Effects of natural enemies on relative fitness of Heliothis virescens genotypes adapted and not adapted to resistant host plants

We investigated the potential of two natural enemies of Heliothis virescens (F.) (Noctuidae) to affect its rate of adaptation to tobacco containing Bacillus thuringiensis Berliner toxin. Larval fitness of two laboratory strains of H. virescens, one adapted to B. thuringiensis toxin and one not adapted, was compared on toxic and nontoxic plants, in the presence of the parasitoid Campoletis sonorensis (Cameron) (Ichneumonidae) or the entomopathogenic fungus Nomuraea rileyi (Farlow) Samson. By exposing larvae to plants and enemies for no more than 24 h, we focussed on the behavioral rather than physiological component of their interaction with toxic plants and natural enemies. Parasitism of H. virescens larvae by C. sonorensis during exposure periods of 1–4 h was lower on toxic plants than nontoxic plants and was lower for nonadapted larvae than for toxin-adapted larvae. Decreased larval feeding damage on toxic versus nontoxic plants, and by nonadapted versus adapted larvae, may explain differences in parasitism, because C. sonorensis locates host larvae using cues from damaged plants. Effects of plant toxicity and larval strain on H. virescens survival were numerically consistent with effects on parasitism, but they were not statistically significant. When mean larval survival is used to estimate fitness of the nonadapted genotype relative to the toxin-adapted genotype, we find that C. sonorensis is expected to delay adaptation to toxic plants. Percent infection by N. rileyi of H. virescens larvae exposed to fungus-treated plants for 24 h was greater when plants were toxic, and was greater for nonadapted larvae than toxin-adapted larvae. There were corresponding decreases in larval survival on toxic compared to nontoxic plants, and of nonadapted compared to adapted larvae. Interaction of effects of plant line and larval strain on survival was significant in the presence of fungus, but not in the absence of fungus, which indicates that the effect of toxic plants on the relative fitness of toxin-adapted and nonadapted larvae was mediated by fungus. As in the interaction with C. sonorensis, behavior of larvae on plants may explain differences in susceptibility to N. rileyi. Because nonadapted larvae moved more than toxin-adapted larvae on toxic plants, nonadapted larvae may have been more likely to encounter a lethal dose of conidia. In contrast with C. sonorensis, N. rileyi, which decreased the fitness of the nonadapted genotype relative to the adapted genotype, is expected to accelerate adaptation to toxic plants.     

Saliva proteins of vector Culicoides modify structure and infectivity of bluetongue virus particles

Bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) are related orbiviruses, transmitted between their ruminant hosts primarily by certain haematophagous midge vectors (Culicoides spp.). The larger of the BTV outer-capsid proteins, 'VP2', can be cleaved by proteases (including trypsin or chymotrypsin), forming infectious subviral particles (ISVP) which have enhanced infectivity for adult Culicoides, or KC cells (a cell-line derived from C. sonorensis). We demonstrate that VP2 present on purified virus particles from 3 different BTV strains can also be cleaved by treatment with saliva from adult Culicoides. The saliva proteins from C. sonorensis (a competent BTV vector), cleaved BTV-VP2 more efficiently than those from C. nubeculosus (a less competent/non-vector species). Electrophoresis and mass spectrometry identified a trypsin-like protease in C. sonorensis saliva, which was significantly reduced or absent from C. nubeculosus saliva. Incubating purified BTV-1 with C. sonorensis saliva proteins also increased their infectivity for KC cells ～10 fold, while infectivity for BHK cells was reduced by 2-6 fold. Treatment of an 'eastern' strain of EHDV-2 with saliva proteins of either C. sonorensis or C. nubeculosus cleaved VP2, but a 'western' strain of EHDV-2 remained unmodified. These results indicate that temperature, strain of virus and protein composition of Culicoides saliva (particularly its protease content which is dependent upon vector species), can all play a significant role in the efficiency of VP2 cleavage, influencing virus infectivity. Saliva of several other arthropod species has previously been shown to increase transmission, infectivity and virulence of certain arboviruses, by modulating and/or suppressing the mammalian immune response. The findings presented here, however, demonstrate a novel mechanism by which proteases in Culicoides saliva can also directly modify the orbivirus particle structure, leading to increased infectivity specifically for Culicoides cells and, in turn, efficiency of transmission to the insect vector.     

