Simon L. Elliot

Hidden diversity behind the zombie-ant fungus Ophiocordyceps unilateralis: four new species described from carpenter ants in Minas Gerais, Brazil.

Background Ophiocordyceps unilateralis (Clavicipitaceae: Hypocreales) is a fungal pathogen specific to ants of the tribe Camponotini (Formicinae: Formicidae) with a pantropical distribution. This so-called zombie or brain-manipulating fungus alters the behaviour of the ant host, causing it to die in an exposed position, typically clinging onto and biting into the adaxial surface of shrub leaves. We (HCE and DPH) are currently undertaking a worldwide survey to assess the taxonomy and ecology of this highly variable species. Methods We formally describe and name four new species belonging to the O. unilateralis species complex collected from remnant Atlantic rainforest in the south-eastern region (Zona da Mata) of the State of Minas Gerais, Brazil. Fully illustrated descriptions of both the asexual (anamorph) and sexual (teleomorph) stages are provided for each species. The new names are registered in Index Fungorum (registration.indexfungorum.org) and have received IF numbers. This paper is also a test case for the electronic publication of new names in mycology. Conclusions We are only just beginning to understand the taxonomy and ecology of the Ophiocordyceps unilateralis species complex associated with carpenter ants; macroscopically characterised by a single stalk arising from the dorsal neck region of the ant host on which the anamorph occupies the terminal region and the teleomorph occurs as lateral cushions or plates. Each of the four ant species collected - Camponotus rufipes, C. balzani, C. melanoticus and C. novogranadensis - is attacked by a distinct species of Ophiocordyceps readily separated using traditional micromorphology. The new taxa are named according to their ant host.

Quantifying the reproduction of Bacillus thuringiensis HD1 in cadavers and live larvae of Plutella xylostella.

The Bacillus cereus group comprises a range of micro-organisms with diverse habits, including gut commensals, opportunistic pathogens and soil saprophytes. Using quantitative microbiological methods we tested whether Bacillus thuringiensis (Bt) could reproduce in cadavers of Plutella xylostella killed by Bt, or in the gut of live insects, or be transmitted vertically from females to their offspring. We also tested whether diverse Bt strains could grow in high nutrient broth at a pH similar to that in the larval midgut. Low levels of reproduction were found in insect cadavers but there was no evidence of vertical transmission, or of significant reproduction in live insects. Four strains of B. thuringiensis var. kurstaki and one of B. thuringiensis var. tenebrionis were found to be capable of growth at high pH. Greater spore recovery rates in frass were found in hosts that were resistant or tolerant of infection. We concluded that that spores recovered in frass represent, in general, an ungerminated fraction of ingested inoculum and that germination rates are reduced in unsuitable hosts.

Density‐dependent prophylactic immunity reconsidered in the light of host group living and social behavior

According to the density-dependent hypothesis (DDP), hosts living at high densities suffer greater risk of disease and so invest more in immunity. Although there is much empirical support for this, especially from invertebrate systems, there are many exceptions, notably in social insects. We propose that (A) density is not always the most appropriate population parameter to use when considering the risks associated with disease and (B) behavioral defenses should be given a greater emphasis in considerations of a hosts repertoire of immune defenses. We propose a complementary framework stressing the connectivity between and within populations as a starting point and emphasizing the costs represented by disease above the risk of disease per se. We consider the components of immune defense and propose that behaviors may represent lower-cost defenses than their physiological counterparts. As group-living and particularly social animals will have a greater behavioral repertoire, we conclude that with group living comes a greater capacity for behavioral immune defense, most particularly for social insects. This may escape our notice if we consider physiological parameters alone.

Ophiocordyceps unilateralis: A keystone species for unraveling ecosystem functioning and biodiversity of fungi in tropical forests?

Ophiocordyceps unilateralis (Ascomycota: Hypocreales) is a specialized parasite that infects, manipulates and kills formicine ants, predominantly in tropical forest ecosystems. We have reported previously, based on a preliminary study in remnant Atlantic Forest in Minas Gerais (Brazil), that O. unilateralis represents a species complex. On each of the four species of infected carpenter ant (Camponotus) collected, the fungus – characterized macroscopically by a single stalk arising from the dorsal neck region on which the sexual structures (stromatal plates) are borne laterally – can readily be distinguished both microscopically and functionally. Here, we describe and discuss the biology, life cycle and infection strategies of O. unilateralis s.l. and hypothesize that there may be hundreds of species within the complex parasitizing formicine ants worldwide. We then address the diversity within related hypocrealean fungi, with particular reference to symbionts (mutualists through to parasites), and argue that the widely-quoted total of extant fungi (1.5 million species) may be grossly underestimated.

