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DNA metabarcoding offers new perspectives, especially with regard to the high-throughput identification and diagnostics of pathogens. Microsporidia are an example of widely distributed, opportunistic and pathogenic microorganisms in which molecular identification is important for both environmental research and clinical diagnostics. We have developed a method for parallel detection of both microsporidian infection and the host species. We designed new primer sets: one specific for the classical Microsporidia (targeting the hypervariable V5 region of small subunit [ssu] rDNA), and a second one targeting a shortened fragment of the COI gene (standard metazoan DNA-barcode); both markers are well suited for next generation sequencing. Analysis of the ssu rDNA data set representing 607 microsporidian species (120 genera) indicated that the V5 region enables identification of >98% species in the data set (596/607). To test the method, we used microsporidians that infect mosquitoes in natural populations. Using mini-COI data, all field-collected mosquitoes were unambiguously assigned to seven species; among them almost 60% of specimens were positive for at least 11 different microsporidian species, including a new microsporidian ssu rDNA sequence (Microsporidium sp. PL01). Phylogenetic analysis showed that this species belongs to one of the two main clades in the Terresporidia. We found a high rate of microsporidian co-infections (9.4%). The numbers of sequence reads for the operational taxonomic units suggest that the occurrence of Nosema spp. in co-infections could benefit them; however, this observation should be retested using a more intensive host sampling. Our results show that DNA barcoding is a rapid and cost-effective method for deciphering sample diversity in greater resolution, including the hidden biodiversity that may be overlooked using classical methodology., (© 2020 John Wiley & Sons Ltd.)

Effective mosquito control is vital to curtail the devastating health effects of many vectored diseases. RNA interference (RNAi)-mediated control of mosquitoes is an attractive alternative to conventional chemical pesticides. Previous studies have suggested that transcripts for inhibitors of apoptosis (IAPs) may be good RNAi targets. To revisit and extend previous reports, we examined the expression of Aedes aegypti (L.) IAPs (AaeIAPs) 1, 2, 5, 6, 9, and a viral IAP-associated factor (vIAF) as well as Anopheles quadrimaculatus Say and Culex quinquefasciatus Say IAP1 homologs (AquIAP1 and CquIAP1) in adult females. Expression profiles of IAPs suggested that some older female mosquitoes had significantly higher IAP mRNA levels when compared to the youngest ones. Minor differences in expression of AaeIAPs were observed in mosquitoes that imbibed a bloodmeal, but the majority of the time points (up to 48 h) were not significantly different. Although in vitro experiments with the Ae. aegypti Aag-2 cell line demonstrated that the various AaeIAPs could be effectively knocked down within one day after dsRNA treatment, only Aag-2 cells treated with dsIAP1 displayed apoptotic morphology. Gene silencing and mortality were also evaluated after topical application and microinjection of the same dsRNAs into female Ae. aegypti. In contrast to previous reports, topical administration of dsRNA against AaeIAP1 did not yield a significant reduction in gene expression or increased mortality. Knockdown of IAP1 and other IAPs by microinjection did not result in significant mortality. In toto, our findings suggest that IAPs may not be suitable RNAi targets for controlling adult mosquito populations.

Microsporidia are highly divergent fungi that are obligate intracellular pathogens of a wide range of host organisms. Here we review recent findings from the genome sequences of mosquito-infecting microsporidian species Edhazardia aedis and Vavraia culicis, which show large differences in genome size, although similar numbers of predicted genes. We also show a video of E. aedis polar tube firing, which is the dramatic mechanism used by microsporidia to deliver the germ cell (sporoplasm) into the host cell to initiate intracellular infection., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The impact of the presence of larval mosquito pathogens with potential for biological control on oviposition choice was evaluated for three mosquito species/pathogen pairs present in Florida. These included Aedes aegypti infected with Edhazardia aedis, Aedes albopictus infected with Vavraia culicis, and Culex quinquefasciatus infected with Culex nigripalpus nucleopolyhedrovirus (CuniNPV). Two-choice oviposition bioassays were performed on each host and pathogen species with one oviposition cup containing infected larvae and the other cup containing uninfected larvae (control). Both uninfected and E. aedis-infected female Ae. aegypti laid significantly fewer eggs in oviposition cups containing infected larvae. Uninfected gravid female Ae. albopictus and Cx. quinquefasciatus oviposited equally in cups containing uninfected larvae or containing larvae infected with V. culicis or CuniNPV, respectively. Gravid female Ae. albopictus infected with V. culicis did not display ovarian development and did not lay eggs. The decreased oviposition by gravid Ae. aegypti in containers containing E. aedis-infected larvae may indicate that the infected larvae produce chemicals deterring oviposition., (© 2013 The Society for Vector Ecology.)

