An immature merocyst of Hepatocystis malayensis and gametocytes of H. brayi were studied with the electron microscope. The merocyst consisted of a highly complex cytoplasmic reticulum ramifying through an amorphous matrix: the entire complex was enclosed by a simple unit membrane. The host cell was apparently destroyed completely during growth of the cyst. Immature gametocytes were highly amoeboid and showed extensive vacuolisation or attenuation of the cytoplasm. The nucleus contained one or two prominent nucleoli. Mature gametocytes had compact cytoplasm and contained pyriform osmiophilic bodies which were believed to function in the release of the parasites from the host cells. Macrogametocytes were distinguished from microgametocytes by cytoplasmic differences in numbers of ribosomes, and cristate mitochondria and in the extent of development of the smooth endoplasmic reticulum. The compact nuclei of the macrogametocytes had inconspicuous DNA but prominent nucleoli whereas those of the microgametocytes were irregular and showed a central aggregate of DNA. In microgametogenesis karyokinesis of the parent nucleus was delayed until axoneme formation was complete. Then the nuclear buds were extruded into emerging microgametes. At fertilisation the plasmalemmas of the two gametes fused and the single axoneme and nucleus of the microgamete moved into the cytoplasm of the macrogamete.
Elasmobranchs display various reproductive modes, which have been key to their evolutionary success. In recent decades there has been a rise in the number of reported cases of foetal abnormalities including fertilised, double-embryos held within one egg capsule, hereafter referred to as twins. Previously, the occurrences of twin egg cases have been reported in two batoid and one shark species. We report the first cases of twins in three species of oviparous elasmobranchs: the undulate ray (Raja undulata), the nursehound (Scyliorhinus stellaris), and the small-spotted catshark (Scyliorhinus canicula). We investigated the genetic relationships between the twins in S. stellaris, and S. canicula using microsatellite markers. Whilst the S. stellaris twins displayed the same genotypes, we found that the S. canicula twin individuals arose through heteropaternal superfecundation. This is the first reported incidence of such a paternity in elasmobranchs. The relationship between environmental change and reproductive strategy in elasmobranchs is unclear and further research is needed to determine its effect on the prevalence and mechanisms of formation of elasmobranch twins.
A total of 341 fertilized and 37 unfertilized oocytes from 63 intracytoplasmic sperm injection (ICSI) treatment cycles were included for retrospective assessment using the Embryoscope time-lapse video system. The second polar body (pb2) extrusion occurred at 2.9±0.1 h (range 0.70-10.15 h) relative to sperm injection. All oocytes reduced in size following sperm injection (p<0.05) with shrinkage ceasing after 2h in the unfertilized and at pb2 extrusion in the fertilized oocytes. Pb2 extrusion was significantly delayed for women aged >38 years compared to those <35 years (3.4±0.2 vs. 2.8±0.1, p<0.01) or 35-38 years (3.4±0.2 vs. 2.8±0.1, p<0.01), but timing was not related to the Day 3 morphological grades (1-4) of subsequent embryos (2.9±0.1, 2.9±0.1, 2.8±0.2 and 3.0±0.1; p>0.05 respectively). A shorter time of first cleavage division relative to either sperm injection or pb2 extrusion is associated with both top grade (AUC=0.596 or 0.601, p=0.006 or 0.004) and usable embryos (AUC=0.638 or 0.632, p=0.000 respectively) on Day 3. In summary, (i) pb2 of human oocytes extrudes at various times following sperm injection, (ii) the timing of pb2 extrusion is significantly delayed when female age >38 years, but not related to subsequent embryo development, (iii) all human oocytes reduce in size following sperm injection, (iv) completion of pb2 extrusion in the fertilized oocytes is a pivotal event in terminating shrinkage of the vitellus, and (v) time to first cleavage division either from sperm injection or pb2 extrusion is a significant predictive marker for embryo quality on Day 3.
