Cyclic and non-cyclic dynamics in populations of the helminth parasites of wood mice, Apodemus sylvaticus

Two populations of Apodemus sylvaticus, one in deciduous woodland and another in coniferous forest in Co. Down, Northern Ireland, were trapped at monthly intervals over 33 months. The dynamics, reproduction and age structure of the mouse populations differed between years with over a 40 fold range of population size. Nine species of helminths were recovered from the alimentary tract and associated organs of the mice. The prevalence and mean worm burden of the cestodes, Catenotaenia lobata, Hymenolepis hibernia and Taenia taeniaeformis varied erratically. The trematodes, Corrigia vitta and to a lesser degree Brachylaima recurva, had seasonal changes in abundance. The nematodes Capillaria murissylvatici and Nematospiroides dubius had strongly seasonal cycles of abundance associated with the onset of host reproduction. Trichuris muris was the rarest helminth. Despite protracted periods of absence this species maintained a seasonal increase in mean worm burden in late winter. The nematode, Syphacia stroma, had a weakly seasonal pattern of dynamics. The bases of these cyclical and non-cyclical dynamics of the helminths of A. sylvaticus are discussed.     

Helminth infections in Apodemus sylvaticus in southern England: interactive effects of host age, sex and year on the prevalence and abundance of infections.

Helminth parasites were studied in the wood mouse, Apodemus sylvaticus, in southern England in September of each of four successive years (1994-1997). Nine species of helminths were recorded: five nematodes (Heligmosomoides polygyrus, Syphacia stroma, Pelodera strongyloides, Trichuris muris, Capillaria murissylvatici), two cestodes (Microsomacanthus crenata, Taenia taeniaeformis) and two trematodes (Corrigia vitta, Brachylaemus recurvum). In total, 134 mice were examined and 91.8% carried at least one species of helminth. The majority of mice carried two to three species (60.5%) and the highest combination was six of the nine species recorded in the study. The patterns of between-year variations in the prevalence and abundance of infection were different for each of the six species for which sufficient quantitative data were available to enable statistical analysis. For H. polygyrus, the most important source of variation arose from between-year differences, host age and the interaction of these factors: abundance increased with host age but in 1995 the age pattern was markedly different from that in the remaining years. The abundance of C. vitta also varied significantly between years but additionally there was a strong independent age effect. For M. crenata, the year x age interaction was significant, indicating that abundance among different age cohorts varied from year to year but there was also a weak significant main effect of age arising from the youngest age cohort carrying no parasites and the oldest age cohort the heaviest infections. For P. strongyloides the only significant factor was between-year variation with 1995 being a year of exceptionally low prevalence and abundance of infection. No significant between-year variation was detected for S. stroma but there was a strong sex effect (males carrying heavier infections) and an age effect (older mice of both sexes carrying heavier infections). The abundance of Trichuris muris varied only in relation to host age, worm burdens growing in intensity with increasing age, but there was also a significant interaction between year and host sex with respect to prevalence. For the remaining three species, the prevalence of infections was too low (< 8.2%) to enable any meaningful interpretation. This analysis emphasizes the need for carefully controlled statistical procedures in aiding the interpretation and the prioritization of the factors affecting worm burdens in wild rodents.     

The trophic impact of small mammals in successional grasslands

The habitat use and diets of small mammals inhabiting grassland plots of different successional ages were investigated by live-trapping and faecal analysis over a period of 16 months. The contribution of the major plant life forms and the structural profile of the vegetation of each plot and the availability of insect prey were assessed. The dominant species of small mammal found on each plot were Sorex araneus, S. minutus, Apodemus sylvaticus and Microtus agrestis . Small mammals were most commonly found in the late and mid-successional stages, reflecting the habitat structure and food availability. A wide range of insects and other invertebrates were eaten by all four species and insects formed 50–62% of the animal prey taken. The incidence of certain prey in the diets differed between plots. The daily consumption rate of invertebrates by shrews and mice was estimated at some 6800 prey per ha. The greatest predatory impact came from shews but A. sylvaticus showed increased consumption of invertebrates in spring. The combined predatory impact of the grassland small mammal community on insect populations alone was estimated to average 0.01% per day and is predicted to be greatest in the mid-successional stages.     

