infected with PEMV. Exposure to adult Aphidius ervi caused Acyrthosiphon pisum to rapidly drop off bean plants and disperse to new hosts, resulting in considerably higher plant infection rates (70%) than that seen in control arenas (25%). The results of this investigation demonstrate that when parasitoids are added to a plant-pathogen-vector system, benefits to the host plant due to reduced herbivore infestation must be balanced against the consequences of parasitoid-induced aphid dispersal and a subsequent increase in the level of plant infection.     

Host-based divergence in populations of the pea aphid: insights from nuclear markers and the prevalence of facultative symbionts

In North America, the pea aphid Acyrthosiphon pisum encompasses ecologically and genetically distinct host races that offer an ideal biological system for studies on sympatric speciation. In addition to its obligate symbiont Buchnera, pea aphids harbour several facultative and phylogenetically distant symbionts. We explored the relationships between host races of A. pisum and their symbiotic microbiota to gain insights into the historical process of ecological specialization and symbiotic acquisition in this aphid. We used allozyme and microsatellite markers to analyse the extent of genetic differentiation between populations of A. pisum on pea, alfalfa and clover in France. In parallel, we examined: (i) the distribution of four facultative symbionts; and (ii) the genetic variation in the Buchnera genome across host-associated populations of A. pisum. Our study clearly demonstrates that populations of A. pisum on pea, clover and alfalfa in France are genetically divergent, which indicates that they constitute distinct host races. We also found a very strong association between host races of A. pisum and their symbiotic microbiota. We stress the need for phylogeographic studies to shed light on the process of host-race formation and acquisition of facultative symbionts in A. pisum. We also question the effects of these symbionts on aphid host fitness, including their role in adaptation to a host plant.     

豌豆蚜唾液蛋白家族的克隆及在不同发育阶段的表达

豌豆蚜Acyrthosiphon pisum是一种重要的刺吸式害虫, 其分泌的唾液对取食寄主植物和传播植物病毒有重要的作用。为了探讨蚜虫唾液蛋白的功能, 本研究克隆了在豌豆蚜唾液腺高表达的一个未知功能的蛋白家族, 该家族包括13个基因, 编码14种蛋白, 其中4个基因在唾液腺高表达。这个家族是蚜虫特有的蛋白家族, 富含半胱氨酸, 有14个半胱氨酸高度保守, 其中6个半胱氨酸形成3个保守的CXXC结构域。通过与基因组比对, 发现这个家族的基因没有内含子, 分布在基因组的9个scaffold上。用半定量逆转录PCR检测了每个成员在豌豆蚜不同发育阶段的表达, 结果显示这个家族没有发育阶段特异性。推测这个家族的表达可能具有组织特异性, 有氧化还原酶或DNA甲基化酶的功能。     

Influence of temperature on pea aphid Acyrthosiphon pisum (Hemiptera: Aphididae) resistance to natural enemy attack

The ability to resist or avoid natural enemy attack is a critically important insect life history trait, yet little is understood of how these traits may be affected by temperature. This study investigated how different genotypes of the pea aphid Acyrthosiphon pisum Harris, a pest of leguminous crops, varied in resistance to three different natural enemies (a fungal pathogen, two species of parasitoid wasp and a coccinellid beetle), and whether expression of resistance was influenced by temperature. Substantial clonal variation in resistance to the three natural enemies was found. Temperature influenced the number of aphids succumbing to the fungal pathogen Erynia neoaphidis Remaudière & Hennebert, with resistance increasing at higher temperatures (18 vs. 28 degrees C). A temperature difference of 5 degrees C (18 vs. 23 degrees C) did not affect the ability of A. pisum to resist attack by the parasitoids Aphidius ervi Haliday and A. eadyi Stary, González & Hall. Escape behaviour from foraging coccinellid beetles (Hippodamia convergens Guerin-Meneville) was not directly influenced by aphid clone or temperature (16 vs. 21 degrees C). However, there were significant interactions between clone and temperature (while most clones did not respond to temperature, one was less likely to escape at 16 degrees C), and between aphid clone and ladybird presence (some clones showed greater changes in escape behaviour in response to the presence of foraging coccinellids than others). Therefore, while larger temperature differences may alter interactions between Acyrthosiphon pisum and an entomopathogen, there is little evidence to suggest that smaller changes in temperature will alter pea aphid-natural enemy interactions.     