The late-aged egg and third-instar life stages of laboratory-reared Malaysian fruit fly, Bactrocera latifrons (Hendel); Mediterranean fruit fly, Ceratitis capitata (Wiedemann); melon fly, B. cucurbitae Coquillett; and oriental fruit fly, B. dorsalis (Hendel), (Diptera: Tephritidae); and the third instars of wild Mediterranean fruit fly were exposed to thermal treatments. A heating block system was used to determine the thermal death kinetics of the four fruit fly species. Treatments consisted of heating the fruit fly life stages to 44, 46, 48, and 50 degrees C and holding for different times ranging from 0 to 120 min depending on the thermal mortality response and time required to obtain 100% mortality for each species and life stage. The 0.5-order kinetic model had the best fit to the survival ratio for all the treatment temperatures and was used to predict lethal times. The thermal death time (TDT) curves showed a tolerance order of Mediterranean fruit fly eggs < or = third instars at 44, 46, and 50 degrees C, third instars < or = eggs at 48 degrees C, and wild third instars < the laboratory-reared third instars. Comparison between Mediterranean fruit fly third instar thermotolerance from Hawaii and Israel showed that Israel Mediterranean fruit fly was more thermotolerant. A comparison of minimum treatment times at a given temperature required to obtain 100% mortality of laboratory-reared Malaysian, Mediterranean (Hawaii and Israel strains), melon, Mexican, and oriental fruit fly eggs or third instars and wild Mediterranean fruit fly (Hawaii strain) eggs or third instars showed that oriental fruit fly was the most thermotolerant among the third instars, and the difference in heat tolerance between third instars and eggs was negligible at 50 degrees C.
Urbanization has caused an increase in favorable habitats for Aedes aegypti (Diptera: Culicidae), given their ability to reproduce in small and often non-degradable artificial water-containers. While much work has been done on Ae. aegypti biology and ecology in urban landscapes, the role of shading on immature stages as an independent factor from temperature, and any possible interactions between these factors, remains unexamined. We assessed how temperature and shading affected egg hatch-rate, larval/pupal mortality, and larval development to adult stage under different factorial temperature (28; 31; 34; 37; 40° C) and shade (0%, 3,100 lux; 40%, 1,860 lux; 75%, 775 lux; 100%, 0 lux) regimes. Hatch-rate was significantly lower at 37° C (57 %), and no eggs hatched at 40° C. There was no significant effect caused by shading on hatchability. Larval and pupal mortality at 37° C was significantly higher (35%) compared to lower temperature groups, while the effects of shading were emergent at low temperatures. Developmental times from hatching to adult emergence were significantly reduced with increasing temperatures and with greater light exposures. The eco-physiological response of Ae. aegypti larvae to temperature and light regimes suggest a photosensitivity previously unstudied in this species.
Moisture plays a major role in the dynamics of mosquito populations, especially those breeding in container habitats. Despite this importance, the role of moisture conditions as they affect oviposition and egg development in Aedes vectors remains largely unexplored. We investigated the effect of exposing gravid female Aedes albopictus mosquitoes and their eggs to different moisture levels (MLs) for various periods on oviposition and hatching. Overall, high-moisture substrates (HMSs; 66% and 72%) provided better environments for egg laying. The timing of initial egg laying was far longer at the lowest substrate moisture level (LSML, 25% and 41.2%) than at HMSs. The numbers of eggs laid were much lower in the drier environments. At LSMLs, gravid females retained increasing numbers of mature eggs until death, and egg retention decreased gradually with increasing ML. The HMSs also provided better environments for larval eclosion. The numbers of eggs hatched were lower at the LSML than the HSML environment. No egg hatching occurred after 1 h exposure to moisture. However, egg hatching occurred by installment, with spontaneous hatching (SH) increasing gradually with increasing ML. High-moisture conditions combined with long exposure (30 h and 48 h) favored SH. These results suggest that Ae. albopictus females can respond to better moisture conditions for increased success of embryonation and larval eclosion. This information may be useful in the colonization of floodwater Aedes species.
