Daniel R. Sutherland

Aquatic eutrophication promotes pathogenic infection in amphibians

The widespread emergence of human and wildlife diseases has challenged ecologists to understand how large-scale agents of environmental change affect host–pathogen interactions. Accelerated eutrophication of aquatic ecosystems owing to nitrogen and phosphorus enrichment is a pervasive form of environmental change that has been implicated in the emergence of diseases through direct and indirect pathways. We provide experimental evidence linking eutrophication and disease in a multihost parasite system. The trematode parasite Ribeiroia ondatrae sequentially infects birds, snails, and amphibian larvae, frequently causing severe limb deformities and mortality. Eutrophication has been implicated in the emergence of this parasite, but definitive evidence, as well as a mechanistic understanding, have been lacking until now. We show that the effects of eutrophication cascade through the parasite life cycle to promote algal production, the density of snail hosts, and, ultimately, the intensity of infection in amphibians. Infection also negatively affected the survival of developing amphibians. Mechanistically, eutrophication promoted amphibian disease through two distinctive pathways: by increasing the density of infected snail hosts and by enhancing per-snail production of infectious parasites. Given forecasted increases in global eutrophication, amphibian extinctions, and similarities between Ribeiroia and important human and wildlife pathogens, our results have broad epidemiological and ecological significance.

PARASITE (RIBEIROIA ONDATRAE) INFECTION LINKED TO AMPHIBIAN MALFORMATIONS IN THE WESTERN UNITED STATES

Parasites and pathogens can influence the survivorship, behavior, and very structure of their host species. For example, experimental studies have shown that trematode parasites can cause high frequencies of severe limb malformations in amphibians. In a broad-scale field survey covering parts of California, Oregon, Washington, Idaho, and Montana, we examined relationships between the frequency and types of morphological abnormalities in amphibians and the abundance of trematode parasite infection, pH, con- centrations of 61 pesticides, and levels of orthophosphate and total nitrate. We recorded severe malformations at frequencies ranging from 1% to 90% in nine amphibian species from 53 aquatic systems. Infection of larvae by the trematode Ribeiroia ondatrae was associated with, and functionally related to, higher frequencies of amphibian limb malfor- mations than found in uninfected populations (#5%). Parasites were concentrated around the basal tissue of hind limbs in infected anurans, and malformations associated with infection included skin webbings, supernumerary limbs and digits, and missing or mal- formed hind limbs. In the absence of Ribeiroia, amphibian populations exhibited low (0- 5%) frequencies of abnormalities involving missing digits or distal portions of a hind limb. Species were affected differentially by the parasite, and Ambystoma macrodactylum , Hyla regilla, Rana aurora, R. luteiventris, and Taricha torosa typically exhibited the highest frequencies of abnormalities. None of the water-quality variables measured was associated with malformed amphibians, but aquatic snail hosts (Planorbella spp.) were significant predictors of the presence and abundance of Ribeiroia infection. Morphological comparisons of adult specimens of Ribeiroia collected from different sites and raised in experimental definitive hosts suggested that all samples represented the same species— R. ondatrae. These field results, coupled with experimental research on the effects of Ribeiroia on amphibians, demonstrate that Ribeiroia infection is an important and widespread cause of amphibian limb malformations in the western United States. The relevance of trematode infection to declines of amphibian populations and the influence of habitat modification on the pathology and life cycle of Ribeiroia are emphasized as areas requiring further research.

Diversity and disease: community structure drives parasite transmission and host fitness

Changes in host diversity and community structure have been linked to disease, but the mechanisms underlying such relationships and their applicability to non-vector-borne disease systems remain conjectural. Here we experimentally investigated how changes in host community structure affected the transmission and pathology of the multi-host parasite Ribeiroia ondatrae, which is a widespread cause of amphibian limb deformities. We exposed larval amphibians to parasites in monospecific or heterospecific communities, and varied host number to differentiate between density- and diversity-mediated effects on transmission. In monospecific communities, exposure to Ribeiroia significantly increased mortality (15%), malformations (40%) and time-to-metamorphosis in toads. However, the presence of tree frogs significantly reduced infection in toads, leading to fewer malformations and higher survival than observed in monospecific communities, providing evidence of parasite-mediated facilitation. Our results suggest that interspecific variation in parasite resistance can inhibit parasite transmission in multi-species communities, reducing infection and pathology in sensitive hosts.

