New record of nucleopolyhedroviruses in tea looper caterpillars in India

Tea production in North-East India hit a record loss due to the widespread severe outbreak of a mixed brood of three species of looper caterpillar pests of geometrid moths (Lepidoptera) in 2008-2010. In addition to Buzura suppressaria, two newly recorded geometrids, viz., Hyposidra infixaria and Hyposidra talaca have caused widespread severe damage in recent years. In the present study we report the nucleopolyhedroviruses (NPV) isolated from the tea looper caterpillar from North-East India. We identified and characterized the NPV by cloning and sequencing a partial segment of polyhedrin gene of virus infected larvae of B. suppressaria, H. talaca and H. infixaria. A comparison of deduced amino acids of polyhedrin gene among H. talaca, H. infixaria and B. suppressaria showed that same strain was found to infect all the three loopers in India, which show high sequence identity with B. suppressaria Chinese isolates. Based on the polyhedrin sequence homology, it is predicted that a variant of B. suppressaria Chinese isolate of NPV found to infect H. talaca, H. infixaria and B. suppressaria in India.     

Interaction between MbMNPV and the braconid parasitoid Habrobracon hebetor (Hym., Braconidae) on larvae of beet armyworm,Spodoptera exigua (Lep., Noctuidae)

The survival of a braconid parasitoid Habrobracon hebetor was investigated on nucleopolyhedrovirus (NPV)-infected Spodoptera exigua larvae. The second-instar larvae were exposed to 30, 51.4 and 180 PIB/mm² of Mamestra brassicae NPV (MbMNPV) as under-LD₅₀, LD₅₀ and over-LD₅₀ values, respectively. They were accessible to be parasitized by H. hebetor after 24, 48 and 72 h post-treatment. Infection of the larvae with MbNPV was deleterious to the survival and parasitism of H. hebetor. The survival of H. hebetor in MbNPV-infected S. exigua larvae was dependent on the interval between viral infection and parasitization, as well as on the treatment dose of MbMNPV; very few adults of parasitoid emerged from infected hosts when host larvae were exposed to 180 PIB/mm² of MbNPV on 72-h interval treatment. The inoculation dose of MbNPV and the timing of parasitoid release had significant effect on the development of H. hebetor on virus-infected hosts. Field applications of virus for biocontrol of S. exigua may lead to substantial mortality of immature parasitoids.     

Transmission of a nuclear polyhedrosis virus inHeliothis zea [Lep.: Noctuidae] larval populations on soybean

Transmission of a nuclear polyhedrosis virus (NPV) inHeliothis zea (Boddie) on soybean was examinated. Artificial infestations ofH. zea were established at densities of 6.5, 19.5 and 58.5 larvae/row-m. Additional larvae infected to die from NPV in the 2nd stage were released into subplots to simulate 5 and 25% mortality levels. Virus transmission from infected to noninfected larvae was correlated with the initial incidence of infection in the population but not the density of larvae/row-m. Deposition of virus on plants from cadavers of larvae that died of virus infection was correlated with the initial incidence of infection in the populations and the density of larvae/row-m. After pupation of larvae in the 1st population, noninfected larvae only were again released to examine transmission of viral inoculum remaining on plants and soil. The percent mortality of larvae collected from the 2nd release was low and did not differ significantly between treatments. The concentration of virus on foliage and in soil after the 2nd release was directly correlated with density of larvae/row-m but not the incidence of infection within the population in the 1st release. This material is based upon work supported in part by the U.S. Department of Agriculture under Agreement N^o 82 CRSR-2-1000.     

Density-related variation in vertical transmission of a virus in the African armyworm