Elucidation of the transmission patterns of an insect-borne bacterium

Quantitative data on modes of transmission are a crucial element in understanding the ecology of microorganisms associated with animals. We investigated the transmission patterns of a gamma-proteobacterium informally known as pea aphid Bemisia-like symbiont (PABS), also known as T-type, which is widely but not universally distributed in natural populations of the pea aphid, Acyrthosiphon pisum. The vertical transmission of PABS to asexual and sexual morphs and sexually produced eggs was demonstrated by a diagnostic PCR-based assay, and the maximum estimated failure rate was 2%. Aphids naturally lacking PABS acquired PABS bacteria administered via the diet, and the infection persisted by vertical transmission for at least three aphid generations. PABS was also detected in two of five aphid honeydew samples tested and in all five siphuncular fluid samples tested but in none of 15 samples of salivary secretions from PABS-positive aphids. However, PABS-negative aphids did not acquire PABS when they were cocultured with PABS-positive aphids; the maximal estimated level of horizontal transmission was 18%. A deterministic model indicated that the force of infection by a horizontal transmission rate of 3% is sufficient to maintain a previously described estimate of the prevalence of PABS-positive aphids (37%), if the vertical transmission rate is 98%. We concluded that PABS infections in A. pisum can be maintained by high vertical transmission rates and occasional horizontal transmission, possibly via the oral route, in the absence of selection either for or against aphids bearing this bacterium.     

Acquisition, Retention and Transmission of Faba Bean Necrotic Yellows Virus by Two of its Aphid Vectors, Aphis craccivora (Koch) and Acyrthosiphon pisum (Harris)

Faba bean necrotic yellows virus (FBNYV) belongs to a new group of plant viruses that have unusually small isometric virions and a multipartite ssDNA genome. It is the causal agent of some virus diseases affecting several food and fodder legumes in west Asia and north Africa. FBNYV is persistently transmitted by various aphid species of which Aphis craccivora appears to be the most significant natural vector. In attempts to obtain a better understanding of factors involved in FBNYV spread under field conditions, the interactions of the virus with A. craccivora and Acyrthosiphon pisum were studied. The two species were efficient vectors and very similar in their minimum acquisition (AAP) and minimum inoculation access feeding periods which ranged from 15 to 30min and 5–15min, respectively. Following an AAP of 72 h and daily serial transfers of individual aphids to single plants, many individuals retained and transmitted the virus throughout their life span (up to 32 days) but at erratic efficiencies. In this persistence experiment A. pisum was a more efficient vector than A. craccivora. For both aphid species no decrease in transmission efficiency was observed, suggesting that nymphs acquired large amounts of FBNYV virions which were not depleted in their hemocoel during the experiment. Based on log-pro-bit analysis, median latency period (LPso) values of 108.8h and 105.0h were calculated for FBNYV in A. craccivora and A. pisum , respectively. FBNYV was not lost during moults and was not passed on to the par-thenogenetic offspring by viruliferous adults. Aphids which acquired FBNYV as adults were strikingly poor vectors as compared to nymphs.     

Infectivity of Pandora neoaphidis (Zygomycetes: Entomophthorales) to Acyrthosiphon pisum (Hom., Aphididae) in response to varying temperature and photoperiod regimes

The infectivity of the pea aphid, Acyrthosiphon pisum , by the aphid-specific entomophthoralean fungus, Pandora neoaphidis , was studied in environmental chambers using computer simulated late-season temperature and photoperiod patterns and an excised fava bean leaf system. A complementary log-log (CLL) time–dose–mortality model was used to model time–dose trends in infectivity of A. pisum by P. neoaphidis as a function of time and dose, and calculate the LC₅₀. The likelihood ratio test based on the CLL model was used to test for differences in levels of aphid infectivity by P. neoaphidis over different photoperiod regimes. Differences in the LC₅₀ values from CLL modelling over different time periods were found among the two regimes of 11- and 16-h photophases at a constant temperature of 20°C, and two regimes at daily fluctuating temperatures from 5.4 to 18.9°C (with half-hourly changes of 0.56°C the mean temperature was 12.12°C). The LC₅₀ for the same time period was lower under the higher temperature conditions than under lower fluctuating temperature conditions. The likelihood test ratio statistics showed no significant difference in the slope parameter for three out of four treatments (two from the constant temperature of 20°C, and one from 5.4 to 18.9°C, irrespective of photophase conditions). This suggests that different temperatures and photophases may change the temporal characteristics of P. neoaphidis , and result in higher or lower infectivity to the pea aphid at certain dosage levels. However, changes in the photoperiod appear to be less important for the infectivity of P. neoaphidis to pea aphid than changes in temperature.     