Relationships between polydnavirus gene expression and host range of the parasitoid wasp Campoletis sonorensis

To evaluate the relationship between immune suppression and host range six lepidopteran species were parasitized by the ichneumonid parasitoid Campoletis sonorensis. Parasitism inhibited the growth of permissive hosts (Heliothis virescens, Helicoverpa zea, and Trichoplusia ni), whereas growth of semi-permissive (Spodoptera exigua, Agrotis ipsilon) and non-permissive hosts (Manduca sexta) was not significantly affected. The 29-36 kDa ovarian protein (OP), responsible for transient immunosuppression in the permissive host H. virescens, bound to and was endocytosed by hemocytes of permissive and non-permissive hosts. Expression of the cysteine-rich polydnavirus gene, VHv1.4, was detected in all the hosts, but declined only in semi- and non-permissive hosts at later times after parasitization. The VHv1.4 protein bound to hemocytes of permissive and semi-permissive hosts, but did not bind to hemocytes of the non-permissive host, M. sexta. Melanization of larval hemolymph was severely inhibited by parasitism in permissive hosts, but was unaffected in M. sexta. In the semi-permissive host, A. ipsilon, hemolymph melanization was transiently inhibited while viral genes were expressed. In conclusion, C. sonorensis OP transiently inhibits encapsulation in all hosts that were tested. The host range of C. sonorensis seems to be determined by whether or not the C. sonorensis ichnovirus (CsIV) is able to establish persistent infections of parasitized larvae to provide long-term suppression of host immunity.     

A comparative cytogenetic study of the tetraploid oat species with the A and C genomes: Avena insularis, A. magna, and A. murphyi

Differential C-banding of chromosomes and in situ hybridization with the probes pTa71 and pTa794 were used for a comparative cytogenetic study of the three tetraploid oat species with the A and C genomes: Avena insularis, A. magna, and A. murphyi. These species were similar in the structure and patterns of C-banding of several chromosomes as well as in the location of the loci 5S rRNA genes and main NOR sites; however, they differed in the number and localization of minor 45S rDNA loci as well as in the morphology and distribution of heterochromatin in some chromosomes. According to the data obtained, A. insularis is closer to A. magna, whereas A. murphyi is somewhat separated from these two species. Presumably, all the three studied species originated from the same tetraploid ancestor, and their divergence is connected with various species-specific chromosome rearrangements. The evolution of A. murphyi is likely to have occurred independently of the other two species.     

Molecular evidence for allopolyploid speciation and a single origin of the western Mediterranean orchid Dactylorhiza insularis (Orchidaceae)

The hybrid origin of the western Mediterranean orchid Dactylorhiza insularis was demonstrated by genetic markers. Allozyme data showed that throughout its range D. insularis has an allotriploid constitution and reproduces apomictically. The parental species of D. insularis were identified as D. romana andD. sambucina; they contributed 2 alleles and 1 allele, respectively, at the allozyme loci studied. The maternal species of D. insularis was D. romana , as inferred from cpDNA ( trn L(UAA) intron). High genetic similarities were found when comparing present populations of D. romana and D. sambucina with their respective genomes 'frozen' in D. insularis. Dactylorhiza insularis showed fixed (or nearly fixed) heterozygosity at 11 out of the 19 loci studied, and poor genetic variation: eight multilocus genotypes were detected at allozyme level. No multilocus genotype differs from the most similar one by more than one allele substitution. All D. insularis individuals showed the same cpDNA haplotype (I) , regardless of their geographic origin and multilocus genotype. The I haplotype is similar, but not identical to that found in D. romana (R). No recurrent formation of D. insularis was observed in hybrid zones between D. romana and D. sambucina , where diploid sexual hybrids (F_1; F_n, backcrosses) were detected. Available data agree with a single origin for D. insularis , which possibly occurred in the present postglacial, when D. romana and D. sambucina , expanding from their glacial refugia, came into contact. The genetic homogeneity found between D. romana and D. markusii , both from their locus classicus , indicates that the latter is a junior synonym of D. romana; on the other hand, D. romana and D. sambucina are well differentiated species ( D_Nei = 0.59).     

