Huffmanela cf. carcharhini (Nematoda: Trichosomoididae: Huffmanelinae) from skin of a sandbar shark, Carcharhinus plumbeus, in the Pacific Ocean

Eggs of Huffmanela cf. carcharhini from the skin of an aquarium-held, juvenile sandbar shark, Carcharhinus plumbeus , from the Pacific Ocean were studied using light and scanning electron microscopy. Grossly, eggs imparted a scribble-like skin marking approximately 130 × 60 mm on the right side of the shark's snout adjacent to its eye and nostril. Fresh (unfixed) eggs were elliptical, 75-95 μm long (xˉ = 85 μm, SD = ±4.5; n = 75), 48-63 μm wide (53 ± 3.4; 75), 8-10 μm in shell thickness (9 ± 1.3; 27), 45-68 μm in vitelline mass length (52 ± 6.9; 8); had a smooth shell surface and nonprotruding polar plugs 8-13 μm wide (10 ± 1.5; 73); lacked thin filaments, superficial envelope, and shell spines; sank in 35 ppt artificial seawater; and did not spontaneously hatch after 12 hr in 35 ppt artificial seawater. Formalin-fixed eggs measured 193 days postfixation were 75-95 μm long (84 ± 3.9; 150), 45-60 μm wide (50 ± 2.2; 150), 5-10 μm in shell thickness (8 ± 1.2; 87), 45-60 μm in vitelline mass length (51 ± 3.0; 92), and 30-40 μm in vitelline mass width (33 ± 2.0; 84), and had nonprotruding polar plugs that were 10-15 μm long (11 ± 1.4; 93) and 8-10 μm wide (9 ± 1.1; 108). Forcibly hatched first-stage larvae (unfixed) were filiform, 188-273 μm long (212 ± 25.5; 13), 8-13 μm wide (10 ± 1.2; 13), and had fine transverse striations. Eggs infected the epidermis only. Histology revealed intra-epithelial inflammation with eosinophilic granulocytes and hyperplasia, plus dermal lymphofollicular hyperplasia associated with the infection. The eggs of H. cf. carcharhini likely undergo considerable ex utero development before being sloughed (unhatched) from the host, along with epidermal cells.     

Experimental life cycle of Lagochilascaris major leiper, 1910 (Nematoda: Ascarididae) in cats (Felis domesticus)

The life cycle of Lagochilascaris major was studied using eggs collected from a natural clinical case in a domestic cat. Twenty-seven white mice (Mus musculaus), 5 hamsters (Mesocricetus auratus), and 1 vesper mouse (Calomys callosus) were orally inoculated with 800-1,300 embryonated eggs. When examined from 73 to 246 days postinoculation (PI), encysted third-stage larvae were seen in skeletal muscles and less frequently in connective tissue, liver, and lungs. Twenty-two of the 23 cats orally inoculated with 40-430 encysted larvae from these rodents, and necropsied from 1 hr to 185 days PI, became infected. Third-stage larvae were located in the stomach, esophagus, and oropharynx from 1 to 24 hr PI. At 48 hr, larvae, from mainly the fourth stage, were only found, unilaterally or bilaterally, inside a "sac" in the region of the semilunar fold of the palatine tonsil at the base of the tongue. Adult worms were found in this location from 10 to 175 days PI. No fistulated abscess to the outside medium was found. Adult worms were also found in the middle ears of 2 cats showing purulent otitis. Eggs in the ear secretion were under different stages of development. Eggs in feces were first observed on days 14 and 15 PI, and 1 cat shed them until 178 days PI. Six infected cats were treated with fenbendazole at 50 mg/kg of body weight for 3 consecutive days, eliminating all the parasites present in the tonsils. The drug was not effective against the parasites present in the middle ear. No stage of the parasite was found in the tissues of 5 cats given 4,000-5,200 eggs orally and examined after 19 and 50 days PI. This indicates that the life cycle of L. major requires an obligate paratenic host and is characterized by heteroxenic cycle.     

