Testicular myxosporidiasis in anurans,with a description of Myxobolus fallax n. sp.

During studies of amphibian sperm cryopreservation, a new species of myxosporidean parasite (Myxozoa, Myxosporae) was observed in the testes of the Australian dwarf green tree frog Litoria fallax (Peters). Myxosporidiasis was found to have no affect on L. fallax body condition or sperm numbers. Myxobolus spores from L. fallax are morphologically distinct from Myxobolus hylae spores (infecting the sympatric Litoria aurea Lesson) and the three previously named (exotic to Australia) Myxobolus species found in anurans. Myxobolus fallax n. sp. is characterised by: pseudocyst white, spherical to ovoid, 141 x 74 to 438 x 337 microm in diameter (mature); plasmodium with spores loosely arranged within interior. Spores ovoid 13.4 +/- 0.5 (12.6-14.6) microm length, 9.5 +/- 0.4 (8.3-10.6) microm width, 6.8 +/- 0.4 (6.5-7.6) microm depth, 1.4 +/- 0.1 (1.3-1.6) length/width; polar capsules broadly pyriform and equal in size 4.2 +/- 0.3 (3.3-4.7) microm length, 2.4 +/- 0.2 (2.1-2.8) microm width; filament coils 7-8, wound tightly and perpendicular to the longitudinal axis of the capsule; polar filament 34 +/- 7.0 (18-50) microm length; intercapsular appendix and sutural ridge folds absent; and iodinophilous vacuole and mucous envelope lacking. In addition to this new species, data from archival samples of M. hylae are provided which show two morphologically distinct spore types. Both appeared rarely in the same pseudocysts and we cautiously retain the single species.     

The effect of the migration of Alosa fallax fallax (Laécpède) into fresh water, on branchial and gut parasites

The prevalence and relative density of branchial and gut parasites taken from Almafullax in the estuary of the River Severn, at the start of the freshwater phase of its spawning migration, were compared with those taken from their spawning grounds in fresh water. Mazocrues alosue showed no significant difference in prevalence between the two habitats, though their relative density did fall significantly. Because of the direct relationship between length and relative density, this difference may be explained by the estuary samples of A. fullux being of fish significantly larger than those from fresh water. Other possible causes are discussed. The prevalence and relative density of M. alosae showed a trend to increase with age of the host. Repeat spawning A. fallax had a significantly greater relative density and prevalence of M. alosue when compared to virgin fish.
Analysis of the sampled population showed that Clavellisu emarginata declined significantly in both prevalence and relative density in fresh water, and this was confined to the younger and first time spawning A. fullux. Thynnuscuris aduncum showed significant reductions in prevalence and relative density of infection in fresh water when compared to the estuarine samples.
The species Hemiurus uppendiculatus and Pomphorhynchus laevis showed no significant difference in prevalence and relative density of infection, and Pronoprymna ventrzcosa no significant difference in prevalence, between the two habitats.     

Relationship between Marteilioides chungmuensis infection and reproduction in the Pacific oyster, Crassostrea gigas

Marteilioides chungmuensis, a protozoan paramyxean parasite, infects the oocytes of the Pacific oyster, Crassostrea gigas. The effects of infection on the reproductive cycle of C. gigas were investigated over two consecutive years at Okayama Prefecture, Japan. In male oysters, gonadal development began during February/March, maturity was achieved in June and spawning activity extended from July to September. In November and December, male oysters were not seen, probably because their gonads regressed to connective tissue and they transformed into undifferentiated oysters. By contrast, female oysters, in which parasite spore formation occurred, were still carrying oocytes until the following March and the spawning process of female oysters took 5 months longer than that of males in epizootic areas. The prevalence of M. chungmuensis infection increased from July to September, when most female oysters had their spawning period, and declined from October to the following April when oysters were at the spent stage. The prevalence of infection increased again in May of the following year and high prevalence was observed in the following July. When prevalence was compared between oysters of different age classes, higher prevalence was detected in older than in younger oysters. Histological examination showed that infected oysters produced oocytes continuously and spawned repeatedly from October to March, during which period healthy oysters were reproductively inactive. Parasites can infect the oocytes of infected oysters throughout the longer spawning period. These observations suggest that M. chungmuensis extends the reproductive period of infected oysters for its own reproductive benefit.     