Effect of temperature on the transmission of orbiviruses by the biting midge,Culicoides sonorensis

The influence of temperature on the likelihood of Culicoides sonorensis Wirth & Jones (Diptera: Ceratopogonidae) transmitting African horse sickness virus (AHSV) serotypes 4 and 6, bluetongue virus (BTV) serotypes 10 and 16 and epizootic haemorrhagic disease of deer virus (EHDV) serotype 1 was investigated. Extrinsic incubation periods (EIP), vector competence and vector survival were determined at 15, 20, 25 and 30 degrees C. The effect of humidity on vector survival was also investigated by maintaining adult C. sonorensis at 40, 75 and 85% r.h. at each temperature. Higher temperatures were associated with a shorter EIP for all virus serotypes except AHSV6, to which C. sonorensis was orally refractory, increased vector competence for AHSV4 and EHDV1, but not for BTV10 or BTV16, and a reduction in vector survival. Humidity interacted with temperature in influencing vector survival, such that at low temperatures, lower humidity (40 and 75% r.h.) was detrimental for survival (up to 18% reduction in longevity), whereas at high temperatures, high humidity (85% r.h.) was detrimental (up to 36% reduction in longevity). In general, the transmission potential of C. sonorensis for AHSV4, EHDV1, BTV10 and BTV16 was greater at higher temperatures, because although vector survival was reduced, this was more than compensated for by the accompanying decrease in duration of the EIP.     

Genetic variation in populations of Culicoides variipennis complex in the six New England states, U.S.A.

Abstract. We investigated the identity and distribution of members of the Culicoides variipennis complex in the six New England states of the U.S.A., a region where bluetongue transmission has not been detected. Analyses of seven polymorphic isozyme-encoding loci showed that only C.v.variipennis , not considered to be a vector of the bluetongue viruses, was present. The populations of C.v.variipennis were significantly more hetero-zygous than C.v.sonorensis and Cv.occidentalis populations from similar studies in the state of California. Estimates of genetic diversity among populations of C.v.variipennis in New England were similar to C.v.sonorensis in the state of Colorado, but were significantly more genetically divergent than California populations of Cv.occidentalis. The impact of these findings on the status of New England as a possible bluetongue-free region for the purpose of international trade in ruminant livestock and their germplasm is discussed.     

In vitro rearing of Campoletis sonorensis, a larval endoparasitoid of Heliothis virescens from egg to third instar in an artificial medium devoid of insect sources

Campoletis sonorensis is a solitary larval endoparasitoid of several Heliothis spp. pests. This study was carried out to develop media devoid of insect sources for in vitro rearing the parasitoid. Trehalose, lysine, threonine, asparagine, glutamine, hydroxyproline, serine, bovine serum albumin, and lactalbumin were beneficial to C. sonorensis. Addition of fresh chicken egg yolk at a low level improved the artificial media. Addition of 20-hydroxyecdysone increased the molting rate, reduced the critical size at molt and decreased development time. Parasitoid development in vitro was dependent on eggs that had been in the host for at least 22 h. Luminosity was also critical for the development of C. sonorensis in vitro. Optimal development occurred in a L14:D10 photoperiod at 43 Lux light intensity. Utilizing the best media and conditions, 100% of the parasitoid larvae reached the second instar and over 37% molted to the third instar. However, no further development occurred.     

Vector competence of Culicoides bolitinos and C. imicola for South African bluetongue virus serotypes 1, 3 and 4