Arthrostoma miyazakiense (Nematoda: Ancylostomatidae) infection in raccoon dogs of Korea and experimental transmission to dogs

Arthrostoma miyazakiense (Nematoda: Ancylostomatidae) is a hookworm species reported from the small intestines of raccoon dogs (Nyctereutes procyonoides) in Japan. Five Korean raccoon dogs (N. procyonoides koreensis) caught from 2002 to 2005 in Jeollanam-do (Province), a southeastern area of South Korea, contained helminth eggs belonging to 4 genera (roundworm, hookworm, whipworm, and Capillaria spp.) and cysts of Giardia sp. in their feces. Necropsy findings of 1 raccoon dog revealed a large number of adult hookworms in the duodenum. These hookworms were identified as Arthrostoma miyazakiense based on the 10 articulated plates observed in the buccal capsule and the presence of right-sided prevulval papillae. Eggs of A. miyazakiense were 60-65 x 35-40 micrometer (av. 62.5 x 35 micrometer), and were morphologically indistinguishable from those of Ancylostoma caninum. The eggs were cultured to infective 2nd stage larvae via charcoal culture, and 100 infective larvae were used to experimentally infect each of 3 mixed-bred puppies. All puppies harbored hookworm eggs in their feces on the 12th day after infection. This is the first report thus far concerning A. miyazakiense infections in raccoon dogs in Korea, and the first such report outside of Japan.     

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.     

Embryonation and Infectivity of Ascaris suum Eggs. A Comparison of Eggs Collected from Worm Uteri with Eggs Isolated from Pig Faeces

Ascaris suum eggs were collected from pig faeces or dissected from worms obtained from the same pigs. Eggs from the two sources were allowed to embryonate in 0.1 N H2SO4, in 1 % buffered formalin or in tap water. The embryonation of the sulphuric acid and water cultures occurred at the same speed, while the formalin cultures developed slightly more slowly. By experimental inoculation of helminthfree pigs and subsequent counting of white spots in the livers and larvae in the lungs day 7 p. i., the infectivity of eggs dissected from worm uteri and embryonated in sulphuric acid (a normal laboratory procedure) was compared with that of eggs collected from faeces and embryonated in water (i.e. more naturally developed eggs). The results suggest that the two types of eggs were equally infective. For this reason the common practice of using Ascaris eggs dissected from worms for experimental infections might be acceptable.     

The development of Eustrongylides tubifex (Nematoda: Dioctophymatoidea) in oligochaetes

Egg development of Eustrongylides tubifex (Nitzsch in Rudolphi, 1819) J?gerski?ld, 1909, was studied at 5, 10, 15, 20, 25, and 30 C. Eggs developed at 20, 25, and 30 C. Development ceased at 0, 5, 10, and 15 C but resumed when temperatures were raised. Eggs contained first-stage larvae in 23-26 days at 25 C. Seven species of aquatic oligochaetes were exposed experimentally to eggs of E. tubifex containing first-stage larvae. Third-stage larvae of E. tubifex developed in the aquatic oligochaetes, Limnodrilus hoffmeisteri Claparède, 1862, and Tubifex tubifex (Müller, 1774). Larvae developed in the ventral blood vessel of oligochaetes. Second-stage larvae were seen first in oligochaetes 30 days postinfection and third-stage larvae were seen 70-109 days postinfection at 25 C. Third-stage larvae in oligochaetes retained the cuticle of the first and second molt.     

Selection, egg viability, and fecundity of the sugarcane borer (Lepidoptera: Crambidae) with tebufenozide.

Two separate attempts to select the sugarcane borer, Diatraea saccharalis (F.), for resistance to tebufenozide were unsuccessful. Both selected colonies were lost after the fourth generation due to a lack of oviposition. Differences were not detected in fecundity or percent egg viability for 5-d-old third instars exposed to concentrations (EC5, EC15, and EC30) of tebufenozide for 7 d. Decreases (P < or = 0.01) in mean female pupal weights were detected in larvae exposed to EC15 and EC30 concentrations. An ovicidal impact using serial dilutions of tebufenozide (10, 100, and 200 ppm) also was detected. Percent viability was reduced from 98% for untreated eggs to 61% for eggs dipped in 10 ppm and below 6% for eggs dipped in > or = 100 ppm. Eggs treated with 200 ppm did not hatch. Though some embryonic development was observed on eggs treated with the high concentrations (100 and 200 ppm), sclerotization of head capsule was not apparent. The ovicidal property of tebufenozide may enhance its effectiveness in controlling populations of the D. saccharalis on an area-wide basis. Fecundity and egg viability were affected in later generations of selection; however, separate studies assessing individuals that were exposed to sublethal concentration (EC5, EC15, and EC30) of tebufenozide as third instars for 7 d in one generation did not detect differences.     

