Introgression of Ivermectin Resistance Genes into a Susceptible Haemonchus contortus Strain by Multiple Backcrossing

Anthelmintic drug resistance in livestock parasites is already widespread and in recent years there has been an increasing level of anthelmintic drug selection pressure applied to parasitic nematode populations in humans leading to concerns regarding the emergence of resistance. However, most parasitic nematodes, particularly those of humans, are difficult experimental subjects making mechanistic studies of drug resistance extremely difficult. The small ruminant parasitic nematode Haemonchus contortus is a more amenable model system to study many aspects of parasite biology and investigate the basic mechanisms and genetics of anthelmintic drug resistance. Here we report the successful introgression of ivermectin resistance genes from two independent ivermectin resistant strains, MHco4(WRS) and MHco10(CAVR), into the susceptible genome reference strain MHco3(ISE) using a backcrossing approach. A panel of microsatellite markers were used to monitor the procedure. We demonstrated that after four rounds of backcrossing, worms that were phenotypically resistant to ivermectin had a similar genetic background to the susceptible reference strain based on the bulk genotyping with 18 microsatellite loci and individual genotyping with a sub-panel of 9 microsatellite loci. In addition, a single marker, Hcms8a20, showed evidence of genetic linkage to an ivermectin resistance-conferring locus providing a starting point for more detailed studies of this genomic region to identify the causal mutation(s). This work presents a novel genetic approach to study anthelmintic resistance and provides a “proof-of-concept” of the use of forward genetics in an important model strongylid parasite of relevance to human hookworms. The resulting strains provide valuable resources for candidate gene studies, whole genome approaches and for further genetic analysis to identify ivermectin resistance loci.     

Reproductive barriers between populations of the cereal rust mite <Emphasis Type="Italic">Abacarus hystrix</Emphasis> confirm their host specialization

Recent studies have shown that certain host populations of the cereal rust mite Abacarus hystrix are highly specialized in their host use and it is likely that reproductive isolation exists between them. Here I verified this expectation by testing for reproductive barriers between ryegrass and quackgrass populations of A. hystrix. I performed reciprocal crosses between individuals from both populations and observed results of crosses. Leaves of the three grass species, ryegrass, quackgrass and wheat, were used as mating arenas. I used two criteria to determine reproductive barriers between strains: the proportion of female progeny and viability of progeny. If studied populations of this haplodiploid species are reproductively isolated a male-biased sex ratio and/or hybrid progeny of reduced viability would be expected. I found that in the presence of quackgrass and ryegrass pre-zygotic barriers between studied populations exists. On wheat I observed asymmetry in reproductive barriers. Between females from quackgrass and males from ryegrass a pre-zygotic barrier existed (only males obtained). However, the opposite reciprocal cross (females from ryegrass and males from quackgrass) produced progeny of both sexes. A male-biased sex ratio and low adult emergence suggests that post-zygotic mechanisms acted here. Low viability of progeny obtained from crosses in which females from ryegrass were engaged suggests that the origin of the female nymph acted as a predictor of hybrid inviability. The pattern of sterility observed in the cereal rust mite indicates that in the presence of three hosts (ryegrass, quackgrass and wheat) pre- and post-zygotic reproductive barriers between quackgrass and ryegrass populations of this mite exist. In addition to host fidelity (which acts as pre-zygotic barrier) there are post-zygotic barriers to gene flow.     

