Prevalence and Molecular Characterization of Ascaridoid Parasites of Philippine Decapterus Species

There are relatively few studies on parasite fauna of marine fishes in Philippine waters. This study aimed to determine the prevalence of marine ascaridoid infection in Decapterus species in Balayan Bay and Tayabas Bay. A total of 371 fishes belonging to three different species of Decapterus (D. tabl [n = 130], D. macrosoma [n = 121], and D. maruadsi [n = 120]) were collected. Ascaridoid parasite larvae were found in all fish host species, with an overall fish infection rate of 22%. The highest infection rate was observed in D. tabl (27.69%), followed by D. macrosoma (19%), and then D. maruadsi (17.50%). Moreover, a higher prevalence of infection was detected in Tayabas Bay (27.57%) than in Balayan Bay (15.59%). Molecular analyses based on the ITS2 and 18S rRNA gene supported the identification of the larvae into two species: Anisakis typica and Raphidascaris (Ichthyascaris) lophii. This is the first report of the genetic identification of these two helminth parasites in Decapterus fish species in the Philippines. Paucity in the database of Philippine marine fish parasites warrants more research efforts, especially concerning economically important fish species with implications to food safety and food security.     

Multiple primer PCR for the identification of anisakid nematodes from Taiwan Strait

There were six major larval anisakid species found in commercial marine fishes caught in the Minnan fishing ground in the Taiwan Strait: Anisakis physeteris, Anisakis pegreffii, Raphidascaris trichiuri, Contracaecum aduncum, Contracaecum muraenesoxi, Contracaecum sp. For rapid identification of the parasite species above, a single and a multiple primer PCR (multiplex PCR) method, using specific primers based on aligned sequences of the internal transcribed spacer ITS-1, 5.8S, and ITS-2 of nuclear ribosomal DNA, were jointly used for the rapid identification of these anisakid larvae. The primers yielded distinct PCR products for each of the anisakid nematodes, providing rapid and accurate tools for identifying anisakid nematodes with distinct geographical distribution.     

Some larval ascaridoids from south-eastern queensland marine fishes

A survey of 123 species of fishes from southeastern Queensland during 1975 revealed in 47 species nine distinct larval types of ascaridoid nematodes: Anisakis Type I, Terranova Types I and II, Contracaecum Types I and II, and Thynnascaris Types I, II, III and IV. These larvae are described and figured.     

Anisakis spp. larvae in three mesopelagic and bathypelagic fish species of the central Mediterranean Sea

In this work 437 fish samples of species belonging to the families Myctophidae (Electrona risso and Diaphus metopoclampus) and Phosichthyidae (Vinciguerria attenuata) were examined for the presence of Anisakidae larvae. The study was performed with fishes in the central Mediterranean Sea, particularly in the Strait of Sicily and in the Strait of Messina. The visual inspection and chloro-peptic analysis revealed the presence of nematode parasites with prevalence values between 2.9% in Electrona risso samples and 5.4% in Vinciguerria attenuata samples. A positive correlation was found between standard length (SL) and prevalence of infestation in D. metopoclampus samples (p < 0.05). The larvae examined were morphologically ascribed, at genus level, to Anisakis morphotypes I and II and molecularly identified as Anisakis pegreffii, Anisakis ziphidarum and Anisakis physeteris, in 67%, 9% and 24% of the fish samples examined. Overall, A. pegreffii and A. ziphidarum larvae were isolated in 14 and 2 specimens of D. metopoclampus respectively, A. physeteris larvae were found in 3 E. risso and 2 V. attenuata. A positive correlation was found between standard length and prevalence of infestation in D. metopoclampus samples (p < 0.05). First information is provided on the presence of Anisakis spp. larvae of the myctophid fish species E. risso, D. metopoclampus and V. attenuata from the Central Mediterranean. It is also confirmed the role of lanternfishes (Myctophidae) as paratenic hosts for Anisakis spp.     