Larvae of the African armyworm, Spodoptera exempta, are darker and more resistant to baculovirus infection when reared in groups (gregarious form) compared to being reared singly (solitary form). Lepidoptera that survive virus challenge as larvae could potentially retain a sublethal virus infection which is then transmitted vertically to the next generation. Here we examine whether gregarious and solitary forms of the armyworm differ in the costs of surviving virus infection and in their capacity to transmit an active baculovirus infection to their offspring. Pupae of larvae reared gregariously that survived virus challenge weighed significantly less than uninfected individuals, but this was not so for those reared solitarily. This did not, however, translate into differences in fecundity, at least under laboratory conditions. As found in previous studies, pre-oviposition period was shorter for solitary than gregarious insects, and it was also shorter for females that had been challenged with virus as larvae. Both the prevalence of egg batches containing larvae that died from nucleopolyhedrovirus (NPV) infection and the proportion of infected larvae within each egg batch were significantly increased (approximately doubled) when parental moths were previously challenged with the virus during their larval state. This demonstrates that horizontal transmission in one generation can elevate vertical transmission to the next generation. Moreover, prevalence of overt infection in the offspring generation was two to three times greater when parental moths were reared solitarily as larvae than when reared gregariously. Disease prevalence and proportional infection were both independent of the sex of the infected parent and whether or not the egg batch was surface-sterilized to remove potential contaminants. This suggests that the eggs are infected internally (transovarial) rather than externally (transovum). These results help to shed light on the observed temporal pattern of virus epizootics in eastern Africa.     

Utilization of the polymerase chain reaction in the diagnosis of nuclear polyhedrosis virus infections of gypsy moth (Lymantria dispar, Lep., Lymantriidae) populations

Abstract:  Three specific DNA probes were used for the detection of the nuclear polyhedrosis (NPV) virus of Lymantria dispar ( Ld NPV) genome. Two of these probes, H2 and H3 were obtained by classical cloning method and one (TR6) by polymerase chain reaction (PCR). These probes, used individually or in a pool in the standard slot–blot hybridizations, were able to detect 10⁹ genome copies. By performing 35 cycles of PCR amplification before hybridization with primers specific to Ld NPV genome on DNA extracted from infected larvae, the sensitivity of the hybridization technique was increased, so that as little as 10 copies of the Ld NPV genome could be detected. Using these methods, L. dispar naturally infected by Ld NPV were identified among field populations in Canada and in the United States near the eastern Canadian border. Using a combination of PCR and hybridization, Ld NPV contamination of egg masses were also detected. By disinfecting the eggs with sodium hypochlorite prior to PCR amplification and hybridization, it was also demonstrated that transmission of viral infection in the natural populations is mainly caused by external contamination of the egg and is unlikely to occur through the transovarial route.     

A Comparison of the Efficacy of Nuclear Polyhedrosis and Granulosis Viruses in Spray and Bait Formulations for the Control of Agrotis segetum (Lepidoptera: Noctuidae) in Maize

Agrotis segetum nuclear polyhedrosis virus (AsNPV) and granulosis virus (AsGV), propagated in laboratory cultures of A. segetum in England and A. ipsilon in Spain, respectively, were applied to plots of maize plants at the one‐ to four‐leaf stage of growth. Plots were arranged in a 6 x 6 Latin square design and infested with second‐instar A. segetum larvae (the common cutworm). Each virus was applied in separate treatments by two application methods; as an aqueous spray containing 0.1% Agral as a wetting agent, and as a bran bait. The NPV was applied at a rate of 4 X 10¹² polyhedra/ha, and the GV at 4 X 10¹³ granules/ha. Soil and plants were sampled for larvae on three occasions following virus treatment: 24 h, 4 days and 11 days. The larvae were reared on diet in the laboratory, until death or pupation, to examine the rate and level of viral infection. Infection data showed 87.5% and 91% NPV infection and 12.5% and 55% GV infection in spray and bait treatments, respectively, in larvae sampled 24 h after treatment. In larvae sampled 4 days after treatment, the results were 78% and 100% NPV infection, and 13% and 6% GV infection. A total of only six larvae were retrieved on day 11. In both treatments larvae infected with AsNPV died significantly more rapidly and at an earlier instar than those infected with AsGV, indicating that AsNPV appears to have better potential as a control agent for A. segetum.     

Characterization of a baculovirus newly isolated from the tea slug moth, <Emphasis Type="Italic">Iragoidae fasciata</Emphasis>

The tea slug moth Iragoidae fasciata (Lepidoptera, Eucleidae) is one of the main insect pests that attack tea bushes. A new nucleopolyhedrovirus (NPV) called Iragoidae fasciata NPV (IrfaNPV) was recently isolated from diseased larvae. An 11,626 bp fragment of the viral genomic DNA containing the polyhedrin gene and other 12 genes was cloned and sequenced. Gene comparison and phylogenetic analysis showed that IrfaNPV is a member of the Group I NPVs. However, the genomic organization of IrfaNPV is highly distinct. In addition, electron microscopy analysis showed that IrfaNPV is a single nucleocapsid NPV (SNPV). An inoculation assay showed that IrfaNPV is semi-permissive in the Trichoplusia ni cell line Tn-5Bl-4. Bioassays on lethal concentration (LC₅₀) and lethal time (LT₅₀) were conducted to test the susceptibility of I. fasciata larvae to the virus.     