The common physical and chemical methods for controlling rat pest are less than satisfactory and inhumane. Immunocontraception approach has been considered more humane and it can be accomplished by inducing the relevant host immune response that block further development of reproductive gametes. ZP3 proteins are known to play very important role during sperm-ovum fertilization. It is a self-antigen and only localized in female ovaries. Therefore, an immunization with ZP3 protein elsewhere will induce a generalize host immune response against ZP3 protein. This study employed rat ZP3 (rZP3) gene prepared from its cDNA of Rattus rattus diardii. It was delivered and expressed in vivo by naked plamid DNA (DrZP3) or recombinant ZP3-Adenovirus (Ad-rZP3). Expression studies in vitro with DrZP3 or Ad-ZP3 showed rZP3 proteins were successfully expressed in Vero cells. Hyperimmune serum against rZP3 that were prepared by immunizing several rats with purified rZP3-pichia yeast fusion protein showed it blocked sperms from binding DrZP3-transfected Vero cells. Female Sprague Dawley rats immunized with DrZP3 demonstrated a long-term effect for significant reduction of fertility up to 92.6%. Ovaries from rats immunized with DrZP3 were severely atrophied with disappearance of primordial follicles from ovarian cortex with an increased in the amount of oocyte-free cell clusters. Female rats immunized with Ad-rZP3 demonstrated 27% reduction of fertility. The infertility induced by Ad-rZP3 is comparatively low and ineffective. This could be due to a strong host immune response that suppresses the recombinant virus itself resulted in minimum rZP3 protein presentation to the host immune system. As a result, low antibody titers produced against rZP3 is insufficient to block oocytes from maturity and fertilization. Therefore, immunization with DrZP3 for immunocontraception is more effective than Ad-rZP3 recombinant adenovirus. It is proposed to explore further on the use of adenovirus or other alternative viruses to deliver ZP3 protein and for the development of enhanced expression of rZP3 in target host.
A potential advantage of group movement in animals is increased locomotion efficiency. This implies a reduced energetic cost for individuals that occur in larger groups such as herds, flocks and schools. When chelonian hatchlings hatch in the underground nest with finite energy for their post-hatching dispersal phase, they face the challenge of minimizing energetic expenditure while escaping the nest. The term 'social facilitation' has been used to describe the combined digging effort of sea turtle hatchlings during nest escape. Given that in a normal clutch, a substantial part of the energy reserve within the residual yolk is used by hatchlings in the digging out process, a decreased cohort size may reduce the energy reserve available to cross the beach and sustain the initial swimming frenzy. This hypothesis was experimentally tested by varying cohort size in hatchling green turtles (Chelonia mydas) and measuring energy expenditure during the nest escape process using open-flow respirometry. The energetic cost of escaping through 40 cm of sand was calculated to vary between 4.4 and 28.3 kJ per individual, the cost decreasing as the number of individuals in the cohort increased. This represents 11-68% of the energy contained in a hatchling's residual yolk at hatching. The reduced energetic cost associated with large cohorts resulted from both a lower metabolic rate per individual and a shortened nest escape time. We conclude that synchronous digging activity of many hatchlings during nest escape evolved not only to facilitate rapid nest emergence but also to reduce the energetic cost to individuals.
Soil contaminated with helminth eggs and protozoan cysts is a potential source of infection and poses a threat to the public, especially to young children frequenting playgrounds. The present study determines the levels of infection of helminth eggs in soil samples from urban and suburban playgrounds in five states in Peninsular Malaysia and identifies one source of contamination via faecal screening from stray animals. Three hundred soil samples from 60 playgrounds in five states in Peninsular Malaysia were screened using the centrifugal flotation technique to identify and determine egg/cyst counts per gram (EPG) for each parasite. All playgrounds, especially those in Penang, were found to be contaminated with eggs from four nematode genera, with Toxocara eggs (95.7%) the highest, followed by Ascaris (93.3%), Ancylostoma (88.3%) and Trichuris (77.0%). In addition, faeces from animal shelters were found to contain both helminth eggs and protozoan cysts, with overall infection rates being 54% and 57% for feline and canine samples, respectively. The most frequently occurring parasite in feline samples was Toxocara cati (37%; EPG, 42.47 ± 156.08), while in dog faeces it was Ancylostoma sp. (54%; EPG, 197.16 ± 383.28). Infection levels also tended to be influenced by season, type of park/playground and the texture of soil/faeces. The occurrence of Toxocara, Ancylostoma and Trichuris eggs in soil samples highlights the risk of transmission to the human population, especially children, while the presence of Ascaris eggs suggests a human source of contamination and raises the issue of hygiene standards and public health risks at sites under investigation.
