Made available in DSpace on 2016-08-15T20:27:58Z (GMT). No. of bitstreams: 1 JosivaniaSP_TESE.pdf: 2216086 bytes, checksum: 4328c3795e67888b55db8e1be380a936 (MD5) Previous issue date: 2015-11-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Small wild mammals, including rodents and marsupials, have ecological and epidemiological importance for being prey and predators in ecological chains, hosts of ecto and endoparasites and pathogens reservoirs. In Northeast Brazil, only a few studies have evaluated the diversity of ectoparasites and their hosts in the Caatinga biome. This study aimed to identify the hosts and their ectoparasites, with morphometric analysis and chaetotaxy and to determine the rates of parasitic Prevalence, Abundance and Medium Intensity of ectoparasites collected from rodents and marsupials, proceeding from the Experimental Station Rafael Fernandes, Rio Grande do Norte, Brazil, at the Universidade Federal Rural do Semi-Árido (UFERSA). The animals were captured between January 2014 and February 2015, in a fragment of 26 hectares, divided into six equally spaced transects (20m). Tomahawk and Sherman traps were distributed in six consecutive nights of each month and in 100 capture stations. The captured animals were identified by morphology and submitted to ectoparasites collection. All the collected ectoparasites were classified based on morphometric characters, chaetotaxy and taxonomic keys, as well as DNA extraction, PCR and sequencing of the amplified products. 78 small mammals were captured, from which 69 were classified as Didelphimorphia order: Gracilinanus agilis (Burmeister, 1854) and Monodelphis domestica (Wagner, 1842); and nine Rodentia: Wiedomys Hershkovitz, 1959, Thrichomys Trouessart, 1880 and Rattus norvegicus Berkenhout, 1769. It was collected ectoparasites identified as: Amblyomma (Koch, 1844), Amblyomma parvum, Aragão 1908, Amblyomma auricularium (Conil, 1878), Ornithodoros mimon Kohls, Clifford e Jones 1969, Ornithodoros Kohls, Clifford e Jones 1837, Quadraseta Brennan, 1970, Eutrombicula Ewing, 1938, Metachiroecius Fain, 1996, Aeromychirus Fain, 1976, Amlistrophorus Fain, 1981, Radfordia Ewing, 1938, Listropsoralges aff. similis, Ornithonyssus pereirai (Fonseca, 1935), Tur lativentralis (Fonseca 1936), Polyplax spinulosa (Burmeister, 1839) and Eogyropus lenti lenti (Werneck 1936). As for the parasitic rates were noted that for the Ixodidae and Argasidae studied, M. domestica presented higher parasitism values for A. auricularium; G. agilis, O. mimon and Thrichomys sp., to A. parvum and A. auricularium. As for mites, the prevalence was the same in M. domestica and to all its studied Listrophoridae. Also for rodents, it was observerd the same prevalence rate for Laelapidae mites and Polyplacidae lice in the Thrichomys sp. All of those records of small mammals and ectoparasites shown are unprecedented to Mossoró, Rio Grande do Norte. This study supports evidences from the literature that ectoparasites use marsupials and wild rodents as hosts for immature stages. Justifies the importance of maintaining a legal reserve area in the Caatinga biome for preservation of these animals. In addition, this research increases knowledge about the occurrence of Acari, Phthiraptera and its ectoparasitic association with the wildlife in Northeast Brazil / Os pequenos mamíferos silvestres, dentre eles os roedores e marsupiais, apresentam importância ecológica e epidemiológica por serem presas e predadores nas cadeias ecológicas, hospedeiros de ecto e endoparasitos e reservatórios de agentes patogênicos. No Nordeste do Brasil, poucos estudos avaliaram a diversidade de ectoparasitos e seus hospedeiros no bioma Caatinga. Objetivou-se identificar os hospedeiros e seus ectoparasitos, realizar morfometria e quetotaxia e determinar os índices parasitários de Prevalência, Abundância e Intensidade Média dos ectoparasitos coletados de roedores e marsupiais, procedentes da Estação Experimental Rafael Fernandes da Universidade Federal Rural do Semi-Árido (UFERSA). Capturou-se animais de janeiro de 2014 a fevereiro de 2015, em um fragmento de 26 hectares da Estação, divididos em seis transectos equidistantes (20m). Foram distribuídas armadilhas Tomahawk e Sherman em seis noites consecutivas de cada mês e em cem estações de captura. Os