Bioassays for comparative infectivity of mermithid nematodes (Romanomermis iyengari,Romanomermis culicivorax and Strelkovimermis spiculatus) for culicine mosquito larvae

Two improved bioassays were developed to establish infectivity baselines for selection experiments using mermithid nematode variants. Comparative infectivity of Romanomermis iyengari, Romanomermis culicivorax and Strelkovimermis spiculatus using larvae of three mosquito spp. Aedes sierrensis, Aedes aegypti and Culex pipiens were evaluated with “plate” and “tray” bioassays at selected intensity of infections. Using the “plate” bioassay, single mosquito larvae were immersed in 2 ml of water within individual depressions of 12-well, polystyrene tissue culture plates. One, three, or five preparasitic juveniles (J2) were added to each well. In the “tray” bioassay, polyethylene trays containing 500 ml water and 100 mosquito larvae were exposed to 500 (5:1, nematode:insect host) or 1000 (10:1) J2s. Percentage infection (PINF, infectivity) and intensity of infection (IINF, #nematodes per infected larvae) number were determined only after emergence of post-parasitic J3 juveniles. Under the bioassay conditions, all three species of nematodes resulted in infections in all mosquito hosts, but R. iyengari exhibited better effectiveness in the parasitism of mosquito larvae. The three species of mosquitoes presented high levels of susceptibility to each of the three species of nematodes, but in general Cx. pipiens and Ae. sierrensis were slightly more susceptible than Ae. aegypti. The “plate” bioassay was more efficient in measurement of infectivity of the mermithid species and in establishing baseline characteristics for these mosquito-parasitic nematodes. The “tray” bioassay was an effective bioassay for large cohorts of both infective juveniles and host larvae and, potential for field interactions.     

Anthelminthic properties of mangostin and mangostin diacetate

Few anthelminthic drugs are available for human use despite the significant burden caused by helminth infections. We studied the activities of mangostin, a major bioactive xanthone isolated from the pericarp and fruit of Garcinia mangostana and of the synthetic derivative mangostin diacetate. Mangostin and mangostin diacetate lacked activity against the nematodes Heligmosomoides polygyrus (third-stage larvae (L3)), Ancylostoma ceylanicum L3, and Trichuris muris adults and showed only low activity against A. ceylanicum adults (IC₅₀s of 91 μg/ml) in vitro. Mangostin showed promising activities (IC₅₀ of 2.9–15.6 μg/ml) against the trematodes Schistosoma mansoni, Echinostoma caproni, and Fasciola hepatica in vitro. Single oral doses (400 mg/kg and 800 mg/kg) of the drugs achieved worm burden reductions ranging from 0 to 38% and 11–54% against S. mansoni and E. caproni in vivo, respectively. Pharmacokinetic studies would be helpful to understand the differences observed between in vitro and in vivo activities and lacking dose–response relationships.     

Hysterothylacium patagonense n. sp. (Nematoda: Anisakidae) from freshwater fishes in Patagonia,Argentina, with a key to the species of Hysterothylacium in American freshwater fishes

A new anisakid nematode, Hysterothylacium patagonense n. sp., is described from the intestine of the temperate bass Percichthys trucha (Cuvier & Valenciennes), a freshwater fish from Lake Aluminé, Patagonia, Argentina. It is characterised mainly by the absence of lateral alae, the length ratio of the caecum and ventricular appendix (1:0.9–1.8), the length of the spicules (0.952–1.292 mm) and the presence of minute spines on the tip of the tail. In the same locality and in Lake Huechulafquen, advanced third-stage and fourth-stage larvae of this parasite were also recorded from the digestive tract of the introduced salmonids, Salvelinus fontinalis (Mitchell), Oncorhynchus mykiss (Walbaum) and Salmo trutta L. This is the second Hysterothylacium species described from freshwater fishes in South America. A key to Hysterothylacium species parasitising American freshwater fishes is provided.     

Potential for integrated control of Culex quinquefasciatus (Diptera: Culicidae) using larvicides and guppies