The impact of co-infection by a nucleopolyhedrovirus and the endoparasitoid Microplitis pallidipes on the total hemocyte count and composition in larval beet armyworm,Spodoptera exigua

The physiological effects of nucleopolyhedrovirus (NPV) infection and parasitism by Microplitis pallidipes (Hymenoptera: Braconidae) on the hemocytes of Spodoptera exigua (Lepidoptera: Noctuidae) larvae were examined. We found that compared to healthy (control) larvae, the total hemocyte count (THC) and granulocyte count in parasitized larvae increased 1 day after parasitization and then decreased, while the plasmatocyte count was not significantly affected for the first 5 days but was significantly enhanced on day 6 after parasitization. In parasitized + infected larvae, both the THC and granulocyte counts began be lower from day 1 compared to parasitized larvae, while the plasmatocyte count was generally lower than in parasitized larvae. Compared to the control, THC, and granulocyte counts of virus-infected larvae were higher 1 day after infection. Compared to that in virus-infected larvae, THC and granulocyte counts in parasitized + infected larvae began to decrease from day 1 while the plasmatocyte count generally decreased. We concluded that the host immune response of cell communities to parasitization by M. pallidipes was elicited during the development of the parasitoid egg, but that immune response was inhibited during larval development of parasitoids in the host body. Meanwhile, we found that NPV infection impeded the regulatory effect of M. pallidipes on host cellular immune responses, and parasitization by M. pallidipes similarly inhibited the host cellular immune response caused by NPV infection.     

Investigating the horizontal transmission of the Cydia pomonella granulovirus (CpGV) in a model system

Repeated applications of Cydia pomonella granulovirus (CpGV) can effectively control the codling moth (CM) in apple orchards. However, it is still unknown whether horizontal transmission of the virus from infected to uninfected larvae contributes to the efficacy of the virus insecticide. Horizontal transmission of CpGV was assayed using detached apples. In experiments using artificially applied virus dots on the apple’s surface or infected CM larvae as virus inoculum, it was found that the likelihood of infection of healthy CM larvae relied mainly on the larval behavior. The amount of virus inoculum, either applied artificially or produced by the infected larvae, impacted the infection rate only to a small degree. In the experiments, CM larvae exhibited a strong preference in entry sites, increasing the chance for horizontal transmission. Depending on the experimental design, horizontal transmission rates of about 40% were observed in laboratory assays.     

Activation of latent virus infections in larvae of Adoxophyes orana (Lepidoptera: Tortricidae) and Barathra brassicae (Lepidoptera: Noctuidae) by foreign polyhedra

The number of larvae containing polyhedra increased when larvae of Adoxophyes orana and Barathra brassicae were fed on polyhedra of nuclear polyhedrosis virus (NPV) of the reciprocal species. Comparison of restriction endonuclease EcoRI cleavage patterns of DNA isolated from polyhedra used as inocula and from polyhedra obtained after cross-inoculation showed that cross infection did not occur. The observations indicate that latent viruses were activated in both insects. Activation of the A. orana latent NPV with polyhedra of a cytoplasmic polyhedrosis virus (CPV) of B. brassicae, and cross-inoculation with an extract prepared from healthy larvae indicated that an activating agent does not have to be a component of nuclear polyhedra.     

The infectivity and host range of Orgyia anartoides nucleopolyhedrovirus

The painted apple moth (PAM), Teia anartoides (Walker) (Lepidoptera: Lymantriidae) made a recent incursion into New Zealand. A nucleopolyhedrovirus (NPV), Orgyia anartoides NPV (OranNPV), originally isolated from PAM in Australia, was tested for its pathogenicity to PAM and a range of non‐target insect species found in New Zealand, to evaluate its suitability as a microbial control for this insect invader. Dosage‐mortality tests showed that OranNPV was highly pathogenic to PAM larvae; mean LT₅₀ values for third instars ranged from 17.9 to 8.1 days for doses from 10² to 10⁵ polyhedral inclusion bodies/larva, respectively. The cause of death in infected insects was confirmed as OranNPV. Molecular analysis established that OranNPV can be identified by PCR and restriction digestion, and this process complemented microscopic examination of infected larvae. No lymantriid species occur in New Zealand; however, the virus had no significant effects on species from five other lepidopteran families (Noctuidae, Tortricidae, Geometridae, Nymphalidae and Plutellidae) or on adult honeybees. Thus, all indications from this initial investigation are that OranNPV would be an important tool in the control of PAM in a future incursion of this species into New Zealand.     

