Major differences in the larval anatomy of the digestive and excretory systems of three Oestridae species revealed by micro-CT.

Oestrid flies (Diptera: Oestridae) do not feed during the adult stage, so they depend on an efficient assimilation and storage of nutrients during their parasitic larval stage. We describe the general morphology and provide volumetric data for the digestive and excretory organs of the three larval instars of the nasal bot fly Oestrus ovis L., using micro-computed tomography. The size of the digestive and excretory organs greatly increased across larval instars. In all instars, the two salivary glands were remarkably large and formed a 'glandular band' by coming together, but without lumina uniting, at their posterior ends. The distal region of the anterior Malpighian tubules was greatly enlarged and full of highly radio-opaque concretions. Moreover, the anatomy of O. ovis third-instar larva was compared to that of two species of, respectively, similar and different feeding habits: Cephenemyia stimulator (Clark) and Hypoderma actaeon Brauer. Whereas the general morphology and arrangement of the digestive and excretory systems of C. stimulator was similar to that of O. ovis, some differences were observed in H. actaeon: a swollen anterior region of the midgut, salivary glands shorter and not forming a 'band' and anterior Malpighian tubules narrowly uniform throughout their entire length.

Internal morphological changes during metamorphosis in the sheep nasal bot fly, Oestrus ovis.

During the larval stage, oestrid flies (Diptera: Oestridae) are obligate parasites, whereas during the adult stage they are free-living and do not feed. Like other cyclorrhaphous flies, oestrids undergo metamorphosis inside an opaque puparium, formed by the contracted and hardened cuticle of the third-instar larva. The present study documents the internal morphological changes taking place during metamorphosis of the sheep nasal bot fly, Oestrus ovis L., using non-invasive, micro-CT-based virtual histology and provides quantitative data of volumetric changes in specific organs. Virtual histological sections allowed visualisation of the progression and completion of the apolyses, which delimit the different intra-puparial stages, and the connection to the tracheal system of a large gas bubble, which plays an essential role during early metamorphosis. Overall, our results show that the sequence of morphological and volumetric changes in tissues and organs is similar to those found in other cyclorrhaphous flies, but they also reveal developmental differences that result in an adult vestigial digestive tract. Future studies could develop non-invasive, reliable methods for aging the intra-puparial forms of different oestrid species of veterinary importance, based on both qualitative and quantitative markers, thus improving our knowledge of their development and the efficiency of control strategies.

Micro-computed tomography visualization of the vestigial alimentary canal in adult oestrid flies.

Oestrid flies (Diptera: Oestridae) do not feed during the adult stage as they acquire all necessary nutrients during the parasitic larval stage. The adult mouthparts and digestive tract are therefore frequently vestigial; however, morphological data on the alimentary canal in adult oestrid flies are scarce and a proper visualization of this organ system within the adult body is lacking. The present work visualizes the morphology of the alimentary canal in adults of two oestrid species, Oestrus ovis L. and Hypoderma lineatum (de Villiers), with the use of non-invasive micro-computed tomography (micro-CT) and compares it with the highly developed alimentary canal of the blow fly Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae). Both O. ovis and H. lineatum adults showed significant reductions of the cardia and the diameter of the digestive tract, an absence of the helicoidal portion of the midgut typical of other cyclorrhaphous flies, and a lack of crop and salivary glands. Given the current interest in the alimentary canal in adult dipterans in biomedical and developmental biology studies, further understanding of the morphology and development of this organ system in adult oestrids may provide valuable new insights in several areas of research.

The immunobiology of myiasis infections--whatever happened to vaccination?

The current state of myiasis vaccine technologies are reviewed mainly in the primary research genera of Lucilia and Hypoderma. The importance of myiasis flies as primary causes of morbidity and mortality in agricultural species and man has not diminished despite the existence of good control strategies. However, the development of vaccines against myiasis infections has been relatively quiescent for more than 10 years despite the rapid development of genomic and proteomic analysis and of skills in data interpretation. The value of vaccine research in an era of chemical primacy is analysed. In fact, recent findings of drug resistance and the impact of animal welfare concerns should mean a renewed interest in alternative controls. The reasons that this has not been true to date are explored and new possibilities discussed.

Life history parameters of the cattle long-nosed sucking louse, Linognathus vituli.