Acute toxicity tests were carried out to determine the effect of three larvicides, spinosad, pirimiphos methyl, and chlorpyrifos, on Culex quinquefasciatus Say, Anopheles gambiae s.s. Giles (Diptera: Culicidae), and guppies, Poecilia reticulata Peters (Pisces: Poeciliidae). Thereafter, larvae of Cx. quinquefasciatus were introduced to P. reticulata in containers of different volumes with low concentrations of each larvicide at established predator to prey densities of 1–35; 5–70 and 10–350 (fish to larvae) respectively. The experiment was replicated six times, and the larval consumption was counted after 24 h. Spinosad and pirimiphos methyl were significantly toxic to Cx. quinquefasciatus, the less susceptible mosquito species. Guppy consumption of Culex larvae was highest at a predator to prey density 5–70. Feeding activity of guppies increased in the spinosad treatment at 49 μg L⁻¹ compared to control and the synthetic larvicides. The synthetic larvicides generally reduced consumption of larvae except for the lowest concentration of pirimiphos methyl which increased it at the highest predator to prey density in a 3 L container. The highest percentage of Culex larvae was consumed by fish in the presence of spinosad at 49 μg L⁻¹ and a predator to prey density of 5–70 in 0.5 L plastic containers. Thus, predator to prey density, container size, type and concentration of larvicide are all important factors to be considered in integrated management of mosquito larvae.     

Temperature and dose effects on the pathogenicity and reproduction of two Korean isolates of Heterorhabditis bacteriophora (Nematoda: Heterorhabditidae)

Out of some isolated Heterorhabditis bacteriophora from Korea, ecological study on two isolates which had different geographical features was investigated. That is, effects of temperature and dose on the pathogenicity and reproduction of two Korean isolates of H. bacteriophora were investigated using Galleria mellonella larvae in the laboratory. The median lethal dose (LD₅₀) decreased with increasing temperature, but increased at 35 °C. The optimal temperatures for infection were 30 °C for H. bacteriophora Jeju strain and 24 °C for H. bacteriophora Hamyang strain. The median lethal time, LT₅₀ of H. bacteriophora Hamyang strain was recorded at 13 °C to 35 °C and that of H. bacteriophora Jeju strain was recorded at 18 °C to 30 °C. The number of established nematodes in G. mellonella larvae was significantly different depending on temperature and dose. When G. mellonella larvae were exposed to 300 infective juveniles (IJs), mortality of G. mellonella gradually increased with exposure time with H. bacteriophora Jeju strain but not with H. bacteriophora Hamyang strain. 87.5% mortality of G. mellonella was recorded by H. bacteriophora Hamyang strain after 1440 min whereas 100% mortality was recorded by H. bacteriophora Jeju strain after 4320 min. The time from infection to the first emergence of nematodes decreased with increasing temperature. Duration of emergence of the two strains in the White traps also decreased with increasing temperature. The highest progeny numbers of H. bacteriophora Jeju strain were 264,602 while those of H. bacteriophora Hamyang strain were 275,744 at the rate of 160 IJs at 24 °C.     

Rapid detection of Trichinella spiralis larvae in muscles by loop-mediated isothermal amplification

Trichinella spiralis is a tissue-dwelling nematode parasite. A loop-mediated isothermal amplification (LAMP) assay was developed and validated for the sensitive and rapid detection of T. spiralis larvae in muscle samples. Sixteen sets of primers were designed to recognise distinct sequences of a conserved gene, a 1.6 kb repetitive element of the Trichinella genome. One set of primers was selected as the most appropriate for rapid detection. The specificity and sensitivity of the primers in LAMP reactions for T. spiralis larvae and muscle samples of mice infected with T. spiralis were determined. Another 10 heterologous parasites were selected for specificity assays. The results showed that target DNA was amplified and visualised by monitoring turbidity and adding calcein detection methods within 70 min at an isothermal temperature of 63 °C. The sensitivity of LAMP with the detection limit of 362 fg/μl was >10 times higher than that for PCR. The designed primers had a good specificity. No cross-reactivity was found with the DNA of any other parasites. The assay was able to detect T. spiralis in all mouse muscle samples infected with 10 T. spiralis larvae on day 20 p.i. We believe this is the first report regarding the application of the LAMP assay for detection of T. spiralis larvae in muscle samples from experimentally infected mice. This method demonstrates a potentially valuable means for the direct detection of T. spiralis larvae in meat inspection.     