A method for single pair mating in an obligate parasitic nematode

Parasitic nematode species have extremely high levels of genetic diversity, presenting a number of experimental challenges for genomic and genetic work. Consequently, there is a need to develop inbred laboratory strains with reduced levels of polymorphism. The most efficient approach to inbred line development is single pair mating, but this is challenging for obligate parasites where the adult sexual reproductive stages are inside the host, and thus difficult to experimentally manipulate. This paper describes a successful approach to single pair mating of a parasitic nematode, Haemonchus contortus. The method allows for polyandrous mating behaviour and involves the surgical transplantation of a single adult male worm with multiple immature adult females directly into the sheep abomasum. We used a panel of microsatellite markers to monitor and validate the single pair mating crosses and to ensure that the genotypes of progeny and subsequent filial generations were consistent with those expected from a mating between a single female parent of known genotype and a single male parent of unknown genotype. We have established two inbred lines that both show a significant overall reduction in genetic diversity based on microsatellite genotyping and genome-wide single nucleotide polymorphism. There was an approximately 50% reduction in heterozygous SNP sites across the genome in the MHco3.N1 line compared with the MoHco3(ISE) parental strain. The MHco3.N1 inbred line has subsequently been used to provide DNA template for whole genome sequencing of H. contortus. This work provides proof of concept and methodologies for forward genetic analysis of obligate parasitic nematodes.     

Interspecific crossing and genetic mapping reveal intrinsic genomic incompatibility between two Senecio species that form a hybrid zone on Mount Etna,Sicily

Studies of hybridizing species can reveal much about the genetic basis and maintenance of species divergence in the face of gene flow. Here we report a genetic segregation and linkage analysis conducted on F₂ progeny of a reciprocal cross between Senecio aethnensis and S. chrysanthemifolius that form a hybrid zone on Mount Etna, Sicily, aimed at determining the genetic basis of intrinsic hybrid barriers between them. Significant transmission ratio distortion (TRD) was detected at 34 (∼27%) of 127 marker loci located in nine distinct clusters across seven of the ten linkage groups detected, indicating genomic incompatibility between the species. TRD at these loci could not be attributed entirely to post-zygotic selective loss of F₂ individuals that failed to germinate or flower (16.7%). At four loci tests indicated that pre-zygotic events, such as meiotic drive in F₁ parents or gametophytic selection, contributed to TRD. Additional tests revealed that cytonuclear incompatibility contributed to TRD at five loci, Bateson–Dobzhansky–Muller (BDM) incompatibilities involving epistatic interactions between loci contributed to TRD at four loci, and underdominance (heterozygote disadvantage) was a possible cause of TRD at one locus. Major chromosomal rearrangements were probably not a cause of interspecific incompatibility at the scale that could be examined with current map marker density. Intrinsic genomic incompatibility between S. aethnensis and S. chrysanthemifolius revealed by TRD across multiple genomic regions in early-generation hybrids is likely to impact the genetic structure of the natural hybrid zone on Mount Etna by limiting introgression and promoting divergence across the genome.     

Biological evaluation of the systematic validity of the African Argas (Persicargas) Arboreus and the Asian-Australian A. (P.) Robertsi (Ixodoidea: Argasidae)

Khalil G. M., Hoogstraal H. and Oliver J. H. Jr. 1980. Biological evaluation of the systematic validity of the African Argas (Persicargas) arboreus and the Asian-Australian A.(P.)robertsi (Ixodoidea: Argasidae). International journal for Parasitology10: 253–259. The closely related A. (P.)arboreus (Ethiopian Faunal Region) and A. (P.) robertsi (Oriental and Australian Faunal Regions) interbreed readily and produce partially fertile progeny. Intrahybrid crosses are much less productive than P₁ homogamic crosses. Results of backcrosses indicate that the progeny of the robertsi ♂ × arboreus ♀ cross are less fertile than the progeny of the reciprocal cross. The fertility of F₁ hybrid progeny is lower than that of the P₁ pure species as expressed in lower female fecundity, egg hatch, and/or viability of immature stages. These 2 species probably originated from a common ancestor and geographic isolation caused genetic incompatibility.     

