Structure, histochemistry, ultrastructure and seasonal variations of the male prostatic complex in the black Myotis bat, Myotis nigricans (Chiroptera: Vespertilionidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Impact of the processes of testicular regression and recrudescence in the prostatic complex of the bat myotis nigricans (Chiroptera: Vespertilionidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Impact of the processes of total testicular regression and recrudescence on the epididymal physiology of the bat myotis nigricans (chiroptera: vespertilionidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Caracterização morfológica do pênis de espécies de morcegos vespertilionídeos dos gêneros Histiotus, Lasiurus e Myotis (Chiroptera, Mammalia)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Caracterização morfológica do pênis de espécies de morcegos vespertilionídeos dos gêneros Histiotus, Lasiurus e Myotis (Chiroptera, Mammalia)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Study of variability and genetic structure of European populations of Myotis emarginatus and Myotis capaccinii (Chiroptera, Vespertilionidae)

The Geoffroy’s bat Myotis emarginatus is mainly present in southern, south-eastern and central Europe (Červerný, 1999) and is often recorded from northern Spain (Quetglas, 2002; Flaquer et al., 2004). It has demonstrated the species’ preference for forest. Myotis capaccinii, confined to the Mediterranean (Guille´n, 1999), is classified as ‘vulnerable’ on a global scale (Hutson, Mickleburgh & Racey, 2001). In general, the species preferred calm waters bordered by well-developed riparian vegetation and large (> 5 m) inter-bank distances (Biscardi et al. 2007). In this study we present the first results about population genetic structure of these two species of genus Myotis. We used two methods of sampling: invasive and non-invasive techniques. A total of 323 invasive samples and a total of 107 non-invasive samples were collected and analyzed. For Myotis emarginatus we have individuated for the first time a set of 7 microsatellites, which can work on this species, started from a set developed on Myotis myotis (Castella et al. 2000). We developed also a method for analysis of non-invasive samples, that given a good percentage of positive analyzed samples. The results have highlighted for the species Myotis emarginatus the presence on the European territory of two big groups, discovered by using the microsatellites tracers. On this species, 33 haplotypes of Dloop have been identified, some of them are presented only in some colonies. We identified respectively 33 haplotypes of Dloop and 10 of cytB for Myotis emarginatus and 25 of dloop and 15 of cytB for Myotis capaccinii. Myotis emarginatus’ results, both microsatellites and mtDNA, show that there is a strong genetic flow between different colonies across Europe. The results achieved on Myotis capaccinii are very interesting, in this case either for the microsatellites or the mitochondrial DNA sequences, and it has been highlighted a big difference between different colonies.

White-Nose Syndrome-Affected Litttle Brown Myotis (Myotis Lucifugus) Increase Grooming and Other Active Behaviors During Arousals from Hibernation

White-nose syndrome (WNS) is an emerging infectious disease of hibernating bats linked to the death of an estimated 5.7 million or more bats in the northeastern United States and Canada. White-nose syndrome is caused by the cold-loving fungus Pseudogymnoascus destructans (Pd), which invades the skin of the muzzles, ears, and wings of hibernating bats. Previous work has shown that WNS-affected bats arouse to euthermic or near euthermic temperatures during hibernation significantly more frequently than normal and that these too-frequent arousals are tied to severity of infection and death date. We quantified the behavior of bats during these arousal bouts to understand better the causes and consequences of these arousals. We hypothesized that WNS-affected bats would display increased levels of activity (especially grooming) during their arousal bouts from hibernation compared to WNS-unaffected bats. Behavior of both affected and unaffected hibernating bats in captivity was monitored from December 2010 to March 2011 using temperature-sensitive dataloggers attached to the backs of bats and infrared motion-sensitive cameras. The WNS-affected bats exhibited significantly higher rates of grooming, relative to unaffected bats, at the expense of time that would otherwise be spent inactive. Increased self-grooming may be related to the presence of the fungus. Elevated activity levels in affected bats likely increase energetic stress, whereas the loss of rest (inactive periods when aroused from torpor) may jeopardize the ability of a bat to reestablish homeostasis in a number of physiologic systems.