A new record of Myxobolus brachysporus and M. israelensis in the tilapia (Oreochromis niloticus) collected from the Nile River,Egypt

The present study was carried out as part of an ongoing general survey for myxosporean parasites infecting tilapias in the River Nile, Egypt. In the present study, 77 Nile tilapia (Oreochromis niloticus) were collected from boat landing sites at Beni-Suef governorate, Egypt and examined for the myxosporean infection. The infection was encountered as a huge number of free spores in the kidney and the spleen. The infection showed a prevalence of 51.9% (40/77) for Myxobolus brachysporus while it was 25.9% (20/77) for Myxobolus israelensis. Mature spores of M. brachysporus were ellipsoidal and measured 8.6 × 13.2 μm. The polar capsules were subcircular with 5–6 filament turns and measured 4.7 × 3.6 μm. Spores of M. israelensis were ellipsoidal in the frontal view and fusiform in the lateral view. Spore measurements were 13.4 μm long and 8.7 μm wide. The polar capsules were elongated with 6–7 filament coils and measured 8.6 × 3.1 μm. The findings presented here proved that tilapia fishes in the Nile River are still suffering from infections with Myxobolus species. Therefore, further studies should be carried out to survey the Myxobolus infection among tilapias under culture conditions to clarify the pathological impacts of this parasite in tilapias aquaculture.     

Trophic structure of common marine species in the Bohai Strait,North China Sea,based on carbon and nitrogen stable isotope ratios

Trophic relationships among common coastal species in the Bohai Strait, North China Sea, were investigated in this study using stable isotope ratios of carbon (¹³C/¹²C) and nitrogen (¹⁵N/¹⁴N) as tracers. During the research cruises of this study, 16 species of invertebrates and 18 species of fishes were collected, as were various food sources (phytoplankton, macroalgae, and sediment). The carbon and nitrogen isotope ratios of all collected samples were measured and used for trophic level analyses. The results showed that the grazers Oregonia gracilis, Notoacmea schrenckii, Chlorostoma rustica, and Chelon affinis, preferentially consumed Zostera marina (p < 0.01), whereas Anthocidaris crassispina preferred Ulva conglobata (p < 0.01). Two trophic models based on nitrogen isotope ratios were built to identify the trophic level of each species. The model that combined food sources was more appropriate than the model that used a single primary producer to identify the relative trophic positions of primary consumers. Scombermorus niphonius, an important fishery resource, was at the top trophic level. The seastar Asterias amurensis was at the highest level among the invertebrates and directly threatened production of shellfish. Based on the trophic level and food source relationships identified in this study, we gave some advice for ecological restoration, such as optimizing the structure of food source distribution, limiting seastar numbers, and improving the applicability of habitat for fishery species.     

Modifying orchard planting sites conserves entomopathogenic nematodes,reduces weevil herbivory and increases citrus tree growth,survival and fruit yield

In Florida, a root weevil pest of citrus, Diaprepes abbreviatus, is more damaging and attains higher population density in some orchards on fine textured, poorly drained “flatwoods” soils than in those on the deep, coarse sandy soils of the central ridge. Previous research revealed that sentinel weevil larvae were killed by indigenous entomopathogenic nematodes (EPNs) at significantly higher rates in an orchard on the central ridge, compared to one in the flatwoods. We hypothesized that filling tree planting holes in a flatwoods orchard with sandy soil from the central ridge would provide a more suitable habitat for EPNs, thereby reducing weevil numbers and root herbivory. Fifty trees were planted in oversized planting holes filled with coarse sand and 50 trees were planted in native soil in a split plot design where whole plots were species of introduced EPNs and split plots were soil type. Each of Steinernema diaprepesi, Steinernema riobrave, Heterorhabditis indica, Heterorhabditis zealandica, or no EPNs were introduced into the rhizospheres in 10 plots of each soil type. During four years, EPN numbers in soil samples and the relative abundance of seven species of nematophagous fungi associated with nematodes were measured three times using real-time PCR. The efficacy of EPNs against sentinel weevil larvae was also measured three times by burying caged weevils in situ. EPN species richness (P = 0.001) and diversity (P = 0.01) were always higher in sand than native soil. Soil type had no effect on numbers of EPNs in samples, but EPNs were detected more frequently (P = 0.01) in plots of sandy soil than native soil in 2011. Two nematophagous fungi species, Paecilomyces lilacinus and Catenaria sp. were significantly more abundant in nematode samples from sandy soil on all three sampling dates. Efficacy of EPNs against weevil larvae was greater in sandy soil inoculated with S. diaprepesi (P = 0.03) in June 2010 and in all treatments in sandy soil in May 2011 (P = 0.03). Sixty-eight percent more adult weevils (P = 0.01) were trapped emerging from native soil during two years than from sandy soil. By May 2011, the cumulative number of weevils emerging from each plot was inversely related (P = 0.01) to the numbers of EPNs detected in plots and to EPN efficacy against sentinels. Three trees in sandy soil died as a result of root herbivory compared to 21 trees in native soil. Surviving trees in sandy soil had trunk diameters that were 60% larger (P = 0.001) and produced 85% more fruit (P = 0.001) than those in native soil. Although it is not possible to characterize all of the mechanisms by which the two soil treatments affected weevils and trees, substitution of sand for native soil was an effective means of conserving EPNs and shows promise as a cultural practice to manage D. abbreviatus in flatwoods citrus orchards with a history of weevil damage to trees.     