Orchis × colemanii hybridization: Molecular and morphological evidence,seed set success,and evolutionary importance

Despite highly specialized pollination strategies, hybridization is a common phenomenon among Mediterranean deceptive orchids. Food-deceptive species sire a progeny of F₁ unfertile plants, which work as a late post-zygotic barrier. Conversely, when pre-zygotic barriers of sexually deceptive (Ophrys) species are absent, the hybrids are fertile and an extensive introgression may occur. Here, we have performed molecular analysis and hand pollination treatments to characterize a hybrid zone of two food-deceptive species, O. mascula and O pauciflora. Hybrids (called O. × colemanii) have shown different amounts of parental nrDNA, strongly supporting that they are F₂ and/or successive hybrid generations. Comparable high levels of reproductive success have been detected in natural conditions and in experimental crosses suggesting the absence of effective reproductive barriers either between hybrids, either between hybrids and parental species. In light of ecological and distributional features of O. × colemanii across its distribution range, we hypothesize that these populations have originated by secondary contact in the periglacial belt of Apennines. Moreover, the rare and localized O. pauciflora could benefit a genetic enrichment by hybridizing with a widespread related species. O. × colemanii is not a dead end population, but may have a role as potential reserve of adaptive variability and is an unusual stage along the speciation process.     

Critical photoperiod and daylength threshold differences between northern and southern populations of the butterfly Limenitis archippus

Laboratory studies show that different photoperiods induce diapause in northern (Vermont) and southern (Maryland) larval strains of the butterfly Limenitis archippus. The northern strain responds to $\text{1}\text{2}\text{hr}$ longer photoperiod thresholds and critical ranges than does the southern one. These responses are correlated with geographic differences in the ambient photoperiod of the two localities. In this facultative diapausing species, third instar larvae construct hibernacula within the basal portions of tubular leaves spun with silk, when daylength approaches either 13·5 hr (Vermont strain) or 13·0 hr (Maryland strain). When reared in total darkness some larvae develop directly to fourth inszar without diapause, although mortality is high. Among both strains different broods exhibit different incidences of diapause. Reciprocal inter-strain hybrids show intermediate diapause responses, suggesting that larval diapause is under the control of multiple genes.     

Reproductive isolating mechanisms contributing to asymmetric hybridization in Killifishes (Fundulus spp.)

When species hybridize, one F1 hybrid cross type often predominates. Such asymmetry can arise from differences in a variety of reproductive barriers, but the relative roles and concordance of pre-mating, post-mating prezygotic, and post-zygotic barriers in producing these biases in natural animal populations have not been widely investigated. Here, we study a population of predominantly F1 hybrids between two killifish species (Fundulus heteroclitus and F. diaphanus) in which >95% of F1 hybrids have F. diaphanus mothers and F. heteroclitus fathers (D♀ × H♂). To determine why F. heteroclitus × F. diaphanus F1 hybrids (H♀ × D♂) are so rare, we tested for asymmetry in pre-mating reproductive barriers (female preference and male aggression) at a common salinity (10 ppt) and post-mating, pre-zygotic (fertilization success) and post-zygotic (embryonic development time and hatching success) reproductive barriers at a range of ecologically relevant salinities (0, 5, 10, and 15 ppt). We found that F. heteroclitus females preferred conspecific males, whereas F. diaphanus females did not, matching the observed cross bias in the wild. Naturally rare H♀ × D♂ crosses also had lower fertilization success than all other cross types, and a lower hatching success than the prevalent D♀ × H♂ crosses at the salinity found in the hybrid zone centre (10 ppt). Furthermore, the naturally predominant D♀ × H♂ crosses had a higher hatching success than F. diaphanus crosses at 10 ppt, which may further increase their relative abundance. The present study suggests that a combination of incomplete mating, post-mating pre-zygotic and post-zygotic reproductive isolating mechanisms act in concert to produce hybrid asymmetry in this system.     

Unilateral incompatibility as a major cause of skewed segregation in the cross between Lycopersicon esculentum and L. pennellii