The incidence of the Pea crab, Pinnotheres pisum in the two types of Mytilus (Mollusca: Bivalvia) from Padstow, south-west England

Significant differences in the infection of M. edulis and the "Padstow type" mussel with P. pisum are recorded, and some possible explanations for these differences are discussed.
Both types of Mytilus from the mid and lower regions of the mussel bed showed heavier infections than mussels higher on the shore. Even so, the differences between the two types were still maintained.
A relationship exists between crab and mussel size, larger crabs being found only in larger hosts. The smallest mussel found to be infected with Pinnotheres measured 3·35 cm in length.
Infection in M. edulis was found to increase with increased size of host, the largest occurring mussels having from 80 to 100% infection. Larger mussels occurred in greater numbers in the low shore. It is assumed that infection in the "Padstow type" would show a similar relationship if sufficient recordings had been available.
The presence of the crab causes gill damage, and infected mussels show considerably lower tissue weights and slightly greater shell weights than uninfected mussels of similar size.
The presence of the crab does not appear to influence the reproductive capacity of the mussel.     

Uptake of Encephalitozoon spp. and Vittaforma corneae (Microsporidia) by different cells

Microsporidia are obligate intracellular parasites infecting a broad range of vertebrates and invertebrates. Various microsporidian species induce different clinical pictures in humans. The reason for this is not clear. It has been speculated that the different microsporidian species are transmitted by various routes, thus causing infections in different organs. Another possibility is that the diverse microsporidia have different tropisms to organ-specific cells, thus causing various diseases. In this study, we investigated the uptake of microsporidian spores by different cells with an immunofluorescence staining technique to investigate whether there is a difference between microsporidian species as well as between different cells. Using this technique, we were able to distinguish between intra- and extracellular microsporidian spores. All examined cell lines were able to internalize microsporidian spores, but the extent of internalization differed significantly between the cells. Although the results showed some patterns that correlate with the distribution of the parasites in humans, the different clinical pictures cannot be sufficiently explained by this phenomenon, so it seems more likely that the various clinical manifestations caused by the different microsporidian species are a consequence of different infection routes rather than of different affinities of the microsporidian species to different cells.     

The Partial Characterization of a Badnavirus Infecting the Greater Asiatic or Water Yam (Dioscorea alata)

A bacilliform virus from Dioscorea alata, designated Dioscorea alata bacilliform virus (DaBV), from Barbados and West Africa and from other Dioscorea spp. from West African, Carribean, Asian and South American countries, has been characterized. The virus was transmitted by the mealybug, Planococcus citri and by mechanical transmission of partially purified preparations to several Dioscorea spp. DaBV was serologically related to a distinct bacilliform virus from Dioscorea bulbifera, to one isolate of sugarcane bacilliform badnavirus and two isolates of banana streak badnavirus (BSV) but was not related to another isolate of BSV or to Kalanchoe top spotting or cacao swollen shoot badnaviruses. The coat protein of DaBV was about 56 kDa and the nucleic acid was double-stranded DNA of about 7.5 kbp, part of which showed distant homology with other badnaviruses. Thus, DaBV is a distinct hitherto uncharacterized badnavirus.     

Temporal habitat variability and the maintenance of sex in host populations of the pea aphid

The evolutionary maintenance of sex, despite competition from asexual reproduction, has long intrigued the evolutionary biologists owing to its numerous apparent short-term costs. In aphids, winter climate is expected to determine the maintenance of sexual lineages in the high latitude zones owing to their exclusive ability to produce frost-resistant eggs. However, diverse reproductive modes may coexist at a local scale where climatic influence is counteracted by microgeographical factors. In this study, we tested the influence of local habitat characteristics on regional coexistence of reproductive modes in the pea aphid, Acyrthosiphon pisum. In the laboratory, the induction of sexual morph production of many pea aphid genotypes from the local fields of annual (pea and faba bean) and perennial (alfalfa and red clover) crops in Western France indicated that A. pisum lineages from annual crops had a significantly higher investment in sexual reproduction than A. pisum lineages from the perennial hosts. We propose that temporal habitat variability exerts a selective pressure to maintain the sexual reproduction in A. pisum. The ecological and evolutionary consequences of the association between the mode of reproduction and the host population on gene flow restriction and on ecological specialization are discussed.