Hibernacula Microclimate and White-nose Syndrome Susceptibility in the Little Brown Myotis (Myotis lucifugus)

The objective of this project was to determine the relationship between hibernacula microclimate and White-nose Syndrome (WNS), an emerging infectious disease in bats. Microclimate was examined on a species scale and at the level of the individual bat to determine if there was a difference in microclimate preference between healthy and WNS-affected little brown myotis (Myotis lucifugus) and to determine the role of microclimate in disease progression. There is anecdotal evidence that colder, drier hibernacula are less affected by WNS. This was tested by placing rugged temperature and humidity dataloggers in field sites throughout the eastern USA, experimentally determining the response to microclimate differences in captive bats, and testing microclimate roosting preference. This study found that microclimate significantly differed from the entrance of a hibernaculum versus where bats traditionally roost. It also found hibernaculum temperature and sex had significant impacts on survival in WNS-affected bats. Male bats with WNS had increased survivability over WNS-affected female bats and WNS bats housed below the ideal growth range of the fungus that causes WNS, Geomyces destructans, had increased survival over those housed at warmer temperatures. The results from this study are immediately applicable to (1) predict which hibernacula are more likely to be infected next winter, (2) further our understanding of WNS, and (3) determine if direct mitigation strategies, such as altering the microclimate of mines, will be effective ways to combat the spread of the fungus.

The tail plays a major role in the differing manoeuvrability of two sibling species of mouse-eared bats (Myotis myotis and Myotis blythii)

Two sympatrically occurring bat species, the greater mouse-eared bat (Myotis myotis (Borkhausen, 1797)) and the lesser mouse-eared bat (Myotis blythii (Tomes, 1857)) (Chiroptera, Vespertillionidae), share numerous similarities in morphology, roosting behaviour, and echolocation and are often difficult to distinguish. However, despite these similarities, their foraging behaviour is noticeably different. Our aim was to examine the extent to which these different foraging strategies reflect morphological adaptation. We assessed whether the morphology of the wing, body, and tail differed between M. myotis and M. blythii. In addition, in a laboratory experiment involving an obstacle course, we compared differences in manoeuvrability by relating them to our morphological measurements. The two species differed in their overall size, wing-tip shape, and tail-to-body length ratio. The generally smaller sized M. blythii performed better in the obstacle course and was therefore considered to be more manoeuvrable. Although differences in wing-tip shape were observed, we found the most important characteristic affecting manoeuvrability in both species to be the tail-to-body length ratio. Additionally, when we compared two bats with injured wing membranes with unharmed bats of the same species, we found no difference in manoeuvrability, even when the wing shape was asymmetric. We therefore postulate that morphometric differences between the two species in their overall size and, more importantly, in their tail-to-body length ratio are the main physical characteristics providing proof of adaptation to different foraging and feeding strategies.

The insect endosymbiont Sodalis glossinidius utilizes a type III secretion system for cell invasion

Sodalis glossinidius is a maternally transmitted secondary endosymbiont residing intracellularly in tissues of the tsetse flies, Glossina spp. In this study, we have used Tn5 mutagenesis and a negative selection procedure to derive a S. glossinidius mutant that is incapable of invading insect cells in vitro and is aposymbiotic when microinjected into tsetse. This mutant strain harbors Tn5 integrated into a chromosomal gene sharing high sequence identity with a type III secretion system invasion gene (invC) previously identified in Salmonella enterica. With the use of degenerate PCR, we have amplified a further six Sodalis inv/spa genes sharing high sequence identity with type III secretion system genes encoded by Salmonella pathogenicity island 1. Phylogenetic reconstructions based on the inv/spa genes of Sodalis and other members of the family Enterobacteriaceae have consistently identified a well-supported clade containing Sodalis and the enteric pathogens Shigella and Salmonella. These results suggest that Sodalis may have evolved from an ancestor with a parasitic intracellular lifestyle, possibly a latter-day entomopathogen. These observations lend credence to a hypothesis suggesting that vertically transmitted mutualistic endosymbionts evolve from horizontally transmitted parasites through a parasitism–mutualism continuum.

Alberti Guilielmi Rothi, medicinae doctoris, physici provincialis ducatus Bremensis, Societatis Halensis Naturae Curiosorum sodalis, Tentamen florae Germanicae.

Tomus III has imprint: In bibliopolio Gleditschiano.

Prevalence and associations of Trypanosoma spp. and Sodalis glossinidius with intrinsic factors of tsetse flies