Hematozoa in endemic subspecies of common kestrel in the Cape Verde Islands

We examined 130 Common Kestrel (Falco tinnunculus) representing two endemic subspecies and nine resident island populations on the Cape Verde archipelago between 1996 and 1999 to study diversity, prevalence, and intensity of hematozoa. Hematozoan diversity was very low; we detected only Plasmodium fallax, a species that is rarely found in Falconoformes, and, possibly, Haemoproteus brachiatus. Moreover, prevalence of Plasmodium fallax was low (1.5%) with a mean intensity of infection of 0.05 protozoa/10(-3) erythrocytes. Only one bird (0.8%) was infected with a gametocyte that was most likely Haemoproteus brachiatus; the intensity in this infected bird was 1.5 protozoa/10(-3) erythrocytes. A single parasite or two parasites were observed in blood smears in four additional birds, but identification to genus was not possible. This is the first record of blood parasites in birds on the Cape Verde Archipelago. The low prevalence of these parasites might be because of arid and less-favorable conditions for the pathogen's vectors. The sedentary nature and high level of isolation of the island kestrel populations are also factors that could decrease the probability of infection.     

Offsetting effects of Wolbachia infection and heat shock on sperm production in Drosophila simulans: analyses of fecundity, fertility and accessory gland proteins

Infection in Drosophila simulans with the endocellular symbiont Wolbachia pipientis results in egg lethality caused by failure to properly initiate diploid development (cytoplasmic incompatibility, CI). The relationship between Wolbachia infection and reproductive factors influencing male fitness has not been well examined. Here we compare infected and uninfected strains of D. simulans for (1) sperm production, (2) male fertility, and (3) the transfer and processing of two accessory gland proteins, Acp26Aa or Acp36De. Infected males produced significantly fewer sperm cysts than uninfected males over the first 10 days of adult life, and infected males, under varied mating conditions, had lower fertility compared to uninfected males. This fertility effect was due to neither differences between infected and uninfected males in the transfer and subsequent processing of accessory gland proteins by females nor to the presence of Wolbachia in mature sperm. We found that heat shock, which is known to decrease CI expression, increases sperm production to a greater extent in infected compared to uninfected males, suggesting a possible link between sperm production and heat shock. Given these results, the roles Wolbachia and heat shock play in mediating male gamete production may be important parameters for understanding the dynamics of infection in natural populations.     

Cannibalism and protective behavior of eggs in Arctic charr (Salvelinus alpinus)

From video recordings of spawning events, we quantified protective and cannibalistic behavior of Arctic charr occurring immediately after spawning. The number of fish cannibalizing on stray eggs was examined regarding (a) whether more than one male shed milt during the spawning event, that is, whether sperm competition occurred, (b) whether the sperm competition included few or many males, that is, the intensity of sperm competition, and (c) the density of fish at the spawning site. Response behavior toward egg cannibalism was also examined among females and dominant males in order to determine any parental investment toward protecting the eggs after spawning. Cannibalistic behavior was seen in almost 50% of the spawnings, and the multiple spawning events showed the highest numbers of fish cannibalizing on eggs. Both the number of males releasing milt and the number of fish approaching the spawning site were positively correlated with egg cannibalism. Sperm competition was, however, not a prerequisite for egg cannibalism. Although we also observed partial filial cannibalism, protective behavior of eggs was seen both among dominant males and females, suggesting that charr actually conduct parental care.     

Activation and killing of Dictyostelium discoideum spores with urea.