animais capturados foram identificados pela morfologia e submetidos à coleta dos ectoparasitos. Estes foram classificados com base em caracteres morfométricos, quetotaxia e chaves taxonômicas, além de extração de DNA, PCR e sequenciamento dos produtos amplificados. Foram capturados 78 pequenos mamíferos, dos quais 69 foram classificados na ordem Didelphimorphia: Gracilinanus agilis (Burmeister, 1854) e Monodelphis domestica (Wagner, 1842); e nove Rodentia: Wiedomys Hershkovitz, 1959, Thrichomys Trouessart, 1880 e Rattus norvegicus Berkenhout, 1769. Coletou-se ectoparasitos identificados como: Amblyomma (Koch, 1844), Amblyomma parvum Aragão, 1908, Amblyomma auricularium (Conil, 1878), Ornithodoros mimon Kohls, Clifford e Jones, 1969, Ornithodoros Kohls, Clifford e Jones 1837, Quadraseta Brennan, 1970, Eutrombicula Ewing, 1938, Metachiroecius Fain, 1996, Aeromychirus Fain, 1976, Amlistrophorus Fain, 1981, Radfordia Ewing, 1938, Listropsoralges aff. similis, Ornithonyssus pereirai (Fonseca, 1935), Tur lativentralis (Fonseca 1936), Polyplax spinulosa (Burmeister, 1839) e Eogyropus lenti lenti Werneck 1936. Quanto aos índices parasitários, observou-se que para os Ixodidae e Argasidae estudados, M. domestica apresentou maiores valores de parasitismo para A. auricularium; G. agilis, para O. mimon e Thrichomys sp., para A. parvum e A. auricularium. Quanto aos ácaros, a prevalência foi igual em M. domestica e para todos os seus Listrophoridae estudados. Igualmente, para os roedores, observou-se mesmo valor de prevalência para os ácaros Laelapidae e para os piolhos Polyplacidae em Thrichomys sp. Todos os registros dos pequenos mamíferos e ectoparasitos apresentados são inéditos para Mossoró, Rio Grande do Norte. Este estudo corrobora evidências da literatura dos ectoparasitos utilizarem marsupiais e roedores silvestres como hospedeiros em fases imaturas. Justifica a importância da manutenção de uma área de reserva legal no bioma Caatinga para conservação destes animais. Em adição, a presente pesquisa aumenta o conhecimento sobre ocorrência de Acari, Phthiraptera e sua associação ectoparasitária de vida selvagem, no Nordeste do Brasil

The study investigated biological and ecological parameters controlling and influencing the production and distribution of the free-swimming larval stages of Lepeophtheirus salmonis (Kroyer, 1838), and to a lesser extent Caligus elongatus Nordmann 1832, in the natural environment The reproductive output of L. salmonis was influenced by seasonal effects. The number of eggs produced per brood showed an inverse relationship with increasing temperature. The number of eggs per brood was also influenced by adult female body size (cephalothorax length), which in itself exhibited an inverse relationship with increasing temperature. Photoperiod had no significant effect upon the number of eggs produced or on adult female size. Mean egg size of L. salmonis varied significantly over the year; larger eggs were produced during the summer months and smaller eggs over the winter. However, factors controlling the size of the eggs were not elucidated. The proportion of viable eggs per L. salmonis ovisac remained constant throughout the year. Large variations in egg number per egg string were found in both L. salmonis and C elongatus populations sampled at one point in time. These were attributed in part to phenotypic variation in adult female size and also the number of broods individual females had produced. Egg viability was not correlated with brood size, but mean egg size was related to the number of eggs per brood. Experimental studies indicated that hatching and development of L. salmonis was highly variable. The percentage of eggs hatched and the time period over which hatching occurred varied markedly, even when held under constant and optimal environmental conditions. Temperature did not affect hatching success or viability of the nauplius I stage, although at higher temperatures the period over which hatching occurred was reduced. Low and medium salinities caused a significant decrease in both hatching success and nauplius viability. Photoperiod had no effect on initiation of hatching. Hatching occurred in a manner similar to that observed in free-living copepods. The nauplii were enclosed by two egg membranes, the outer one