The fungal matrices of <Emphasis Type="Italic">Ophiostoma ips</Emphasis> hinder movement of the biocontrol nematode agent, <Emphasis Type="Italic">Deladenus siricidicola</Emphasis>, disrupting management of the woodwasp, <Emphasis Type="Italic">Sirex noctilio</Emphasis>

Deladenus (=?Beddingia) siricidicola (Tylenchida: Neotylenchidae) is the most effective biocontrol agent used against the invasive wood wasp, Sirex noctilio (Fabricius) (Hymenoptera: Siricidae). The nematodes feed and reproduce on the wood-inhabiting fungus, Amylostereum areolatum (Chaillet ex Fr.) Boidin (Russulales: Amylostereaceae) and parasitise larvae of S. noctilio. In the nematode biocontrol program, the nematodes are inoculated into herbicide-weakened ‘trap trees’. Recent declines in nematode parasitism of S. noctilio in Australia have coincided with an increased incidence of an exotic bark beetle, Ips grandicollis (Eichhoff) (Coleoptera: Curculionidae), attacking trap trees and vectoring a wood-inhabiting fungus, Ophiostoma ips (Rumbold) Nannfelt (Ophiostomatales: Ophiostomataceae), which may inhibit migration of the nematode within the tree to the detriment of S. noctilio biocontrol. Several in vitro and in vivo experiments were conducted to investigate the effect of fungal interactions on the ability of D. siricidicola to locate and reproduce on A. areolatum. Deladenus siricidicola showed preference to A. areolatum in the presence and absence of O. ips, but the presence of O. ips negatively affected the choice response and the number of eggs laid by the nematodes. Deladenus siricidicola was unable to survive and reproduce on O. ips. Results give a clearer understanding of the choice response of D. siricidicola in I. grandicollis infested trees, explaining the disruptive impact of bark beetles on biocontrol of S. noctilio, an effect that could extend from Australia to other important pine growing countries.     

Horizontal transmission of a parasitic nematode from a non-native to a native woodwasp?

In eastern North America, the exotic invasive woodwasp, Sirex noctilio, attacks pines (Pinus spp.) and often shares larval habitat with the native woodwasp, Sirex nigricornis. The parasitic nematode, Deladenus siricidicola, has been used widely in the southern hemisphere as a biological control agent because it sterilizes female S. noctilio. This nematode was introduced accidentally to North America along with S. noctilio. Historical reports indicate nematode-woodwasp fidelity: the parasitic nematode, D. siricidicola, exclusively infects S. noctilio, and the native nematode, Deladenus proximus, exclusively infects S. nigricornis. From two sites in southern Ontario, separated by 225 km, we collected woodwasps from three Pinus sylvestris, and identified the nematode species present in the abdomens of infected wasps. Both wasp species co-occurred in all three trees. D. siricidicola was present in the haemocoel, but not inside the eggs, of infected S. noctilio and S. nigricornis. This evidence suggests horizontal transmission of D. siricidicola likely occurred from S. noctilio to S. nigricornis.     

Putative source of the invasive Sirex noctilio fungal symbiont, Amylostereum areolatum, in the eastern United States and its association with native siricid woodwasps

Two genotypes of the fungal symbiont Amylostereum areolatum are associated with the invasive woodwasp Sirex noctilio first found in North America in 2004. S. noctilio is native to Europe but has been introduced to Australasia, South America and Africa where it has caused enormous losses in pine plantations. Based on nucleotide sequence data from the intergenic spacer region (IGS) of the nuclear ribosomal DNA, the A. areolatum genotypes found in North America are most similar to genotypes found in Europe, and not to genotypes from the southern hemisphere. Although two IGS strains of A. areolatum were found in North America it cannot be stated whether A. areolatum was introduced to North America from Europe once or twice based on our study. Genetic groupings formed by sequencing data were in most cases supported by vegetative compatibility groups (VCGs). Other siricid woodwasp species in the genus Sirex are native to North America. The North American native Sirex edwardsii emerging from the same tree as S. noctilio carried the same strain of A. areolatum as S. noctilio. The North American native Sirex sp. ‘nitidus’ collected outside the geographical range of S. noctilio carried a unique strain within A. areolatum. Our findings of A. areolatum in the native North American species, S. sp. ‘nitidus’, contrast with the previous view that A. areolatum was not present in North America before the accidental invasion of S. noctilio.     