Amphibian deformities and Ribeiroia infection: an emerging helminthiasis

Since their widespread appearance in the mid-1990s, malformed amphibians have evoked fear, as well as fascination within the scientific and public communities. Recent evidence from field and laboratory studies has implicated infection by a digenetic trematode--Ribeiroia ondatrae--as an important cause of such deformities. Ribeiroia spp. have a complex life cycle involving planorbid snails, amphibians and water birds. Under natural conditions, malformations might promote parasite transmission by increasing the susceptibility of infected amphibians to predation by definitive hosts. However, with respect to the recent outbreak of deformities, we suggest that exogenous agents (e.g. pesticides, nutrient run-off, introduced fishes) might be interacting with Ribeiroia, resulting in elevated infection levels, and we highlight the need for studies incorporating multiple stressor dynamics to further explore this problem.

Defense reactions of mosquitoes to filarial worms: Comparative studies on the response of three different mosquitoes to inoculated Brugia pahangi and Dirofilaria immitis microfilariae

Abstract The melanization response of Aedes trivittatus and the Rockefeller (RKF) and black-eyed Liverpool (LVP) strains of Aedes aegypti against intrathoracically inoculated Dirofilaria immitis and Brugia pahangi microfilariae (mff) was investigated. All mff of either species were melanized in A. trivittatus following Day 2 postinoculation, and the response of this species was significantly more rapid and effective than either strain of A. aegypti. The refractory RKF strain had a significantly greater response against both D. immitis and B. pahangi than the highly susceptible LVP strain, but data suggest that the increased responsiveness was due to a physiologic incompatibility in RKF A. aegypti, thereby resulting in a greater mortality and subsequent melanization of inoculated mff. Inoculation of large numbers of mff overloaded the defense capabilities of A. aegypti (LVP), but not those of A. trivittatus. The melanization response against D. immitis mff was effectively reduced for up to 4 days in A. aegypti (LVP), but for only 1 day in A. trivittatus, when mosquitoes were maintained on a 0.3 m sucrose diet containing from 0.1 to 1.0% (w/v) phenylthiourea.

ADDING INFECTION TO INJURY: SYNERGISTIC EFFECTS OF PREDATION AND PARASITISM ON AMPHIBIAN MALFORMATIONS

We explored the importance of interactions between parasite infection and predation in driving an emerging phenomenon of conservation importance: amphibian limb malformations. We suggest that injury resulting from intraspecific predation in combination with trematode infection contributes to the frequency and severity of malformations in salamanders. By integrating field surveys and experiments, we evaluated the individual and combined effects of conspecific attack and parasite (Ribeiroia ondatrae) infection on limb development of long-toed salamanders (Ambystoma macrodactylum). In the absence of Ribeiroia, abnormalities involved missing digits, feet, or limbs and were similar to those produced by cannibalistic attack in experimental trials. At field sites that supported Ribeiroia, malformations were dominated by extra limbs and digits. Correspondingly, laboratory exposure of larval salamanders to Ribeiroia cercariae over a 30-day period induced high frequencies of malformations, including extra digits, extra limbs, cutaneous fusion, and micromelia. However, salamander limbs exposed to both injury and infection exhibited 3-5 times more abnormalities than those exposed to either factor alone. Infection also caused significant delays in limb regeneration and time-to-metamorphosis. Taken together, these results help to explain malformation patterns observed in natural salamander populations while emphasizing the importance of interactions between parasitism and predation in driving disease.

ULTRASTRUCTURE OF THE MELANIZATION RESPONSE OF AEDES TRIVITTATUS AGAINST INOCULATED DIROFILARIA IMMITIS MICROFILARIAE

Scanning and transmission electron microscopy revealed that intrathoracically-inoculated microfilariae (mff) of Dirofilaria immitis elicited a rapid and effective immune response in the hemocoel of Aedes trivittatus mosquitoes. Hemocyte lysis and melanization of inoculated mff began immediately following exposure to the hemolymph environment. Initial melanin accumulation occurred at any site along the surface of mff and rapidly increased in thickness. Hemocyte encapsulation generally described for insects did not occur, but hemocytes might be necessary for activation of the melanization response. Although intact hemocytes were never abundant, those that were present seemed to show an active secretion of membrane-bound vacuoles directed toward mff. Activated hemocytes were in close association, but never in direct contact with the parasite, and were most commonly seen in various stages of lysis. Numerous cell remnants were noted throughout the developing melanin capsule. Parasites were completely melanized by 24 hr postinoculation (PI). By about 3 days PI, a membrane began to form around deposited melanin and hemocyte remnants. This developed into a double membrane-like structure of 25-30 nm thickness and resulted in the enclosure and isolation of the mff, melanin deposits, and cellular remnants from hemolymph components. It is suggested that this membrane functions as a boundary to isolate the melanized parasite and prevents additional hemocyte involvement.