Cattle sucking lice, Linognathus vituli (L.) (Phthiraptera: Linognathidae), were obtained from naturally infected cattle and maintained within 'arenas' affixed to the backs of cattle confined in controlled environment chambers maintained at a constant temperature of 15 °C. Temperatures measured within the arenas at an ambient temperature of 15 °C were constant at about 34 °C and only slightly above the temperature on nearby skin. The effect of temperature on egg development was determined using a gradient of temperatures between 25 °C and 41 °C. Eggs did not develop at temperatures of < 26 °C or > 39 °C. Survival of eggs was highest at temperatures of 30 °C and 35 °C. The earliest hatch was observed at 5 days post-oviposition (at 33-35 °C). Development was extended to as long as 13 days at the lower temperatures. Kaplan-Meier survival probabilities were compared for lice kept at two densities in the arenas and showed there to be no effect of density on louse survival. Similarly, the mean number of eggs/louse/day over an 8-day period was not influenced by louse density.

A look into the Medical and Veterinary Entomology crystal ball.

Medical and Veterinary Entomology (MVE) represents a leading periodical in its field and covers many aspects of the biology and control of insects, ticks, mites and other arthropods of medical and veterinary importance. Since the first issue of the journal, researchers working in both developed and developing countries have published in MVE, with direct impact on current knowledge in the field. An increasing number of articles dealing with the epidemiology and transmission of vector-borne pathogens have been published in MVE, reflecting rapid changes in vector distribution, pathogen transmission and host-arthropod interactions. This article represents a gaze into the crystal ball in which we identify areas of increasing interest, discuss the main changes that have occurred in the epidemiology of parasitic arthropods since the first issue of MVE, and predict the principal scientific topics that might arise in the next 25 years for scientists working in medical and veterinary entomology.

The mitochondrial genome of the common cattle grub, Hypoderma lineatum.

The mitochondrial DNA of the cattle grub Hypoderma lineatum (de Villers) (Diptera: Oestridae) was completely sequenced. The entire molecule was 16,354 bp long and presented a heavy bias towards A + T, which accounted for 77.8% of the whole genome. Hypoderma lineatum genes were organized in the same order and orientation as in the mitochondrial genomes available for other species belonging to the Oestroidea superfamily and compared in this study [Chrysomya putoria (Wiedemann), Cochliomyia hominivorax (Coquerel), Lucilia sericata (Meigen) and Dermatobia hominis (L.)], except for the occurrence of a 102-bp non-coding region partially present in other species. The complete sequence of H. lineatum will represent a useful dataset to evaluate the evolutionary pattern of mtDNA within Oestroidea by using molecular information in diagnostic, taxonomic and evolutionary studies.

Biodiversity and extinction versus control of oestrid causing myiasis in Mediterranean area.

Oestrid larvae causing myiasis display a wide degree of biodiversity, in terms of species of domestic and wild mammals infected and anatomical sites. The presence in some regions of southern Europe of a high number of different species of oestrids in domestic animals stimulated interest in exploring the basis of such degree of parasitic biodiversity in the Mediterranean region. However, broad spectrum anti-parasitic treatments (e.g. macrocyclic lactones) constitute a critical factor for the selection of species of Oestrids and for the maintenance of their biodiversity in a given area. The dynamic equilibrium that oestrid larvae have established with the host and the environment as well as the span of biodiversity they represent may be considered to be at odds with maintaining animal welfare and reducing animal production losses.

Evaluation of an antigen capture ELISA for the early diagnosis of Hypoderma lineatum in cattle under field conditions.

An antigen capture or sandwich ELISA (sELISA) was evaluated for the diagnosis of Hypoderma lineatum in cattle under field conditions in northwestern Spain. The kinetics of circulating hypodermin C (HyC) and specific antibodies during the course of natural infestation were determined in a group of 10 Frisian calves. In addition, oesophagi and blood samples were taken from 105 cows at a slaughterhouse in order to compare three methods for the diagnosis of H. lineatum: sandwich ELISA for the detection of the antigen HyC (sELISA), indirect ELISA for the detection of antibodies anti-HyC (iELISA) and the detection of first instars (L1) in the oesophagus. In naturally infested cattle, HyC was present in circulation at low levels during the early and late phases of the infestation. However, in the middle phase, coinciding with the presence of L1 in the oesophagus, two peaks of increased HyC concentration were observed. Specific antibodies increased progressively until the first appearance of larvae in warbles on the back. There was no correlation between antigen or antibody levels and the number of grubs in the back. Prevalence of first instars in the oesophagi of slaughtered cows was 21.9% (23/105). The percentage of cattle that were positive for circulating antigen was slightly higher (24.8%), suggesting the recent destruction of migrating larvae in some animals. However, there was no correlation between the number of L1 and HyC levels. With the iELISA, 79% of the animals were positive to Hypoderma, which means that a high percentage of those animals have been exposed to the parasite but they had no apparent current infestation. The sELISA is a good tool to follow larval development within the host; however, the episodic elevation of HyC levels limits the usefulness of this test for the early diagnosis of Hypoderma under field conditions.