bursting within the ovisac, the inner one after the ovisac membrane has split. Swelling of the egg and its subsequent hatching was attributed to osmotic effects, with water being taken up from the external environment. Development was also highly dependent upon both temperature and salinity. At 5'C, nauplius 11 stages failed to enter the moult to the copepodid stage. At 7.5'C, although moulting was initiated, in a large proportion of cases it was not successfully completed. At I O'C, development to the copepodid stage was successful. Nauplii only developed successfully to the copepodid stage at salinities of 25%o or greater. Copepodids raised under optimal conditions then exposed to a range of salinities had a greater salinity tolerance than nauplii. Biochemical analysis of the eggs of L. salmonis revealed that lipids constituted a large proportion of their dry weight. Naupliar stages contained a discrete area containing lipid which decreased in size over time, suggesting that the free-swimming larval stages utilised this as an energy reserve. Rate of depletion was faster in nauplii held at higher temperatures. Longevity, activity and infectivity of the infective stage decreased with age. However, both spontaneous and stimulus dependent activity ceased many hours before death and both activity and longevity were affected by temperature. Infectivity of I day old L. salmonis copepodids was higher than 7 day old larvae, and was considered to be related to the size of the energy reserves. The settlement and distribution pattern of copepodids did not change with age of copepodid, the majority being recorded from the fins. All three L. salmonis free-swimming larval stages demonstrated a "hop and sink" swimming pattern. The velocity and duration of both passive sinking and active swimming was recorded for both nauplii and copepodids. Although greater periods of time were spent passively sinking, the speeds obtained during both upward spontaneous and stimulated swimming meant that a net upward movement of larvae in the water column occurred. At higher temperatures spontaneous swimming activity increased, whilst low salinities caused a cessation of such ability. L. salmonis larvae were positively phototactic and negatively geotactic. As well as their positive responses to light intensity, the nauplius 11 and copepodid stages reacted positively to blue-green spectral wavelengths. Moulting times were relatively short, although the larvae were not able to swim during such periods. No relationship was found between the level of lipid reserves and the overall buoyancy of the larvae. Naupliar stages of both L. salmonis and C. elongalus were obtained from the water column as a result of a plankton sampling programme at a commercial Atlantic salmon farm. No copepodid stages of either species were found. There was no difference in the vertical distribution of the two L. salmonis naupliar stages. Live larvae tended to aggregate between 0 and 5m in depth, with no diurnal vertical migration. Dead nauplii, and those with low lipid reserves, were found deeper in the water column. Naupliar stages, and in particular the first larval stage, were concentrated in number within cages indicating that the cages have a retentive characteristic. A novel control method in the form of a commercially available light lure was tested. Though increasing the numbers of free-living copepods captured, it had no effect on the numbers of L. salmonis naupliar or copepodid stages obtained in plankton samples. The present study has therefore provided valuable data concerning the biology and ecology of the free-swimming larval stages of sea lice, in what was a comparatively poorly understood area.