Schistosoma malayensis n. sp., a member of the Schistosoma japonicum complex is described from Rattus muelleri in Peninsular Malaysia and 2 strains are characterized. The only morphological differences noted among adults from natural hosts were that S. malayensis are in general smaller than S. mekongi and S. japonicum. But these differences may be the result of host-induced variations and therefore are of little taxonomic value. To minimize the effects of host-induced variations, adult worms recovered from laboratory mice with similar worm burdens at 50-56 days postinfection were compared. These comparisons revealed only minor morphometric differences among these 3 species. Schistosoma malayensis eggs from naturally and experimentally infected hosts are most similar to those of S. mekongi, with eggs of both species being, in general, smaller than those of S. japonicum. The egg index for S. malayensis is usually higher than for S. japonicum and lower than for S. mekongi. Differences were noted in the developmental rates in mice for 2 isolates of S. malayensis, S. mekongi, and S. japonicum (Philippine strain), but relatively large differences observed between isolates of S. malayensis indicate that, in this case, the developmental rate is not a useful taxonomic character. Schistosoma malayensis is erected principally on the basis of differences, reported elsewhere, in the life histories and in the electrophoretic migration patterns of isoenzymes of adult worms as compared to S. mekongi and S. japonicum. These comparisons indicate that S. malayensis is more closely related to S. mekongi than to S. japonicum.
Three cases of schistosomiasis in 2 Filipinos and one Chinese in Sabah are reported. Diagnosis was based on incidental histological findings of Schistosoma japonicum-like ova in the liver and rectal biopsies. As these 3 patients are immigrants to Sabah, it is assumed that they are imported cases, and that Sabah has been free of the disease from 1970 to 1977.
Descriptions of the eggs of Mansonia uniformis, Ma. indiana and Ma. annulifera are provided with the aid of scanning electron micrographs. Eggs of these three species, although similar in shape and colour, are covered by outer chorionic reticulum and tubercles which provide reliable morphological character for their identification. Size, distribution and number of lobes on the large tubercles present in the region between the anterior tube and posterior region, are important distinguishing features. Measurements of egg sizes and other chorionic differences are also discussed.
The genus Rhitymna Simon, 1897 is revised by means of new material. Four new species are described: R. gerdmangel spec. nov. (Thailand, Malaysia; male, female), R. merianae spec. nov. (Indonesia: Bali; male), R. flores spec. nov. (Indonesia: Flores; male, female), R. senckenbergi spec. nov. (Philippines; male). The male of R. plana Jäger, 2003 and the female of R. tangi Quan Liu, 2012 are described for the first time. Rhitymna simoni Jäger, 2003 is recognised as junior synonym of R. cursor (Thorell, 1894) comb. nov., the latter transferred from the genus Olios Walckenaer, 1837. New records are given for further Rhitymna species, among them new country or island records for R. verruca (Wang, 1991) (Thailand), R. tangi Quan Liu, 2012 (Laos), R. plana Jäger, 2003 (Cambodia), R. pinangensis (Thorell, 1891) (Thailand), R. deelemanae Jäger, 2003 (Bali). The number of cheliceral bristles close to the fang base is recognised as size dependent, therefore without true phylogenetic signal. Two main types of copulatory organs within the genus are recognised and discussed. R. gerdmangel spec. nov. has a special biology as it lives exclusively in bamboo. Holes made by beetles or woodpeckers are used to enter the bamboo stem. Spiders hide during the day and lay their eggs in bamboo internodes.
Eggs of temperate Aedes albopictus populations are cold hardy and can diapause, but tropical populations are not cold hardy and cannot diapause. Heterozygotes possess intermediate diapause and cold hardiness. Males of a tropical strain from Malaysia with a distinctive genetic marker were released into an existing temperate population in East St. Louis, Illinois. Subsequent egg samples from the release site had genetic marker frequency of up to 24%. Reduced cold hardiness and decreased diapause incidence were also observed in the release site population. No such changes occurred at a nearby control site. The rank order of overwintering survival of eggs at the release site was: Aedes triseriatus > temperate Ae. albopictus > hybrid temperate/tropical Ae. albopictus > tropical Ae. albopictus. Eggs collected from the release population the next summer showed total absence of the genetic marker; presumably carriers were removed by the winter.