Effect of temperature on woodwasp (Sirex noctilio F.) development and parasitism by the entomopathogenic nematode,Deladenus siricidicola

The woodwasp, Sirex noctilio, is a significant global pest of exotic pine plantations in the Southern Hemisphere and now threatens native pine forests in North America. Management in Australia relies on biocontrol using the nematode, Deladenus (= Beddingia) siricidicola (Bedding), which infects and sterilises females who then further disperse the nematode. This pest is spreading into warmer regions in Australia and South America and coupled with the threat of global climate change, there is uncertainty as to how increasing temperatures will affect the biocontrol program. S. noctilio within nematode-inoculated wood were reared at four temperatures (24, 25.3, 26.6 and 28 °C) to investigate the effects of elevated temperatures on wasp development (emergence time, sex ratio and size), development of eggs (number, size, and maturation) and infection by the nematode. At 24 °C, which reflects current field temperature, S. noctilio were bigger in size and all the eggs were normal and all were infected with nematodes. Modest rises in temperature reflecting climate change scenarios resulted in smaller sized S. noctilio, disrupted egg development and maturation, and lowered the nematode sterilisation rate in females. Reduced S. noctilio female body size and egg infection will likely compromise biocontrol by D. siricidicola in its current distribution, but disrupted egg development may act directly on the pest, limiting dispersal of S. noctilio into subtropical pine plantations and adaptation to climate change.     

Influence of Nematode Parasitism,Body Size,Temperature, and Diel Period on the Flight Capacity of <Emphasis Type="Italic">Sirex noctilio</Emphasis> F. (Hymenoptera: Siricidae)

Sirex noctilio F. (Hymenoptera: Siricidae) is a woodwasp of pine trees that has recently invaded and established in North American forests. Although S. noctilio has had a limited impact in North America to date, there is some concern that it could have a significant impact on pine plantations, especially in the southeastern U.S.A. Moreover, there are few data on the flight capacity of male S. noctilio. We found no association between parasitism by D. siricidicola and whether or not S. noctilio initiated flight on the flight mill. Male wasps that were parasitized by nematodes were heavier than non-parasitized males, but there was no significant difference in mass between parasitized and non-parasitized females. We also examined the flight capacity of male and female S. noctilio in relation to nematode parasitism, body mass, temperature (for only males), and diel period. Body mass, temperature, and diel period affected flight in S. noctilio such that wasps were generally observed to fly faster, farther, and more frequently if they were heavier, flying at warmer temperatures, and flying during the photoperiod. The fact that nematode-parasitized male wasps were found to fly farther than the non-parasitized males is consistent with the hypothesis that nematode parasitism does not negatively affect the flight capacity of S. noctilio.     

A double-edged sword: Amylostereum areolatum odors attract both Sirex noctilio (Hymenoptera: Siricidae) and its parasitoid,Ibalia leucospoides

The Eurasian woodwasp, Sirex noctilio, is an invasive pest of pines in temperate regions of the Southern Hemisphere, and has been introduced to North America. A parasitoid, Ibalia leucospoides, has been broadly employed for biological control of this pest. Volatiles emitted from the fungal symbiont of S. noctilio, Amylostereum areolatum, are reliable cues for S. noctilio and I. leucospoides females to optimize their foraging behavior (host location and host habitat finding) in a chemically complex environment. The headspace volatiles of A. areolatum, were analyzed using coupled gas chromatography-mass spectrometry (GC-MS) and GC-electroantennographic detection (GC-EAD) for both S. noctilio and I. leucospoides females. Analyses revealed that both species could detect several fungal volatiles. In olfactometer bioassays, S. noctilio females were attracted to a 4-component blend of 6-methyl-5-hepten-2-one, trans-3-hexenyl acetate, linalool, and geraniol, while the addition of ρ-anisaldehyde to the blend was necessary for attraction of I. leucospoides females. The results of trap catches in field experiments confirmed that these fungal volatiles in combination with host tree volatiles are attractive to both species, although the release rate of the fungal volatiles is important. These volatiles can serve as a basis for the development of improved lures for both species.     