Skewed segregations are frequent events in segregating populations derived from different interspecific crosses in tomato. To determine a basis for skewed segregations in the progeny of the cross between Lycopersicon esculentum and L. pennellii, monogenic segregations of 16 isozyme loci were analyzed in an F₂ and two backcross populations of this cross. In the F₂, 9 loci mapping to chromosomes 1, 2, 4, 9, 10 and 12 exhibited skewed segregations and in all cases there was an excess of L. pennellii homozygotes. The genotypic frequencies at all but one locus were at Hardy-Weinberg equilibria. In the backcross populations, all except two loci exhibited normal Mendelian segregations. No post-zygotic selection model could statistically or biologically explain the observed segregation patterns in the F₂ and backcross populations. A pre-zygotic selection model, assuming selective elimination of the male gametophytes during pollen function (i.e., from pollination to karyogamy), could adequately explain the observed segregations in all three populations. The direction of the skewed segregations in the F₂ population was consistent with that expected based on the effects of unilateral incompatibility reactions between the two species. In addition, the chromosomal locations of 5 of the 9 markers that exhibited skewed segregations coincided with the locations of several known compatibility-related genes in tomato. Multigenic unilateral incompatibility reactions between L. esculentum pollen and the stigma or style of L. pennellii (or its hybrid derivatives) are suggested to be the major cause of the skewed segregations in the F₂ progeny of this cross.     

The Role of Late-Acting Self-Incompatibility and Early-Acting Inbreeding Depression in Governing Female Fertility in Monkshood,Aconitum kusnezoffii

Reduced seed yields following self-pollination have repeatedly been observed, but the underlying mechanisms remain elusive when self-pollen tubes can readily grow into ovaries, because pre-, post-zygotic late-acting self-incompatibility (LSI), or early-acting inbreeding depression (ID) can induce self-sterility. The main objective of this study was to differentiate these processes in Aconitum kusnezoffii, a plant lacking stigmatic or stylar inhibition of self-pollination. We performed a hand-pollination experiment in a natural population of A. kusnezoffii, compared seed set among five pollination treatments, and evaluated the distribution of seed size and seed set. Embryonic development suggested fertilization following self-pollination. A partial pre-zygotic LSI was suggested to account for the reduced seed set by two lines of evidence. The seed set of chase-pollination treatment significantly exceeded that of self-pollination treatment, and the proportion of unfertilized ovules was the highest following self-pollination. Meanwhile, early-acting ID, rather than post-zygotic LSI, was suggested by the findings that the size of aborted selfed seeds varied continuously and widely; and the selfed seed set both exhibited a continuous distribution and positively correlated with the crossed seed set. These results indicated that the embryos were aborted at different stages due to the expression of many deleterious alleles throughout the genome during seed maturation. No signature of post-zygotic LSI was found. Both partial pre-zygotic LSI and early-acting ID contribute to the reduction in selfed seed set in A. kusnezoffii, with pre-zygotic LSI rejecting part of the self-pollen and early-acting ID aborting part of the self-fertilized seeds.     

Silencing of dominant genes in heterozygous genotypes of interspecific hybrids <Emphasis Type="Italic">Fagopyrum esculentum</Emphasis> Moench. × C2026 <Emphasis Type="Italic">F. homotropicum</Emphasis> Ohnishi

Fagopyrum homotropicum Ohnishi is a very polymorphic self-pollinating species with homostylous flowers, which morphologically different lineages are differ also in ability to hybridize with F. esculentum Moench. (closely related outcrosser with heterostyly). A lineage C2026 F. homotropicum diverged from F. esculentum with forming noticeable pre-zygotic and post-zygotic barriers: the most successful interspecific crossing F. esculentum × C2026 resulted wrinkled hybrid seeds germinated in Petri dishes. These interspecific hybrids and backcrosses F. esculentum × F₁, being heterozygous at the loci DET/det, SHT/sht and a homostyly gene of F. homotropicum, in our experiments often formed phenotype like a recessive homozygote for at least one of these genes, i.e., dominant alleles were silenced. Apparently, these effects can be caused by disorders of epigenetic regulation associated with the divergence of hybridized species. Such disorders, especially those that occur at the stage of seed development, represent one of the main experimentally confirmed mechanisms of pre-zygotic isolation between species. Apparently, F. esculentum and the lineage C2026 of F. homotropicum represent an example of intermediate stage of post-zygotic isolation development process which based on epigenetic deregulation of gene expression in the hybrids. Sometimes it may be revealed not only at the stage of seed development, but also at later stages of ontogenesis.     