Disease Dynamics in a Specialized Parasite of Ant Societies

Coevolution between ant colonies and their rare specialized parasites are intriguing, because lethal infections of workers may correspond to tolerable chronic diseases of colonies, but the parasite adaptations that allow stable coexistence with ants are virtually unknown. We explore the trade-offs experienced by Ophiocordyceps parasites manipulating ants into dying in nearby graveyards. We used field data from Brazil and Thailand to parameterize and fit a model for the growth rate of graveyards. We show that parasite pressure is much lower than the abundance of ant cadavers suggests and that hyperparasites often castrate Ophiocordyceps. However, once fruiting bodies become sexually mature they appear robust. Such parasite life-history traits are consistent with iteroparity– a reproductive strategy rarely considered in fungi. We discuss how tropical habitats with high biodiversity of hyperparasites and high spore mortality has likely been crucial for the evolution and maintenance of iteroparity in parasites with low dispersal potential.

A standardizable protocol for infection of Rhodnius prolixus with Trypanosoma rangeli, which mimics natural infections and reveals physiological effects of infection upon the insect

Trypanosoma rangeli is a protozoan parasite that shares hosts - mammals and triatomines - with Trypanosoma cruzi, the etiological agent of Chagas disease. Although T. rangeli is customarily considered to be non-pathogenic to human hosts, it is able to produce pathologies in its invertebrate hosts. However, advances are hindered by a lack of standardization of infection procedures and these pathologies need documentation. To establish a suitable, and standardizable, infection protocol, the duration of the fourth instar was evaluated in nymphs infected by injection into the thorax with different concentrations of parasites, and compared with nymphs infected naturally (i.e. orally). We demonstrate that delays in moult were attributable to the presence of the parasite in the haemolymph (vs. the gut) and propose that the protocol presented here simulates closely natural infections. This methodology was then used for the evaluation of physiological parameters and several hitherto unreported effects of T. rangeli infection on Rhodnius prolixus were revealed. Haemolymph volume was greater in infected than uninfected nymphs but this alteration could not be attributed to water retention, since infected insects lost the same amount of water as controls. However, we found that lipid content and fat body weight were both increased in insects infected by T. rangeli. We propose that this is due to the parasites sequestration of host blood lipids and carrier proteins. With these findings, we have taken a few first steps to unravelling physiological details of the host-parasite interaction. We suggest future directions towards a fuller understanding of mechanistic and adaptive aspects of triatomine-trypanosomatid interactions.

Trypanosoma cruzi, Etiological Agent of Chagas Disease, Is Virulent to Its Triatomine Vector Rhodnius prolixus in a Temperature- Dependent Manner

It is often assumed that parasites are not virulent to their vectors. Nevertheless, parasites commonly exploit their vectors (nutritionally for example) so these can be considered a form of host. Trypanosoma cruzi, a protozoan found in mammals and triatomine bugs in the Americas, is the etiological agent of Chagas disease that affects man and domestic animals. While it has long been considered avirulent to its vectors, a few reports have indicated that it can affect triatomine fecundity. We tested whether infection imposed a temperature-dependent cost on triatomine fitness. We held infected insects at four temperatures between 21 and 30°C and measured T. cruzi growth in vitro at the same temperatures in parallel. Trypanosoma cruzi infection caused a considerable delay in the time the insects took to moult (against a background effect of temperature accelerating moult irrespective of infection status). Trypanosoma cruzi also reduced the insects’ survival, but only at the intermediate temperatures of 24 and 27°C (against a background of increased mortality with increasing temperatures). Meanwhile, in vitro growth of T. cruzi increased with temperature. Our results demonstrate virulence of a protozoan agent of human disease to its insect vector under these conditions. It is of particular note that parasite-induced mortality was greatest over the range of temperatures normally preferred by these insects, probably implying adaptation of the parasite to perform well at these temperatures. Therefore we propose that triggering this delay in moulting is adaptive for the parasites, as it will delay the next bloodmeal taken by the bug, thus allowing the parasites time to develop and reach the insect rectum in order to make transmission to a new vertebrate host possible.