Lack of fidelity revealed in an insect–fungal mutualism after invasion

Symbiont fidelity is an important mechanism in the evolution and stability of mutualisms. Strict fidelity has been assumed for the obligate mutualism between Sirex woodwasps and their mutualistic Amylostereum fungi. This assumption has been challenged in North America where the European woodwasp, Sirex noctilio, and its fungal mutualist, Amylostereum areolatum, have recently been introduced. We investigate the specificity of the mutualism between Sirex and Amylostereum species in Canada, where S. noctilio co-infests Pinus with native Sirex nigricornis and its mutualist, Amylostereum chailletii. Using phylogenetic and culture methods, we show that extensive, reciprocal exchange of fungal species and strains is occurring, with 75.3 per cent of S. nigricornis carrying A. areolatum and 3.5 per cent of S. noctilio carrying A. chailletii. These findings show that the apparent specificity of the mutualism between Sirex spp. and their associated Amylostereum spp. is not the result of specific biological mechanisms that maintain symbiont fidelity. Rather, partner switching may be common when shifting geographical distributions driven by ecological or anthropogenic forces bring host and mutualist pairs into sympatry. Such novel associations have potentially profound consequences for fitness and virulence. Symbiont sharing, if it occurs commonly, may represent an important but overlooked mechanism of community change linked to biological invasions.     

Biochemistry of Anhydrobiosis in Beddingia siricidicola,a Biological Control Agent of Sirex noctilio

Proto-anhydrobiosis of the nematode, Beddingia siricidicola, was achieved by incubation in polyethylene glycol or various concentrations up to 4 M of glycerol. The associated changes in the levels of glycerol, unbound proline, trehalose, lipids, and glycogen were determined by alkylation strategies, followed by gas chromatography or gas chromatography/mass spectrometry. The level of glycerol reached 8.9% of dry weight, proline 2.4% of dry weight, and trehalose 8.0% of dry weight within B. siricidicola that were incubated in 1.5 M glycerol over 6 d, while glycerol reached 17.9% of dry weight after incubation for the same period in 4 M glycerol. Movement was thereby reduced but the nematodes from 1.5 M glycerol revived after a few minutes upon rehydrating and they were able to avoid osmotic damage by rapidly excreting the glycerol, much of it being expelled within the first hour. The potential for storage and transport of this nematode for the biological control of the pine-killing wasp, Sirex noctilio, was greatly improved when nematode suspensions were maintained in 1.5 M glycerol under refrigeration.     

Fidelity Among Sirex Woodwasps and Their Fungal Symbionts

We report that associations between mutualistic fungi and their economically and ecologically important woodwasp hosts are not always specific as was previously assumed. Woodwasps in the genus Sirex engage in obligate nutritional ectosymbioses with two species of Amylostereum, a homobasid\iomycete genus of white rot fungi. In the present study, the Amylostereum species and genotypes associated with three species of Sirex native to eastern North America and one relatively recent invasive Sirex from Europe were investigated by comparing intergenic spacer regions (IGS). Sirex spp. were sampled over 6 years from 23 sites in six US states, ranging from Maine in the northeast to Louisiana in the southeast, to obtain samples of Amylostereum from mycangia of adult females. Two of the native Sirex species (Sirex nigricornis and Sirex nitidus) were associated with either Amylostereum chailletii or Amylostereum areolatum, refuting the hypothesis of strict species-specific relationships. However, the invasive Sirex noctilio and the native Sirex cyaneus were each collected with only A. areolatum or A. chailletii, respectively, although S. noctilio was associated with two different IGS genotypes of A. areolatum and S. cyaneus occurs sympatrically with the other native Sirex. In Pinus, the preferred host tree of S. nigricornis and S. noctilio, these species co-occurred in 25.9 % of trees sampled, and horizontal transmission of fungal strains from S. noctilio to S. nigricornis was documented, although only in one tree. The extent that further spread and establishment of S. noctilio will alter the composition of symbionts carried by native Sirex is unknown but will depend in part on the degree of flexibility in these host–symbiont associations.     