Control of the cattle louse Bovicola bovis with the fungal pathogen Metarhizium anisopliae.

The effects of the entomopathogenic fungus Metarhizium anisopliae (Metschnikoff) were evaluated against the common louse parasite of cattle, Bovicola bovis (Piaget) (Trichodectidae: Ischnocera). Two different concentrations and formulations of conidial suspensions were applied to contained populations of adult female lice. In vitro, lice immersed in suspensions of M. anisopliae formulated in 0.03% Tween 80 developed infections; at the highest concentration (1x10(8)conidia ml-1) a mean of 71% (+/-11.52%, 95% C.I.) of lice became infected. Lice exposed to the Tween 80 only in vitro, showed high levels of survival and zero infection. In vivo, fungal conidia were applied to louse populations contained in 7 cm diameter circular arenas glued to the backs of Holstein cattle, maintained in controlled climate conditions. Conidia were formulated in either Tween 80 or silicone oil. The treatment with M. anisopliae resulted in high levels of infection and there was no overall difference between the two formulations in the number of infections observed. At the highest concentration (1x10(8)conidia ml-1) a mean of 73% (+/-15.57%, 95% C.I.) lice became infected. It is concluded that the strategic seasonal use of a fungal pathogen on cattle, applied in early winter, may be of value in suppressing the winter increase in abundance, preventing the population increasing to clinically significant levels.

Skin immune responses in cattle after primary and secondary infections with Hypoderma lineatum (Diptera: Oestridae) larvae.

Cellular immune responses were examined in the skin of cattle after primary and secondary experimental infections with Hypoderma lineatum larvae. Skin biopsies were taken at 0, 6, 12, 48 and 96 h post-infection (h.p.i.). In primary infected animals the penetration of Hypoderma larvae was characterized by moderate inflammatory responses. The pattern of cellular changes in previously infected animals suggested an allergic or rheumatic process, probably as a consequence of the development of a type III hypersensitivity reaction against the second or successive reinfections. Perivascular infiltration with CD3(+) T lymphocytes was marked in infected groups, but especially in previously infected animals, with a significant increase with respect to uninfected controls at 48 h.p.i. B cells remained close to control values during primary infection, while they increased significantly 12 h.p.i. in reinfected animals. IgG(+) plasma cells were also very abundant during secondary infections, with significant differences from primary infected and uninfected animals from 6 h.p.i. onwards. These results suggest that secondary infections allow cellular responses which may be effective in killing some of the entering larvae, resulting in a degree of resistance.

Parasites and behavior: an ethopharmacological analysis and biomedical implications.

Parasites and disease are increasingly recognized as agents of behavioral, ecological and evolutionary importance having a variety of influences on their hosts other than the more obvious pathological and immunological changes. Parasites can have significant behavioral effects even when parasitism is sub-clinical with these effects proposed to either benefit the parasite (parasite 'manipulation'), benefit the host, or to simply arise as side-effects of the infection (parasitic 'constraints'). However, until relatively recently little attention has been paid to the neuromodulatory substrates that mediate these behavioral changes. Ethopharmacology incorporates an evolutionary approach to the study of behavior with pharmacological analysis of neuromodulatory mechanisms. As such, this approach is appropriate for, and has been applied to, the analysis of the effects of ectoparasites (e.g. biting and blood-feeding flies) and endoparasites (e.g. protozoa, nematodes) on a number of behaviors (e.g. pain inhibition, learning and memory, responses to predators and anxiety, mate selection) in selected host-parasite systems. Ethopharmacology suggests a promising direction by which neuromodulatory mechanisms that underlie the effects of parasites on behavior, including that of humans, can be addressed.

Impaired discrimination of and aversion to parasitized male odors by female oxytocin knockout mice.

