Life cycle of Amblyospora indicola (Microspora: Amblyosporidae), a parasite of the mosquito Culex sitiens and of Apocyclops sp. Copepods

The life cycle of Amblyospora indicola, a parasite of the mosquito Culex sitiens, was revealed by field observations and laboratory infection experiments conducted in Australia. In northern Queensland, infected C. sitiens larvae were often found breeding in association with two cyclopoid copepods: Apocyclops dengizicus and an undescribed species of the same genus. The latter species was found to be an intermediate copepod host of this microsporidium whereas A. dengizicus was not. One complete cycle of the parasite extends over two mosquito generations (by transovarial transmission from females with binucleate spores to their eggs) and by horizontal transmission between mosquitoes and copepods. The latter involves horizontal transmission from mosquitoes to copepods via meiospores produced in larval fat body infections and horizontal transmission from copepods to mosquitoes via uninucleate spores produced within infected copepods. Uninucleate clavate spores were formed in Apocyclops sp. nov. copepods 7-10 days after exposure to larval meiospores and were infectious to larvae of a microsporidian-free colony of C. sitiens. The development of A. indicola within mosquito larvae exposed to infected copepods is similar to that of A. dyxenoides infecting C. annulirostris. It proceeds from stages with a single nucleus to diplokaryotic binucleate cells in oenocytes. These stages persist through pupation to adult emergence after which time a proportion of male mosquitoes and female mosquitoes may develop binucleate spores without the need for a blood meal. A proportion of both male and female larval progeny of infected females with binucleate spores develop patent fat body infections via transovarial transmission and die in the fourth larval instar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epizootiological studies of Amblyospora camposi (Microsporidia: Amblyosporidae) in Culex renatoi (Diptera: Culicidae) and Paracyclops fimbriatus fimbriatus (Copepoda: Cyclopidae) in a bromeliad habitat

The epizootiology of Amblyospora camposi was studied in a natural population of Culex renatoi, a bromeliad-inhabiting mosquito, and its intermediate host, Paracyclops fimbriatus fimbriatus, over a 2-year period. Twenty Eryngium cabrerae plants were sampled monthly from January 2003 to January 2005 and the prevalence of A. camposi in P.f. fimbriatus and Cx. renatoi populations was determined. The monthly prevalence rates of meiospore infections in Cx. renatoi larvae never exceeded 5.5% and was detected in 50% of the monthly samples. Meiospores were available in plants over the course of the study at a mean concentration of 2 x 10(4) meiospores/ml. Within each plant the parasite was maintained by horizontal transmission. P.f. fimbriatus with vegetative stages and mature spores were found regularly in bromeliads suggesting efficient meiospore infectivity to field copepod populations. The mean concentration of spores from copepods found in plants was 8 x 10(2) spores/ml. Infections in copepods were detected in 54% of the monthly samples with a prevalence rate ranging from 0.55 to 17.4% and an overall average of 5.1%. Vegetative stages in fourth instar mosquito larvae (probably derived from the horizontal pathway via spores formed in copepods) were detected in 12.5% of the monthly samples with an overall prevalence rate of 1.1%. Infections in female and male adults were detected in 20.8% of the monthly samples with an overall average of 4.1% and 6.8%, respectively.     