Haemonchus contortus and Trichostrongylus colubriformis did not adapt to long-term exposure to sheep that were genetically resistant or susceptible to nematode infections

We tested the hypothesis that Haemonchus contortus and Trichostrongylus colubriformis would adapt to long-term exposure to sheep that were either genetically resistant or susceptible to H. contortus. Sheep genotypes were from lines with 10 years prior selection for low (resistant, R) or high (susceptible, S) faecal worm egg count (WEC) following H. contortus infection. Long-term exposure of H. contortus and T.colubriformis to R or S genotypes was achieved using serial passage for up to 30 nematode generations. Thus, we generated four nematode strains; one strain of each species solely exposed to R sheep and one strain of each species solely exposed to S sheep. Considerable host genotype differences in mean WEC during serial passage confirmed adequate nematode selection pressure for both H. contortus (R 4900 eggs per gram (epg), S 19,900 epg) and T. colubriformis (R 5300 epg, S 13,500 epg). Adaptation of nematode strain to host genotype was tested using seven cross-classified tests for H. contortus, and two cross-classified and one outbred genotype test for T. colubriformis. In the cross-classified design, where each strain infects groups of R, S or randomly bred control sheep, parasite adaptation would be indicated by a significant host genotype by nematode strain interaction for traits indicating parasite reproductive success; specifically WEC and, for H. contortus strains, packed cell volume. We found no significant evidence of parasite adaptation to host genotype (P > 0.05) for either the H. contortus or T. colubriformis strains. Therefore, we argue that nematodes will not adapt quickly to sheep bred for nematode resistance, where selection is based on low WEC, although selecting sheep using a subset of immune functions may increase adaptation risk. Our results support the hypothesis that nematode resistance is determined by many genes each with relatively small effect. In conclusion, selection of sheep for nematode resistance using WEC should be sustainable in the medium to long-term.     

Floral scent in natural hybrids of Ipomopsis (Polemoniaceae) and their parental species

Background and Aims

Floral traits, such as floral volatiles, can contribute to pre-zygotic reproductive isolation by promoting species-specific pollinator foraging. When hybrid zones form, floral traits could also influence post-zygotic isolation. This study examined floral volatiles in parental species and natural hybrids in order to explore potential scent mediation of pre-zygotic and post-zygotic isolation.

Methods

Floral bouquets were analysed for the sister species Ipomopsis aggregata and I. tenuituba and their natural hybrids at two contact sites differing in both hybridization rate and temporal foraging pattern of hawkmoth pollinators. Floral volatiles were quantified in diurnal and nocturnal scent samples using gas chromatography–mass spectrometry.

Key Results

The bouquets of parental species and hybrids showed qualitative overlap. All flowers emitted similar sets of monoterpenoid, sesquiterpenoid, aliphatic and benzenoid compounds, but separated into groups defined by multivariate analysis of quantitative emissions. The parental species differed most strikingly in the nitrogenous compound indole, which was found almost exclusively in nocturnal bouquets of I. tenuituba. Natural hybrid bouquets were highly variable, and showed emission rates of several compounds that appeared transgressive. However, indole emission rates were intermediate in the hybrids compared with rates in the parents. Volatile bouquets at the contact site with lower hybridization did not show greater species specificity in overall scent emission, but I. tenuituba presented a stronger indole signal during peak hawkmoth activity at that site.

Conclusions

The two species of Ipomopsis differed in patterns of floral bouquets, with indole emitted in nocturnal I. tenuituba, but not in I. aggregata. Natural hybrid bouquets were not consistently intermediate between the parents, although hybrids were intermediate in indole emission. The indole signal could potentially serve as a hawkmoth attractant that mediates reproductive isolation both before and after hybrid formation.     