A major cost of social behavior is the increased risk of exposure to parasites, with animals utilizing social information to recognize and avoid infected conspecifics. In mice, females can discriminate between infected and uninfected males on the basis of social cues, displaying aversive responses to the odors of infected males. In the present study, using female mice whose gene for oxytocin (OT) has been selectively deleted (OT knockout mice (OTKO)), we show that at least one normal allele for OT is required for the mediation of the recognition and avoidance of parasitized males. Female wild type (OTWT) and heterozygous (OTHZ) mice distinguished between the odors of individual males infected with the louse, Polyplax serrata, and uninfected males while the KO mice did not. Exposure to the odors of infected males induced analgesia in OTWT and OTHZ females, with OTKO females displaying attenuated analgesia. OTWT and OTHZ females, but not the OTKO females, also distinguished between the odors of novel and familiar infected males and modulated their analgesic responses on the basis of prior familiarity. In an odor choice test, OTWT and OTHZ females displayed a marked initial choice for the odors of uninfected males, whereas the OTKO females showed no consistent choice. This impairment was specific to the odors of infected males. OTKO females displayed normal analgesic responses to another aversive social odor, that of a stressed male, and an aversive non-social odor, that of a cat. The OTKOs had normal non-social olfactory memory, but were impaired in their social odor memory. These findings indicate that a normal OT gene comprises an essential part of the central recognition mechanism whereby females can both reduce the transmission of parasites to themselves and select for parasite-free males.

Discrimination by female mice between the odours of parasitized and non-parasitized males.

The detection and avoidance of parasitized males has been proposed to be a component of female mate choice. We investigated whether or not female laboratory mice, Mus musculus domesticus, could discriminate between parasitized and non-parasitized males on the basis of odour. Female mice were given a choice between the urine and other odorous secretions of either a male mouse sub-clinically infected for five days with the naturally occurring, enteric, single host, protozoan parasite, Eimeria vermiformis, or an uninfected male. Females showed a marked preference for the odours of non-parasitized male mice over those of the parasitized males; as measured by number of investigations, time spent per investigation, and total investigation time of the odours in a choice situation. Female mice also displayed an overwhelming initial, or first choice, preference for the odours of the non-parasitized male mice over those of the parasitized males. These observations show that female mice can distinguish between the odours of parasitized and non-parasitized males, and discriminate against parasitized males on the basis of odour. We suggest that the detection and avoidance of infected males by female mice through odour cues may function to reduce parasite transmission and potentially serve as a component of female mate selection or choice.

Parasitized female mice display reduced aversive responses to the odours of infected males.

The present study showed that parasites influence both the responses of uninfected females to males and the responses of female hosts to infected males. In female laboratory mice one of the consequences of exposure to the olfactory cues associated with an infected male was a reduction of the reactivity to a thermal surface, i.e. pain inhibition or analgaesia. Uninfected oestrous and non-oestrous female mice displayed marked analgaesic responses after exposure to the odours of males infected with either the enteric single-host nematode parasite, Heligmosomoides polygyrus, or the protozoan parasite, Eimeria vermiformis. The uninfected oestrous females distinguished between infected and physically stressed males, displaying a greater analgaesic response to the odours of infected males. These analgaesic responses and their anxiety/ fearfulness-associated behavioural correlates could elicit either a reduced interest in, or avoidance of, parasitized males by females. Oestrous female mice infected with H. polygyrus displayed a reduced analgaesic response to the odours of the infected males and differentially responded to the odours of males infected with either the same (H. polygyrus) or a different parasite (E. vermiformis). An exposure time of 1 min elicited minimal responses to the odours of males infected with the same parasite, H. polygyrus, and an attenuated, though significant, non-opioid peptide-mediated analgaesic response to males infected with E. vermiformis. An exposure time of 30 min elicited similar markedly reduced endogenous opioid peptide-mediated analgaesic responses to the odours of both of the categories of infected males. The responses to the odours of a stressed male were, however, unaffected by the parasitic infection. The reduced analgaesic responses of the parasitized females to the odours of infected males may involve either enhanced odour familiarity and responses to group odour templates and/or neuromodulatory shifts resulting in reduced fearfulness and potentially greater interest in the infected males.

Analgesic responses of male mice exposed to the odors of parasitized females: effects of male sexual experience and infection status.

The present study shows that parasites influence both the responses of males to infected females and the responses of male hosts to females. Male mice exposed for 30 min to the odors of females infected with the nematode parasite Heligmosomoides polygyrus displayed a naloxone-sensitive, opioid-mediated analgesia, whereas males exposed for 1 min showed a shorter duration and lower amplitude naloxone-insensitive "nonopioid" analgesia that involved serotoninergic (5-HT) and excitatory amino acid (N-methyl-D-aspartate [NMDA] receptor) systems. The male mice distinguished between the odors of infected and physically stressed females, displaying greater analgesia after exposure to the odors of infected than stressed females. The analgesic responses to the odors of infected females were also affected by the males' prior sexual experience; sexually experienced males exhibited significantly greater analgesia than sexually naive males. In contrast, male mice infected with H. polygyrus failed to show a nonopioid analgesia after exposure to the odors of infected females and displayed a markedly lower level of opioid analgesia than uninfected mice. These results show that male mice can discriminate between the odors of parasitized and nonparasitized females and find the odors of parasitized estrous females aversive.