The optimal conditions for activation of Dictyostellium discoideum spores are an 8 M urea treatment for 30 min. The lag between activation and swelling is 45 min. Lower concentrations of urea do not activate entire spore populations. Incubating spores in 8 M urea for 60 min or treatment with 10 M urea for 30 min results in a lengthening of the post-activation lag and a decrease in the final percentage of germination. Urea-activated spores can be deactivated by azide, cyanide, osmotic pressure, and low-temperature incubation. Activated spores do not germinate if incubated in 1 M urea for 24 h but will complete germination upon resuspension in urea-free buffer. Shocking spores at 45 degrees C in 8 M urea or incubating spores in 4-8 M urea for 10 h at 23.5 degrees C causes inactivation. When suspended in urea-free buffer, a larger percentage of these dead spores release spheroplasts through a longitudinal split in the spore case. Sequential enzyme treatment of spheroplasts with cellulase and pronase causes them to release lysable protoplasts. The data of these experiments suggest that shedding of the outer and middle wall layers during physiological spore swelling may be a physical process rather than an enzymatic one.     

Nosema ceranae in age cohorts of the western honey bee (Apis mellifera)

Nosemaceranae intensity (mean spores per bee) and prevalence (proportion of bees infected in a sample) were analyzed in honey bees of known ages. Sealed brood combs from five colonies were removed, emerging bees were marked with paint, released back into their colonies of origin, and collected as recently emerged (0-3 days old), as house bees (8-11 days old), and as foragers (22-25 days old). Fifty bees from each of the five colonies were processed individually at each collection date for the intensity and prevalence of N. ceranae infection. Using PCR and specific primers to differentiate Nosema species, N. ceranae was found to be the only species present during the experiment. At each collection age (recent emergence, house, forager) an additional sample from the inner hive cover (background bees=BG) of each colony was collected to compare the N. ceranae results of this sampling method, commonly used for Nosema spore quantification, to the samples comprised of marked bees of known ages. No recently emerged bees exhibited infection with N. ceranae. One house bee out of the 250 individuals analyzed (prevalence=0.4%) tested positive for N. ceranae, at an infection level of 3.35×10(6) spores. Infection levels were not statistically different between the recently emerged (mean=0 spores/bee) and house bees (mean=1.34×10(4) spores/bee) (P=0.99). Foragers exhibited the highest prevalence (8.3%) and infection intensity (mean=2.38×10(6) spores/bee), with a range of 0-8.72×10(7) spores in individual bees. The average infection level across all foragers was significantly higher than that of recently emerged bees (P=0.01) and house bees (P=0.01). Finally, the prevalence of Nosema in infected bees was found to be positively correlated with the infection intensity in the sample.     

Sperm competition affects male behaviour and sperm output in the rainbow darter

Rainbow darters, Etheostoma caeruleum, are promiscuous fish with moderate rates of group spawning (between one and five males may simultaneously mate with one female). In this study, I examined male sperm output and male willingness to spawn under different levels of sperm competition intensity. One male and one female were allowed to spawn in an aquarium where they had visual and olfactory access to one of four treatments: four males, one male, zero males, or one female. Theory predicts that males should reduce sperm output when there are more than the average number of males at a group spawning (four-male treatment) and should increase sperm output when there are fewer than average males at a group spawning (one-male treatment). Mean sperm output did not differ among treatments. However, males released more sperm when spawning in the presence of competing males (four-male and one-male treatments pooled) than when spawning in the absence of competing males (zero-male and one-female treatments pooled). Males were also most likely to forego spawning opportunities when sperm competition intensity was high. Furthermore, male willingness to spawn was size dependent. Large males were more likely to forego spawning opportunities under high sperm competition intensity. Large males may be better off waiting for future spawning opportunities when there is a lower potential for sperm competition intensity.     