Many vertebrate and insect viruses possess antiapoptotic genes that are required for their infectivity. This led to the hypothesis that apoptosis is an innate immunoresponse important for limiting virus infections. The role of apoptosis may be especially important in insect antiviral defense because of the lack of adaptive immunity. However, the cellular mechanism that elicits apoptosis in response to viral infection in insects has not been determined. Using an in vivo infection system with the mosquito baculovirus CuniNPV (Culex nigripalpus nucleopolyhedrovirus), we demonstrated that michelob_x (mx), the mosquito ortholog of Drosophila proapoptotic gene reaper, is specifically induced in larval midgut cells following viral infection. Interestingly, the dynamics of mx induction corresponds with the outcome of the infection. In the permissive mosquito C. quinquefasciatus, a slow induction of mx failed to induce prompt apoptosis, and the infected cells eventually undergo necrosis with heavy loads of encapsulated viruses. In contrast, in the refractory mosquito Aedes aegypti, a rapid induction of mx within 30  min p.i. is followed by apoptosis within 2-6  h p.i., suggesting a possible role for apoptosis in limiting viral infection. When the execution of apoptosis was delayed by caspase inhibitors, viral gene expression became detectable in the A. aegypti larvae.

The release of infected mosquitoes or other arthropods by bioterrorists, i.e., arboterrorism, to cause disease and terror is a threat to the USA. A workshop to assess mosquito control response capabilities to mount rapid and effective responses to eliminate an arboterrorism attack provided recommendations to improve capabilities in the USA. It is essential that mosquito control professionals receive training in possible responses, and it is recommended that a Council for Emergency Mosquito Control be established in each state to coordinate training, state resources, and actions for use throughout the state.

Ultra-low-volume (ULV) and thermal fog aerosol dispersals of pesticides have been used against mosquitoes and other insects for half a century. Although each spray technology has advantages and disadvantages, only 7 studies have been identified that directly compare their performance in the field. US military personnel currently operating in hot-arid environments are impacted by perpetual nuisance and disease vector insect problems, despite adulticide operations using modern pesticide-delivery equipment such as ULV. None of the identified comparative studies has looked at the relative feasibility and efficacy of ULV and thermal fog equipment against mosquitoes in hot-arid environments. In this study we examine the impact of ULV and thermal fog applications of malathion against caged sentinel mosquitoes in the field in a warm temperate area of Florida, followed by a similar test in a hot-dry desert area of southern California. Patterns of mortality throughout 150 m x 150 m grids of sentinel mosquitoes indicate greater efficacy from the thermal fog application in both environments under suboptimal ambient weather conditions. We discuss the implications of these findings for future military preventive medicine activities and encourage further investigations into the relative merits of the 2 technologies for force health protection.

alpha-Mangostin derived from mangosteen was identified as a mosquito sterol carrier protein-2 inhibitor via high throughput insecticide screening, alpha-Mangostin was tested for its larvicidal activity against third instar larvae of six mosquito species, and the median lethal concentration values range from 0.84 to 2.90 ppm. The residual larvicidal activity of alpha-mangostin was examined under semifield conditions. The results indicated that alpha-mangostin was photolytic with a half-life of 53 min in water under full sunlight exposure. The effect of alpha-mangostin on activities of major detoxification enzymes such as P450, glutathione S-transferase, and esterase was investigated. The results showed that alpha-mangostin significantly elevated activities of P450 and glutathione S-transferase in larvae, whereas it suppressed esterase activity. Toxicity of alpha-mangostin against young rats was studied, and there was no detectable adverse effect at dosages as high as 80 mg/kg. This is the first multifaceted study of the biological activity of alpha-mangostin in mosquitoes. The results suggest that alpha-mangostin may be a lead compound for the development of a new organically based mosquito larvicide.