Coelomomyces dodgei: Establishment of an in vivo laboratory culture

An in vivo laboratory culture of the fungus Coelomomyces dodgei (Chytridiomycetes: Blastocladiales) was established, using the copepod Cyclops vernalis as an intermediate host and the larvae of the mosquito Anopheles quadrimaculatus as the definitive host. The culture was perpetuated by infecting copepods and mosquitoes using separate procedures. Copepods were infected by being combined with dehiscing sporangia. Patently infected copepods, which contained either light amber, bright orange, or both light amber and bright orange mycelia, were collected daily beginning 12 days later. Mosquitoes were infected by combining 100 first-instar larvae for 48 hr with a mixture of 12 infected copepods, four of each of the above types. The mean rate of infection for the first 100 trials was 41%. When groups of 100 first-, second-, third-, and fourth-instar larvae were exposed to a similar mixture of infected copepods for 48 hr, the mean rates of infection were 37.4, 27.0, 17.8, and 2.4%, respectively. Observations and experimental evidence suggest that the differentially pigmented mycelia found in infected copepods are gametophytes which develop into gametangia that subsequently release gametes of opposite mating types, the light amber gametangia producing female and the bright orange gametangia producing male gametes. Zygotes resulting from the fusion of these gametes lead to the infection of mosquito larvae. Thus, C. dodgei appears to have an Euallomyces type of life cycle with sporophyte and gametophyte generations alternating between mosquito and copepod hosts, respectively, with differentially pigmented sexual structures present in the gametophyte phase.     

Distribution of Kroeyerina elongata (Kroyeriidae: Siphonostomatoida, Copepoda) in the olfactory sacs of the blue shark, Prionace glauca

The infection pattern of Kroeyerina elongata (Kroyeriidae, Copepoda) in the olfactory sacs of the blue shark, Prionace glauca, was investigated using 4,722 copepods from 54 olfactory sacs. Copepod prevalence and mean intensity of infection per olfactory sac were 94.0 and 91.1%, respectively, and the most intensely infected olfactory sac and shark hosted 218 and 409 copepods, respectively. There were significant linear relationships between the number of female and total copepods per left olfactory sac and shark fork length as well as between the numbers of female, male, and total copepods per shark and mean olfactory sac width and cumulative olfactory sac width. Female copepods typically outnumbered males within olfactory sacs (mean intensity = 65.7 and 26.3, respectively), and no statistical differences were detected between the numbers of copepods inhabiting the left and right olfactory sacs. Copepods were not evenly distributed within olfactory sacs. Typically, female copepods occupied olfactory chambers located centrally along the length of the olfactory sac, while males infected lateral olfactory chambers nearest the naris. The orientation of most copepods (84.6%) suggested positive rheotaxis relative to the path of water through the olfactory sac. Within olfactory chambers, most mature females (68.2%) infected the first third of the peripheral excurrent channel and the adjacent fringe of olfactory lamellae, while most males (91.7%) infected the olfactory lamellae, and the 4 larval females collected were attached within the lamellar field and grasped by males. Based on the observed infection patterns and the pattern of water flow throughout the olfactory sac, a hypothesis regarding the life cycle of K. elongata is advanced wherein infective copepodids are swept into the olfactory sac from the surrounding sea and initially colonize the olfactory lamellae. Copepodids feed and mature among the olfactory lamellae, and adult males search for mates and copulate with young females among the olfactory lamellae. Inseminated females move to the peripheral excurrent channels to mature and produce ovisacs. Hatching ovisacs release free-swimming nauplii into the excurrent water flow to be swept into the milieu, where they can molt into infective copepodids that may infect new hosts.     

Transovarial and transphasic transmissions of Borrelia by the taiga tick Ixodes persulcatus (Ixodidae)

Three generations of the taiga tick Ixodes persulcatus, the descendants from naturally infected females, have been examined by means of dark field and phase contrast microscopies and indirect immunofluorescent reactions with monoclonal antibodies. Location of borreliae in oocytes was examined by means of electron microscopy. The examined ticks derived from 9 females collected in the Novgorod Province, from 6 females of 1st laboratory generation and 5 females of the 2nd generation. In total, 250 larvae, 178 nymphs, 59 females and 70 males of three consequent generation have been examined. Almost 100% of descendants of naturally infected females were infected with Borrelia burgdorferi s. l. and similar infection rate was observed in unfed tick larvae collected in field conditions. The borreliae received transovarially to larvae of the 1st generation then were transmitted to 100% nymphs and imago of this generation and two next generations.     

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.     

Life cycle of Gnathostoma nipponicum Yamaguti, 1941.