Trypanosomiasis has been identified as a neglected tropical disease in both humans and animals in many regions of sub-Saharan Africa. Whilst assessments of the biology of trypanosomes, vectors, vertebrate hosts and the environment have provided useful information about life cycles, transmission, and pathogenesis of the parasites that could be used for treatment and control, less information is available about the effects of interactions among multiple intrinsic factors on trypanosome presence in tsetse flies from different sites. It is known that multiple species of tsetse flies can transmit trypanosomes but differences in their vector competence has normally been studied in relation to individual factors in isolation, such as: intrinsic factors of the flies (e.g. age, sex); habitat characteristics; presence of endosymbionts (e.g. Wigglesworthia glossinidia, Sodalis glossinidius); feeding pattern; host communities that the flies feed on; and which species of trypanosomes are transmitted. The purpose of this study was to take a more integrated approach to investigate trypanosome prevalence in tsetse flies. In chapter 2, techniques were optimised for using the Polymerase Chain Reaction (PCR) to identify species of trypanosomes (Trypanosoma vivax, T. congolense, T. brucei, T. simiae, and T. godfreyi) present in four species of tsetse flies (Glossina austeni, G. brevipalpis, G. longipennis and G. pallidipes) from two regions of eastern Kenya (the Shimba Hills and Nguruman). Based on universal primers targeting the internal transcribed spacer 1 region (ITS-1), T. vivax was the predominant pathogenic species detected in flies, both singly and in combination with other species of trypanosomes. Using Generalised Linear Models (GLMs) and likelihood ratio tests to choose the best-fitting models, presence of T. vivax was significantly associated with an interaction between subpopulation (a combination between collection sites and species of Glossina) and sex of the flies (X2 = 7.52, df = 21, P-value = 0.0061); prevalence in females overall was higher than in males but this was not consistent across subpopulations. Similarly, T. congolense was significantly associated only with subpopulation (X2 = 18.77, df = 1, P-value = 0.0046); prevalence was higher overall in the Shimba Hills than in Nguruman but this pattern varied by species of tsetse fly. When associations were analysed in individual species of tsetse flies, there were no consistent associations between trypanosome prevalence and any single factor (site, sex, age) and different combinations of interactions were found to be significant for each. The results thus demonstrated complex interactions between vectors and trypanosome prevalence related to both the distribution and intrinsic factors of tsetse flies. The potential influence of the presence of S. glossinidius on trypanosome presence in tsetse flies was studied in chapter 3. A high number of Sodalis positive flies was found in the Shimba Hills, while there were only two positive flies from Nguruman. Presence or absence of Sodalis was significantly associated with subpopulation while trypanosome presence showed a significant association with age (X2 = 4.65, df = 14, P-value = 0.0310) and an interaction between subpopulation and sex (X2 = 18.94, df = 10, P-value = 0.0043). However, the specific associations that were significant varied across species of trypanosomes, with T. congolense and T. brucei but not T. vivax showing significant interactions involving Sodalis. Although it has previously been concluded that presence of Sodalis increases susceptibility to trypanosomes, the results presented here suggest a more complicated relationship, which may be biased by differences in the distribution and intrinsic factors of tsetse flies, as well as which trypanosome species are considered. In chapter 4 trypanosome status was studied in relation to blood meal sources, feeding status and feeding patterns of G. pallidipes (which was the predominant fly species collected for this study) as determined by sequencing the mitochondrial cytochrome B gene using DNA extracted from abdomen samples. African buffalo and African elephants were the main sources of blood meals but antelopes, warthogs, humans, giraffes and hyenas were also identified. Feeding on multiple hosts was common in flies sampled from the Shimba Hills but most flies from Nguruman had fed on single host species. Based on Multiple Correspondence Analysis (MCA), host-feeding patterns showed a correlation with site of sample collection and Sodalis status, while trypanosome status was correlated with sex and age of the flies, suggesting that recent host-feeding patterns from blood meal analysis cannot predict trypanosome status. In conclusion, the complexity of interactions found suggests that strategies of tsetse fly control should be specific to particular epidemic areas. Future studies should include laboratory experiments that use local colonies of tsetse flies, local strains of trypanosomes and local S. glossinidius under controlled environmental conditions to tease out the factors that affect vector competence and the relative influence of external environmental factors on the dynamics of these interactions.

Prospects for control of African trypanosomiasis by tsetse vector manipulation

The extensive antigenic variation phenomena African trypanosomes display in their mammalian host have hampered efforts to develop effective vaccines against trypanosomiasis. Human disease management aims largely to treat infected hosts by chemotherapy, whereas control of animal diseases relies on reducing tsetse populations as well as on drug therapy. The control strategies for animal diseases are carried out and financed by livestock owners, who have an obvious economic incentive. Sustaining largely insecticide-based control at a local level and relying on drugs for treatment of infected hosts for a disease for which there is no evidence of acquired immunity could prove extremely costly in the long run. It is more likely that a combination of several methods in an integrated, phased and area-wide approach would be more effective in controlling these diseases and subsequently improving agricultural output. New approaches that are environmentally acceptable, efficacious and affordable are clearly desirable for control of various medically and agriculturally important insects including tsetse. Here, Serap Aksoy and colleagues discuss molecular genetic approaches to modulate tsetse vector competence.