The optimum distance and duration of ultraviolet (UV) irradiation for the complete inactivation of African catfish Clarias gariepinus egg nucleus was investigated in this study. The UV light was suspended above the unfertilized eggs at four distances (5, 10, 20 and 30 cm) and for five durations (1, 2, 3, 4 and 5 min). Then, the irradiated eggs were activated with sperm from diploid C. gariepinus and cold shocked at 5°C for 5 min just moments before cell cleavage. Ploidy analysis was performed using karyotype chromosome counting. The results obtained suggested that the further the distance, the better the hatchability rate, however prolonged duration seemed to significantly reduced hatchability. All treatments with surviving progenies at the end of the study showed evidence of successfully diploid gynogen (2n = 56) induction at different percentages. However, the optimal protocol that gave a moderately high hatchability/survival rate and completely induced gynogens was exposure of the eggs to UV irradiation at 20 cm for 1 min. It was concluded that the distance and duration of UV irradiation affects gynogenetic induction in African catfish C. gariepinus.
SummaryThis study investigated the breeding parameters and embryogenic development of diploid and heat shock-induced triploid eggs of Anabas testudineus (Bloch, 1792). To this effect, broodstocks of A. testudineus were induced to spawn using the Ovaprim® hormone. After fertilization, the eggs were divided into two groups and one portion heat shocked at 41°C (for 3 min), at approximately 4 min after fertilization. Results of fertilization, hatchability, as well as the sequence and timing of embryogenic development were collated from three breeding trials. Fertilization percentages were similar in both treatments (≈90%) while hatchability was higher in the diploid eggs (79.56%) than the triploid induced eggs (50.04%). Both treatments had the same sequence of embryogenetic stages; however, the timing of development was significantly delayed in the triploids (i.e. beyond the 2-cell stages) as compared with the observations in the control group (diploid eggs). Consequently, hatching time was 5 h faster in the diploid eggs [i.e. 18 hours post fertilization (hpf)] compared with the triploid induced eggs (23 hpf). The most critical stage of embryonic development in which mass mortality occurred in the different treatments was the somite stage. The status of triploid hatchlings was affirmed using erythrocyte morphology in 2-month-old fingerlings.
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.
Polyplocia nebulosa sp. nov. is described based on male and female imagos from Malaysia, Sabah State. Nymphs provisionally assigned to this species are also described. This species can be readily separated from P. vitalisi by the color pattern of the wings, with light brown longitudinal veins, narrow dark brown clouds on cross veins and margins of wings tinged with brown. The male genitalia are distinct from those of other species of Polyplocia: penes are broad, T-shaped, with large lateral projecting lobes, which are apically rounded, and with a small dorsolateral spine on each lobe; the styliger plate is short and not projected posteriorly. Eggs of the genus are described for the first time using scanning electron microscopy. Eggs are 265-267 µm in length, 170-186 µm in width, barrel-shaped, without polar caps or other attachment structures, with one visible micropyle and with chorion forming an irregular mesh with raised ridges (mesh between 3.8-8 µm). A key to male adults of Polyplocia is provided and additional records of Polyplocia from Thailand are given.
Angiostrongylus malaysiensis is a potential zoonotic parasite, which reported to co-occur with A. cantonensis in human cerebrospinal fluid. It is a heteroxenous nematode that primarily develops through the early larval stages in gastropods and attains sexual maturity within rats. This study was conducted to determine the host species responsible for the reservoir of A. malaysiensis and investigate the risk factor for transmission among the hosts in Kuala Lumpur, Malaysia. Sampling was conducted in six recreational parks. The rats were trapped alive using steel wire traps with bait, while the gastropods were collected by active searching. The rats were euthanized and dissected to collect any adult worms observed. The molecular detection of A. malaysiensis was performed by PCR on gastropod tissue samples. Biotic and landscape factors were recorded for risk factor analysis. In total, 82 rats and 330 gastropods were collected throughout the study. Overall, 3.64% of gastropods and 32.9% of rats were infected with A. malaysiensis. Rattus tiomanicus (Malayan wood rat) and Parmarion martensi (Yellow-shelled semi-slug) were found as important hosts for A. malaysiensis. Host species, sampling site and macrohabitat type are risk factors associated with the prevalence of A. malaysiensis infection in rats. For gastropods, host species and sampling site are risk factors that correlate with the parasite detection. In total, 128 adult A. malaysiensis were recovered from the infected rats. The mean intensity of infection with adult A. malaysiensis was 4.65 for Rattus rattus complex and 4.90 for R. tiomanicus. Adult worms were found in the pulmonary artery or right ventricle, while eggs and first-stage larvae were found in capillaries of the caudal lung lobe. Infected lungs showed extravasated red blood cells in the alveolar spaces. The pulmonary arteries in the infected lung lobe were thickened. Kepong Metropolitan Park is the hotspot area for A. malaysiensis in Kuala Lumpur. These results provide essential information for public health officials to develop targeted interventions to reduce the transmission of A. malaysiensis in urban areas, particularly in recreational parks.