Formulation and optimisation of various nuclear polyhedrosis virus isolates and assessment of their insecticidal activity against Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) larvae

Helicoverpa armigera (Hubner) is a pest that poses serious threat to the tomato crop in India. Larvae of this species are susceptible to the nucleopolyhedrovirus (NPV), which has attracted interest as a potential biocontrol agent. Rearing of larvae in natural diet resulted in less number of pupae, while in artificial diet more pupae and healthy adults were obtained. The NPV was tested for its insecticidal action against H. armigera in natural and artificial diets. The bioassay results of inoculated H. armigera nucleopolyhedrosis virus (HaNPV) with different concentrations indicate that the 4.0 g/l dosage caused maximum mortality (70.3% and 60.54%), and minimum mortality 46.83% and 44.08% was recorded in the 0.5 g/l dosage under laboratory and pot culture conditions, respectively. Polyhedral inclusion bodies (PIBs) were estimated using a standard haemocytometer. According to the estimate, purified PIBs were found at a concentration of 1 × 10⁹/ml. The different formulation of NPV, i.e. wettable fine powder with a hue of white, had a particle size of 100 μm with a concentration of 1 × 10⁸/ml. The encapsulated product was found to be in the form of beads and was red in colour with a concentration of 1 × 10⁸ PIBs/ml. The effectiveness of NPV against H. armigera larvae was noted at maximum LC₅₀ values at a concentration of 1 × 10⁹/ml. Qualitative analysis estimates showed that protein polyhedrin and enzyme chitinase had a molecular weight of 33 and 45 kDa, respectively. Viral DNA was isolated from NPV-infected larvae and was then separated using SDS-PAGE. The estimated polyhedrin with a molecular weight of 33 kDa, which is responsible for the increased mortality percent of larvae, was confirmed by the bioassay. Hence it was concluded that NPV is ecofriendly in the management of pests on tomato and other crops.     

Foraging in a pathogen reservoir can lead to local host population extinction: a case study of a Lepidoptera-virus interaction

In 1990, natural infestations of the polyphagous vapourer moth, Orgyia antiqua (Lepidoptera: Lymantriidae) in lodgepole pine plantations in northern Scotland, were studied to ascertain the role of host foraging behaviour on the prevalence of nucleopolyhedrovirus (NPV; Baculoviridae) infection in the population. Aerial dispersal of early instar larvae (L1–L3) from the tree canopy onto heather foliage at the forest understorey, with subsequent relocation back onto the tree as late-instar larvae (L4–L6) appeared to play a significant role in the development of a widespread virus epizootic in which approximately 80% of L4–L6 individuals succumbed to disease. Bioassays of foliage 1 year later showed that the distribution of NPV followed a pronounced vertical gradient through the forest canopy culminating in high concentrations of virus in the forest understorey. Experimental systems comprising potted pine trees positioned above heather bases showed that NPV infections could be acquired by early stage larvae following dispersal from the tree and feeding on the undercanopy vegetation, then translocated to the tree component for secondary transmission to susceptible tree-feeding individuals. Behavioural studies indicated that the tendency for first-, second- and third-instar larvae to disperse to the understorey was probably not influenced by larval density on the tree but was strongly dependent on larval instar. In contrast, the tendency for larvae to relocate from the understorey heather to the tree was affected by both larval density and larval instar, suggesting that both these factors may significantly affect virus acquisition, translocation and transmission in the host population. In the present study, the heather understorey appeared to act as a pathogen reservoir in which virus could persist between host generations. Spatial heterogeneity in virus distribution combined with host foraging behaviour (dispersal and feeding) resulted in the pathogen playing a major role in host population dynamics over an extended time period (3 years). The reservoir theory is supported by the observation that similar dynamics were not observed in O. antiqua populations at neighbouring sites which lacked understorey food plants. Received: 8 June 1998 / Accepted: 5 October 1998     