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-06-01T11:44:36Z No. of bitstreams: 1 ralphmaturanopinheiro.pdf: 530518 bytes, checksum: c7705e2c22719ac685677b3a3ed3fe26 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-07-02T12:54:45Z (GMT) No. of bitstreams: 1 ralphmaturanopinheiro.pdf: 530518 bytes, checksum: c7705e2c22719ac685677b3a3ed3fe26 (MD5) / Made available in DSpace on 2016-07-02T12:54:45Z (GMT). No. of bitstreams: 1 ralphmaturanopinheiro.pdf: 530518 bytes, checksum: c7705e2c22719ac685677b3a3ed3fe26 (MD5) Previous issue date: 2012-02-03 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A bionomia de Chelopistes meleagridis fora do hospedeiro foi observada com o objetivo de compreender aspectos relacionados ao ciclo de vida desta espécie. Para isto, adultos de C. meleagridis foram coletados e colocados em condições controladas para se reproduzir, oferecendo-se pena como alimento. Da prole destes adultos, foi observado o desenvolvimento de 150 indivíduos desde o ovo até a fase adulta. Para 75 destes, foi oferecida a dieta composta de pena, enquanto para os outros 75 a dieta foi composta de pena e pele do hospedeiro (peru, Meleagris gallopavo). Ao verificar que a dieta “pena + pele” foi a que resultou no maior número de adultos, foram observadas a fertilidade, fecundidade e a longevidade de piolhos criados in vitro desde o primeiro ínstar alimentados com esta dieta. Valores altos relacionados à reprodução desta espécie foram encontrados em relação a outros piolhos da subordem Ischnocera, destacando-se: número de ovos produzidos por dia e número de ovos produzidos por fêmeas durante a vida (médias de 2,54 e 26,61 ovos, respectivamente, para fêmeas selvagens e 2,11 e 29,33 ovos, respectivamente, para fêmeas criadas in vitro.). A inclusão de pele na dieta foi determinante para o desenvolvimento até o estágio adulto, uma vez que 48% dos piolhos alimentados com essa dieta atingiram a fase adulta. Quando foi oferecido apenas pena, 1,3% dos piolhos atingiram a maturidade. O tempo de desenvolvimento de machos e fêmeas foi semelhante (média de 29,38 dias) sem haver diferença na proporção sexual dos adultos. / Bionomics of the large louse turkey Chelopistes meleagridis off host was evaluated to comprehend aspects related to life cycle. To this, adults was collected and put in controlled conditions for reproduce, offered feathers as food. From offspring of these adults were observed the development of 150 lice from eggs to adults. To 75 of them was given only feathers as resource of food while to others 75 lice the diet was composed by feathers and skin debris from the natural host, Meleagris gallopavo. When verified that diet composed by feather + skin resulted in more adults, was evaluated fertility, fecundity and longevity of this specie reared in vitro from first nymphs to adults feed with this diet. High values related to reproduction of this specie were found in comparison with others avian Ischnocera, like: number of eggs produced by day and number of egg produced during the lifespan of females (means of 2.54 and 26.61, respectively to wild females and 2.11 and 29.33 respectively to females reared in vitro). The addition of skin in diet was determinant to development to adult stage, once time 48% fed with this diet reared to adults while the lice feed with feathers only 1.3 % of then became adult stage. The time of development of males and females was the same (29 days). The sex rate of adults reared in vitro was 1:1.

Abstract The life histories of ectoparasites are shaped by both host and off-host environment. A suitable host is primarily needed during reproduction, whereas juvenile stages outside the host are directly exposed to environmental variability. Viviparity, i.e. the development of an embryo inside the body of the mother resulting in large offspring size, increases offspring survival. The production of large offspring has its consequences in terms of high variation in offspring age and in the environment that each young individual will face. I used a viviparous ectoparasite, the deer ked (Lipoptena cervi), to investigate the consequences of long reproductive lifespan and varying offspring environment on offspring life-histories and seasonal adaptations. Offspring life-histories varied seasonally. I showed that the resources provided by the deer ked females determine offspring performance throughout its off-host period. Offspring size increased towards the spring and the end of the reproductive period, and simultaneously offspring survival and cold tolerance increased. Seasonal variation in offspring size did not reflect the resources that would guarantee offspring survival during the longest diapause or the highest cold tolerance during the harshest winter period. Diapause intensity varies with birth time according to the expected length of the winter ahead. However, the deer ked pupae, regardless of their