Genetic and morphological heterogeneity in small rodent whipworms in southwestern Europe: characterization of Trichuris muris and description of Trichuris arvicolae n. sp. (Nematoda: Trichuridae)

Genetic and morphological variability of whipworms Trichuris Roederer, 1761 (Nematoda: Trichuridae), parasites of small rodents in southwestern Europe, was studied. Isozyme patterns of natural populations of nematodes parasitizing rodent species of the Muridae (Apodemus sylvaticus, Apodemus flavicollis, Mus musculus) and Arvicolidae (Clethrionomys glareolus, Microtus agrestis, Microtus arvalis) were analyzed at 6 putative loci. Two diagnostic loci were found in T. muris from Muridae and from Arvicolidae. Thus, the existence of 2 species of Trichuris restricted to different host families was indicated. They included Trichuris muris Schrank, 1788, originally described as being from mice, and Trichuris arvicolae n. sp., parasitizing the above species of Arvicolidae. The morphological variability of both species was compared. Although ranges of all morphological characters of the new species overlapped with those of T. muris, stepwise discriminant analysis yielded a 100% accurate classification of females when using vagina length and egg size. Males of T. muris and T. arvicolae cannot be separated entirely. A set of 6 variables yielded 95.7% discrimination; the most discriminating variables were spicule size and body width.     

Impairment of primary expulsion of Trichuris muris in mice concurrently infected with Nematospiroides dubius

Primary immune expulsion of Trichuris muris was markedly delayed by concurrent infection with Nematospiroides dubius. Maximum delay of expulsion was dependent on size and timing of N. dubius infection relative to T. muris infection. In NIH mice infection with 400 N. dubius larvae immediately before or after T. muris infection was found to be most effective in suppressing expulsion. Infection on day 8 of T. muris infection, when mice are sensitized to T. muris, also impaired expulsion. From this evidence it is suggested that the larvae of N. dubius are immunosuppressive and that the efferent role of the immune response to T. muris is inhibited. The results are discussed in terms of non-specific immunosuppression and their relevance to the tropical disease situation is emphasized.     

Developmental modulation of immunity: changes within the feeding period of the fifth larval stage in the defence reactions of Manduca sexta to infection by Photorhabdus

In insect pathogen interactions, host developmental stage is among several factors that influence the induction of immune responses. Here, we show that the effectiveness of immune reactions to a pathogen can vary markedly within a single larval stage. Pre-wandering fifth-stage (day 5) larvae of the model lepidopteran insect Manduca sexta succumb faster to infection by the insect pathogenic bacterium Photorhabdus luminescens than newly ecdysed fifth-stage (day 0) caterpillars. The decrease in insect survival of the older larvae is associated with a reduction in both humoral and cellular defence reactions compared to less developed larvae. We present evidence that older fifth-stage larvae are less able to over-transcribe microbial pattern recognition protein and antibacterial effector genes in the fat body and hemocytes. Additionally, older larvae show reduced levels of phenoloxidase (PO) activity in the cell-free hemolymph plasma as well as a dramatic decrease in the number of circulating hemocytes, reduced ability to phagocytose bacteria and fewer melanotic nodules in the infected tissues. The decline in overall immune function of older fifth-stage larvae is reflected by higher bacterial growth in the hemolymph and increased colonization of Photorhabdus on the basal surface of the insect gut. We suggest that developmentally programmed variation in immune competence may have important implications for studies of ecological immunity.     

Population regulation in ticks: the role of acquired resistance in natural and unnatural hosts

Attachment, engorgement and subsequent development of successive infestations of Ixodes trianguliceps larvae and nymphs on natural hosts, Apodemus sylvaticus, and unnatural hosts, laboratory mice, are compared. In laboratory mice, primary infestations above a threshold level of about 10 ticks elicit an immunological response which reduces, in a density-dependent manner, the rate of successful tick engorgement during subsequent infestations. In contrast, in A. sylvaticus successive infestations of larvae result in unchanged or slightly improved survival through to nymphs. The relevance of these results to the concept of host-parasite co-evolution and to tick population regulation is discussed.     

Time-course of Giardia muris infection in male and female immunocompetent mice

The time-course of acute Giardia muris infection was compared in male and female immunocompetent BALB/c mice that had not previously been exposed to the parasite. No sex-related difference was observed in the time-course of the infection in these mice. Sexually mature mice of both sexes excreted substantial numbers of G. muris cysts (greater than 10(4)/2 hr) over a longer period than did sexually immature mice.     