The relative toxicities of 8 repellents (DMP, Rutgers 612, DEET, IR3535, Picardin, PMD, AI3-35765, and AI3-37220) were evaluated by topical application against females of Aedes aegypti, Culex quinquefasciatus, Anopheles quadrimaculatus, and An. albimanus. Based on 24-h LD50 values, the most toxic repellent against all 4 mosquito species was AI3-37220, with values of 0.25, 0.20, 0.16, and 0.11 microg/mg for the listed 4 mosquito species, respectively. The least toxic of the 8 repellents tested was DMP, with LD50 values of 5.40, 4.72, 2.50, and 1.83 microg/mg, respectively. Based on the 24-h LD50 values, An. albimanus was the most susceptible species. The findings of the study reported herein provide a comprehensive examination of the toxicities of 4 currently used, 2 formerly used, and 2 experimental repellents against 4 mosquito species.

The effects of temperature, pH, and NaCl concentrations on the infectivity of zoospores of Leptolegnia chapmanii (Argentine isolate) were determined for Aedes aegypti and Culex pipiens under laboratory conditions. Zoospores of L. chapmanii were infectious at temperatures between 10 and 35 degrees C but not at 5 or 40 degrees C. At the permissive temperatures, mortality rates in young instars were much higher than in older instars and larvae of Ae. aegypti were more susceptible to L. chapmanii than larvae of Cx. pipiens. At 25 degrees C, Ae. aegypti larvae challenged with L. chapmanii zoospores resulted in 100% infection at pH levels ranging from 4 to 10. Larvae of Cx. pipiens exposed to similar pH and zoospore concentrations resulted in increasing mortality rates from 62% to 99% at pH 4 to 7, respectively, and then decreased to 71% at pH 10. Aedes aegypti larvae exposed to L. chapmanii zoospores in NaCl concentrations ranging from 0 to 7 parts per thousand (ppt) at 25 degrees C resulted in 100% mortality while mortality rates for Cx. pipiens decreases from 96% in distilled water to 31.5% in water with 6 ppt NaCl. Control Cx. pipiens larvae died when exposed at a NaCl concentration of 7 ppt. Vegetative growth of L. chapmanii was negatively affected by NaCl concentrations. These results have demonstrated that the Argentinean isolate of L. chapmanii tolerated a wide range of temperatures, pH, and salinity, suggesting that it has the potential to adapt to a wide variety of mosquito habitats.

There are several types of viral pathogens that cause disease in mosquitoes with most belonging to 4 major groups. The most common viruses of mosquitoes are the baculoviruses (NPVs) (Baculoviridae: Nucleopolyhedrovirus) and cytoplasmic polyhedrosis viruses (CPVs) (Reoviridae: Cypovirus). The other major types of viruses in mosquitoes are represented by the densoviruses (DNVs) (Parvoviridae: Brevidensovirus) and the iridoviruses (MIVs) (Iridoviridae: Chloriridovirus). Baculoviruses, densoviruses and iridoviruses are DNA viruses while cypoviruses are the main RNA viruses in mosquitoes. This chapter presents an overview of the recent advancements in the study of mosquito pathogenic viruses and discusses how this new understanding of virus-mosquito interactions can be used to develop novel research and control strategies.

CuniNPV is a pathogen of Culex mosquitoes, vectors of West Nile virus and other forms of encephalitis. Successful development of CuniNPV requires an efficient production system and formulated product that incorporates magnesium, an essential component for transmission. It may be possible to develop mosquito baculoviruses as a new type of biopesticide by microencapsulating the virus and magnesium into formulations that would be effective regardless of the water quality. In addition, this new insight on transmission may facilitate the discovery and development of additional baculoviruses for the control of other important mosquito vectors. Biological mining of the CuniNPV genome and investigations to understand virus-mosquito interactions at the molecular level offer exciting possibilities for the development of novel mosquito control strategies and tools. Understanding the molecular mechanisms of infection will provide the opportunity to devise new control strategies, for example, compromising the defensive systems of the mosquito (proteases for the peritrophic matrix) or exploiting receptors used by the virus to specifically deliver toxins to mosquito larvae via the midgut. As additional baculovirus genomes become available, comparative genomics could lead to a more informed understanding of how the virus exploits its host as well as the factors responsible for the genus-specific host range of most known mosquito baculoviruses.