age, overwinter at an opportunistic diapause, which may be terminated rapidly only by an exposure to high temperature. Contrary to general observations, photoperiod has no role in regulating the seasonal shifts of the deer ked. Neither is high cold tolerance associated only with diapause, but it remains high through four seasons, also in the active developmental and adult stages. I also evaluated the effects of life-history variation on the invasion potential of the deer ked. I conducted a large-scale transplant experiment to test the survival and pupal development at and beyond the current range. I found that the lower spring and summer temperatures and the shorter growth season in the north cause a deterioration in pupal performance and shorten the flight period. However, the colder climate may not totally prevent further spread. A more important factor that will affect deer ked invasion is host availability, and especially in Finland, the density of the moose population. Seasonal variation in offspring life histories in viviparous ectoparasites differs from the variation patterns reported in most invertebrates. This may be due to the extremely large offspring size and to the fact that maternally derived resources determine offspring performance through the entire off-host period. Variation in offspring performance is thus determined by maternal resources and seasonal variation in the condition of the moose. / Tiivistelmä Useimpien ulkoloisten elinkierto on riippuvainen isännästä ja isännän ulkopuolella kasvavien jälkeläisten kohtaamista ympäristöoloista. Viviparia eli jälkeläisen kehitys naaraan sisällä ja siitä usein seuraava suuri jälkeläiskoko parantavat jälkeläisten selviytymistä. Suurten jälkeläisten tuottaminen pitkällä aikavälillä johtaa siihen, että eri-ikäiset jälkeläiset kohtaavat vuodenajasta riippuen hyvin erilaiset olosuhteet. Väitöstyössäni tarkastelin Suomessa nopeasti yleistyneen hirvieläinten ulkoloisen, hirvikärpäsen (Lipoptena cervi), avulla, mitä seurauksia viviparialla, pitkällä lisääntymiskaudella ja ympäristön vuodenaikaisvaihtelulla on jälkeläisten elinkierto-ominaisuuksiin. Väitöskirjatyössäni havaitsin, että hirvikärpäsjälkeläisten elinkiertopiirteet vaihtelevat jälkeläisen syntymäajan mukaan. Osoitin myös, että hirvikärpäsnaaraan jälkeläisilleen tarjoamat ravintovarat määrittelevät jälkeläisten isännästä riippumattoman elinkierron aikaisen menestyksen. Jälkeläisten keskimääräinen koko kasvoi lisääntymiskauden edetessä talvesta kohti kevättä, jolloin myös selviytyminen ja kylmänsietokyky paranivat. Jälkeläiskoon vuodenaikaisvaihtelu ei näin ollen vastaa jälkeläisten tarvitsemia resursseja suhteessa talvehtimisen pituuteen eikä korreloi koville talvipakkasille altistumisen todennäköisyyden kanssa. Lepotilan keston määrittelevä diapaussin syvyys vaihteli syntymävuodenaikaa vastaavasti. Diapaussin havaitsin kuitenkin olevan ensisijaisesti opportunistinen, jolloin pelkästään korkea lämpötila voi nopeasti päättää lepotilan kaikenikäisillä yksilöillä. Vastoin yleisiä käsityksiä valorytmi ei vaikuta diapaussin säätelyyn. Vastoin ennakko-odotuksia kylmänsietokyky säilyy korkeana vuoden ympäri ja kaikissa tutkituissa elinkierron vaiheissa. Sovelsin tutkimieni elinkiertopiirteiden vaikutusta myös lajin invaasiokykyyn, ja tutkin istutuskokeen avulla koteloiden selviytymistä ja kehitystä nykyisellä esiintymisalueella ja sen pohjoispuolella. Alhaisemmat kevät- ja kesälämpötilat sekä lyhyempi kasvukausi vähentävät aikuiseksi selviytymistä ja lyhentävät lentoaikaa syksyllä. Ilmastotekijöiden suhteen hirvikärpänen voisi esiintyä nykyistä pohjoisempana. Tärkein tekijä hirvikärpäsen leviämistä tarkasteltaessa on kuitenkin sopivien isäntäeläimien saatavuus ja Suomen oloissa erityisesti hirven eli hirvikärpäsen pääisännän kannan tiheys. Tutkimukseni perusteella ulkoloisten vivipariasta seuraava jälkeläisten elinkiertopiirteiden ajallinen vaihtelu eroaa muiden selkärangattomien vastaavasta vaihtelusta. Yhtenä syynä eroihin lienee se, että hirvikärpäsnaaras tuottaa erityisen suuria jälkeläisiä ja että jälkeläiset puolestaan ovat täysin riippuvaisia emon antamista resursseista. Emon lisääntymisresurssit ja hirven kunnon vuodenaikaisvaihtelu vaikuttavat mahdollisesti siihen, minkälaisia jälkeläisiä hirvikärpänen milloinkin kykenee tuottamaan.