Costs of cannibalism in the presence of an iridovirus pathogen of Spodoptera frugiperda

Abstract.  1. The costs of cannibalism were examined in larvae of Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) in the presence of conspecifics infected by a lethal invertebrate iridescent virus (IIV). The hypothesis of a positive correlation between insect density and the likelihood of disease transmission by cannibalism was examined in laboratory microcosms and a field experiment.
2. Transmission was negligible following peroral infection of early instars with purified virus suspensions or following coprophagy of virus-contaminated faeces excreted by infected insects. In contrast, 92% of the insects that predated infected conspecifics acquired the infection and died prior to adult emergence in the laboratory. Diseased larvae were more likely to be victims of cannibalism than healthy larvae.
3. The prevalence of cannibalism was density dependent in laboratory microcosms with a low density (10 healthy insects + one infected insect) or high density (30 healthy insects + one infected insect) of insects, and field experiments performed on maize plants infested with one or four healthy insects + one infected insect.
4. Cannibalism in the presence of virus-infected conspecifics was highly costly to S. frugiperda ; in all cases, insect survival was reduced by between ≈ 50% (laboratory) and ≈ 30% (field) in the presence of the pathogen. Contrary to expectations, the prevalence of disease was not sensitive to density because cannibalism resulted in self-thinning. As infected individuals are consumed and disappear from the population, the prevalence of disease will be determined by the timescale over which transmission can be achieved, and the rate at which individuals that have acquired an infection become themselves infectious to conspecific predators.     

Intestinal helminths infection of rats (Ratus norvegicus) in the Belgrade area (Serbia): the effect of sex, age and habitat

SUMMARY: Gastrointestinal helminths of Norway rat (Rattus norvegicus) from the Belgrade area were studied as a part of a wider ecological research of rats in Serbia (data on the distribution, population ecology, economic and epizoothiological-epidemiological importance, and density control). Rats were captured from May 2005 to July 2009 at both urban and suburban-rural sites. Of a total of 302 trapped rats 48% were males and 52% females, with 36.5% and 38.8% of juvenile-subadult individuals, per sex respectively. Intestinal helminth infection was noted in 68.5% of rats, with a higher prevalence in male hosts and in adult individuals. Higher numbers of infected juveniles-subadults were noted in suburban-rural habitats, while an opposite tendency was noted in adult rats. Seven helminth species were recovered, of which five were nematode (Heterakis spumosa, Nippostrongylus brasiliensis, Capillaria sp., Trichuris muns and Syphacia muris) and two cestode species (Hymenolepis diminuta and Rodentolepis fraterna). The most prevalent parasites were Heterakis spumosa (36.7%) and Hymenolepis diminuta (30.5 %). Sex and habitat-related differences were noted in the prevalence of infection with Capillaria sp. and Trichuris muris, while there were no age-related differences in the prevalence of infection with any individual helminth species. Significantly higher prevalence of infection was noted in summer as compared to spring or winter, with a tendency to be higher in autumn as compared to spring. The only significant difference in the prevalence of infection between habitat-related was noted during spring. H. spumosa was most prevalent in summer, while H. diminuta and N. brasiliensis in autumn. The mean intensity of infection with H. spumosa, R. fraterna, S. muris and T muris was higher in autumn than in the other seasons, while N. brasiliensis and Capillaria sp. occured in winter. No more than four helminth species were found in one host.     

Asynchrony in larval development of the pine beauty moth, Panolis flammea, on an introduced host plant may affect parasitoid efficacy

In the United Kingdom, Panolis flammea (Den. and Schiff.) (Lepidoptera: Noctuidae) is an important pest species of the introduced lodgepole pine but not of its natural host Scots pine. The timing of P. flammea larval growth must be synchronized with its host tree if the larvae are to succeed. We collected field data during 1990 which revealed that the phenological window starts earlier in Scots pine and is shorter than that observed in lodgepole pine. The larvae are found in the field earlier and within a narrower time frame within a Scots pine forest than in a lodgepole pine forest. The larval developmental period is significantly longer on lodgepole pine than on Scots pine. The synchrony/asynchrony of P. flammea to its natural host (Scots pine) and an introduced tree (lodgepole pine) results in the parasitoids having a different impact on the larvae of the two hosts. At any one time, the host plant, caterpillars and parasitoids are more synchronous on the ancestral Scots pine than on lodgepole pine, resulting in a higher percentage of larvae in the optimal instar for parasitism at that time. In lodgepole pine, the percentage of suitable instars available to parasitoids is lower at any given time. The information presented here furthers our understanding of the possible mechanisms for the observed differential population dynamics of the insect on Scots pine and lodgepole pine in the UK. Handling editor: Robert Glinwood.     