Iridoviruses (IVs) are classified into five genera: Iridovirus and Chloriridovirus, whose members infect invertebrates, and Ranavirus, Lymphocystivirus, and Megalocytivirus, whose members infect vertebrates. Until now, Chloriridovirus was the only IV genus for which a representative and complete genomic sequence was not available. Here, we report the genome sequence and comparative analysis of a field isolate of Invertebrate iridescent virus type 3 (IIV-3), also known as mosquito iridescent virus, currently the sole member of the genus Chloriridovirus. Approximately 20% of the 190-kbp IIV-3 genome was repetitive DNA, with DNA repeats localized in 15 apparently noncoding regions. Of the 126 predicted IIV-3 genes, 27 had homologues in all currently sequenced IVs, suggesting a genetic core for the family Iridoviridae. Fifty-two IIV-3 genes, including those encoding DNA topoisomerase II, NAD-dependent DNA ligase, SF1 helicase, IAP, and BRO protein, are present in IIV-6 (Chilo iridescent virus, prototype species of the genus Iridovirus) but not in vertebrate IVs, likely reflecting distinct evolutionary histories for vertebrate and invertebrate IVs and potentially indicative of genes that function in aspects of virus-invertebrate host interactions. Thirty-three IIV-3 genes lack homologues in other IVs. Most of these encode proteins of unknown function but also encode IIV3-053L, a protein with similarity to DNA-dependent RNA polymerase subunit 7; IIV3-044L, a putative serine/threonine protein kinase; and IIV3-080R, a protein with similarity to poxvirus MutT-like proteins. The absence of genes present in other IVs, including IIV-6; the lack of obvious colinearity with any sequenced IV; the low levels of amino acid identity of predicted proteins to IV homologues; and phylogenetic analyses of conserved proteins indicate that IIV-3 is distantly related to other IV genera.

The two major groups of pathogenic viruses in mosquitoes are the occluded viruses, represented by baculoviruses and cypoviruses, and the non-occluded viruses, represented by the densoviruses and the iridoviruses. Baculoviruses, densoviruses, and iridoviruses are DNA viruses, while cypoviruses are the major group of RNA viruses reported from mosquitoes. Research on mosquito pathogenic viruses has been limited, in part, due to the inability to effectively transmit them to the larval mosquito host. Recently, there have been tremendous advancements in the ability to transmit mosquito baculoviruses and cypoviruses with the finding that transmission is mediated by divalent cations. Oral transmission of both baculoviruses and cypoviruses to mosquito larvae is enhanced by magnesium and inhibited by calcium ions. The current status of transmission for each of the major groups is reviewed with emphasis on the common role of divalent cations in transmission of the distantly related baculoviruses and cypoviruses.

A novel cypovirus has been isolated from the mosquito Uranotaenia sapphirina (UsCPV) and shown to cause a chronic infection confined to the cytoplasm of epithelial cells of the gastric ceca and posterior stomach. The production of large numbers of virions and inclusion bodies and their arrangement into paracrystalline arrays gives the gut of infected insects a distinctive blue iridescence. The virions, which were examined by electron microscopy, are icosahedral (55 to 65 nm in diameter) with a central core that is surrounded by a single capsid layer. They are usually packaged individually within cubic inclusion bodies (polyhedra, approximately 100 nm across), although two to eight virus particles were sometimes occluded together. The virus was experimentally transmitted per os to several mosquito species. The transmission rate was enhanced by the presence of magnesium ions but was inhibited by calcium ions. Most of the infected larvae survived to adulthood, and the adults retained the infection. Electrophoretic analysis of the UsCPV genome segments (using 1% agarose gels) generated a migration pattern (electropherotype) that is different from those of the 16 Cypovirus species already recognized. UsCPV genome segment 10 (Seg-10) showed no significant nucleotide sequence similarity to the corresponding segment of the other cypoviruses that have previously been analyzed, and it has different "conserved" termini. A BLAST search of the UsCPV deduced amino acid sequence also showed little similarity to Antheraea mylitta CPV-4 (67 of 290 [23%]) or Choristoneura fumiferana CPV-16 (33 of 111 [29%]). We conclude that UsCPV should be recognized as a member of a new Cypovirus species (Cypovirus 17, strain UsCPV-17).

