Effect of seasonal abiotic conditions and field margin habitat on the activity of Pandora neoaphidis inoculum on soil

The ability of the aphid pathogenic fungus Pandora neoaphidis to remain active in the absence of a resting stage through a combination of continuous infection and as conidia deposited on soil was assessed alongside the potential for planted field margins to act as a refuge for the fungus. P. neoaphidis was able to infect the pea aphid, Acyrthosiphon pisum, when maintained under controlled conditions that simulated those that occur seasonally in the UK. Although there was a significant inverse relationship between temperature and time-to-kill, with death occurring after 4.2, 6.9 and 13.6 days when maintained under fluctuating summer, autumn and winter temperatures, respectively, there were no additional statistically significant effects of photoperiod. The activity of inoculum on soil was indirectly assessed by baiting with A. pisum. Under controlled conditions P. neoaphidis remained active on soil and was able to infect aphids for up to 80 days. However, the percentage of aphids that became infected decreased from 76% on day 1 to 11% on day 80. Whereas there was little difference in the activity of conidia that had been maintained at 4 degrees C and 10 degrees C, activity at 18 degrees C was considerably reduced. Under field conditions the activity of inoculum was strongly influenced by season. On day 49 there was little or no activity during spring, summer or winter. However, during autumn a mean proportion of 0.08 aphids still became infected with P. neoaphidis. Margin type did not affect the activity of conidia nor was there a difference in activity between blocks that had regenerated naturally and those that had been planted. These results suggest that P. neoaphidis can infect aphids and remain active on soil under the abiotic conditions that occur seasonally in the UK and that this fungus may be able to persist annually without a resting stage.     

Chytrid mycoparasitism of entomophthoralean azygospores

Mycoparasitism – when one fungus parasitizes another – has been reported to affect Beauveria bassiana and mycorrhizal fungi in the field. However, mycoparasitism of any fungi in the Order Entomophthorales has never been reported before now. The majority of entomophthoralean species persist as resting spores (either zygospores or azygospores) in the environment and dormant entomophthoralean resting spores (whether formed as zygospores or azygospores) are thought to be especially well adapted for survival over long periods due to their thick double walls. Entomophthoralean resting spores can accumulate in the soil as large reservoirs of inoculum which can facilitate the onset and development of epizootics. We report parasitism of azygospores of the gypsy moth pathogen Entomophaga maimaiga caged in soil from southern Ohio by the chytrid fungus Gaertneriomyces semiglobifer. G. semiglobifer had previously been isolated from soil samples from North America, Europe and Australia or horse manure from Virginia. After isolation and identification of G. semiglobifer, azygospores of E. maimaiga exposed to zoospores of G. semiglobifer exhibited high levels of mycoparasitism and G. semiglobifer was subsequently reisolated from mycoparasitized resting spores. We discuss the importance of this finding to the epizootiology of insect diseases caused by entomophthoralean fungi.     

Response of the entomopathogenic fungus Pandora neoaphidis to aphid-induced plant volatiles

We used a model plant-aphid system to investigate whether the aphid-specific entomopathogenic fungus Pandora neoaphidis responds to aphid-induced defence by the broad-bean plant, Vicia faba. Laboratory experiments indicated that neither in vivo sporulation, conidia size nor the in vitro growth of P. neoaphidis was affected by Acyrthosiphon pisum-induced V. faba volatiles. The proportion of conidia germinating on A. pisum feeding on previously damaged plants was significantly greater than on aphids feeding on undamaged plants, suggesting a direct functional effect of the plant volatiles on the fungus. However, there were no significant differences in the infectivity of P. neoaphidis towards A. pisum feeding on either undamaged V. faba plants or plants previously infested with A. pisum. Therefore, these results provide no evidence to suggest that P. neoaphidis contributes to plant indirect defence strategies.     

Pandora neoaphidis transmission and aphid foraging behaviour

Pandora neoaphidis is an aphid-specific entomopathogen that produces infective conidia. As aphid movement increases, so does the likelihood of contact with conidia. Volatile distress signals released in response to aphid infestation as an indirect defence against herbivory may affect aphid foraging and, therefore, the fungus-aphid interaction. In this study, two different methods were used to investigate the effect of plant volatiles and P. neoaphidis-sporulating cadavers on (1) the colonisation of Vicia faba plants by Acyrthosiphon pisum and (2) P. neoaphidis transmission. This study indicates that A. pisum does not avoid bean plants containing P. neoaphidis and that transmission of conidia occurs during plant colonisation and, to a lesser extent, during in situ feeding. Although significantly more aphids were recovered from damaged plants compared to undamaged plants, the likelihood of infection was not affected by previous infestation by aphids.     