Microbiological monitoring in inbred mouse foundation stocks in Japan

Microbiological monitoring on 128 inbred mouse foundation stocks consisted of common 10 inbred strains and inbred strains originated from outbred dd mice was performed by cooperation of 24 organizations. A total of 881 mice were divided into 647 conventional animals from 95 colonies and 234 barrier-sustained animals from 33 colonies. Three viral, one mycoplasmal, 6 bacterial, one fungal and 3 parasitic agents selected as monitoring microbes according to the proposed selection standards. Among conventional colonies, 84.2% were positive for at least one agent. The highest detection rate was 44.2% for S. obvelata, followed by P. pneumotropica and S. muris, P. aeruginosa, G. muris, Sendai virus, M. pulmonis, MHV and E. coli O115a, c: K (B). Of these agents, only one microbe, P. aeruginosa, was detected in barrier-sustained colonies (36.4%), thus the efficacy of barrier system for the microbiological quality control of the inbred mouse foundation stocks was actually demonstrated. The positive rates of MHV (6.3%) and Sendai v. (16.8%) were significantly low compared with those in experimental mouse colonies. Positivity for parasites was rather high and they were infested together with other pathogens in many cases. Thus parasites including G. muris, S. muris and S. obvelata were regarded as useful indicators to see microbiological contaminations in conventional mice. There observed no strain difference in susceptibility to pathogens except for C57BL/6 and AKR mice which seemed to be high antibody responders to MHV.     

A study of the internal parasites of small rodents from woodland areas in Surrey

The occurrence of helminth parasites and leptospires from the Long-tailed field mouse, Apodemus sylvaticus , the Bank vole, Clethrionomys giareolus and the Short-tailed vole, Microtus agrestis has been studied from four woodland areas in Surrey. The composition of the helminth fauna of these rodents, particularly A. sylvaticus and C. giareolus , differs from that of previous studies carried out in the British Isles in consisting largely of cestodes and fewer species of digeneans and nematodes. On the other hand the degree of infection of the rodents with nematodes as in the case of A. sylvaticus infected with the trichostrongylid Nematospiroides dubius and theoxyurid Syphacia stroma and of C. giareolus with thecap-illarid Capillaria muris sylvatici is greater than the cestode and digenean infections and is probably linked with the monoxenous nature of the nematode life cycles.
Differences in the number, incidence and degree of infection of mice and voles with species of helminths is undoubtedly linked with differences in the feeding habits of the respective hosts but the present study has also confirmed that infection levels in both leptospires and helminths are influenced by the age/sex relationships of the host together with season and habitat. The relationship between the distribution of rodents infected with leptospires and some helminths suggests similarities in the mode of their transmission.     

转双抗虫基因741杨的抗虫效应及其规律研究

通过室内饲虫试验 ,应用昆虫的总死亡率、各龄期死亡率、各龄历期及发育速率、昆虫体重增长速率等指标 ,测定了转双抗虫基因 74 1杨的抗虫性。研究结果表明 ,转基因抗虫杨不同株系对昆虫的致死效应有显著差异 ;不同昆虫种类和同一昆虫幼虫的不同龄级对转基因杨株系的敏感性有明显差异 ,4种测试昆虫的敏感性强弱依次为 :杨小舟蛾 >美国白蛾 >杨扇舟蛾 >舞毒蛾 ,随着昆虫幼虫龄级的增加 ,昆虫对转基因杨株系的敏感性逐渐降低 ;高抗虫株系主要表现为对低龄幼虫的强致死作用 ,中等抗虫株系表现为对各龄幼虫稍低的但是持续的致死效应 ;各转基因杨株系对测试昆虫的生长和发育都有明显抑制作用 ,可导致昆虫的发育历期延长、发育速率和体重增长速率降低。     