Developmental stage affects eggshell-breaking strength in two ground-nesting birds: the partridge (Alectoris rufa) and the quail (Coturnix japonica)

We examined the relationship between embryo development and egg hardness in two ground nesting bird species, the red-legged partridge (Alectoris rufa; n = 165 eggs) and the quail (Coturnix japonica; n = 148 eggs). For both species, we observed a strong effect of developmental stage on egg hardness. Eggs near hatching were significantly weaker than unincubated eggs (partridge: 18 and 23 N, respectively, and the quail 7 and 10 N, respectively). We additionally explored the effect of incubation on egg hardness in a control sample of non-fertilised quail eggs (i.e., without embryo development). The control eggs maintained in the incubator for the full incubation time (17 days) were significantly harder (7-9 N) than eggs containing fully developed chicks (5-7 N). Thus, the incubation conditions of high temperature and humidity alone seem not to have a significant effect on egg hardness, and support the important effect of calcium uptake.     

Effects of Beauveria bassiana and Metarhizium anisopliae on mortality, fecundity and egg fertility of Tetranychus evansi

Abstract:  The susceptibility of various developmental stages of Tetranychus evansi Baker & Pritchard (eggs, larvae, protonymphs, deutonymphs and adults) to the entomopathogenic fungi Metarhizium anisopliae (Metschnikoff) Sorokin and Beauveria bassiana (Balsamo) Vuillemin was evaluated under laboratory conditions. Three concentrations (3.0 × 10⁶, 1.0 × 10⁷ and 1.0 × 10⁸ conidia/ml) of both fungi were used for each stage. The effect of fungal infection on fecundity and egg fertility was also investigated using both fungal species. Deutonymphs that survived the infection and developed into adult females were allowed to oviposit. Adults and deutonymphs were more susceptible to fungal infection than larval and protonymphal stages at all the concentrations. Nevertheless, the concentration level influenced the mortality of the different mite stages. Eggs were also susceptible to fungal infection and mortality was dose-dependent. Fungus-treated female mites laid fewer eggs than the controls but there was no significant difference in egg hatchability between the treatments.     

Laboratory evaluation of entomopathogenic fungi against larvae and adults of onion maggot (Diptera: Anthomyiidae)

Laboratory experiments were done to measure the susceptibility of larvae and adults of the onion maggot, Delia antiqua (Meigen) (Diptera: Muscidae: Anthomyiidae) to 27 isolates of entomopathogenic fungi from four genera [Beauveria Vuillemin, Lecanicillium (Petch) Zare & W. Gams, Metarhizium Sorokin, and Paecilomyces Bainier]. A novel bioassay was developed for D. antiqua larvae by using a diet based on mixed vegetable powder. When evaluated in a virulence screen, the fungal isolates caused less mortality of D. antiqua larvae than adults. Only three isolates caused > 50% mortality of larvae, whereas 12 isolates caused > 50% mortality of adults. Fungal species was a statistically significant factor affecting the mortality of larvae but not of adults. The fungal isolates causing the most mortality of larvae tended to belong to Metarhizium anisopliae (Metschnikoff) Sorokin. Two M. anisopliae isolates (389.93 and 392.93) were evaluated in dose-response bioassays. The median lethal concentrations of the isolates against larvae were 6.1 x 10(7) conidia ml(-1) for isolate 389.93 and 7.6 x 10(7) conidia ml(-1) for isolate 392.93. The emergence of adult flies from pupae was reduced at high concentrations of conidia (3.0 x 10(8) and 1.0 x 10(8) conidia ml(-1)). The median lethal concentrations of the isolates against adults were 1.7 x 10(7) and 4.0 x 10(7) conidia ml(-1), respectively. Some of the fungal isolates examined may have potential as biological control agents of larvae of D. antiqua and related species.     