Female-biased infection and transmission of the gastrointestinal nematode Trichuris arvicolae infecting the common vole, Microtus arvalis

Previous studies addressing the importance of host gender in parasite transmission have shed light on males as the more important hosts, with the higher transmission potential of males being explained by the fact that they often harbour higher parasite loads than females. However, in some systems females are more heavily infected than males and may be responsible for driving infection under such circumstances. Using a wild population of common voles (Microtus arvalis), we showed that females were more frequently infected by the intestinal nematode Trichuris arvicolae than males (i.e. prevalence based on the presence of eggs in the faeces) and that females were shedding greater numbers of parasite eggs per gram of faeces (EPG) than males. By applying an anthelmintic treatment to either male or female voles, we demonstrated that treating females significantly reduced parasite burdens (i.e. prevalence and EPG) of both male and female hosts, while treating males only reduced parasite burden in males. These findings indicate that in this female-biased infection system females play a more important role than males in driving the dynamics of parasite transmission.     

Life history, pathology, and description of Kudoa ovivora n. sp. (Myxozoa, Myxosporea): an ovarian parasite of Caribbean labroid fishes

We describe Kudoa ovivora n. sp. from ovaries of bluehead wrasse, Thalassoma bifasciatum, and record its presence in 6 species (Labroidei) collected in the San Blas Islands. Panama. Kudoa ovivora spores are quadrate with rounded edges in apical view, oval-shaped with apical valve extensions in side view (mean spore dimensions: length 6.5 microns, width 7.7 microns, thickness 6.9 microns; mean polar capsule dimensions: length 2.1 microns, width 1.5 microns). This is the first Kudoa species from gonads of fishes. Prevalence of infection varied among labrids (Thalassoma bifasciatum, Halichoeres bivittatus, Halichoeres garnoti, Halichoeres poevi), with T. bifasciatum exhibiting the greatest prevalence. Density of infection, measured as percent infected eggs, also varied among species with highest densities occurring in H. garnoti. Kudoa ovivora may not require an intermediate host because fishes fed infected tissue developed more infections than unfed fish. Infected eggs are inviable and larger and heavier than uninfected eggs. Infected eggs contain more organic and inorganic material, indicating that K. ovivora increases resource allocation to eggs. Therefore, infected females may have reduced growth, fecundity, and/or spawning activity. Because males were uninfected and all identified hosts are protogynous sequential hermaphrodites, further studies of K. ovivora may provide new insights on the costs/benefits of sex change.     

Weak population differentiation in northern European populations of the endangered anadromous clupeid Alosa fallax

Examination of genetic structure among different spawning populations of the endangered twaite shad Alosa fallax showed statistically significant but weak differentiation estimated using nine microsatellite loci. Results point to substantial gene flow among the majority of individual estuary/river spawning locations, and no evidence that habitat disruption has led to increased local genetic drift.     

Prevalence of Myxobolus cerebralis infections among genetic lineages of Tubifex tubifex at three locations in the Madison River, Montana

Host biodiversity can impact disease risk and influence the transmission of parasitic disease. Stream sediment-dwelling worms, Tubifex tubifex (Clitellata: Oligochaeta), are the definitive host of the parasite Myxobolus cerebralis (Myxozoa: Myxosporea), which causes whirling disease in salmonid fishes. Genetic diversity of T. tubifex is correlated with host susceptibility to M. cerebralis , and mitochondrial Lineage III is generally shown to be more likely to be infected and produce the triactinomyxon (TAM) spores than other lineages. We determined the mitochondrial lineage, relative abundance, and prevalence of infection of T. tubifex collected at 3 sites in the Madison River, Montana, where previous study had shown variation in whirling disease prevalence and severity in caged trout fry. We also compared visual identification of TAMs released from cultured worms with a molecular genetic assay (diagnostic polymerase chain reaction [PCR]) for parasite detection of both infected and uninfected worms. We estimated that mitochondrial Lineage III was most abundant at the site previously shown to have high fish disease and was also most likely to be infected. The 2 techniques for detecting parasite infection did not always agree, and the likelihood of PCR (+) and spore (-) was not significantly different from PCR (-) and spore (+). Differences in the relative infection prevalence for these 2 lineages may explain the wide range of infection in natural streams.     