The objective was to evaluate the effect of the interval between ovarian hyperstimulation and laparoscopic ovum pick-up (LOPU) on quality and developmental competence of goat oocytes before and after in vitro maturation (IVM) and intracytoplasmic sperm injection (ICSI). Estrus was synchronized with an intravaginal insert containing 0.3g progesterone (CIDR) for 10d, combined with a luteolytic treatment of 125 microg cloprostenol 36 h prior to CIDR removal. Ovaries were hyperstimulated with 70 mg FSH and 500 IU hCG given im 36, 60, or 72 h prior to LOPU (n=15, 16, and 7 does, respectively). For these groups, oocyte retrieval rates (mean+/-S.E.M.) were 24.7+/-2.9, 54.5+/-4.7, and 82.8+/-4.6% (P<0.001), and the proportions of cumulus-oocyte complexes (COC) with more than five layers of cumulus cells were 29.7+/-8.3, 37.6+/-6.9, and 37.3+/-7.0% (P<0.001). The proportion of IVM oocytes was highest at 72 h (82.1+/-2.8%; P<0.05), with no significant difference between 36 and 60 h (57.3+/-8.9% and 69.0+/-8.4%). Cleavage rates of ICSI embryos were 4.2+/-4.2, 70.9+/-8.4, and 78.9+/-8.2% with LOPU 36, 60, and 72 h post FSH/hCG (P<0.01), with a lower proportion of Grade-A embryos (P<0.05) following LOPU at 36 h compared to 60 and 72 h (29.7+/-8.3%, 37.6+/-6.9%, and 37.3+/-7.0%). In summary, a prolonged interval from FSH/hCG to LOPU improved oocyte retrieval rate and oocyte quality. Therefore, under the present conditions, LOPU 60 or 72 h after FSH/hCG optimized yields of good-quality oocytes for IVM and embryo production in goats.
Plasmid DNA (pDNA)-based vaccines have emerged as effective subunit vaccines against viral and bacterial pathogens. In this study, a DNA vaccine, namely plasmid internal ribosome entry site-HN/F, was applied in ovo against Newcastle disease (ND). Vaccination was carried out using the DNA vaccine alone or as a mixture of the pDNA and dextran-spermine (D-SPM), a nanoparticle used for pDNA delivery. The results showed that in ovo vaccination with 40 μg pDNA/egg alone induced high levels of antibody titer (P<0.05) in specific pathogen-free (SPF) chickens at 3 and 4 weeks postvaccination compared to 2 weeks postvaccination. Hemagglutination inhibition (HI) titer was not significantly different between groups injected with 40 μg pDNA + 64 μg D-SPM and 40 μg pDNA at 4 weeks postvaccination (P>0.05). Higher antibody titer was observed in the group immunized with 40 μg pDNA/egg at 4 weeks postvaccination. The findings also showed that vaccination with 40 μg pDNA/egg alone was able to confer protection against Newcastle disease virus strain NDIBS002 in two out of seven SPF chickens. Although the chickens produced antibody titers 3 weeks after in ovo vaccination, it was not sufficient to provide complete protection to the chickens from lethal viral challenge. In addition, vaccination with pDNA/D-SPM complex did not induce high antibody titer when compared with naked pDNA. Therefore, it was concluded that DNA vaccination with plasmid internal ribosome entry site-HN/F can be suitable for in ovo application against ND, whereas D-SPM is not recommended for in ovo gene delivery.
The diamondback moth (DBM) Plutella xylostella (L.) has traditionally been managed using synthetic insecticides. However, the increasing resistance of DBM to insecticides offers an impetus to practice integrated pest management (IPM) strategies by exploiting its natural enemies such as pathogens, parasitoids, and predators. Nevertheless, the interactions between pathogens and parasitoids and/or predators might affect the effectiveness of the parasitoids in regulating the host population. Thus, the parasitism rate of Nosema-infected DBM by Cotesia vestalis (Haliday) (Hym., Braconidae) can be negatively influenced by such interactions. In this study, we investigated the effects of Nosema infection in DBM on the parasitism performance of C. vestalis. The results of no-choice test showed that C. vestalis had a higher parasitism rate on non-infected host larvae than on Nosema-treated host larvae. The C. vestalis individuals that emerged from Nosema-infected DBM (F1) and their progeny (F2) had smaller pupae, a decreased rate of emergence, lowered fecundity, and a prolonged development period compared to those of the control group. DBM infection by Nosema sp. also negatively affected the morphometrics of C. vestalis. The eggs of female C. vestalis that developed in Nosema-infected DBM were larger than those of females that developed in non-infected DBM. These detrimental effects on the F1 and F2 generations of C. vestalis might severely impact the effectiveness of combining pathogens and parasitoids as parts of an IPM strategy for DBM control.