Ovicidal and larvicidal activity against Aedes aegypti and Anopheles gambiae complex mosquitoes of essential oils extracted from three spontaneous plants of Burkina Faso

Essential oils extracted from dried leaves of three spontaneous plants naturally growing in Burkina Faso, i.e. Cymbopogon proximus, Lippia multiflora and Ocimum canum, exhibited larvicidal activity by the WHO standard protocol against 3rd and 4th instar F1-larvae of field-collected mosquitoes vectors of human disease, namely Aedes aegypti and members of the Anopheles gambiae complex, An. arabiensis and An. gambiae. The median lethal concentration (LC50) for Ae. aegypti and An. gambiae s.l. larvae ranged between 53.5-258.5 ppm and 61.9-301.6 ppm, respectively. The LC90 estimates ranged 74.8-334.8 ppm for Ae. aegypti, and 121.6-582.9 ppm for An. gambiae s.l. Ovicidal activity against eggs of An. gambiae s.l. was also demonstrated. The LC50 values for An. gambiae s.l. eggs ranged between 17.1-188.7 ppm, while LC90 values ranged between 33.5-488 ppm. Lippia multiflora showed the highest activity against An. gambiae s.l. eggs and Ae. aegypti larvae, whereas no difference was found among C. proximus and L. multiflora in their activity against An. gambiae s.l. larvae. Of the three plants, essential oils from O. canum had the lowest activity against both eggs and larvae. Eggs were more susceptible than larvae. Ae. aegypti larvae were more susceptible than larvae of An. gambiae s.l.     

Biological studies of the Australian predatory mite Typhlodromips montdorensis (Schicha) (Acari: Phytoseiidae), a potential biocontrol agent for western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae)

Abstract The biology of the Australian phytoseiid mite Typhlodromips montdorensis is described from material collected in Queensland and South Australia in 1994−1996. At 25°C, when fed on cumbungi ( Typha sp.) pollen, the life cycle was completed in approximately 7 days, with an intrinsic rate of natural increase ( r _m ) of 0.32. Female−male pairs produced a mean total of 52.7 eggs within 28 days of oviposition. Females that were deprived of males after first mating stopped laying eggs after 7−19 days; however, if another male was added, they resumed egg laying and produced, on average, a total of 49.4 eggs. The sex ratio was 2.24 females to one male. At 25°C, fecundity on a diet of thrips larvae (first-instar Frankliniella schultzei Trybom) was high, ranging from 2.72 to 3.58 eggs per day on the third day, depending on previous diet. Consumption rate of thrips was also high, with an average of 7.23−14.44 first-instar larvae eaten per day on the third day, depending again on previous diet and also on number of thrips larvae made available. The species was also observed to feed on: (i) broad mite, Polyphago­tarsonemus latus (Banks); (ii) tomato russet mite, Aculops lycopersici (Massee); and (iii) two-spotted mite, Tetranychus urticae Koch. No diapause was observed under conditions of 25°C, 8 h light and 10°C, 16 h dark. Eggs were sensitive to low humidity, with 50% failing to hatch below 70.8% relative humidity. This species is of interest as a candidate biological control agent for thrips, broad mite and tomato russet mite in protected crops.     