Symbionts mediate oviposition behaviour in invasive and native woodwasps

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A comparison of control results for the alien invasive woodwasp,Sirex noctilio,in the southern hemisphere

1 Sirex noctilio has resulted in one of the most damaging biological invasions of pine forestry in the southern hemisphere. 2 An intensive, integrated control programme has been developed for this pest and is generally considered very successful. However, a critical comparison of S. noctilio infestations and control efforts throughout the southern hemisphere reveals that control has not been uniformly effective. Of particular concern is the apparently unexplained variability in establishment and success of biological control agents, including various parasitic wasps and the parasitic nematode Deladenus siricidicola. 3 An overlooked aspect in the establishment of the biological control interventions for S. noctilio is the potential influence of genetic bottlenecks on the ability of the agents to adapt to different environments and different populations of S. noctilio. 4 Despite established biological control agents, stress in forests caused by silvicultural practices or the environment will predispose trees to heavy infestation. Unfortunately, improved silvicultural practices are not always economically feasible and environmental changes are often unavoidable. 5 Sirex noctilio continues to pose a serious threat to pine forestry in many areas. Despite extensive earlier research into a single integrated control for S. noctilio, it is important to recognize that such a strategy will probably require significant local adaptation in new areas of infestation and over time.     

Multiple introductions of <Emphasis Type="Italic">Sirex noctilio</Emphasis> (Hymenoptera: Siricidae) in northeastern North America based on microsatellite genotypes,and implications for biological control

The invasive woodwasp Sirex noctilio (Hymenoptera: Siricidae) has been moved from Eurasia into regions in the Southern Hemisphere, where extensive tree mortality has occurred in pines (Pinus spp.) introduced for forestry. More recently this woodwasp was found in northeastern North America, where pines are native, and it is a species of concern due to the economic importance of pines. Understanding the genetic diversity of North American S. noctilio points to new areas of inquiry, particularly regarding the ability of parasitic nematodes to sterilize woodwasps, which could provide control methods in the US and/or Canada. We investigated the genetic diversity of 924 S. noctilio from nine populations from New York and Pennsylvania (US), Ontario (CA), and Queensland (AU) using nine microsatellite loci. To avoid inflating the number of populations estimated by Bayesian inference, we measured the full-sibling relationships of woodwasps within 13 trees and removed all but one member of each full-sib family from the genetic analysis, resulting in a final sample size of 741 S. noctilio. Within a tree, on average 39% of woodwasps did not have a full sibling, and there were 5.6 families with at least two full-sibling members per tree. The mean family size across trees was 1.9 when single offspring (i.e., no full siblings) were included. Given the short time span since invasion, variation within North American S. noctilio is likely due to differences among founding genotypes. Genetic analyses support the hypothesis that at least two separate introductions occurred. Within North America, genetic distance measures were greatest between a site in southwestern Ontario and all other sites, suggesting that this population could represent a separate introduction event. Two methods of Bayesian clustering also support this idea; they detected 4 or 5 distinct genetic clusters with little admixture between the southwestern Ontario site and other North American populations. The wasps from Australia, where biological control with nematodes has been successful, showed low genetic diversity and clustered with the southwestern Ontario population in one out of two Bayesian analyses. Within the Ontario subset of samples, high woodwasp activity level (i.e., attack and mortality of trees) was associated with one genetic cluster more strongly than another. Population variation should be taken into account in studies of S. noctilio spread and management within North America.     

Rhizosphere Colonization and Control of Meloidogyne spp. by Nematode-trapping Fungi

The ability of nematode-trapping fungi to colonize the rhizosphere of crop plants has been suggested to be an important factor in biological control of root-infecting nematodes. In this study, rhizosphere colonization was evaluated for 38 isolates of nematode-trapping fungi representing 11 species. In an initial screen, Arthrobotrys dactyloides, A. superba, and Monacrosporium ellipsosporum were most frequently detected in the tomato rhizosphere. In subsequent pot experiments these fungi and the non-root colonizing M. geophyropagum were introduced to soil in a sodium alginate matrix, and further tested both for establishment in the tomato rhizosphere and suppression of root-knot nematodes. The knob-forming M. ellipsosporum showed a high capacity to colonize the rhizosphere both in the initial screen and the pot experiments, with more than twice as many fungal propagules in the rhizosphere as in the root-free soil. However, neither this fungus nor the other nematode-trapping fungi tested reduced nematode damage to tomato plants.     