A new microhylid, Kalophrynus yongi, Is described from the Cameron Highlands of Peninsular Malaysia. Morphologically, the new species differs from all known congeners by having a very stout forelimb with a humeral spine in males. Acoustically, it resembles K. baluensis and K. heterochirus and sharply differs from K. interlineatus, K. pleurostigma, K. palmatissimus, and K. nubicola.
The reproductive and developmental characteristics of the nereidid polychaete, Neanthes glandicincta Southern, 1921, commonly recorded in tropical estuaries in the Indo-West Pacific, were examined from Malaysia (the mangrove area of Kuala Ibai, Terengganu) and Thailand (the Lower Songkhla Lagoon) on the east coast of the Malay Peninsula. Epitokous metamorphosis of fully mature males and females and their reproductive swimming behaviour were recorded based on ten Malaysian epitokous specimens, which were collected at night during spring tides in a period of January 2018 to March 2019. Six Thailand epitokes were obtained in February and March 2006 by the laboratory rearing of immature worms. Epitokous metamorphosis is characterised by the enlargement of eyes in both sexes, division of the body into three parts and modification of parapodia with additional lobes in the mid-body of males, and replacement of atokous chaetae in the mid-body by epitokous natatory chaetae, completely in males and incompletely in females. The diameter of coelomic unfertilised eggs in females was 100-140 µm. After fertilisation, each egg formed a jelly layer, inside which embryonic development progressed. Trochophores hatched out of the jelly layer, entering a short free-swimming larval phase followed by demersal life at the early stage of 3-chaetiger nectochaeta one day after fertilisation. Then, the larvae entered benthic life as juveniles, crawling on the bottom, at the late stage of 3-chaetiger nectochaeta two days after fertilisation. The results indicate that N. glandicincta has an annual life cycle, which is usually completed within an estuary with limited larval dispersal ability.
We describe unique reproductive features of two threatened Galliformes species, the Malaysian peacock-pheasant (Polyplectron malacense) and Bornean peacock-pheasant (P. schleiermacheri). These features pose challenges to successful captive breeding to build up sustainable populations for conservation purposes. Single egg clutches in both species mean that reproductive rates are low and population increase is slow. The very rounded eggs of Bornean Peacock-pheasant cause difficulties for egg orientation in incubators. Single chicks with special requirements are poorly suited to rearing by broody bantams and successful husbandry requires increased human time, commitment, and skills.
Forty-five years ago a naturally attenuated tick-borne flavivirus, Langat (LGT) strain TP21, was recovered from ticks in Malaysia. Subsequently, it was tested as a live attenuated vaccine for virulent tick-borne encephalitis viruses. In a large clinical trial its attenuation was confirmed but there was evidence of a low level of residual virulence. Thirty-five years ago further attenuation of LGT TP21 was achieved by multiple passages in eggs to yield mutant E5. To study the genetic determinants of the further attenuation exhibited by E5 and to allow us to manipulate the genome of this virus for the purpose of developing a satisfactory live attenuated tick-borne flavivirus vaccine, we recovered infectious E5 virus from a full-length cDNA clone. The recombinant E5 virus (clone 651) recovered from a full-length infectious cDNA clone was more attenuated in immunodeficient mice than that of its biologically derived E5 parent. Increase in attenuation was associated with three amino acid substitutions, two located in the structural protein E and one in nonstructural protein NS4B. Subsequently an even greater degree of attenuation was achieved by creating a viable 320 nucleotide deletion in the 3'-noncoding region of infectious full-length E5 cDNA. This deletion mutant was not cytopathic in simian Vero cells and it replicated to lower titer than its E5-651 parent. In addition, the E5 3' deletion mutant was less neuroinvasive in SCID mice than its E5-651 parent. Significantly, the deletion mutant proved to be 119,750 times less neuroinvasive in SCID mice than its progenitor, LGT strain TP21. Despite its high level of attenuation, the E5 3' deletion mutant remained highly immunogenic and intraperitoneal (ip) inoculation of 10 PFU induced complete protection in Swiss mice against subsequent challenge with 2000 ip LD50 of the wild-type LGT TP21.