Chronic Giardia muris infection in anti-IgM-treated mice. I. Analysis of immunoglobulin and parasite-specific antibody in normal and immunoglobulin-deficient animals

To investigate the role of B cells and antibody in the immune response of mice to the murine intestinal parasite Giardia muris, we used mice treated from birth with rabbit anti-IgM antisera (aIgM). Such mice developed in serum and in gut secretions extreme Ig deficiency (IgM, IgA, and IgG) relative to control animals. The aIgM-treated mice showed no anti-G. muris antibody in serum or in gut wash material. Infections of G. muris in these mice were chronic, with a high load of parasite present in the small bowel, as reflected by prolonged cyst excretion (greater than 11 wk) and high trophozoite counts. In contrast, normal, untreated mice or NRS-treated animals developed anti-parasite IgA and IgG antibody in serum, demonstrated IgA antibody against the parasite in gut washings, and expelled the parasite within 9 wk. These effects of aIgM treatment on the murine response to primary infection with G. muris were demonstrated in two strains of mice: BALB/c and (C57BL/6 X C3H/He) F1. It was also observed that the response to G. muris infection in untreated animals was characterized by higher than normal total secretion of IgA into the gut and a concomitant increase in the serum polymeric IgA level. Mice treated with aIgM had a marked decrease of both monomeric and polymeric IgA in serum, and little detectable IgA in the intestinal lumen. These experiments provide the first demonstration that anti-IgM treatment suppresses a specific intestinal antibody response to antigen, and provide evidence that B cells and antibody play a role in the development of an effective response to a primary infection with G. muris in mice.     

Intestinal parasites of conventionally maintained BALB/c mice and Mastomys coucha and the effects of a concomitant schistosome infection

A longitudinal study was carried out to identify the spectrum of intestinal parasites present in conventionally maintained BALB/c mice and Mastomys coucha and to determine the effects of concomitant schistosome infections on their parasite status. Six parasites were observed during the course of the study, namely the nematodes Aspiculuris tetraptera and Syphacia obvelata, Entamoeba muris and the flagellates Trichomonas muris, Spironucleus muris and Chilomastix spp. Although the 2 rodents shared common facilities, the overall prevalences of S. obvelata, T. muris and S. muris were significantly higher in M. coucha than BALB/c mice. BALB/c mice with concomitant schistosome infection had increased prevalences of E. muris, T. muris and S. muris. In M. coucha, in contrast, there were no significant increases in parasite prevalences. Infection intensities of T. muris and S. muris were significantly greater in M. coucha than BALB/c mice. Concomitant schistosome infection resulted in increased intensities of T. muris infection in BALB/c mice only. The influence of immune status in determining the susceptibilities of rodents to environmentally transmitted parasites is discussed.     

Seasonal variations in infestations of parasites in a Wood mouse Apodemus sylvaticus population in the West of Ireland

Seasonal variation in most of the metazoan parasites of a Wood mouse population was investigated by a statistical analysis of their frequencies in 11 samples of about 30 mice trapped regularly over one year. Numbers of the flea Ctenophthalmus nobilis , most mesostigmatid mites and the beetle Leptinus testaceus were correlated with breeding in the mouse population and may have been directly linked with it as all ofthese arthropods are common in Wood mouse nests. The tick Ixodes ricinus was abundant over the summer but scarce in winter. Its preponderance on male hosts is doubtless because they cover more ground than females. Flukes were commonest in summer, probably as a result ofthe period of intensive feeding on invertebrates by the mice which usually occurs in early summer. Female mice were more heavily infected, perhaps because of their increased food requirements in the breeding season. Abundance of flukes declined with the turnover in the mouse population in autumn. The infestation pattern of the nematode Capillaria muris sylvatici is similar and the possibility of an invertebrate vector is discussed. While older mice were generally more heavily infected with helminths than were young, this was least marked in the tapeworm Cataenotaenia lobata (which most likely has a mite as an intermediate host and may be picked up as soon as juveniles start grooming) and the roundworm Syphacia stroma (eggs of which are almost certainly transferred by body contact between hosts)     

Tick-borne zoonotic bacteria in wild and domestic small mammals in northern Spain