A cost of Wolbachia-induced sex reversal and female-biased sex ratios: decrease in female fertility after sperm depletion in a terrestrial isopod

A number of parasites are vertically transmitted to new host generations via female eggs. In such cases, host reproduction is an intimate component of parasite fitness and no cost of the infection on host reproduction is expected to evolve. A number of these parasites distort host sex ratios towards females, thereby increasing either parasite fitness or the proportion of the host that transmit the parasite. In terrestrial isopods (woodlice), Wolbachia bacteria are responsible for sex reversion and female-biased sex ratios, changing genetic males into functional neo-females. Although sex ratio distortion is a powerful means for parasites to increase in frequency in host populations, it also has potential consequences on host biology, which may, in turn, have consequences for parasite prevalence. We used the woodlouse Armadillidium vulgare to test whether the interaction between Wolbachia infection and the resulting excess of females would limit female fertility through the reduction in sperm number that they receive from males. We showed that multiple male mating induces sperm depletion, and that this sperm depletion affects fertility only in infected females. This decrease in fertility, associated with male mate choice, may limit the spread of Wolbachia infections in host populations.     

Characterization of a peptide from skin secretions of male specimens of the frog, Leptodactylus fallax that stimulates aggression in male frogs

During the breeding season of the mountain chicken frog Leptodactylus fallax, fighting between males results in the emergence of dominant animals that subsequently attract females to nesting sites. A peptide, termed Leptodactylus aggression-stimulating peptide (LASP), was isolated from norepinephrine-stimulated skin secretions from male specimens of L. fallax that was not present in skin secretions obtained from females. The primary structure of the peptide was established as: Gly-Leu-Trp-Asp-Asp-Leu-Lys-Ala-Ala-Ala-Lys-Lys-Val-Val-Ser-Ser-Leu-Ala-Ser-Ala-Ala-Ile-Glu-Lys-Leu NH2. LASP had no pheromone-like action on females but had a chemoattractive effect on males and stimulated aggressive behaviors, such as rearing and leaping. It is suggested that this peptide may play an important role in initiating the competitive male-male interactions that are associated with the onset of reproductive behavior in L. fallax.     

Vertical transmission of Nosema fumiferanae (Microsporidia: Nosematidae) and consequences for distribution, post-diapause emergence and dispersal of second-instar larvae of the spruce budworm, Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae)

We examined vertical transmission of Nosema fumiferanae in the eastern spruce budworm, Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae), and how it affects overwintering distribution and survival and spring emergence and dispersal of second-instar larvae in outbreak populations. Females containing 5.0 x 10(5) spores or more consistently produced 100% infected progeny. Transmission efficiency was still 50% at burdens as low as 0.2 x 10(5) spores per moth. Infection intensity in offspring increased with maternal spore load but became highly variable above 25 x 10(5) spores per female. Nosema multiplied in second instars for at least 1 month after they entered dormancy, regardless of temperature (2 degrees C versus 21 degrees C). Infection did not affect the distribution of overwintering larvae in a white spruce canopy. Dormancy survival between late-summer and the following spring was lower in families from infected females and was negatively correlated with larval infection intensity. Infection delayed larval emergence from hibernacula in the spring and resulted in delayed dispersal of emerged larvae, at least when parasite prevalence and infection intensities were high. Infected larvae were less successful in establishing feeding sites after dispersal. Our results underscore the potential of Nosema infection to negatively affect processes early in the budworm life cycle.     

Worker genetic diversity and infection by Nosema apis in honey bee colonies.

The hypothesis that parasites and pathogens select for polyandry in eusocial Hymenoptera was tested, using the honey bee Apis mellifera and its microsporidian parasite Nosema apis. Five honey bee colonies with low and five with high worker genetic diversity were infected with N. apis spores. At 54-56 days after inoculation, parasite spores in the workers' midguts were counted to determine whether there was a greater variation of infection intensity (spore counts per worker) in high-diversity colonies than in low-diversity ones. In all colonies there were two discrete sets of workers, with few or many parasite spores. To compare the variations of infection intensity between two colony groups, coefficients of variation were calculated for all workers examined, and for the slightly infected and strongly infected workers. The percentages of slightly infected workers in the low- and high-diversity groups were also compared. None of the comparisons between low- and high-diversity colonies showed significant differences, therefore no relation was found between honey bee workers' genetic diversity and their infection with N. apis.     