The life cycle of Gnathostoma nipponicum was examined by field survey and by experimental infection of animals with the larvae. Naturally infected larval G. nipponicum were found in loaches, catfish, and snakes. Experimentally, loaches, killifishes, frogs, salamanders, mice, and rats were successfully infected with the early third-stage larvae of G. nipponicum obtained from copepods (the first intermediate host), whereas snakes, quails, and weasels were not. Frogs, snakes, quails, and rats were experimentally infected with the advanced third-stage larvae (AdL3) from loaches. These results reveal that some species of fishes, amphibians and mammals can act as the second intermediate host and that some species of reptiles, birds and mammals can act as a paratenic host. The life cycle was completed in weasels, the definitive host, which were infected with AdL3 from loaches and started to evacuate eggs of G. nipponicun in faeces on days 65-90 postinfection.     

Shallow males, deep females: sex-biased differences in habitat distribution of the freshwater calanoid copepod Arctodiaptomus alpinus

In a small, circular high-altitude karst lake situated in the northeastern calcareous Alps of Austria the copepod Arctodiaptomus alpinus (Imhof) develops each spring from resting eggs after the lake basin fills in early spring. After mid-summer, late developmental stages and adults concentrate in a thin layer near the sediment throughout the lake basin. During daytime an average 86% and 71% of female and male copepods respectively resided near the bottom. Males exhibited stronger diel vertical migrations than females and part of the population concentrated in near-surface water at night, but the majority of copepods remained at the sediment during darkness. Macro-photographs and core samples revealed that the proportion of epibenthic male A. alpinus decreased constantly from >70% in 3 m depth to <20% below the 8-m isobath. The proportion of ovigerous females increased with depth, whereas non-ovigerous females were comparatively more abundant in shallow water. Concurrently the highest frequencies of copulating copepods were detected in areas of high male density. Above the 3-m isobath male A. alpinus formed dense swarms in scattered patches of the macrophyte Ranunculus eradicatus (>93% males; maximum abundance: 362 600 individuals m⁻²). Adults and larvae of the alpine newt Triturus alpestris are the top predators in this fishless alpine lake, and A. alpinus constitutes a major component of their diet. However, predator densities and different predation rates on male and female copepods were not sufficient to explain the observed horizontal distribution of A. alpinus . We argue that the optimization of successful sexual encounters in copepods is the ultimate driving force behind this segregation of both sexes of A. alpinus ('mate encounter hypothesis').     

The behavioural responses and tolerance of freshwater benthic cyclopoid copepods to hypoxia and anoxia

The response of four benthic cyclopoid copepods,Acanthocyclops viridis (Megacyclops viridis) (Jurine, 1820),Macrocyclops albidus (Jurine),Eucyclops agilis (Koch, Sars) (Eucyclops serrulatus) (Fischer, 1851) andParacyclops fimbriatus (Fischer), to hypoxia and anoxia was investigated. All of these species died within six hours when confronted by totally anoxic conditions, but all survived four days at oxygen saturation levels as low as 25%. Males succumbed to the effects of anoxia more rapidly than the larger females of each species, and larger species survived for shorter periods than smaller species. In artificially stratified columns, where the lower layer was anoxic, all four species displayed an upward migratory response towards oxygenated conditions. Where the artificial hypolimnion was hypoxic, however, the migratory response was not observed. The results suggest that some benthic copepods cope with seasonal anoxia in eutrophic stratified lakes by migration rather than the various physiological adaptations shown by planktonic and semi-planktonic species.     

Epizootiology of Amblyospora stimuli (Microsporidiida: Amblyosporidae) infections in field populations of a univoltine mosquito, Aedes stimulans (Diptera: Culicidae), inhabiting a temporary vernal pool.