Efficient control of sea lice is a major challenge for the sustainable production of farmed Atlantic salmon (Salmo salar (Linnaeus, 1758)). These marine ectoparasites feed on mucus, skin and blood of their hosts, thereby reducing the salmon’s growth rate and overall health. In the northern hemisphere, the most prevalent species is Lepeophtheirus salmonis (Krøyer, 1837). In 2006, global costs of sea lice infections are estimated to have exceeded €300 million, with the majority spent on a limited number of chemical delousing agents. Emamectin benzoate (EMB; SLICE®), an avermectin, has been widely used since its introduction in 2000, due to its convenient administration as an in-feed medication and its high efficacy against all parasitic stages of L. salmonis. However, over-reliance on a single or limited range of medicines favours the emergence of drug resistance and, as a result, the efficacy of this compound in treating L. salmonis has decreased in recent years, as reported from e.g. Chile, Norway, Scotland and Canada. Declining efficacy underlines the need for an improved understanding of the molecular mechanisms underlying EMB drug resistance in L. salmonis. Elucidation of these mechanisms would allow for improved monitoring tools, earlier detection of developing resistance, extended usability of current delousing agents and development of new parasiticides. The work described in this thesis sets out to examine the molecular mechanisms underlying EMB resistance in L. salmonis. In earlier studies, research in nematodes and arthropods has linked drug efflux transporters belonging to the family of ATP-binding cassette (ABC) transporters to ivermectin (IVM) resistance, a parasiticide with high chemical similarity to EMB. ABC transporters such as permeability glycoprotein (P-gp), transport a wide range of substrates, including drugs, and have been suggested to provide a potential molecular mechanism through which EMB resistance might be mediated in sea lice. As an example of such mechanisms, increased expression of P-gp is one of the causative factors for drug resistance in human cancer cells and avermectin resistance in nematode parasites such as Caenorhabditis elegans or Haemonchus contortus. Initial research involved screening for novel salmon lice P-gps that might contribute to EMB resistance. A novel P-gp, SL-PGY1, was discovered using a combined bioinformatic and molecular biological approach. The expression was compared in two well-characterised L. salmonis strains differing in their susceptibility to EMB (S = susceptible, R = resistant). Prior to EMB exposure, mRNA levels did not differ from each other, while, after 24 h exposure, a 2.9-fold increase in SL-PGY1 mRNA expression was observed in the R strain. SL-PGY1 appears not to be a major factor contributing to reduced EMB susceptibility, although it could play a role, as expression levels increased upon exposure to EMB. A further four additional drug transporters (ABC C subfamily) were also discovered showing high homology to multidrug-resistance proteins (MRP). The relative expression levels of each MRP was compared in the strains S and R, before and after exposure to EMB. No significant changes were found in their expression patterns. If ABC drug transporters mediate the efflux of EMB and thereby reduce the intracellular concentrations of the drug in exposed animals, the inhibition of those ABC drug transporters was expected to lead to higher intracellular levels of EMB. This could result in an enhanced toxic effect when EMB is co-administered with an inhibitor. Two known inhibitors of human P-gps and MRPs, cyclosporin A (CSA) and verapamil (VER), were co-administered with EMB. CSA increased the toxic effect of EMB in both tested strains, implying that the targets of CSA are expressed at comparable levels and that they may be part of the mechanism conferring EMB resistance. VER increased the toxic effect of EMB in the R strain, but had no significant effects on the S strain. This implies that the expression of factors inhibited by VER differs between the two L. salmonis strains. It is hypothesised that a number of ABC transporters with distinct, yet overlapping patterns of inhibitor specificity are affected by those inhibitors. The search for drug-resistance conferring genes was complemented with a systematic, genome-wide survey of ABC transporters in L. salmonis to find additional members of this important gene family. Next-generation high-throughput RNA sequencing (RNA-seq) was employed to assemble