Effect of fungal infection on the reproductive potential of aphids and their progeny

The effect of infection by Pandora neoaphidis and Beauveria bassiana on the reproductive potential of the pea aphid, Acyrthosiphon pisum, and their progeny was assessed. Infection by either P. neoaphidis or B. bassiana reduced the number of nymphs produced within 24 h of inoculation and over the entire infection period compared to uninfected aphids. However, infection by either P. neoaphidis or B. bassiana for 24 or 72 h did not alter the intrinsic rate of increase of the host aphid's progeny. Therefore, fungal infection appears to have no indirect effects on the fitness of the host's progeny.     

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.     

Observation on the initial inoculum source and dissemination of Entomophthorales-caused epizootics in populations of cereal aphids

A total number of 1092 migratory alates were trapped from air in wheat grown area of Yuanyang County, Henan Province from early April through May 2002 in order to confirm the source and dissemination of entomophthoralean inocula to cause epizootics of cereal aphids. Those included 415 Sitobion avenae, 642 Rhopalosiphum padi, 22 Metopolophium dirhodum, and 13 Schizaphis graminum. The trapped alates were daily collected and individually reared for 7 days on wheat plants in laboratory. Of those 341 alates died of fungal infection, taking 31.2% in the trapped alates. These included 224 S. avenae, 106 R. padi, 8 M. dirhodum, and 3 S. graminum. Deaths of all infected alates occurred during the first 5 days and 78.9% of the deaths occurred within the first 3 days. Individual examination under microscope proved that all deaths were attributed to entomophthoralean fungi. Of those Pandora neoaphidis accounted for 84.6%, Conidiobolus obscurus for 9.9%, and Entomophthora planchoniana for 5.5%. Four alate deaths died of cross infection of P. neoaphidis and C. conidiobolus. Based on the high infection rate of the migratory alates trapped from air and the field occurrence of epizootics in populations of cereal aphids during the trapping period, Entomophthorales-caused epizootics were likely disseminated by infected alates through their flight and colonization. This makes it reasonable to interpret worldwide distribution of aphid epizootics, particularly caused by P. neoaphidis that has no resting spores discovered.     

Observation on the initial inoculum source and dissemination of Entomophthorales-caused epizootics in populations of cereal aphids

A total number of 1092 migratory alates were trapped from air in wheat grown area of Yuanyang County, Henan Province from early April through May 2002 in order to confirm the source and dissemination of entomophthoralean inocula to cause epizootics of cereal aphids. Those included 415 Sitobion avenae, 642 Rhopalosiphum padi, 22 Metopolophium dirhodum, and 13 Schizaphis graminum. The trapped alates were daily collected and individually reared for 7 days on wheat plants in laboratory. Of those 341 alates died of fungal infection, taking 31.2% in the trapped alates. These included 224 S. avenae, 106 R. padi, 8 M. dirhodum, and 3 S. graminum. Deaths of all infected alates occurred during the first 5 days and 78.9% of the deaths occurred within the first 3 days. Individual examination under microscope proved that all deaths were attributed to entomophthoralean fungi. Of those Pandora neoaphidis accounted for 84.6%, Conidiobolus obscurus for 9.9%, and Entomophthora planchoniana for 5.5%. Four alate deaths die     

Plant waxy bloom on peas affects infection of pea aphids by Pandora neoaphidis

This study examined the effects of the surface wax bloom of pea plants, Pisum sativum, on infection of pea aphids, Acyrthosiphon pisum, by the fungal pathogen Pandora neoaphidis. In prior field surveys, a higher proportion of P. neoaphidis-killed pea aphids (cadavers) had been observed on a pea line with reduced wax bloom, as compared with a sister line with normal surface wax bloom. Laboratory bioassays were conducted in order to examine the mechanisms. After plants of each line infested with aphids were exposed to similar densities of conidia, the rate of accumulation of cadavers on the reduced wax line was significantly greater than on the normal wax bloom line; at the end of the experiment (13d), the proportion of aphid cadavers on the reduced wax line was approximately four times that on the normal wax bloom line. When plants were exposed to conidia first and then infested with aphids, the rate of accumulation of cadavers was slightly but significantly greater on the reduced wax line, and infection at the end of the experiment (16d) did not differ between the lines. When aphids were exposed first and then released onto the plants, no differences in the proportion of aphid cadavers were observed between the pea lines. Greater infection of pea aphid on reduced wax peas appears to depend upon plants being exposed to inoculum while aphids are settled in typical feeding positions on the plant. Additional experiments demonstrated increased adhesion and germination by P. neoaphidis conidia to leaf surfaces of the reduced wax line as compared with normal wax line, and this could help explain the higher infection rate by P. neoaphidis on the reduced wax line. In bioassays using surface waxes extracted from the two lines, there was no effect of wax source on germination of P. neoaphidis conidia.     