A MOLECULAR PHYLOGENETIC STUDY OF THE GENUS RIBEIROIA (DIGENEA): TREMATODES KNOWN TO CAUSE LIMB MALFORMATIONS IN AMPHIBIANS

Species of Ribeiroia (Trematoda: Psilostomidae) are known to cause severe limb malformations and elevated mortality in amphibians. However, little is known regarding the number of species in this genus or its relation to other taxa. Species of Ribeiroia have historically been differentiated by slight differences among their larval stages. To better understand the systematics and biogeography of this genus and their potential relevance to the distribution of malformed amphibians, specimens identified as Ribeiroia were collected across much of the known range, including samples from 5 states in the United States (8 sites) and 2 islands in the Caribbean (Puerto Rico and Guadeloupe). A cercaria from East Africa identified as Cercaria lileta (Fain, 1953), with attributes suggestive of Ribeiroia (possibly R. congolensis), was also examined. The intertranscribed spacer region 2 (ITS-2) of the ribosomal gene complex was sequenced and found to consist of 429 nucleotides (nt) for R. ondatrae (United States) and 427 nt for R. marini (Caribbean), with only 6 base differences noted between the 2 species. The ITS-2 region of C. lileta (429 nt) aligned closely with those of the 2 other Ribeiroia species in a phylogenetic analysis that included related trematode genera. This evidence suggests that a third Ribeiroia species exists in tropical Africa. Variation in ITS-2 within R. ondatrae was nonexistent among the 8 populations from North America. Our study further suggests that Ribeiroia spp. originally parasitized Biomphalaria sp., and that a host switch to a closely related snail, Helisoma sp., may have occurred in the lineage represented by R. ondatrae. However, relationships within the Echinostomatidae are not understood well enough to make any robust conclusions at this time.

Defense reactions of mosquitoes to filarial worms: Role of the microfilarial sheath in the response of mosquitoes to inoculated Brugia pahangi microfilariae

Abstract The melanization response of Aedes trivittatus and A. aegypti (black-eyed Liverpool strain) against intrathoracically inoculated sheathed and chemically exsheathed Brugia pahangi microfilariae (mff) was assessed daily through 5 days postinoculation (PI). Response of A. aegypti against exsheathed mff was significantly reduced on all days compared with the response against sheathed mff, and a significantly greater percentage of exsheathed mff were alive through 4 days PI than were sheathed mff. The melanization response of A. trivittatus was nearly 100% effective against either sheathed or exsheathed mff by Day 2 PI. When mff were allowed to migrate through A. aegypti midguts in vitro before inoculation into intact A. aegypti, nearly 94% ( 120 128 ) of the parasites recovered had avoided the response and were developing. Penetration of A. trivittatus midguts in vitro by mff before inoculation into intact A. trivittatus did not prevent a melanization response. Inoculation of mff into A. trivittatus following A. aegypti midgut penetration, however, resulted in almost 60% ( 98 171 ) of the mff avoiding the response and developing as normal L1 larvae after 5 days PI. The possibility of mff acquiring host antigens during midgut penetration and therefore avoiding recognition as nonself by mosquitoes, and (or) the possibility of the midgut environment modifying or stimulating mff to inhibit the response of mosquitoes are discussed.

Midgut barrier as a possible factor in filarial worm vector competency in Aedes trivittatus

Abstract Studies were conducted on the ability of microfilariae (mff) of Brugia pahangi to penetrate midguts of Aedes trivittatus and Aedes aegypti (black-eyed Liverpool strain) in vitro. At 12–14 days postexposure (PE) to microfilaremic jirds, A. trivittatus exhibited a significantly lower prevalence and mean intensity of infection than A. aegypti . Nearly 50% of the mff ingested by A. aegypti , but only 7.5% of the mff ingested by A. trivittatus , penetrated through midguts within 2 hr PE. Examination of 4-μm plastic sections of midguts revealed few quantifiable differences in rate or extent of peritrophic membrane formation between the two mosquito species at 0, 30, 60, 90, or 120 min and 6, 12, or 24 hr PE. At 2 hr PE, blood meals of A. trivittatus appeared more coagulated than did blood meals from A. aegypti . Feeding both mosquito species on heparinized or citrated blood in membrane feeders resulted in significant increases in the percentage of mff penetrating the midgut of A. aegypti , but a significantly reduced percentage of mff penetrating the midgut of A. trivittatus . A significant decrease in percentage of mff penetrating midguts of both mosquito species resulted when mosquitoes were exposed to blood containing EDTA.