Faecal samples were collected from 48 randomly selected smallholder goat farms in northern Peninsular Malaysia. The nematode eggs extracted were tested for resistance to thiabendazole using the egg hatch assay technique. Thiabendazole resistance was found on 19% of farms tested.
Gene reassortment has proved useful in improving yields of influenza A antigens of egg-based inactivated vaccines, but similar approaches have been difficult with influenza B antigens. Current regulations for influenza vaccine seed viruses limit the number of egg passages and as a result resultant yields from influenza B vaccine seed viruses are frequently inconsistent. Therefore, reliable approaches to enhance yields of influenza B vaccine seed viruses are required for efficient vaccine manufacture. In the present study three stable cold-adapted (ca) mutants, caF, caM and caB derived from seasonal epidemic strains, B/Florida/4/2006, B/Malaysia/2506/2004 and B/Brisbane/60/2008 were prepared, which produced high hemagglutinin antigen yields and also increased viral yields of reassortants possessing the desired 6:2 gene constellation. The results demonstrate that consistent improvements in yields of influenza B viruses can be obtained by cold adaptation following extended passage. Taken together, the three ca viruses were shown to have potential as donor viruses for the preparation of high-yielding influenza B vaccine viruses by reassortment.
Since a safe and effective mass vaccination program against dengue fever is not presently available, a good way to prevent and control dengue outbreaks depends mainly on controlling the mosquito vectors. Aedes aegypti mosquito populations can be monitored and reduced by using ovitraps baited with organic infusions. A series of laboratory experiments were conducted which demonstrated that the bacteria in bamboo leaf infusion produce volatile attractants and contact chemical stimulants attractive to the female mosquitoes. The results showed that the female mosquitoes laid most of their eggs (59.9 ± 8.1 vs 2.9 ± 2.8 eggs, P<0.001) in bamboo leaf infusions when compared to distilled water. When the fresh infusion was filtered with a 0.45 μm filter membrane, the female mosquitoes laid significantly more eggs (64.1 ± 6.6 vs 4.9 ± 2.6 eggs, P<0.001) in unfiltered infusion. However when a 0.8 μm filter membrane was used, the female laid significantly more eggs (62.0 ± 4.3 vs 10.1 ± 7.8 eggs, P<0.001) in filtrate compared to a solution containing the residue. We also found that a mixture of bacteria isolated from bamboo leaf infusion serve as potent oviposition stimulants for gravid Aedes mosquitoes. Aedes aegypti laid significantly more eggs (63.3 ± 6.5 vs 3.1 ± 2.4 eggs, P<0.001) in bacteria suspension compared to sterile R2A medium. Our results suggest microbial activity has a role in the production of odorants that mediate the oviposition response of gravid mosquitoes.
Toxocara canis is a major parasite that infects many animals with high risk of human infections. This study aims at assessing the immunization with gamma radiationattenuated infective stage on rats challenged with non-irradiated dose. Level of vaccine protection was evaluated in liver and lung regarding parasitological, histopathological, biochemical and molecular parameters. Fifty rats were enrolled in three groups: group A (10 rats) as normal control; group B (20 rats) subdivided into subgroup B1 (infected control) and subgroup B2 infected then challenged after 14 days with the same dose of infection (challenged infected control); and group C (20 rats) subdivided into subgroup C1 vaccinated with a dose of 800 gray (Gy) gamma-radiated infective eggs (vaccine control) and subgroup C2 vaccinated then challenged on 14th day with same number of infective eggs (vaccinated-challenged). Tissues were stained with Haematoxylin and Eosin (H and E) for histopathological studies. Biochemical studies through detection of nitric oxide (NO) and Caspase-3 were conducted. Extent of DNA damage by Comet assay was assessed. Vaccinated-challenged subgroup revealed a marked reduction in larvae in tissues with mild associated histological changes. In addition there was accompanied reduction of NO, Casepase-3 level and DNA damage compared to the control infected group. It could be concluded that vaccination of rats with a dose of 800Gy gamma radiation-attenuated infective stage improves immune response to challenge infection and drastically reduces the morbidity currently seen.
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.
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.