Transmission of Pandora neoaphidis in the presence of co-occurring arthropods

Transmission of the entomopathogenic fungus Pandora neoaphidis to the nettle aphid Microlophium carnosum was assessed in the presence of arthropods that co-exist with the fungus within the habitat but do not compete for aphid hosts. The presence of a parasitoid significantly enhanced transmission, and transmission rates were similar for both enemy and non-enemy parasitoids. Although herbivory of nettle leaves by Peacock butterfly (Inchis io) caterpillars indirectly reduced the number of M. carnosum by >30% due to a reduction in leaf area for feeding, the addition of I. io significantly increased transmission of P. neoaphidis in the remaining aphids. It is likely that enhanced transmission in the presence of A. rhopalosiphii and I. io is due to disturbance and subsequent movement of the aphid, resulting in contact with conidia deposited on the leaf surface. The presence and impact of co-occurring arthropods should be taken into consideration when assessing the transmission of fungal entomopathogens.     

Sitobion avenae alatae infected by Pandora neoaphidis: their flight ability, post-flight colonization, and mycosis transmission to progeny colonies

Epizootics caused by the obligate Entomophthorales pathogen Pandora neoaphidis may result from more than one possible means of fungal dissemination among host aphids, but we hypothesize that wide dispersal of the fungus is most likely to be associated with the flight behavior of migratory alates. We tested this hypothesis in a simulation experiment by assessing the flight capability of Sitobion avenae alates infected with P. neoaphidis and the potential of their post-flight survival, colonization, and mycosis transmission to progeny. A total of 281 alates were inoculated with P. neoaphidis, individually flown for up to 5h and 9km in a computer-monitored flight mill system and then reared for 10 days on wheat seedlings. The infected alates were capable of surviving on average for 2.9 days (range 1-7 days) and leaving 4.6 nymphs prior to deaths. Transmission of fungal infection within progeny colonies occurred after the mother alates died from P. neoaphidis mycosis. The level of contagious infection among the nymphs reached up to 16.8% within 7 days but varied with the survival time of the infected mother alates after flight. Based on stepwise polynomial regression analysis, progeny colony size was highly correlated with the interactions of flight time with both post-flight survival time and the number of nymphs left per alate before death (r2 = 0.997). Progeny mortality on day 5 after colonization was inversely correlated with post-flight survival time (r2 = 0.949) whereas infection on day 7 was correlated with flight distance and an interaction of post-flight survival time with fecundity of the infected alates (r2 = 0.970). Progeny mortality observed on day 10 was merely correlated to mortality observed on day 5 (r2 = 0.946). These results indicate a successful transmission of alate-borne P. neoaphidis to progeny colonies and further support our hypothesis on the means of primary dispersal of aphid epizootics by migratory alates in a geographically wide range.     

Impact of the entomopathogenic fungus Verticillium lecanii on development of an aphid parasitoid, Aphidius colemani

The parasitoid Aphidius colemani developed normally (approximately 90% adult emergence) when its cotton aphid (Aphis gossypii) host was treated with Verticillum lecanii conidia 5 or 7 days after parasitization. Fungus exposure 1 day before or up to 3 days after parasitization, however, reduced A. colemani emergence from 0 to 10%. Also, numbers of spores and mycelial fragments in aphid homogenates were much higher in aphids exposed to the fungus up to 3 days after parasitization than in aphids treated after 5 or 7 days. Our results suggest that the parasitoid and fungus may be used together for aphid biocontrol as long as fungus applications are timed to allow late-instar development of the parasitoid.     

Geographical distribution and host range of entomophthorales infecting the green spruce aphid Elatobium abietinum Walker in Iceland.