Behavioural response of the woodwasp Sirex noctilio to volatile emissions of its fungal symbiont

The wood‐boring wasp, Sirex noctilio, is a global invasive pest that infects and kills pine trees by inoculating spores of a symbiotic fungus (Amylostereum areolatum) at oviposition. Wasp larvae depend on fungal growth to feed, while the fungus relies on female wasps to initially condition the pine tree by inoculating a phytotoxic venom and for dispersal. Wasp larvae use the fungus as an external gut for the digestion of lignocellulosic compounds resulting in a strong correlation between fungal growth inside the wood and wasp fitness. This study explores the hypothesis that female wasps will use fungal volatiles as a synomone in the process of locating suitable oviposition areas (i.e. trees). Using a Y‐tube olfactometer, adult female wasp behaviour was assessed towards fungal and pine tree volatiles (i.e. positive control). Our results are the first to demonstrate attraction of female S. noctilio towards volatiles of their fungal symbiont. Furthermore, the positive response towards these volatiles seems to be stronger than the response to a known attractant (i.e. pine volatiles). These results could be an important contribution to improving baits for monitoring and control purposes. Further work is needed, mainly oriented towards the identification of the volatiles that trigger the observed attraction response and their possible synergistic effects with tree volatiles.     

Pathology,distribution, morphological and genetic identity of Deladenus proximus (Tylenchida: Neotylenchidae) a parasitic nematode of the woodwasp,Sirex nigricornis in the eastern United States

The woodwasp, Sirex nigricornis (Hymenoptera: Siricidae), is solitary and utilizes a symbiotic fungus to extract nourishment from pine trees to feed its larvae. The woodwasp has a brief adult life, but the larvae develop for 1–3 years in the tree xylem. Infections with the nematode Deladenus proximus have been documented in the native woodwasp, S. nigricornis in the eastern United States and Canada. These nematodes appear to sterilize female woodwasps; however, the extent of the pathology and other aspects of the biology of D. proximus remain unknown. In this study we examined the effects of D. proximus on S. nigricornis using fresh – not preserved – specimens. Between 2009 and 2012, a total of 1639 woodwasps were examined for internal nematodes from emerging sites in Illinois, Louisiana and South Carolina. From this total, only 112 individuals were infected with the nematode D. proximus, with varying prevalence across localities and years. Nematodes were found inside every egg of infected females, as well as the hemocoel and the mycangia. Morphometric analyses of mycetophagous reared adult nematodes suggest that a single species is present in localities from Arkansas, Illinois, Louisiana, New York and South Carolina. The screening of the mitochondrial cytochrome oxidase subunit 1 (cox1) of these organisms is consistent with this pattern in that all of these individuals belong to a single clade. Deladenus proximus appears to be an efficient sterilizer, yet its prevalence is relatively low. Experimental infections of the invasive Sirex noctilio are recommended to test the viability of using this nematode as a biological control agent.     

Factors influencing parasitism of Sirex noctilio (Hymenoptera: Siricidae) by the nematode Deladenus siricidicola (Nematoda: Neotylenchidae) in summer rainfall areas of South Africa

Control of the invasive wasp, Sirex noctilio Fabricius using the parasitic nematode Deladenus siricidicola Bedding is a well known example of a successful classical biological control program. Despite its wide-scale success, this control method has recently had poor success in the summer rainfall areas of South Africa. Data from previous studies showed variation in nematode parasitism from inoculated trees (inoculation success) between different tree sections and among inoculation times. They also pointed to moisture content of the wood or virulence of the nematode as the most likely underlying factors influencing variations in inoculation success. The results from our study showed that the highest levels of parasitism was obtained from early inoculations and from the bottom sections of trees, where moisture content of the wood was highest, supporting the hypothesis that moisture content influences parasitism. However, even when moisture content was adequate, average inoculation success remained below 25% and was often 0%, suggesting that there are other barriers to inoculation success. Different sources from which the nematodes were produced did not influence inoculation success, indicating that nematode virulence is most likely not the cause of the low success. Another interesting finding was that parasitized wasps were larger than unparasitized wasps. Background parasitism was present despite the poor success with past inoculations, but the data also suggest that the natural build-up of this population could be constrained by the same factors that influence inoculations.     