Quantification of Reproductive Isolating Barriers Between Two Naturally Hybridizing Killifish Species

Understanding the relative importance of various reproductive barriers to the early stages of speciation is an essential question in evolutionary biology. The closely related killifishes Fundulus heteroclitus and F. grandis occasionally hybridize in a small region in coastal Northeastern Florida showing that while barriers to reproduction exist, they are incomplete. The objective of this study was to elucidate barriers to reproduction between F. heteroclitus and F. grandis in the lab, as well as to quantify their strengths and relative contributions to reproductive isolation. Pre-zygotic (mating and fertilization) and post-zygotic (hatching) barriers were investigated by performing a variety of choice and no-choice laboratory mating experiments. Under no-choice conditions, barriers to mating had the greatest influence on hybrid production in F. grandis, whereas hatching barriers contributed to the majority of reproductive isolation in F. heteroclitus. Under choice conditions, however, pre-zygotic barriers had the greatest influence on hybrid production in both species. The total reproductive isolation that was observed in females of each species was stronger in F. heteroclitus than in F. grandis, and was nearly complete in F. heteroclitus females under choice conditions and was of moderate strength in F. grandis females. These results reveal an asymmetry in the potential gene flow between these two species, with F. grandis being more likely to hybridize than F. heteroclitus in the absence of environmental influences. No-choice backcrosses were also conducted and showed that at least some F1 hybrids are fertile. The observation that pre-zygotic barriers tend to be stronger than post-zygotic barriers in the early stages of speciation is consistent with similar studies in other organisms.     

Incipient speciation revealed in Anastrepha fraterculus (Diptera; Tephritidae) by studies on mating compatibility, sex pheromones, hybridization, and cytology

It has long been proposed that the nominal species Anastrepha fraterculus is a species complex and earlier studies showed high levels of pre-zygotic isolation between two laboratory strains from Argentina and Peru. Further experiments were carried out on the same populations and on their reciprocal hybrids, including pre- and post-zygotic isolation studies, pheromone analysis, and mitotic and polytene chromosome analysis. A high level of pre-zygotic isolation had been maintained between the parental strains despite 3 years of laboratory rearing under identical conditions. The level of pre-zygotic isolation was reduced in matings with hybrids. There were also differences in other components of mating behaviour. There were quantitative and qualitative differences in the sex pheromone of the two strains with the hybrids producing a mixture. The pre-zygotic isolation barriers were complemented by high levels of post-zygotic inviability and sex ratio distortion, most likely not due to Wolbachia , although there was evidence of some cytoplasmic factor involved in sex ratio distortion. Analysis of polytene chromosomes revealed a high level of asynapsis in the hybrids, together with karyotypic differences between the parental strains. The combined results of the present study indicate that these two strains belong to different biological entities within the proposed A. fraterculus complex.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 97 , 152–165.     

Solanum lycopersicoides gene introgression to tomato,Lycopersicon esculentum,through the systematic avoidance and suppression of breeding barriers

Summary While Lycopersicon esculentum and Solanum lycopersicoides have been successfully hybridized, attempts at further direct gene introgression have been unsuccessful due to the presence of incompatibility barriers. A systematic study of the initial hybridization and subsequent backcrosses has identified multiple barriers to introgression. These barriers are expressed as pollen tube inhibition in the upper style and lower pistil, and failures in syngamy, zygote development, and sporogenesis. Upper style cross-incompatibility barriers were successfully avoided by bud pollinations using a stigma complementation procedure to allow pollen germination on otherwise unreceptive stigmas. The inhibition of pollen tube growth was observed in the lower pistil. A combination of environmental, plant, and genetic manipulations facilitated consistent pollen tube growth to the ovule micropyles in all crosses attempted. Failures at syngamy and early zygote formation proved to be the most difficult barriers to overcome — these were particularly severe in crosses to F₁ hybrid plants. Progeny were obtained in all crossing combinations attempted except in the initial hybridization with S. lycopersicoides as the pistillate parent. Although the strong pre-zygotic barriers were overcome in this cross, further progress was restricted by post-zygotic failures. The capability to overcome pre-zygotic barriers and to excise and culture very young embryos has allowed plantlet recovery from male sterile F₁ plants. Partially pollen-fertile F₁ plants were recovered when relatively large F₁ populations were generated from different _{S. lycopersicoides accessions}. In general, barriers to introgression diminished with increased backcrossing, though exceptions were noted. Progeny from the second backcross to L. esculentum possessed adequate fertility to set self-seed under field conditions. Although all backcross progeny were developed from only a few F₁ individuals, considerable genetic variability was recovered for fruit and vegetative characteristics. Potentially useful levels of disease resistance, particularly to Botrytis cinerea, were also recovered.     