Detrimental impacts of the dinoflagellate Karenia mikimotoi in Fujian coastal waters on typical marine organisms

Blooms of the toxic dinoflagellate Karenia mikimotoi (K. mikimotoi) have occurred frequently in the East China Sea in recent decades and were responsible for massive mortalities of abalones in Fujian coastal areas in 2012, however, little is known about the effects of these blooms on other marine organisms. In this study, the toxic effects and the possible mechanisms of toxicity of K. mikimotoi from Fujian coastal waters on typical marine organisms at different trophic levels, including zooplankton (Brachionus plicatilis, Artemia salina, Calanus sinicus, and Neomysis awatschensis) and aquaculture species (Penaeus vannamei and Scophthalmus maximus) were investigated. At a bloom density of 3 × 10⁴ cells/mL, the Fujian strain of K. mikimotoi significantly affected the tested organisms, which had mortality rates at 96 h of 100, 23, 20, 97, 33, and 53%, respectively. Moreover, the intact cell suspension was toxic to all tested species, whereas cell-free culture and the ruptured cell suspension had no significant effects on the tested organisms. Possible mechanisms for this toxic effect, including reactive oxygen species (ROS) and hemolytic toxins, were evaluated. For K. mikimotoi, 0.014 ± 0.004 OD/(10⁴ cells) superoxide (O₂⁻) and 3.00 ± 0.00 nmol/(10⁴ cells) hydrogen peroxide (H₂O₂) were measured, but hydrogen peroxide did not affect rotifers at that concentration, and rotifers were not protected from the lethal effects of K. mikimotoi when the enzymes superoxide dismutase and catalase were added to counteract the ROS. The lipophilic extract of K. mikimotoi had a hemolytic effect on rabbit erythrocytes but exhibited no significant toxicity. These results suggest that this strain of K. mikimotoi can have detrimental effects on several typical marine organisms and that its toxicity may be associated with intact cells but is not related to ROS or hemolytic toxins.     

Analysis of internal transcribed spacer1 (ITS1) region of rDNA for genetic characterization of Paramphistomum sp.

Paramphistomosis is the most prevalent disease of domestic ruminants, causing heavy economic loss in many countries across the world. The morphological identification of these parasites is difficult, therefore molecular characterization is used to discriminate Paramphistomum species. The present study was conducted to identify Paramphistomum sp. at Mardan District, Khyber Pakhtunkhwa (KPK), Pakistan. All samples of these rumen flukes were collected from buffalo. The gDNA was isolated from the adult parasites and the ITS1 region was amplified for the sequence analysis. All flukes had 100% similarity and there was no intraspecific variation. The Blast results showed that all flukes were P. cervi as they form a single cluster with P. cervi reported from China. The results of the ITS1 sequences of the present study with reference sequencing from China showed eight specific SNPs. This was the first study in which P. cervi was genetically characterized through the ITS1 region of rDNA at District Mardan, Pakistan. It can also be used as a marker for the genetic identification of Paramphistomum species.     

The parasite fauna of <Emphasis Type="Italic">Arctogadus glacialis</Emphasis> (Peters) (Gadidae) from western and eastern Greenland

In all 155 specimens of the high Arctic codfish Arctogadus glacialis examined for metazoan parasites, 55 specimens were from southern and northern Baffin Bay, western Greenland, and 100 specimens from north-eastern Greenland and Scoresby Sound. A total of 20 parasite taxa were recorded. A new myxozoan Gadimyxa arctica was found in southern Baffin Bay and Scoresby Sound. The gadid myxozoan Zschokkella hildae, the digeneans Gonocerca phycidis and Lecithaster gibbosus, the gill copepod Haemobaphes cyclopterina and third-stage larvae of the nematodes Anisakis simplex and Hysterothylacium aduncum were found in Scoresby Sound only. The digenean Hemiurus levinseni and third-stage larvae of the nematode Contracaecum sp. were found at all four stations. The nematodes Ascarophis spp. were found at three stations. The parasite fauna of A. glacialis from Scoresby Sound was very similar to that of 50 specimens of the closely related Boreogadus saida from the same area.     