Learning from others to cope with biting flies: social learning of fear-induced conditioned analgesia and active avoidance.

Although fear conditioning has received extensive attention, little is known about the roles of social learning whereby an individual may learn and acquire the fear responses of another. The authors examined individually and socially mediated acquisition of fear and analgesia to the natural aversive stimulus of biting flies. Exposure to biting flies elicited in individual naive mice analgesia and active self-burying to avoid the flies. When exposed 24 hr later to flies whose biting parts were removed, but not to nonbiting house flies, these mice displayed conditioned analgesia and self-burying. This "one-trial" conditioned analgesia and avoidance was also acquired through social learning without direct individual experience with biting flies. Naive "observer" mice that witnessed other "demonstrator" mice being attacked by biting flies exhibited analgesia and self-burying 24 hr later to altered flies.

Learning to cope with biting flies: rapid NMDA-mediated acquisition of conditioned analgesia.

A 30-min exposure to intact biting flies (stable flies) induced an opioid-mediated analgesia in fly-naive male deer mice, whereas exposure to either altered biting flies whose biting mouthparts were removed or nonbiting house flies had no significant effects. However, mice that were previously exposed to intact stable flies for 30 min exhibited significant analgesia when exposed 24-168 hr later to stable flies whose biting parts were removed, but not to nonbiting house flies. Administration of the specific N-methyl-D-aspartate (NMDA) antagonist NPC 12626 to fly-naive mice before exposure to intact flies, although not significantly reducing the analgesic response, blocked the subsequent conditioned analgesia. Naloxone, which blocked the intact biting fly-induced analgesia, did not alter the acquisition of the conditioned analgesic response to the altered stable flies. This demonstrates an NMDA-mediated acquisition of conditioned analgesia to a natural aversive stimulus.

NMDA-mediated social learning of fear-induced conditioned analgesia to biting flies.

Although fear conditioning has received extensive neurobiological attention little is known about social learning whereby one individual may learn and acquire the fear responses of another. A 30 min exposure to intact biting flies (stable fly, Stomoxys colcitrans L.) elicits in individual fly-naive mice analgesia and active self burying responses to avoid the flies. Fly-naive observer mice that witnessed other demonstrator mice being attacked by biting flies exhibited analgesia and self-burying to avoid flies when exposed 24 h later to altered flies whose biting mouth parts were removed. The opiate antagonist naloxone, while reducing the analgesic responses elicited by exposure to a fly-stressed demonstrator, did not affect either the subsequent conditioned analgesia or self-burying. However, the specific NMDA receptor antagonist NPC 12626, given to observers prior to, but not after, presentation of fly attacked demonstrators blocked the socially determined conditioned analgesia and self burying avoidance. This supports NMDA involvement in the mediation of the social transmission and long-term (24h) retention of conditioned analgesia and fear.

Multiple opioid system involvement in the mediation of parasitic-infection induced analgesia.

Although parasite modification of host behaviour is well established, little is known about the mechanisms underlying such effects. The present study examined the relationships between subclinical infection with the enteric sporozoan parasite, Eimeria vermiformis, nociceptive responses and endogenous opioid systems in male mice. Infected mice displayed significant analgesia which increased through the prepatent period [oocyst formation (pre-infective); days 1-7 post-infection (PI)], reached a maximum with the onset of patency (onset oocyst shedding and infectivity; days 7-8 PI) and declined during patency (oocyst shedding), with response latencies declining to basal levels with the cessation of oocyst production and infectivity (day 15 PI). The increasing nociception during the prepatent period (day 4 PI) was associated with kappa opioid mechanisms, being reduced by the kappa antagonist, nor-binaltorphimine, and insensitive to either the delta antagonist, ICI 174,864, or the general, predominantly mu antagonist, naloxone. Maximum analgesia (day 7 PI) associated with the onset of patency (infectivity) was sensitive to both the kappa and mu antagonists, but insensitive to the delta antagonist, while the declining analgesia during patency (day 10 PI) was reduced by the mu and delta antagonists, but was insensitive to the kappa antagonist. These results indicate that mu, delta and kappa opioid systems are involved in the mediation of subclinical parasitic infection-induced analgesia and likely other associated parasite-induced modifications of host behaviour.