Microsporidia in mosquitoes can be divided into two categories based on their life cycles and host-parasite relationships. Some species of microsporidia exhibit simple life cycles with one spore type responsible for oral (horizontal) transmission. They affect only one generation of the mosquito and are not usually host or tissue specific. Brachiola algerae and Vavraia culicis are examples of species isolated from mosquitoes with relatively straightforward life cycles (one spore type) and simple host-parasite relationships. B. algerae and a close relative of V. culicis have also been isolated from a vertebrate (human) host but sources for these infections are unknown. In contrast to B. algerae and V. culicis, polymorphic (heterosporous) microsporidia in mosquitoes are characterized by complex life cycles involving multiple spore types responsible for horizontal and vertical transmission. They affect two generations of the mosquito and some involve an obligate intermediate host. These microsporidia are generally very host and tissue specific with complex developmental sequences comprised of unique stages and events. The microsporidium Edhazardia aedis is a pathogen of Aedes aegypti and does not require an intermediate host. The developmental cycle of E. aedis is characterized by four sporulation sequences, two in the parental host and two in the filial generation. Recent speculation relative to the source of B. algerae human infection have implicated infected mosquitoes and raised concerns about the safety of mosquito microsporidia in general. The subject of this review is to compare and contrast three species of microsporidia from mosquitoes, two with broad host ranges (B. algerae and V. culicis) and one specific to mosquitoes (E. aedis). This review describes features that distinguish mosquito-parasitic microsporidia with simple life cycles and broad host ranges from truly mosquito-specific microsporidian parasites with complex life cycles.

In this report we present data on biology, gross pathology, ultrastructure, and host range studies of a naturally occurring nucleopolyhedrovirus from the mosquito Uranotaenia sapphirina (UrsaNPV). Development of this virus was restricted to nuclei of epithelial cells in posterior midgut and distal gastric caecum. Occlusion bodies contained numerous singly enveloped rod-shaped virions. Early occlusion bodies were irregularly shaped and seemed to subsequently coalesce to form larger polyhedra. Mature occlusion bodies had a unique dumbbell shape, and lacked a polyhedron envelope and crystalline structure. Developmental and structural features of UrsaNPV were generally similar to other mosquito NPVs, with major differences in occlusion body shape and size. Transmission tests showed that only members of Uranotaenia (Ur. sapphirina and Ur. lowii) were susceptible to this virus. Transmission was facilitated by magnesium. Field collected Ur. sapphirina larvae had a relatively high rate of dual infections with UrsaNPV and UrsaCPV (cypovirus).

Amblyospora species and other aquatic Microsporidia were isolated from mosquitoes, black flies, and copepods and the small subunit ribosomal RNA gene was sequenced. Comparative phylogenetic analysis showed a correspondence between the mosquito host genera and their Amblyspora parasite species. There is a clade of Amblyospora species that infect the Culex host group and a clade of Amblyospora that infect the Aedes/Ochlerotatus group of mosquitoes. Parathelohania species, which infect Anopheles mosquitoes, may be the sister group to the Amblyospora in the same way that the Anopheles mosquitoes are thought to be the sister group to the Culex and Aedes mosquitoes. In addition, by sequence analysis of small subunit rDNA from spores, we identified the alternate copepod host for four species of Amblyospora. Amblyospora species are specific for their primary (mosquito) host and each of these mosquito species serves as host for only one Amblyospora species. On the other hand, a single species of copepod can serve as an intermediate host to several Amblyospora species and some Amblyospora species may be found in more than one copepod host. Intrapredatorus barri, a species within a monotypic genus with Amblyospora-like characteristics, falls well within the Amblyospora clade. The genera Edhazardia and Culicospora, which do not have functional meiospores and do not require an intermediate host, but which do have a lanceolate spore type which is ultrastructurally very similar to the Amblyospora spore type found in the copepod, cluster among the Amblyospora species. In the future, the genus Amblyospora may be redefined to include species without obligate intermediate hosts. Hazardia, Berwaldia, Larssonia, Trichotuzetia, and Gurleya are members of a sister group to the Amblyospora clades infecting mosquitoes, and may be representatives of a large group of aquatic parasites.