Abstract .The susceptibility of field-collected Culicoides bolitinos to infection by oral ingestion of bluetongue virus serotypes 1, 3 and 4 (BLU 1, 3 and 4) was compared with that of field-collected C. imicola and laboratory reared C. variipennis sonorensis . The concentration of the virus per millilitre of bloodmeal was 10^5.0 and 10^6.0TCID₅₀ for BLU 4 and 10^7.2TCID₅₀ for BLU 1 and 3. Of 4927 C. bolitinos and 9585 C. imicola fed, 386 and 287 individual midges survived 10 days extrinsic incubation, respectively. Midges were assayed for the presence of virus using a microtitration assay on BHK-21 cells and/or an antigen capture ELISA. Infection prevalences for the different serotypes as determined by virus isolation ranged from 22.7 to 82.0% in C. bolitinos and from 1.9 to 9.8% in C. imicola; infection prevalences were highest for BLU 1, and lowest for BLU 4 in both species. The mean log₁₀ TCID₅₀ titre of the three BLU viruses per single fly was higher in C. bolitinos than in C. imicola . The results suggested that C. bolitinos populations are capable vectors of the BLU viruses in South Africa. A high correlation was found between virus isolation and ELISA results for the detection of BLU 1, and less for BLU 4; the ELISA failed to detect the presence of BLU 3 in infected flies. The C. v. sonorensis colonies had a significantly lower susceptibility to infection with BLU 1, 3 and 4 than C. bolitinos and C. imicola . However, since infection prevalence of C. v. sonorensis was determined only by ELISA, this finding may merely reflect the insensitivity of this assay at low virus titres, compared to virus isolation.     

Adaptations of Drosophila and yeasts: their interactions with the volatile 2-propanol in the cactus-microorganism-Drosophila model system

The interactions of yeasts growing in decaying cactus tissue with and without 2-propanol were studied with respect to the costs and benefits provided to three cactophilic Drosophila species (D. mojavensis, D. arizonensis and D. buzzatii). Two common cactus yeasts, Candida sonorensis and Cryptococcus cereanus, which can tolerate and metabolize 2-propanol, provide benefits to the three Drosophila species in the presence of the alcohol, as compared with another common cactus yeast, Pichia cactophila, which has less tolerance and cannot metabolize 2-propanol. Because 2-propanol is commonly found in decaying cactus tissue and C. sonorensis and Cr. cereanus are also frequently recovered from the rotting tissue being utilized by the Drosophila species, the interactions described here are viewed as a possible adaptation in which the yeast provides benefits to one of its vectors by metabolism of 2-propanol in the habitat.     

Feeding and survival of Culicoides sonorensis on cattle treated with permethrin or pirimiphos-methyl

The persistence of permethrin (5% a.i.) and pirimiphos-methyl (27% a.i.), applied to the dorsum of calves in the field against Culicoides sonorensis Wirth and Jones (Diptera: Ceratopogonidae), was estimated using a hair-blood-feeding bioassay in the laboratory. Hair clippings were taken before treatment and 3, 7, 14, 21, 28, 42 and 56 days after treatment from the dorsum, side and belly of treated and control calves. Laboratory-reared insects were allowed to feed through thin hair layers and a parafilm membrane on sheep blood warmed using a water-jacketed feeder. Some intoxication after exposure to hair was noted up to 28 days after treatment with permethrin and up to 14 days after treatment with pirimiphos-methyl. Hair from the dorsum caused more intoxication for a longer period than hair from other body regions. Permethrin and pirimiphos-methyl applied to the back did not significantly reduce overall engorgement (body regions pooled) after treatment. Permethrin residues on hair remained far higher on the back than other body regions and were related to insect intoxication and reduction in engorgement in the laboratory. Residues on belly hair never exceeded 12p.p.m. and did not result in significantly reduced feeding at any time. Engorged insects that exhibited sublethal intoxication from feeding through permethrin-treated hair did recover and matured numbers of eggs comparable to controls. Field trials using treated and control calves and enclosure nets showed that dorsal applications of 5% permethrin were not effective in reducing engorgement, despite some intoxication. Vacuum samples from a calf showed that C. sonorensis fed primarily on the belly. A 0.2% permethrin application on the belly (250 ml) did result in > 80% reduction of C. sonorensis in the enclosure nets at 3 and 7 days after treatment, but activity had subsided by 10 days after treatment. The utility of insecticidal treatments for suppression of this vector is discussed.     