The epizootiology of the microsporidium Amblyospora stimuli was studied in natural populations of a univoltine mosquito, Aedes stimulans, inhabiting a temporary vernal pool over an 18-year period. The yearly prevalence of benign oenocytic infections in adult females was variable, ranging from 1.0 to 9.6% (mean = 5.1%). The yearly prevalence of transovarially transmitted meiospore infections in larval populations was consistently lower but less variable, ranging from 1.3 to 5.9% (mean = 3.5%). Meiospore infections in F(1)-generation larvae were significantly correlated with infections in parental-generation females, thus suggesting that larval infection rates could be substantially increased if methods were available to facilitate transmission of A. stimuli to a larger portion of the female population via inundative or inoculative release of infected copepods. No correlation was found when infections in filial-generation adult females were measured against meiospore infections in larvae from the preceding year. Analysis of yearly prevalence data using Fine's Fundamental Vertical Transmission Equation revealed low rates of horizontal transmission from the intermediate copepod host to female larvae in most years, ranging from 0.1 to 8.7% (mean = 3.1%). A. stimuli is enzootic, persists at a very low level, and has minimal impact on Ae. stimulans populations at this site. The low incidence rate of horizontal transmission to larvae appears to be due largely to a paucity of copepods and is a major factor that limits the abundance and subsequent proliferation of A. stimuli in Ae. stimulans populations at this locale. Results support the view that host-parasite cospeciation is an important mechanism of evolution in this group of mosquito/copepod microsporidia.     

Effect of Acanthocephala infection on the reproductive potential of crustacean intermediate hosts

The effect of a naturally acquired infection by three acanthocephalan parasites Dentitruncus truttae, Echinorhynchus truttae, and Polymorphus minutus on the reproductive potential of their intermediate host, Echinogammarus tibaldii (Amphipoda) from Lake Piediluco (Centre of Italy) was assessed. During May 2007, 1135 amphipods were collected from two different samplings and examined for larval helminths. Forty-five amphipods were infected and of those, 16 were infected with D. truttae (intensity = 1-3 larvae), 15 with E. truttae (intensity = 1-2 larvae), and 14 with P. minutus (intensity = 1 larva). The sex ratio was nearly 1:1 in all examined amphipods. One female infected with D. truttae contained six eggs in the brood pouch and another female infected with E. truttae contained five eggs. However, none of the eight female amphipods harbouring P. minutus larva contained eggs in their brood pouch. Uninfected females of the same size and body length as that of the infected females contained between 20 and 32 eggs. No acanthocephalan species were found to co-occur.     

Population dynamics and epidemiology of Toxocara canis in Danish red foxes

Toxocara canis, an intestinal helminth of canids with zoonotic potential, was found in 618 (59%) of 1,040 red foxes (Vulpes vulpes) collected from all Danish provinces (1997-2002). The prevalence and average worm burden were significantly higher for cubs than older foxes and in males than in females. A multiple logistic regression demonstrated that the prevalence was influenced significantly by sex and age of foxes in addition to location, season, and year of collection. The highest prevalence and worm burden were found in rural areas. The size and number of female worms was positively correlated to the fecal egg excretion. The length and fecundity of the worms was significantly higher in male foxes, and a general intensity dependence was suggested from a negative correlation between worm numbers and worm lengths. As compared to intestinal recovery of worms, somatic larvae were recovered from 20% of muscle samples and fecal eggs in 41% of fecal samples. The consistent finding of T. canis larvae in somatic tissues of naturally infected foxes is new.     

Effects of Bacillus thuringiensis on Adults of Cotesia plutellae (Hymenoptera: Braconidae), a Parasitoid of the Diamondback Moth, Plutella xylostella (Lepidoptera: Plutellidae)

To complement existing information on the mortality of larvae of the wasp Cotesia plutellae attacking moth caterpillars infected with Bacillus thuringiensis (Bt) we tested the direct and indirect effects of the bacterium on adult wasp longevity and oviposition behaviour. In one experiment with female parasitoids, mean longevity (SEM) was not significantly different between females exposed to Bt (1.98 +/- 0.08 days) and those not exposed (2.18 +/- 0.13 days). In a second experiment with both males and females, Bt treatment did not significantly effect either male or female parasitoids exposed to Bt. To observe the possible effects of Bt on oviposition behaviour of C. plutellae each of 10 females were given five larvae that had been treated with Bt and five untreated larvae at the same time. All parasitoids were observed to make oviposition attempts in both untreated and treated larvae. Upon dissection of the host larvae, one or more C. plutellae eggs were found in each of the larvae in which a parasitoid attempted oviposition. There was no effect of Bt treatment on parasitoid oviposition. The mean number of ovipositions in treated larvae (4.3 +/- 0.3) was not significantly different from untreated larvae (4.7 +/- 0.2).     