a reference transcriptome from pooled total RNA of salmon lice at different development stages. The transcriptome was assembled against the L. salmonis genome and annotated. Thirty-nine putative ABC transporters were found. Of further interest were transcripts of the subfamily B, C and G, as they contain drug-transporting ABC proteins. For the ABC B subfamily, one full (SL-PGY1) and three half transporter transcripts were found. Only full transporters are known to transport drugs and SL-PGY1 is apparently not a major factor contributing to EMB resistance. Fourteen ABCC sequences were found – 11 MRPs and 3 homologues to sulfonylurea receptors. Of interest are MRPs, as they contribute to drug detoxification in humans and invertebrates. Four MRPs had been identified previously and their expression ratios did not differ between S and R strain parasites. Seven sequences belonging to ABCG subfamily were found. However, none of the L. salmonis ABCG transcripts identified showed sufficient homology to known drug transporters in other species. With the currently limited understanding of the mechanisms conferring EMB resistance, monitoring the susceptibility of L. salmonis subpopulations is essential. Dose-response bioassays are currently widely used. Tests with pre-adult II or adult parasites requires relatively large numbers of parasites (~150) to conduct this type of bioassay, which may not always be available. Addressing this issue, we tested the feasibility of a single-dose bioassay (requiring fewer test animals than dose-response bioassays) to discriminate between L. salmonis strains with differing EMB susceptibility. This alternative approach uses time-course toxicity analysis, where the toxic effect of EMB is monitored over time. After clearly defining the effect criteria, we found that it is possible to discriminate between those L. salmonis strains. However, while requiring fewer test animals, time course toxicity analysis is more labour-intensive, but the alternative design can be suitable under certain circumstances. The work reported here has provided new knowledge concerning the mechanisms of EMB resistance in sea lice. Several novel putative drug transporters have been identified, an important first step toward unravelling the complex interactions of genes involved in EMB resistance in this commercially important parasite.

Human body lice, Pediculus humanus humanus, and head lice, Pediculus humanus capitis, have been hematophagous ectoparasites of humans for thousands of years. Despite being ecotypes, only body lice are known to transmit bacterial diseases to humans, and it appears that lower humoral and cellular immune responses allow body lice to possess a higher vector competence. We previously observed that the transcription level of the defensin 1 gene was up-regulated only in head lice following oral challenge of Bartonella quintana, a causative agent of trench fever, and also that body lice excreted more viable B. quintana in their feces. In this study, we first investigated this differential immune response by performing RNAi to knockdown defensin 1 by dsRNA injection. B. quintana was orally infected 72 h after injection and proliferation was compared at 2 hours (day 0) and day 4 post-infection. At day 0, bacterial cell numbers increased 1.5-fold in defensin 1 (Def1(-)) knocked down head lice compared with non-knocked down, pQE30-dsRNA injected, head lice control. At day 4, Def1(-) knocked down head lice had 2.55-fold more bacterial cells than control head lice and 1.65-fold greater than body lice, indicating that defensin 1 was active in reducing B. quintana cell number in non-knocked down head lice. Second, the levels of cytotoxic reactive oxygen species (ROS) generated by the epithelial cells of the alimentary tract were measured using two general indictors of ROS in both body and head lice at day 1 and day 4 following B. quintana challenge. Challenged body lice showed a 42% and 34% increase in ROS, whereas head lice showed a 70% and 22% increase at day 1 using CM-H2DCFDA and HPF as general indicators, respectively. On day 4, all challenged lice showed similar ROS levels except for body lice which maintained their ROS levels (40% increase using CM-H2DCFDA). Head lice are likely to have multiple immune and/or non-immune factors that suppress B. quintana proliferation, and the production of sustained ROS levels and/or the single knockdown of Defensin 1 is not enough to increase B. quintana proliferation in head lice to that seen in body lice.