Systematic differences in the population density of green spruce aphid, Elatobium abietinum in a provenance trial of Sitka spruce, Picea sitchensis

Quantitative bioassay techniques were used to measure the susceptibility of Heliothis armigera to three nuclear polyhedrosis viruses (NPVs): H. armigera singly-enveloped NPV (HaSNPV), H. zea SNPV (HzSNPV) and H. armigera multiply-enveloped NPV (HaMNPV). Viruses were identified by EcoRI restriction endonuclease analysis. Electrophoretic profiles of DNA fragments revealed that the HaSNPV isolate was a previously undescribed genotypic variant. Bioassays with neonate and 6-day-old larvae measured small but significant differences in virulence between the three viruses. HzSNPV was the most virulent for neonate larvae with a median lethal dose (LD₅₀) of five polyhedra. HaMNPV was least virulent for 6-day-old larvae, with a LD₅₀ of 1400 polyhedra compared with 640–670 polyhedra for HaSNPV and HzSNPV. In addition, the median lethal time (LT₅₀) for infection with HaMNPV in neonate larvae was approximately 1·7 days longer than for the other viruses. Although they varied in virulence, each of the three viruses was sufficiently virulent to have considerable potential as a microbial control agent of H. armigera.     

Effects of single and mixed infections with wild type and genetically modified Helicoverpa armigera nucleopolyhedrovirus on movement behaviour of cotton bollworm larvae

Naturally occurring insect viruses can modify the behaviour of infected insects and thereby modulate virus transmission. Modifications of the virus genome could alter these behavioural effects. We studied the distance moved and the position of virus‐killed cadavers of fourth instars of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) infected with a wild‐type genotype of H. armigera nucleopolyhedrovirus (HaSNPV) or with one of two recombinant genotypes of this virus on cotton plants. The behavioural effects of virus infection were examined both in larvae infected with a single virus genotype, and in larvae challenged with mixtures of the wild‐type and one of the recombinant viruses. An egt‐negative virus variant caused more rapid death and lower virus yield in fourth instars, but egt‐deletion did not produce consistent behavioural effects over three experiments, two under controlled glasshouse conditions and one in field cages. A recombinant virus containing the AaIT‐(Androctonus australis Hector) insect‐selective toxin gene, which expresses a neurotoxin derived from a scorpion, caused faster death and cadavers were found lower down the plant than insects infected with unmodified virus. Larvae that died from mixed infections of the AaIT‐expressing recombinant and the wild‐type virus died at positions significantly lower, compared to infection with the pure wild‐type viral strain. The results indicate that transmission of egt‐negative variants of HaSNPV are likely to be affected by lower virus yield, but not by behavioural effects of egt gene deletion. By contrast, the AaIT recombinant will produce lower virus yields as well as modified behaviour, which together can contribute to reduced virus transmission under field conditions. In addition, larvae infected with both the wild‐type virus and the toxin recombinant behaved as larvae infected with the toxin recombinant only, which might be a positive factor for the risk assessment of such toxin recombinants in the environment.     

Field evaluation of eco-friendly biocontrol package for the management of pod borer,Helicoverpa armigera Hubner,and fusarium wilt in chickpea,Cicer arietinum

Helicoverpa armigera is a serious pest of chickpea and causes great damage to crop. Extensive and indiscriminate use of insecticides has led to the development of resistance in H. armigera. Among the several alternative methods for management of H. armigera, the nuclear polyhedrosis virus (NPV) is promising, whereas Trichoderma sp. have shown promising results against chickpea wilt. The experiments to evaluate biocontrol package against H. armigera and wilt disease under field conditions were conducted. Lowest H. armigera larval population (0.71 larvae/plant) was recorded in chemical control, which was at a par with biocontrol package (0.91 larvae/plant), and both the treatments were significantly better than control. Lowest per cent pod damage (3.85%) was recorded in chemical control followed by biocontrol treatment (5.08%) and unsprayed control (8.61%). The yields from biocontrol package (13.45 q/ha) and chemical control (15.37 q/ha) were significantly higher than unsprayed control (10.7 q/ha). There was no disease incidence in all treatments in both 2008 and 2009.     