Thelazia lacrymalis in horses in Kentucky and observations on the face fly (Musca autumnalis) as a probable intermediate host.

Eyes from 114 (30.3%) of 376 dead horses, examined from 3 April 1975 to 3 April 1976, were naturally infected with adult Thelazia lacrymalis; 1 horse was also infected with 1 male Thelazia skrijabini. Adult T. lacrymalis from dead horses were successfully transferred mechanically to the eyes of 3 of 4 Shetland ponies raised helminth-free. Larvae from gravid female T. lacrymalis underwent development in experimentally infected, laboratory-raised face flies (Musca autumnalis) and third-stage larvae ranging from 1.82 to 2.94 mm in total length were recovered at 12 to 15 days postexposure. A total of 866 naturally occurring face flies were collected from the head region of horses. Twelve of the face flies harbored larval stages of Thelazia spp. One of the larvae resembled third-stage T. lacrymalis that were recovered from the experimentally infected, laboratory-raised face flies. Introduction of 3 third-stage larvae from 1 face fly onto the cornea of a pony raised helminth-free resulted in the recovery of 1 male T. skrjabini 242 days later. In addition to the eyeworm larvae, other parasites recovered from the face flies included Heterotylenchus autumnalis, hypopi of astigmatid mites and a first instar beetle (Coleoptera: Rhipiphoridae). Data from these investigations indicate the likelihood that face flies are an intermediate host for T. lacrymalis and probably other species of Thelazia in this part of the country.     

An experimental investigation of a direct life-cycle in Reighardia sternae (Diesing, 1864), a pentastomid parasite of the herring gull (Larus argentatus).

A direct life-cycle in Reighardia sternae, a cephalobaenid pentastomid of gulls was investigated: the work was prompted by a report of eggs and larvae recovered from the stomach and intestine of a naturally infected gull. Infective pentastomid eggs were obtained by surgically transplanting maturing female Reighardia, taken from freshly shot wild gulls, into captive recipients. Faecal material from birds thus artificially infected was collected daily and examined for eggs. Eggs were force fed to 33 hand-reared (from eggs or nestlings) juvenile gulls which were selected at random and sacrificed at intervals thereafter and examined for pentastomids. One hour after infection, primary larvae appear in the body cavity where they moult immediately. They grow steadily and by 27-35 days are sexually differentiated, and by 66 days have copulated. Fertilized females take a further 116 days to produce eggs by which time they are 7-6 cm long. The complex migrations undertaken by developing larvae in the gull, and the problems of the mechanism of direct transmission, are discussed.     

Distribution,diversity and density of wolbachial infections in cladocerans and copepods from Thailand

Species of the genus Wolbachia comprise a group of Rickettsia-like, maternally-inherited bacteria that cause several reproductive alterations in arthropod hosts. The best known are cytoplasmic incompatibility and feminization. Here, the first systematic surveys of wolbachial infections in cladocerans and copepods from six geographic regions of Thailand, including Northern, Northeastern, Western, Central, Eastern and Southern are reported. Using gene amplification assays with wsp and groE primers, wolbachiae were detected in 239 (4 spp.) of 1885 (57 spp.) copepods and cladocerans from all regions of Thailand surveyed. Screening results obtained with wsp primers or groE primers were similar in all cases. The presence of wolbachiae was only detected in copepods, not in cladocerans. Sex ratio analyses of the progeny of two species of copepods, Mesocyclops aspericornis and Mesocyclops thermocyclopoides, naturally or artificially infected with wolbachiae showed infection causes feminization (female-bias). The relative density if infection in naturally infected populations of three copepod species, M. thermocyclopoides, Heliodiaptomus elegans and Neodiaptomus blachei, were determined using real-time quantitative PCR assay based on the wsp gene. The density of wolbachiae in M. thermocyclopoides was significantly higher than in the other two species. These results suggest that wolbachial infections are distributed throughout Thailand, and that possibly the natural occurrence of these in copepods may be due to their predation on mosquito larvae. This apparent novel biology may have importance as a genetic drive system for control of vector borne diseases in the future.     