The fumarate reductase reaction of Haemonchus contortus and the mode of action of some anthelmintics

Prichard R. K. 1973. The fumarate reductase reaction of Haemonchus contortus and the mode of action of some anthelmintics. International Journal for Parasitology3: 409–417. Fumarate reductase activities in both thiabendazole susceptible and tolerant strains of Haemonchus contortus have been determined using spectrophotometric and radioisotopic methods. The effects of the anthelmintics, thiabendazole, cambendazole, 1-tetramisole, morantel tartrate, mebendazole and disophenol on the fumarate reductase system of these strains were assayed. Thiabendazole inhibited the fumarate reductase system of the susceptible strain, but no effect could be observed in the tolerant strain with a concentration of thiabendazole of 2 × 10^?3 M. Cambendazole inhibited the system in both strains, but the thiabendazole tolerant strain was significantly less susceptible to cambendazole than the thiabendazole susceptible strain. 1-tetramisole, morantel tartrate and disophenol inhibited the fumarate reductase system in both strains. Mebendazole was not found to affect the fumarate reductase system in either strain. The significance of the fumarate reductase system and its associated phosphorylation in the metabolism of helminths and in the susceptibility of helminths to anthelmintics is discussed.     

Protoplast fusion and genetic recombination in Metarhizium anisopliae

Using two strains of Metarhizium anisopliae var. minor designated E6 and RJ from different origins, inter-strain crosses were readily performed by orthodox methods from mutants of strain E6 through the parasexual cycle. However, in most cases crosses between mutants of strain RJ or between RJ and E6 mutant strains were not achieved. Protoplast fusion was carried out in an attempt to cross such strains. Protoplasts were obtained after treatment of mycelium with lytic enzymes using 0.7 m KCl as osmotic stabilizer. Regeneration frequency varied from 1.2 to 3.0%. Poly (tethylene glycol) was used for the fusion of protoplasts and fusion frequencies varied from 0.9 to 44.1 in 10⁵ protoplasts according to the cross. Sectors which emerge from fusion products were analysed and recombinants were obtained even from crosses between mutant strains which could not be crossed by hyphal fusion. In this way protoplast fusion proved to be a valuable tool for further studies of genetics and breeding of Metarhizium anisopliae.     

Mycorrhizal associations and reproductive isolation in three closely related Orchis species

Background and Aims

The maintenance of species boundaries in sympatric populations of closely related species requires some kind of reproductive isolation that limits gene flow among species and/or prevents the production of viable progeny. Because in orchids mycorrhizal fungi are needed for seed germination and subsequent seedling establishment, orchid–mycorrhizal associations may be involved in acting as a post-mating barrier.

Methods

We investigated the strength of post-mating barriers up to the seed germination stage acting between three closely related Orchis species (Orchis anthropophora, O. militaris and O. purpurea) and studied the role of mycorrhizal fungi in hybridization by burying seed packets of pure and hybrid seeds. After retrieval and assessment of seed germination, the fungi associating with protocorms originating from hybrid and pure seeds were determined and compared with those associating with adult individuals using DNA array technology.

Results

Whereas pre-zygotic post-mating barriers were rather weak in most crosses, post-zygotic post-mating barriers were stronger, particularly when O. purpurea was crossed with O. anthropophora. Germination trials in the field showed that seed germination percentages of hybrid seeds were in most cases lower than those originating from pure crosses. In all species pair combinations, total post-mating reproductive isolation was asymmetric. Protocorms associated with a smaller range of fungal symbionts than adult plants, but there was considerable overlap in mycorrhizal associations between protocorms and their respective parents.

Conclusions

Our results suggest that mycorrhizal associations contribute little to reproductive isolation. Pre-mating barriers are probably the main factors determining hybridization rates between the investigated species.