Ovipositional Synergists and Artificial Diets for Trichogramma australicum Girault (Hymenoptera: Trichogrammatidae)

We studied ovipositional synergists and artificial diets for rearing Trichogramma australicum. Artificial "diet A" included 40% haemolymph of Helicoverpa armigera (Hübner) final instar larvae, 30% of a 10% malt solution in deionised water, 20% chicken egg yolk and 10% Neisenheimer's salt solution with 76 units Penicillin and 76 units Streptomycin/ml diet. Artificial "diet B" was identical except Grace's insect medium® replaced Neisenheimer's salt solution. the number of T. australicum larvae in artificial eggs filled with diet A and diet B was not significantly different (P > 0.05). Significantly fewer (P < 0.05) larvae developed to pupae and adults in artificial eggs filled with diet A. Quantity and quality of artificial diet affected the mortality of T. australicum larvae reared in vitro. Ovipositional synergists included 10% gelatine solution, 10% polyvinyl alcohol solution or 1% agar solution in deionised water. Synergist test-solutions were individually smeared on the external surface of artificial eggs (hemispherical depressions in plastic membrane). Eggs laid and number of T. australicum larvae produced were significantly higher in artificial eggs smeared with gelatine than artificial eggs smeared with polyvinyl alcohol, agar or non-smeared (control) eggs.     

Variable colonization of chickens perorally inoculated with Escherichia coli O157:H7 and subsequent contamination of eggs.

Challenging 1-day-old White Leghorn chicks perorally with 2.6 x 10(1) to 2.6 x 10(5) Escherichia coli O157:H7 bacteria per chick resulted in cecal colonization at all levels. Two of six chicks inoculated with only 2.6 x 10(1) E. coli O157:H7 bacteria carried 10(3) to 10(4) E. coli O157:H7 bacteria per g of cecal tissue when sacrificed 3 months postinoculation. E. coli O157:H7 colonization persisted at least 10 to 11 months when chicks were administered 10(8) E. coli O157:H7 bacteria. Eggs from five hens that were fecal shedders of E. coli O157:H7 until the termination of the study (10 to 11 months) were assayed for E. coli O157:H7. The organism was isolated from the shells of 14 of 101 (13.9%) eggs but not from the yolks and whites. Considering that chicks can be readily colonized by small populations of E. coli O157:H7 and continue to be long-term shedders, it is possible that chickens and hen eggs can serve as vehicles of this human pathogen.     

Cryopreservation of common carp (Cyprinus carpio) sperm in 1.2 and 5 ml straws and occurrence of haploids among larvae produced with cryopreserved sperm

Experiments were carried out on the cryopreservation of common carp (Cyprinus carpio) sperm in order to test the suitability of using 1.2 and 5 ml straws and to investigate the ploidy of malformed larvae found among the hatched progeny. In the first set of experiments, the effect of freezing time was investigated on the hatch rate of embryos. The highest hatch rate for 1.2 ml straws was 69+/-16% at the freezing time of 4 min, and 39+/-27% for 5 ml straws at 5 min. In the second set, the effect different egg volumes fertilized with one straw of sperm on the hatch rate and the rate of malformed larvae was investigated. The highest hatch rate with 1.2 ml straws (86+/-12%) was observed when 10 g of eggs were fertilized with one straw, whereas with 5 ml straws the hatch rate was highest (65+/-18%) when 40 g of eggs were fertilized. The highest rate of malformed larvae (15+/-9%) was found in the control, whereas the highest rate of malformed larvae among the groups fertilized with cryopreserved sperm (13+/-7%) was found in the 1x dose group fertilized with 5 ml straw. The chromosome numbers of malformed larvae were investigated and haploids were found among those hatched from eggs fertilized with cryopreserved sperm whereas only diploids were found in the controls.     

In vitro development of the fish parasite Hysterothylacium aduncum from the third larval stage recovered from a host to the third larval stage hatched from the egg

Anisakids are parasitic nematodes of fish worldwide, producing economic and human health concerns. It is thus important to ascertain their in vitro life cycle in laboratory studies. Here we describe the in vitro development of third-stage larvae (L3) of Hysterothylacium aduncum isolated from blue whiting Micromesistius poutassou, to the hatching of L3 from eggs obtained from H. aduncum worms grown in GLIT medium (a modified mixture of Yaeger's LIT [Liver Infusion Tryptose] and Grace's media) at pH 4.0, 13 degrees C and with 5% CO2 in air. Under these conditions, L3 recovered from fish developed to mature adults (3.4 to 6.2 cm in length), with oviposition starting from Day 26 in culture. Fertilized eggs (mean size 64 x 52 microm) had a thick, rugose eggshell and were larger than unfertilized ones (mean size 49 x 42 microm), whose eggshells were thin and smooth. Eggs laid during the first and second week of oviposition, and maintained in 2.8% NaCl solution at 13 degrees C, developed to L3. Under these maintenance conditions, between 20.6 and 52.5% of the eggs laid during the first week developed into larvae. Motile larvae, enclosed in a sheath, hatched from between 2 and 11% of these eggs. The larvae started to hatch 23 d after deposition. These larvae were 144 to 215 microm in length, enclosed in a 237 to 305 microm-long sheath. This GLIT culture medium may help to study the biology of this and other anisakids.     