Entomophthora planchoniana and Neozygites fresenii caused infection in populations of the green spruce aphid, Elatobium abietinum, in Iceland. On this aphid species En. planchoniana was exclusively found in the western part of Iceland, while N. fresenii was exclusively found in the eastern part of Iceland. This discrete and nearly nonoverlapping geographical distribution correlates with the distribution of two different populations of El. abietinum found in Iceland. On other aphid species N. fresenii, En. planchoniana, Pandora neoaphidis, and Conidiobolus obscurus were documented throughout the country. Transmission experiments showed that Pa. neoaphidis and En. planchoniana could infect the eastern population of El. abietinum, although they have never been found on this population in nature. This strongly indicates that there is little or no interaction among El. abietinum, other aphids, and their respective entomophthoralean fungi in the field. Furthermore, this study is the first to record epizootics caused by N. fresenii and En. planchoniana in the subpolar region.     

Influence of the aphid pathogen Pandora neoaphidis on the foraging behaviour of the aphid parasitoid Aphidius ervi

Abstract.  1. The parasitoid Aphidius ervi and the entomopathogenic fungus Pandora neoaphidis both require successful invasion of an aphid host to complete their life cycle. A shorter developmental period allows P. neoaphidis to out-compete A. ervi. Aphidius ervi may reduce this fitness cost by avoiding aphid colonies containing P. neoaphidis . Here the response of A. ervi towards P. neoaphidis was assessed using sequential experiments designed to replicate different stages of parasitoid foraging behaviour.
2. Entry rate experiments showed that A. ervi entered aphid colonies containing P. neoaphidis -sporulating cadavers and that there was no significant difference in the attraction of A. ervi to aphid-damaged Vicia faba plants containing either healthy Acyrthosiphon pisum or P. neoaphidis -sporulating cadavers.
3. Observational behavioural experiments indicated that the presence of P. neoaphidis did not affect the search time or total foraging time of A. ervi on V. faba plants infested with either healthy A. pisum or P. neoaphidis -sporulating cadavers.
4. In Petri dish bioassays using aphids infected with P. neoaphidis over a period of 120 h, A. ervi showed no difference in attack rate against uninfected aphids or living aphids infected with P. neoaphidis for 1, 24, 48, 72, or 96 h. However, sporulating cadavers (120 h infection) were not attacked.
5.  Aphidius ervi appears only able to detect the presence of P. neoaphidis once the host is dead and sporulation has started. The fitness of A. ervi may therefore be severely reduced when foraging in P. neoaphidis -infected aphid colonies.     

Intraguild interactions between the entomopathogenic fungus Pandora neoaphidis and an aphid predator and parasitoid at the population scale

The interactions that occur between the entomopathogenic fungus Pandora neoaphidis and a predator (Coccinella septempunctata) and a parasitoid (Aphidius ervi) were assessed in microcosm and polytunnel experiments. Transmission of P. neoaphidis to the pea aphid, Acyrthosiphon pisum, was enhanced in the presence of both C. septempunctata and A. ervi in microcosm experiments done under fixed abiotic conditions. In contrast, the reproductive success of A. ervi was reduced in the presence of P. neoaphidis. Despite the increased fungal transmission in the presence of C. septempunctata, there was no additional decrease in the aphid population indicating that P. neoaphidis is functionally redundant in the presence of the coccinellid. In polytunnel experiments the reproductive success of A. ervi was not affected by P. neoaphidis. These results do not support those of the microcosm and may be due to the more natural abiotic conditions in the polytunnel reducing the competitive advantage of the fungus. Microcosms therefore provide an arena in which the interactions between fungal pathogens and other aphid-natural enemies can be assessed however, further assessments at increased spatial scales under more natural abiotic conditions are also required to accurately determine the outcome of these interactions.     

Apparent competition between two species of aphid via the fungal pathogen Erynia neoaphidis and its interaction with the aphid parasitoid Aphidius ervi

Abstract 1. Motivated by a community study on aphids and their fungal pathogens, three hypotheses were tested experimentally to investigate the influence of the fungal pathogen, Erynia neoaphidis Remaudière and Hennebert, on aphid population and community ecology.
2. Field experiments were performed in 2 years to test whether two susceptible aphid species on different host plants might interact through the shared fungal pathogen. No strong pathogen-mediated indirect interactions (apparent competition) between populations of pea aphid Acyrthosiphon pisum Harris and nettle aphid Microlophium carnosum Buckton were detected.
3. In the first of the field experiments, pea aphids exposed to the fungus showed a weak tendency to produce more winged dispersal morphs than control populations not exposed to the fungus. In a laboratory test, however, no support was found for the hypothesis that the presence of volatiles from fungus-infected cadavers promotes production of winged offspring.
4. The response of the pea aphid parasitoid Aphidius ervi Halliday to colonies containing hosts infected 1 and 3 days previously was assessed. Wasps initiated fewer attacks on 1-day-old infected colonies than on healthy colonies, with the numbers on 3-day-old fungus-infected colonies intermediate.     