Host sex preferences and transmission success by the water mite Unionicola foili (Acari: Unionicolidae) parasitic on the midge Chironomus tentans (Diptera: Chironomidae)

This study examined whether ecoparasitic larval Unionicola foili exhibited a sex bias when infecting laboratory populations of the host insect Chironomus tentans and whether an association with male or female midges increased the likelihood of larval mites returning to the aquatic habitat. When laboratory populations of C. tentans were exposed to larval U. foili, there was a higher prevalence of mites among female hosts at emergence (17 of 30 males vs. 25 of 30 females infected by mites). However, there was no significant difference in the distribution or abundance of larvae among infected male (mean = 2.3 larvae per host) and female (mean = 2.6 larvae per host) midges. Larval mites parasitizing both male and female chironomids were more likely to return to water than could be expected by chance. Mite larvae infesting female C. tentans were more likely to return to water when female hosts deposited egg masses in water, suggesting that oviposition plays an important role in cueing larvae parasitizing female midges to detach. The mechanism responsible for increasing the likelihood that mites parasitizing male hosts return to water remains unclear. Future studies will address the possibility of parasite-mediated changes in host behavior.     

Laboratory experiments on infection rates of Amblyospora dyxenoides (Microsporida: Amblyosporidae) in the mosquito Culex annulirostris

Laboratory observations were made of the microsporidian parasite Amblyospora dyxenoides in its natural mosquito host, Culex annulirostris. There were no differences in the numbers of eggs laid and in the proportions which hatched between infected and uninfected females, indicating that the parasite did not affect fecundity. Unlike other species of Amblyospora which have been studied the development of binucleate spores in adult mosquitoes increase with age of the host in both sexes and in females it proceeds independently of egg development and blood feeding. The same trend is apparent for adult mosquitoes which acquired the infection in the larval stage by horizontal transmission from the intermediate copepod host as well as for mosquitoes which acquired oenocytic infections by transovarial transmission. There was considerable variation in the proportion of mosquitoes which became infected after exposure to A. dyxenoides infected copepods. Infections in larval progeny of female mosquitoes infected via spores produced in copepods ranged from 0 to 100% in individual batches and averaged 45.6% with meiospore infections, 19.3% with oenocytic infections, with the remaining 35.7% being uninfected. Similar variability was observed in the progeny of infected female mosquitoes in the second generation after exposure to infected copepods. During experiments in which the microsporidium was maintained in C. annulirostris through 9 successive transovarially transmitted cycles (by selectively rearing the progeny of females infected with binucleate spores after an initial exposure to infected copepods) the proportion of infected progeny with oenocytic infections increased from 25 to around 50% whereas the incidence of meiospore infections declined from 50 to 10%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Horizontal transmission of Parathelohania anophelis to the copepod, Microcyclops varicans, and the mosquito, Anopheles quadrimaculatus

The copepod Microcyclops varicans was infected with Parathelohania anophelis by unincleate meiospores from a field-collected fourth instar Anopheles quadrimaculatus larva. Large numbers of unincleate, pyriform spores developed in the copepod. These spores were fed to early instar A. quadrimaculatus larvae, infecting both males and females, resulting in the production of cylindrical, binucleate spores in the adult. These spores were responsible for vertical transmission, through the eggs, to the larvae. The original spore type collected from the field was found in the male progeny from the infected females. Another P. anophelis-infected mosquito colony was established by feeding spores from a single, field-collected, infected copepod to A. quadrimaculatus larvae. The microsporidium was continuously maintained by vertical transmission in newly established infected colonies.