A natural population of Psorophora ferox (Humbold, 1820) infected with the microsporidium Amblyospora ferocis Garcia et Becnel, 1994 was sampled weekly during a seven-month survey in Punta Lara, Buenos Aires Province, Argentina. The sequence of development of A. ferocis in larvae of P. ferox leading to the formation of meiospores followed the developmental pathway previously reported for various species of Amblyospora. The natural prevalence of A. ferocis in the larval population of P. ferox ranged from 0.4% to 13.8%. Spores were detected in the ovaries of field-collected females of P. ferox and were shown to be responsible for transovarial transmission of A. ferocis to the next generation of mosquito larvae in laboratory tests. These spores were binucleate and slightly pyriform in shape. The prevalence of A. ferocis in the adult population ranged from 2.7% to 13.9%. Data on effects of the infection on female fecundity showed that infected field-collected adults of P. ferox laid an average of 47.6 +/- 6.5 eggs of which 35.8% +/- 4.1% hatched. Uninfected field-collected adults of P. ferox laid 82.8 +/- 6.8 eggs of which 64.1% +/- 5.5% hatched. Six species of copepods living together with P. ferox were fed meiospores from field-infected larvae but none became infected. Horizontal transmission of A. ferocis to P. ferox larvae remains unknown.

The infectivity and pathogenicity of newly discovered baculovirus, CuniNPV (family Baculoviridae, genus Nucleopolyhedrovirus) originally isolated from the mosquito Culex nigripalpus Theobald, was evaluated in laboratory bioassys against thirteen species and four genera of mosquitoes native to the northeastern U.S. Purified virus at a dosage rate of 1.6 x 10(7) occlusion bodies/ml with 10 mM Mg2+ added was used in exposures with second through fourth instars at temperatures ranging from 17 to 27 degrees C. High infection rates and accompanying mortality were achieved in Cx. pipiens L. (83.0-14.4%), Cx. pipiens f. molestus (80.4% infection), and Cx. salinarius Coquillett (48.0-43.1%). Cx. restuans Theobald was also susceptible but infection rates were lower (21.3-12.5%). The gross pathology associated with infection was identical to that reported in Cx. nigripalpus. Infected larvae were lethargic and were often suspended at the water surface. Development of CuniNPV was observed in the nuclei of the midgut epitheial cells in the gastric caeca and posterior region of the stomach of host larvae. One hundred percent mortality was observed in all larvae that exhibited gross symptoms of infection within 4-d p.i. Cx. territans Walker (subgenus Neoculex Dyar) was the only Culex mosquito that was not susceptible. No infections were obtained with any species of Aedes [Ae. vexans (Meigen)], Culiseta [Culiseta morsitans (Theobald)] or Ochlerotatus [Ochlerotatus canadensis (Theobald), Oc. cantator (Coquillett), Oc. communis (De Geer), Oc. excrucians (Walker), Oc. japonicus (Theobald), Ochlerotatus stimulans (Walker), and Ochlerotatus triseriatus (Coquillett)]. The host range of CuniNPV appears to be restricted to Culex mosquitoes within the subgenus Culex. An inhibitory effect on transmission of CuniNPV was observed when a liver powder/Brewer's yeast mixture was used as a source of food reinforcing the critical role of Mg2+ and sensitivity of the infection process to the presence other divalent cations (Cu2+, Fe2+, and Zn2+) in the larval medium that interfered with the infection process. The high infectivity and pathogenicity of CuniNPV for the principal vectors of West Nile virus in North America make CuniNPV an attractive candidate for future development as a biopesticide.

Edhazardia aedis was transmitted horizontally to its natural host, Aedes aegypti, and to 6 alternate hosts: Ae. albopictus, Ae. triseriatus, Ae. taeniorhynchus, Anopheles quadrimaculatus, Orthopodomyia signifera and Toxorhynchites rutilus rutilus. The microsporidium produced both binucleate and uninucleate spores in all susceptible hosts. Transovarial transmission, however, was only successful in Ae. aegypti. Therefore, while E. aedis can infect a variety of mosquito species from diverse genera, it is specific for its natural host, Ae. aegypti. Five other mosquito species were not susceptible to E. aedis.

The susceptibility of common nontarget aquatic organisms to the microsporidium Edhazardia aedis was investigated in the laboratory. Eight predacious species along with 9 scavengers and filter feeders were tested. The nontarget organisms were not susceptible to infection by E. aedis and there was no appreciable mortality. To measure the relative safety of E. aedis to nontarget organisms, a simple mathematical expression was employed where risk is defined as the product of the probability of exposure and the result of exposure (infection) expressed as P(e)P(i). In these laboratory tests, the probability of exposure was fixed at 1 (maximum challenge) and the probability of infection was determined to be 0. Therefore, the risk associated with release of E. aedis into the environment is considered to be negligible under these conditions. The true risk for nontarget organisms to E. aedis can only be determined by careful evaluation of controlled field studies in the natural habitat of the target host.