Prospective field study of transovarial dengue‐virus transmission by two different forms of Aedes aegypti in an urban area of Bangkok,Thailand

A prospective field study was conducted to determine transovarial dengue‐virus transmission in two forms of Aedes aegypti mosquitoes in an urban district of Bangkok, Thailand. Immature Aedes mosquitoes were collected monthly for one year and reared continuously until adulthood in the laboratory. Mosquitoes assayed for dengue virus were processed in pools and their dengue virus infection status was determined by one‐step RT‐PCR and nested‐PCR methods. Of a total 15,457 newly emerged adult Ae. aegypti, 98.2% were dark and 1.8% of the pale form. The results showed that the minimum infection rate (MIR) by transovarial transmission (TOT) of dengue virus during the one‐year study ranged between 0 to 24.4/1,000 mosquitoes. Dengue virus TOT increased gradually during the hot summer months, reaching a peak in April‐June, while dengue cases peaked in September, a rainy month near the end of the rainy season. Therefore, mosquito infections due to TOT were prevalent four months before a high incidence of human infections. TOT dengue virus infections occurred in both forms of Ae. aegypti. All four dengue serotypes were detected, with DEN‐4 predominant, followed by DEN‐3, DEN‐1, and DEN‐2, respectively.     

Epizootics caused by a nuclear polyhedrosis virus in populations of spodoptera exigua in southern Spain

A nuclear polyhedrosis virus (NPV) has been identified as the etiological agent causing epizootics in natural populations of Spodoptera exigua in the south of Spain. The incidence of this NPV has been determined from samples of larvae collected from sunflower fields and vegetable greenhouses at two sites located 500 km apart. The NPV was the only disease agent identified at both sites. Through the period surveyed the percentage of NPV‐infected larvae increased from 4.3% to 23% and from 22.5% to 100% in S. exigua larvae collected from sunflower fields and vegetable greenhouses respectively. No mortality attributed to the NPV was found in Spodoptera littoralis, Heliothis armigera, Peridroma saucia and Plusia gamma larvae collected in the same survey from the same sites. This is the first report of epizootics caused by NPV in S. exigua populations in field and greenhouse crops in Europe.     

Production and efficacy of baculviruses

Several baculvirusus of nuclear polyhedrosis virus (NPV) have been produced and tested for microbial control of various Lepidoptera spp. To date, there are three registered preparations of NPV that are exempt from the requirement of tolerance by the Environmental Protection Agency (EPA) in the United States (US). The first and only commercially available viral preparation used in agriculture was developed by Sandoz, Inc. under the name of Elcar® for control of Heliothis spp. on cotton. The other two baculovirus preparations were developed and registered by the US Department of Agriculture (USDA) for control of Douglas-fir tussock moth and gypsy moth on forests. Several methods are being used for production of NPV viruses: (1) field collection of diseased larvae, (2) laboratory rearing of insects followed by infection with viral inoculum, (3) tissue culture. and (4) tissue culture and mass rearing larvae. Recent progress in mass production of insect virus points toward the adoption of tissue culture with the whole organism technology for production of a standardized viral product. The practical usefulness of various baculovirus preparations has been demonstrated for protection of forests from defoliation by various lepidopterous species. In agriculture, Elcar® has been successfully marketed and has been very well received for use in integrated pest management on cotton. Recent development also demonstrated that use of adjuvants further increase the efficacy of Elcar® against Heliothis spp. on cotton.     

Ability of transovarially and subsequent transstadially infected Ixodes hexagonus ticks to maintain and transmit Borrelia burgdorferi in the laboratory

In a previous study, transstadial and transovarial survival of Borrelia burgdorferi in Ixodes hexagonus and transmission to laboratory mice via the bite of infected females were demonstrated. Here, we report the ability of I. hexagonus progeny infected transovarially to maintain and transmit the spriochaete to the host.Ticks were examined for spirochaetes by direct immunofluorescence antibody test. I. hexagonus larvae derived from the parental transstadially infected females were fed on two white mice: 21/54 (38.9%) of these ticks examined as unfed nymphs were infected. I. hexagonus nymphs were fed on three white mice and examined for spirochaetes after moulting as adults: 7/25 (28%) were found to harbour the spirochaete. The success of B. burgdorferi transmission to the mice by larval and nymphal I. hexagonus was determined by xenodiagnosis using I. ricinus larvae: 20/50 (40%) and 30/99 (30.3%) of the I. ricinus larvae fed on the mice infected by I. hexagonus larvae and nymphs respectively became infected.This study shows that B. burgdorferi can be maintained through transovarial and subsequent transstadial transmissions in I. hexagonus.