Impact of a novel species of Nosema on the southwestern corn borer (Lepidoptera: Crambidae)

A study was undertaken to elucidate the impact of an undescribed Nosema sp. on the southwestern corn borer (SWCB; Diatraea grandiosella Dyar). The Nosema sp. (isolate 506) included in the study was isolated from an overwintering SWCB larva in Mississippi. It was highly infectious per os, with a median infective dose of 2.0 x 10(3) spores per larva. Even at the highest dosage tested (10(7) spores per larva), minimal mortality (< or = 3%) was observed in infected larvae, pupae, and adults reared in the laboratory on an artificial diet. However, infected pupae (0- and 7-d-old) were smaller, and the time to adult eclosion from pupation was slightly increased. Furthermore, the number of eggs produced by infected SWCB female moths substantially decreased (32%), and this effect was most pronounced on day 2, when the greatest number of eggs were oviposited by infected and noninfected moths. For eggs produced by infected females mated with infected males, hatch was slightly decreased by 16 and 15% for eggs laid on days 2 and 3, respectively. In addition, egg hatch was reduced in eggs oviposited by noninfected females mated with infected males on day 3. A low prevalence of infection (< 6%) was observed in the F1 generation originating from infected females mating with noninfected males, from noninfected females mating with infected males, and from infected females mating with infected males. Nosema 506 spores were observed in the proximity of reproductive tissues of infected female and male moths. Spores also were detected on the chorion surface and within eggs laid by infected females. Furthermore, 1-11% of larvae hatching from surface-sterilized eggs were infected by Nosema 506 indicating a transovarial mechanism of transmission.     

Male genital tract chlamydial infection: implications for pathology and infertility

Chlamydia trachomatis infections are prevalent worldwide, but current research, screening, and treatment are focused on females, with the burden of disease and infertility sequelae considered to be a predominantly female problem. The prevalence of chlamydial infection, however, is similar in males and females. Furthermore, a role for this pathogen in the development of male urethritis, epididymitis, and orchitis is widely accepted. The role of Chlamydia in the development of prostatitis is controversial, but we suggest that Chlamydia is an etiological agent, with incidences of up to 39.5% reported in patients with prostatitis. Infection of the testis and prostate is implicated in a deterioration of sperm, possibly affecting fertility. Chlamydia infections also may affect male fertility by directly damaging the sperm, because sperm parameters, proportion of DNA fragmentation, and acrosome reaction capacity are impaired with chlamydial infection. Furthermore, the proportion of male partners of infertile couples with evidence of a Chlamydia infection is greater than that documented in the general population. An effect of male chlamydial infection on the fertility of the female partner also has been reported. Thus, the need for a vaccine to protect both males and females is proposed. The difficulty arises because the male reproductive tract is an immune-privileged site that can be disrupted, potentially affecting spermatogenesis, if inappropriate inflammatory responses are provoked. Examination of responses to infection in humans and in experimental animal models suggest that an immunoglobulin A-inducing vaccine will be able to target the male reproductive tract effectively while avoiding harmful inflammatory responses that may impair fertility.     

AFLP analysis of the genetic diversity of Meloidogyne chitwoodi and M. fallax, major agricultural pests

M. chitwoodi and M. fallax populations are clustered and separated from the other species studied. The genetic diversity observed for M. incognita, M. arenaria, M. javanica, M. hapla, and M. mayaguensis correlates well with the previously validated species. Two main groups can be identified within the M. chitwoodi/M. fallax cluster, the first group comprises only M. chitwoodi populations whereas the second group is made of M. chitwoodi and M. fallax populations. Moreover, M. chitwoodi displays a higher genetic diversity than M. fallax and is characterised by the presence of several clusters.