Age-grading the biting midge Culicoides sonorensis using near-infrared spectroscopy

Age-grading of insects is important in the control and monitoring of both insect populations and vector-borne diseases. Microscopy and morphological techniques exist to age-grade most blood-feeding flies, but these techniques are laborious, often destructive to the insects, and slow. Near-infrared spectroscopy (NIRS) can be automated and is a non-destructive technique for age-grading. We applied NIRS techniques to age-grade females of the biting midge, Culicoides sonorensis Wirth & Jones (Diptera: Ceratopogonidae), the vector of bluetongue and other arboviruses in North America. Female flies of five known age cohorts (1, 3, 6, 9 and 12 days post-emergence) from three laboratory colonies were used. The data indicate that NIRS can be used to differentiate age groups of C. sonorensis .     

Phylogenetic relationships among bisexual and unisexual lineages of the weevil Scepticus insularis (Coleoptera: Curculionidae) on Hokkaido, Northern Japan

We examined the geographic distributions and phylogenetic relationships of bisexual and unisexual (parthenogenetic) forms of the weevil Scepticus insularis on Hokkaido Island, northern Japan. Unisexual beetles were widespread throughout Hokkaido, whereas bisexuals were found only in three remote areas. Bisexuals (females and males) and unisexual females occurred sympatrically in two areas. We determined nucleotide sequences for part of the mitochondrial NADH dehydrogenase subunit 2 (ND2; 633 bp) gene for 104 individuals, and for part of the nuclear internal transcribed spacer region 2 (ITS2; 360 bp) for 91 individuals. In an ND2 gene tree, haplotypes of S. insularis fell into two distinct clades (A and B), which were genetically differentiated from one another by 9.1% nucleotide sequence divergence. Haplotypes of females identified as unisexual were all in clade A, whereas those of females identified as bisexual belonged to clade B. Haplotypes of males were in clade B, except for two males having a clade-A haplotype. Circumstantial evidence suggests that these two males were produced by unisexual females. The ND2 tree suggests that the current unisexual form of S. insularis on Hokkaido was of a single origin. In contrast, a gene tree for ITS2 haplotypes show no clear divergence between the two modes of reproduction, with two major haplotypes shared by unisexual females, bisexual females, and males. This incongruence between the nuclear and mitochondrial phylogenies may be attributable to occasional gene flow between the unisexual and bisexual lineages through males occasionally produced by unisexual females, but our results do not exclude the possibility that the two lineages share polymorphic ancestral ITS2 haplotypes.     

Rates of bluetongue virus transmission between Culicoides sonorensis and sheep

Abstract.  Two experiments were undertaken to estimate the transmission rates of bluetongue virus (BTV) serotype 1 between a biting midge vector, Culicoides sonorensis (Wirth & Jones) (Ceratopogonidae), and a natural host, sheep. In an experiment to measure the transmission rate from vector to host (V→H), six batches of one, five and 20 intrathoracically infected midges were fed on a total of 18 bluetongue (BT)-naïve sheep. The sheep were then monitored for 21 days for clinical signs of BT, viraemia and antibody response. All sheep fed on by five or 20 midges and five of six sheep fed on by just one midge showed signs of BT, were viraemic and developed antibody. The sixth sheep fed on by a single infected midge did not show signs of BT or have detectable viraemia; it did, however, develop a weak antibody response. A bite from a single infected midge is therefore able to transmit BTV to naïve sheep with 80–100% efficiency. Sheep fed upon by larger numbers of infected midges took less time to reach maximum viraemia and developed stronger antibody responses. Sheep exposed to greater amounts of BTV in feeding midges developed a higher level of viraemia and stronger antibody responses. In a second experiment to measure the transmission rate from host to vector (H→V), batches of up to 500 uninfected female C. sonorensis fed every 1–2 days on two experimentally infected sheep during the course of infection. Of 3929 engorged midges that were individually titrated after surviving the extrinsic incubation period, only 23 (0.6%) were infected with BTV. Viraemia in the sheep extended for up to 19 days post-inoculation. No infected midges, however, were detected from 14 days post-infection.     