The prevalence and diversity of tick-borne zoonotic bacteria (Borrelia spp., Anaplasma phagocytophilum, Coxiella burnetii, and spotted fever group rickettsiae) infecting 253 small mammals captured in the Basque Country (Spain) were assessed using PCR and reverse line blot hybridization. Trapping sites were selected around sheep farms (study 1, 2000 to 2002) and recreational parks (study 2, 2003 to 2005). The majority of the studied mammals (162) were wood mice (Apodemus sylvaticus), but six other different species were also analyzed: yellow-necked mice (Apodemus flavicollis), shrews (Crocidura russula and Sorex coronatus), bank voles (Clethrionomys glareolus), domestic mice (Mus domesticus), and moles (Talpa europaea). The results showed an infection rate ranging from 10.7% to 68.8%, depending on the small mammal species. One C. russula shrew and one A. sylvaticus mouse gave positive reactions for A. phagocytophilum, and C. burnetii was detected in two domestic mice and one A. sylvaticus mouse in a farm. The DNA of Borrelia spp. was detected in 67 animals (26.5%), most of them presenting positive hybridization with the probe for Borrelia sp. strain R57, the new Borrelia species previously detected in small mammals in our region. Furthermore, a second PCR and reverse line blot hybridization specific for B. burgdorferi sensu lato revealed the presence of Borrelia afzelii in 6.3% of C. glareolus voles and 14.3% of S. coronatus shrews. All small mammals were negative for spotted fever group rickettsiae. These results highlight the relevance of small mammals as reservoirs of some zoonotic bacteria.     

Does the ‘investment principle’ model explain moulting strategies in lepidopteran larvae?

Abstract.  The aim of the present study was to evaluate the applicability of a general optimality model of insect development in the case of lepidopteran larvae. According to the model, larvae moult mainly to increase the size of mouthparts, which is assumed to limit consumption rates. The assumptions of this model being met, one should expect a dependence of growth rates on head capsule size, constancy of absolute growth rates within an instar and higher growth rates in older instars. The validity of these predictions is tested on two species of lymantriid moths. Head capsule size has only a weak effect on growth rates in one of the species, and no effect in the other. Absolute growth rates tend to increase during the development within an instar. Higher growth rates of older instar larvae are explainable by an allometric relationship that extrapolates from growth within the preceding instar. Thus, there is no evidence of an extra increase in growth performance attributable to moulting into the subsequent instar. These results support the idea that growth rates of lepidopteran larvae are limited by nutrient absorption rates rather than by the rates of consumption. There is a considerable cost of moulting in terms of lost growing time: the larvae would double their weights during an instar if they were able to develop without the premoult decrease in growth rates. The investment principle model does not appear to be directly usable to explain moulting strategies in the lepidopteran larvae studied. However, it may well be applicable after some adjustment (i.e. assuming that larvae should moult before consumption rates become limiting).     

Response of Peyer's patch lymphocyte subsets to Giardia muris infection in BALB/c mice. I. T-cell subsets

Initial cellular events in the intestinal immune response occur within Peyer's patches and are subject to complex regulation by T cells. The aim of this study was to analyze the response of murine Peyer's patch T, T-helper (Th), and T-suppressor (Ts) cells to Giardia muris infection. Immunocompetent BALB/c mice were infected with G. muris cysts and, at serial times during the infection, Peyer's patch leukocyte suspensions were incubated with fluorescent monoclonal antibodies that identified murine leukocytes, T, Th, or Ts lymphocytes. These suspensions were examined by flow cytometry to quantify each T-cell subset as a percentage of total leukocytes. Total Peyer's patch leukocytes more than doubled in number during the course of G. muris infection and returned to control levels as the infection was cleared. The percentages of Peyer's patch Th and Ts lymphocytes were 34.1 +/- 0.8% (mean +/- SEM) and 6.2 +/- 0.3%, respectively, in the absence of infection, and did not change significantly during Giardia infection. The Th/Ts ratio in Peyer's patches was 5.6 +/- 0.2 in uninfected BALB/c mice and also did not change significantly during clearance of G. muris. We conclude that Peyer's patch leukocytes double in number in response to G. muris infection in immunocompetent mice, G. muris infection does not lead to altered percentages of Peyer's patch T, Th, or Ts lymphocytes, and clearance of G. muris infection is associated with a Peyer's patch Th/Ts ratio of greater than 5.