Suppression of Rotylenchulus reniformis on Cotton by the Nematophagous Fungus ARF

The reniform nematode, Rotylenchulus reniformis Linford &Oliveira, has become a serious threat to cotton (Gossypium hirsutum L.) production in the United States during the past decade. The objective of this study is to isolate fungi from eggs of R. reniformis and select potential biological control agents for R. reniformis on cotton. Soil samples were collected from cotton fields located in Jefferson County, Arkansas. Eight genera of fungi were included in the 128 fungal isolates obtained, and among them were five strains of the nematophagous fungus ARF. The mtDNA RFLP pattern, colony growth characteristics, and pathogenicity indicate the five ARF isolates represent one described strain and one new strain. Light and electron microscopic observations suggest ARF is an active parasite of R. reniformis, with parasitism ranging from 48% to 79% in in vitro tests. Three greenhouse experiments demonstrated ARF successfully suppressed the number of reniform nematodes during the first and second generation of the nematode. Reductions in numbers of R. reniformis on the roots for the seven application rates of 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% ARF were 87%, 92%, 94%, 96%, 97%, 98%, and and 98%, respectively.     

Do Native Insects and Associated Fungi Limit Non-Native Woodwasp,Sirex noctilio,Survival in a Newly Invaded Environment?

Sirex noctilio F. (Hymenoptera: Siricidae) is an introduced pest of pines (Pinus spp.) in several countries in the Southern Hemisphere. Although S. noctilio is established in North America (first discovered in 2004), it has not been a destructive pest there so far, where forest communities more closely resemble those in its native Eurasian range—where it is not a pest. To investigate the influence of the existing community of associated insects (competitors + natural enemies) and fungi (vectored by insects) on S. noctilio survival in North America, we examined stage-specific mortality factors and their relative importance, generating life tables drawn from experimentally-manipulated and natural cohorts of Sirex spp. (mostly S. noctilio, but some native S. nigricornis F.). For both natural and experimentally-manipulated cohorts, factors which acted during the earliest Sirex life stages, most likely tree resistance and/or competition among fungal associates, were paramount in dictating woodwasp survival. Experimentally-manipulated life tables revealed that protection from the community of associates resulted in a significantly, and substantially larger (>15x) S. noctilio F1 generation than exposure to it. Seventy percent of generation mortality in the exposed cohort was due to tree resistance or unknown causes early in larval development, which could have included competition among other bark- or wood-inhabiting insects and/or their fungal associates. Only 46% of generation mortality in the protected cohort was due to tree resistance and/or unknown causes. Parasitoids, particularly endoparasitoids (Ibalia spp.), showed limited ability to control S. noctilio, and reduced the experimentally-established cohort by only 11%, and natural cohorts an average of 3.4%. The relative importance of tree resistance vs. competition with bark- and wood-borers in reducing S. noctilio survival remains unclear. Tree resistance and/or competition likely contribute more than natural enemies in maintaining the S. noctilio population in North America below damaging levels.     

Effect of Two Bark Beetle-Vectored Fungi on the On-Host Search and Oviposition Behavior of the Introduced Woodwasp Sirex noctilio (Hymenoptera: Siricidae) on Pinus sylvestris Trees and Logs

Sirex noctilio’s fungal symbiont, Amylostereum areolatum, is required for its offspring’s development. The symbiont is a weak competitor with bark beetle-vectored fungi so it would be beneficial to the woodwasp if it avoided ovipositing in substrate colonized by these competitors. The response of S. noctilio to the presence of two beetle-vectored fungi, Leptographium wingfieldii and Ophiostoma minus, inoculated into living trees, and to L. wingfieldii and A. areolatum inoculated into cut logs was investigated. The wasp avoided areas with L. wingfieldii; there were fewer signs of oviposition activity and drilling in these zones. There was no significant response to O. minus or A. areolatum. Female woodwasps can detect the presence of some fungi and make choices about oviposition sites that benefit their offspring.     

Tripius gyraloura n. sp. (Aphelenchoidea: Sphaerulariidae) parasitic in the gall midge Lasioptera donacis Coutin (Diptera: Cecidomyiidae)

A new nematode, Tripius gyraloura n. sp., is described from the arundo gall midge, Lasioptera donacis Coutin (Diptera: Cecidomyiidae). This gall midge is being considered as a biological control agent for use in North America against the introduced giant reed Arundo donax (L.) (Poaceae: Cyperales). Thus the present study was initiated to investigate a nematode parasite that was unknown at the time studies with L. donacis were initiated. The new species has a rapid development in the fly host and the mature parasitic female nematodes evert their uterine cells in the hosts’ hemolymph. Because large numbers of nematodes sterilise the host, eradication of the parasite from laboratory colonies of the midge may be necessary before populations of the fly are released.