The natural diet of the mud crab Scylla olivacea (Herbst, 1896) in Pichavaram mangroves,India

Food and feeding habits of mud crab Scylla olivacea (Herbst, 1896) in Pichavaram mangroves was investigated quantitatively and qualitatively for a period of two years from June 2010 to May 2012. Gut contents from 1737 specimens comprising 843 males and 894 females in the size range between 45 mm and 148 mm were examined. Crustaceans form the predominant food item in a majority of size groups in terms of percentage wet weight and frequency of occurrence, while molluscs showed a preference in few size groups. The other dietary items includes fishes, detritus, mud and sand and miscellaneous. Gut content analysis revealed no significant variation between the quantities of food consumed by both sexes. Feeding intensity was higher in juveniles and subadults of both sexes than that of adults, revealing a greater preference to feed on fast moving prey such as crustaceans and fishes. The results of the present study indicate that S. olivacea in Pichavaram mangroves exhibited a clear preference for crustaceans.     

Evaluation of red seaweed Gracilaria arcuata as dietary ingredient in African catfish,Clarias gariepinus

The aim of this study was to evaluate the use of dried marine seaweed, Gracilaria arcuata for the first time as dietary ingredient in partial substitution of fishmeal on the growth performance, feed utilization and body composition of African catfish, Clarias gariepinus. Four experimental diets were formulated: D1 as a control group; D2; D3 and D4 which included 10%, 20% and 30% G. arcuata meal respectively. One hundred and eighty African catfish weighing 9.62 ± 0.42 g, (mean ± SE) was divided into four groups corresponding to the different feeding regimes. The final body weight of the fishes showed significant differences (P < 0.05) between the control (D1); D2 and other treated groups D3 and D4, with weights of 66.98, 59.60, 47.34 and 30.73 g recorded for D1, D2, D3 and D4, respectively. Significant differences (P < 0.05) were also evident in weight gain, specific growth rate, and feed utilization between treatment and control groups. However, no significant differences (P > 0.05) were observed between the control group and fishes fed D2 for all previous parameters. Protein productive value, protein efficiency ratio, daily dry feed intake and total feed intake were also significantly lower in fish fed with a diet containing G. arcuata than in the control group and D2 which contains 10% of G. arcuata. Overall, the results of the experiment revealed that African catfish fed a diet with G. arcuata included in 20% and 30% levels showed poorer growth and feed utilization than the control group and D2. However, the study recommended that C. gariepinus can accept this ingredient up to 10% in their diets. More defined experiments therefore seem to be necessary in order to determine the maximum level of this marine seaweed in diets with amino acid supplementation for African catfish.     

Haemocytes immunity of rose sawfly,Arge ochropus (Hym.: Argidae) against entomopathogenic nematodes,Steinernema carpocapsae and Heterorhabditis bacteriophora

The rose sawfly, Arge ochropus (Gmelin), is one of the most destructive pests attacking rose bushes in northern Iran. In the present study, the haemocyte reactions of A. ochropus larvae were assessed against two entomopathogenic nematodes, Steinernema carpocapsae (Weiser) and Heterorhabditis bacteriophora Poinar. With injection of infective juveniles of H. bactriophora into A. ochropus larvae, the total haemocyte count showed alternating fluctuations at all time intervals. Encapsulation occurred 22 hours post injection (hpi) of H. bacteriophora infective juveniles into the rose sawfly larvae, while melanization was observed after 24 h. In the case of S. carpocapsae, initial attachment of the haemocytes was detected at 18 hpi and complete encapsulation at 24 hpi; no melanization was observed. In general, findings showed strong immune responses of the rose sawfly larvae against H. bacteriophora, while these reactions were weakened for S. carpocapsae. Therefore, it is concluded that S. carpocapsae has more ability to overcome the host defence system, and this research provides a new window on its potential as a biocontrol agent.