Parasitoid–Pathogen–Pest Interactions of Chelonus insularis, Campoletis sonorensis, and a Nucleopolyhedrovirus in Spodoptera frugiperda Larvae

In this study we examined interactions between two solitary endoparasitoids, the braconid Chelonus insularis and the ichneumonid Campoletis sonorensis, and a multiple-enveloped nucleopolyhedrovirus infecting Spodoptera frugiperda larvae. We examined whether ovipositing females minimize interference by discriminating amongst hosts and examined the outcome of within-host competition between parasitoid species and between the parasitoids and the virus. The egg–larval parasitoid Ch. insularis did not discriminate between virus-contaminated and uncontaminated S. frugiperda eggs; all S. frugiperda larvae that emerged from surface-contaminated eggs died of viral infection prior to parasitoid emergence. The larval parasitoid C. sonorensis also failed to discriminate between healthy and virus-infected S. frugiperda larvae or between larvae unparasitized or parasitized by Ch. insularis. Host larvae parasitized in the egg stage by Ch. insularis were suitable for the development of C. sonorensis when they were multiparasitized by C. sonorensis as first, second, third, and fourth instars, whereas emergence of Ch. insularis was dramatically reduced (by 85 to 100%) in multiparasitized hosts. Nonspecific host mortality was significantly higher in multiparasitized hosts than in singly parasitized hosts. The development time and sex ratio of C. sonorensis in multiparasitized host larvae were unaffected by the presence of Ch. insularis larval stages. Both Ch. insularis parasitized and nonparasitized larvae of the same instar (second, third, or fourth instars) had a similar quantitative response to a challenge of virus inoculum. All host larvae that ingested a lethal dose of virus were unsuitable for Ch. insularis development. In contrast, C. sonorensis did not survive in hosts that ingested a lethal virus dose immediately after parasitism, but parasitoid survival was possible with a 2-day delay between parasitism and viral infection and the percentage of parasitoid emergence increased significantly as the interval between parasitism and viral infection increased. The development time of C. sonorensis was significantly reduced in virus-infected hosts compared to conspecifics that developed in healthy hosts. C. sonorensis females that oviposited in virus-infected hosts did not transmit the virus to healthy hosts that were parasitized subsequently. Field applications of virus for biocontrol of S. frugiperda may lead to substantial mortality of immature parasitoids, although field experiments have not yet demonstrated such an effect.     

Parasitism-linked block of host plasma melanization

When parasitized by the Ichneumonid parasitoid Campoletis sonorensis, larvae of the Noctuid moth, Heliothis virescens, are unable to mount an effective immune response against parasitoid eggs. Defensive melanization of plasma and cellular encapsulation of parasite eggs are dramatically inhibited by infection with the symbiotic immunosuppressive C. sonorensis ichnovirus (CsIV). This study demonstrates that the CsIV-mediated inhibition of melanization is associated with reduction in the enzymatic activity and protein titer of key enzymes in the melanization pathway, phenoloxidase, dopachrome isomerase, and DOPA decarboxylase. Inhibition of the synthesis of key melanization enzymes leads to reductions in the melanization substrates l-dihydroxyphenylalanine, N-acetyldopamine, and N-beta-alanyl dopamine from millimolar to nanomolar levels in parasitized larvae. By contrast, concentration of a precursor catecholamine, dopamine, rises fourfold in these larvae. Thus in CsIV-infected larvae, enzymatic deficiencies in the melanization pathway lead to reduced concentrations of specific enzyme substrates, causing failure of melanization in parasitized insects.     

A possible mechanism for the physiological suppression of conspecific eggs and larvae following superparasitism by solitary endoparasitoids

Competition for possession of a host by internal solitary parasitoids has been attributed to physical combat and physiological suppression, but the mechanisms that result in what has been referred to as physiological suppression is poorly understood. Some insights are provided by the studies reported here using the solitary endoparasitoid, Campoletis sonorensis (Cameron). Embryos of C. sonorensis less than ten hours old rarely hatch in various artificial media, while embryos twenty hours or older generally hatch. These results suggest that young embryos in which the embryonic membranes have not yet formed are only able to develop in a narrow range of environments represented by the nonparasited hemolymph. In contrast, embryos in which the embryonic membranes are formed are able to develop in a wide range of environments represented by parasitized hemolymph which has been shown by a number of studies to change. These ideas were given support by studies reported here, where young and older eggs were incubated singly or paired. We suggest the general changes in the hemolymph of a parasitized host become unfavorable for the development of newly oviposited eggs.