Attracted to the enemy: Aedes aegypti prefers oviposition sites with predator-killed conspecifics

Oviposition habitat choices of species with aquatic larvae are expected to be influenced by both offspring risk of mortality due to predation, and offspring growth potential. Aquatic predators may indirectly influence growth potential for prey by reducing prey density and, for filter-feeding prey, by increasing bacterial food for prey via added organic matter (feces, partially eaten victims), creating the potential for interactive effects on oviposition choices. We tested the hypothesis that the mosquito Aedes aegypti preferentially oviposits in habitats with predatory Toxorhynchites larvae because of indirect effects of predation on chemical cues indicating bacterial abundance. We predicted that A. aegypti would avoid oviposition in sites with Toxorhynchites, but prefer to oviposit where bacterial food for larvae is abundant, and that predation by Toxorhynchites would increase bacterial abundances. Gravid A. aegypti were offered paired oviposition sites representing choices among: predator presence; the act of predation; conspecific density; dead conspecific larvae; and bacterial activity. A. aegypti preferentially oviposited in sites with Toxorhynchites theobaldi predation, and with killed conspecific larvae, but failed to detect preferences for other treatments. The antibiotic tetracycline eliminated the strongest oviposition preference. Both predation by Toxorhynchites and killed larvae increased bacterial abundances, suggesting that oviposition attraction is cued by bacteria. Our results show the potential for indirect effects, like trophic cascades, to influence oviposition choices and community composition in aquatic systems. Our results suggest that predators like Toxorhynchites may be doubly beneficial as biocontrol agents because of the attraction of ovipositing mosquitoes to bacterial by-products of Toxorhynchites feeding.

Yet More "Weeds" in the Garden: Fungal Novelties from Nests of Leaf-Cutting Ants

Background Symbiotic relationships modulate the evolution of living organisms in all levels of biological organization. A notable example of symbiosis is that of attine ants (Attini; Formicidae: Hymenoptera) and their fungal cultivars (Lepiotaceae and Pterulaceae; Agaricales: Basidiomycota). In recent years, this mutualism has emerged as a model system for studying coevolution, speciation, and multitrophic interactions. Ubiquitous in this ant-fungal symbiosis is the “weedy” fungus Escovopsis (Hypocreales: Ascomycota), known only as a mycoparasite of attine fungal gardens. Despite interest in its biology, ecology and molecular phylogeny—noting, especially, the high genetic diversity encountered—which has led to a steady flow of publications over the past decade, only two species of Escovopsis have formally been described. Methods and Results We sampled from fungal gardens and garden waste (middens) of nests of the leaf-cutting ant genus Acromyrmex in a remnant of subtropical Atlantic rainforest in Minas Gerais, Brazil. In culture, distinct morphotypes of Escovopsis sensu lato were recognized. Using both morphological and molecular analyses, three new species of Escovopsis were identified. These are described and illustrated herein—E. lentecrescens, E. microspora, and E. moelleri—together with a re-description of the genus and the type species, E. weberi. The new genus Escovopsioides is erected for a fourth morphotype. We identify, for the first time, a mechanism for horizontal transmission via middens. Conclusions The present study makes a start at assigning names and formal descriptions to these specific fungal parasites of attine nests. Based on the results of this exploratory and geographically-restricted survey, we expect there to be many more species of the genus Escovopsis and its relatives associated with nests of both the lower and higher Attini throughout their neotropical range, as suggested in previous studies.

Effects of Infection by Trypanosoma cruzi and Trypanosoma rangeli on the Reproductive Performance of the Vector Rhodnius prolixus

The insect Rhodnius prolixus is responsible for the transmission of Trypanosoma cruzi, which is the etiological agent of Chagas disease in areas of Central and South America. Besides this, it can be infected by other trypanosomes such as Trypanosoma rangeli. The effects of these parasites on vectors are poorly understood and are often controversial so here we focussed on possible negative effects of these parasites on the reproductive performance of R. prolixus, specifically comparing infected and uninfected couples. While T. cruzi infection did not delay pre-oviposition time of infected couples at either temperature tested (25 and 30°C) it did, at 25°C, increase the e-value in the second reproductive cycle, as well as hatching rates. Meanwhile, at 30°C, T. cruzi infection decreased the e-value of insects during the first cycle and also the fertility of older insects. When couples were instead infected with T. rangeli, pre-oviposition time was delayed, while reductions in the e-value and hatching rate were observed in the second and third cycles. We conclude that both T. cruzi and T. rangeli can impair reproductive performance of R. prolixus, although for T. cruzi, this is dependent on rearing temperature and insect age. We discuss these reproductive costs in terms of potential consequences on triatomine behavior and survival.