Transient host paralysis as a means of reducing self-superparasitism in koinobiont endoparasitoids

The term ‘idiobiont’ refers to those parasitoid species that permanently paralyse their hosts during parasitism, causing the cessation of host growth and development. This is in contrast to koinobiont parasitoids, which allow their hosts to continue developing after being parasitized. While no koinobiont species induce permanent paralysis in their hosts, a minority of koinobionts induce a temporary paralysis that does not interfere with overall host growth and development. We characterized transient paralysis induction in two koinobiont aphid parasitoids in the genus Binodoxys (Hymenoptera: Aphidiinae). Both Binodoxys species induced transient paralysis in Aphis glycines, with paralysis time ranging between 4.5 and 8 min (depending upon parasitoid species and host instar). In a separate experiment, B. communis was capable of inducing transient paralysis in nine aphid species. We addressed two hypotheses potentially explaining the adaptive value of temporary host paralysis in experiments using A. nerii, which is readily accepted but engages in strong defensive behaviour. The first hypothesis is that paralysis increases oviposition success by interfering with host defences and the second is that it aids in the avoidance of self-superparasitism. Paralysed aphids were more likely to be rejected by B. communis than were aphids that had never been stung or that had recovered from paralysis. This result supports the avoidance-of-self-superparasitism hypothesis and is inconsistent with the hypothesis that transient paralysis increases oviposition success of B. communis.     

A PCR-based tool for the cultivation-independent monitoring of Pandora neoaphidis

Pandora neoaphidis is one of the most important fungal pathogens of aphids and has a great potential for use in biocontrol. Little is known on how this fungus persists in an area and in particular on its overwintering strategies. It is hypothesized that natural areas play an important role for survival and that soil may serve as a source of inoculum for new aphid populations in spring. To test these hypotheses, a cultivation-independent PCR-based diagnostic tool was developed, that allows the detection of P. neoaphidis in the environment. Two P. neoaphidis specific PCR primer pairs were designed, targeting sequences in the ribosomal RNA gene cluster. Specificity of both primer pairs was demonstrated with P. neoaphidis and non-target close entomophthoralean relatives. Moreover, single amplicons of expected sizes were obtained with both primer pairs from various environmental sample types, including aphid cadavers, plant material, and soil. The PCR-based diagnostic tool was applied to investigate the persistence of P. neoaphidis in soil samples obtained in 2004/2005 from a nettle field harboring infected aphids in fall 2004. P. neoaphidis was detected in every sample collected in November 2004 and March 2005, suggesting an overwintering stage of P. neoaphidis in top soil layers. The developed cultivation-independent PCR-based tool will be valuable for further investigation of the ecology of P. neoaphidis and for the development and future implementation of management strategies against aphids involving conservation biocontrol.     

The survival ofZoophthora radicans (Zygomycetes: entomophthorales) isolates as hyphal bodies in mummified larvae ofPlutella xylostella (Lep.: Yponomeutidae)

The survival of three isolates ofZoophthora radicans (NW 250, NW 253 & NW 182) as hyphal bodies in dried larvae ofPlutella xylostella stored at 4, 10 and 20°C and 20% R.H was determined. After storage at 20°C, the production of conidia by all isolates was unaffected after 2 weeks but diminished increasingly after 4 and 8 weeks and was entirely lost after 16 weeks. By comparison conidium production at 10°C was unaffected after 16 weeks (isolates NW 250 and NW 182) and, 24 weeks (NW 253) of storage though it declined rapidly in all isolates thereafter. At 4°C many conidia were produced by all isolates even after 34 weeks of storage. These results are consistent with work on other entomophthoralean fungi in dried cadavers suggesting that this may be a common survival strategy in these fungi. NW 250, 253 and 182 were isolated fromP. xylostella in Malaysia and Taiwan, where conditions allow the host to remain active throughout the year. None produced resting sporesin vivo orin vitro but as hosts are always available the ability to survive short dry periods is probably more important than long-term survival for which resting spores are most adapted.