Eggs and larvae of Nototheniops larseni from the Spongocoel of a Hexactinellid sponge near Hugo island,Antarctic Peninsula

Summary A mass of adhesive fish eggs was found within the spongocoel of a hexactinellid (glass) sponge, Rosella nuda, collected by bottom trawl near Hugo Island, Antarctic Peninsula in early September. Eggs were hatched in the ship's laboratory and the larvae identified as Nototheniops larseni (Lonnberg). Eggs and early larvae are described. Potential implications of the use of sponges by antarctic fishes as refuges and spawning sites are discussed.Contribution No. 795 from the School of Fisheries University of Washington, Seattle, Wash     

Three new species of Huffmanela Moravec, 1987 (Nematoda: Trichosomoididae) from the gills of marine fish off New Caledonia

Three new species of Huffmanela, here described from eggs only, are reported from the gills of marine fish caught off Nouméa, New Caledonia. Eggs of Huffmanela branchialis n. sp., from Nemipterus furcosus (Nemipteridae), are 45-52 (mean 48) microm in length and 23-30 (mean 25) microm in width, with thin shells. Each egg is enclosed in a thin membrane forming a spindle-shaped envelope 53-85 (mean 63) microm in length. Eggs of H. filamentosa n. sp., from Gymnocranius grandoculis (Lethrinidae), are 48-53 (mean 50) microm in length and 25-30 (mean 27) microm in width, with thin shells. Each egg bears a few long (150 microm), thin filaments. Eggs of these two new species were compared to those of H. paronai Moravec & Garibaldi, 2000, which are redescribed. Eggs of H. ossicola n. sp. were found within the branchial arch bone of Bodianus loxozonus (Labridae) and also filled the spinal chord bone and other bones. This is the first species of Huffmanela reported from bone tissue. Eggs are large, 72-88 (mean 79) microm in length and 32-40 (mean 36) microm in width, with a very thick shell. Each egg is covered with numerous filaments enclosed in a thin envelope. Fresh eggs were unembryonated, but embryos were visible after incubation in seawater. The three new species can be distinguished from other species of Huffmanela by size and the nature of the egg covering. Egg morphology of and their location in the host suggest different life-cycles: those of the first two species (small eggs, thin shells, egg covering possibly favouring flotation) are released from the gill mucosa with the turnover of living tissues and immediately continue their life-cycle, but eggs of H. ossicola (large eggs, thick shell) are only available for the continuation of the life-cycle after the host's death.     

Fish eggs and larvae in the diet of herring Clupea harengus membras Linnaeus, 1758 and the sprat Sprattus sprattus balticus (Schneider, 1904) (Clupeidae) in the Southeastern Baltic Sea

The qualitative and quantitative composition of fish eggs and larvae in the stomach contents of Baltic herring and sprat was examined. The occurrence of fish eggs and larvae in the diet of herring was markedly higher, compared to sprat. Eggs of four and larvae of nine fish species were found in the herring stomachs. The prey species composition of the herring and sprat diets was related to the ichthyoplankton composition and to the degree of overlapping of the prey and predator distributions. Sprat eggs were the predominant prey in the herring diet in May and sprat larvae prevailed in August. The daily ration estimates indicate that the sprat larvae consumption by herring in August exceeded the sprat egg consumption in May by many times. Cod eggs in the herring stomachs were only found at the optimal oxygen content and salinity for the cod eggs in the near-bottom water layer.