Progress in developing a new detection method for the harmful algal bloom species,Karenia brevis,through multiwavelength spectroscopy

Multiwavelength spectroscopy is a rapid analytical technique that can be applied to detect, identify, and quantify microorganisms such as Karenia brevis, the species known for frequent red-tide blooms in Florida's coastal waters. This research will report on a model-based interpretation of UV–vis spectra of K. brevis. The spectroscopy models are based on light scattering and absorption theories, and the approximation of the frequency-dependant optical properties of the basic constituents of living organisms. Absorption and scattering properties of K. brevis, such as cell size/shape, internal structure, and chemical composition, are shown to predict the spectral features observed in the measured spectra. The parameters for the interpretation model were based upon both reported literature values, and experimental values obtained from live cultures and pigment standards. Measured and mathematically derived spectra were compared to determine the adequacy of the model, contribute new spectral information, and to establish the proposed spectral interpretation approach as a new detection method for K. brevis.     

Proteomic study of a model causative agent of harmful algal blooms, Prorocentrum triestinum II: the use of differentially expressed protein profiles under different growth phases and growth conditions for bloom prediction

Simultaneous comparison of differentially expressed protein profiles of Prorocentrum triestinum grown under different growth phases and growth conditions indicated the presence of phase-specific and stress-responsive proteins, respectively. Correlation studies on these proteins in relation to cell division phasing patterns and to models of phytoplankton growth inferred the possible functions. Most notable among these proteins were groups of proteins thought to trigger or mediate cells through specific phases of division of this alga, e.g., BP1, BP2, PB1, PB2, and PB3. Other proteins (e.g., group 1 proteins) thought to be responsible for maintaining and supporting cell concentration under adverse conditions were found. Furthermore, another group of proteins (group 2 proteins) thought to be stress-responsive were also detected. Taken overall, these differentially expressed proteins provided important information for uncovering various protective and adaptive mechanisms in the dinoflagellate's life cycle. These proteins have the potential to serve as "indicator proteins" for rapid assessment of the nutritional or metabolic status of these phytoplankton cells,and monitoring the differential expression of these phase-specific proteins and stress-specific proteins could be an important biomarker for bloom prediction.     

Some observations on harmful algal bloom (HAB) events along the coast of Guangdong,southern China in 1998

The year 1998 was an unusual year for Guangdong Province and Hong Kong, both in southern China, as the frequency and intensity of harmful algal blooms (HAB) were much higher than usual. This paper describes the causative organisms found associated with these blooms and speculates on the possible causes of these blooms, including the effects of increased temperature, reduced salinity, eutrophication and meteorological and oceanographic events on the initiation and spread of these blooms.     

Satellite remote sensing of harmful algal blooms: A new multi-algorithm method for detecting the Florida Red Tide (Karenia brevis)

In a continuing effort to develop suitable methods for the surveillance of Harmful Algal Blooms (HABs) of Karenia brevis using satellite radiometers, a new multi-algorithm method was developed to explore whether improvements in the remote sensing detection of the Florida Red Tide was possible. A Hybrid Scheme was introduced that sequentially applies the optimized versions of two pre-existing satellite-based algorithms: an Empirical Approach (using water-leaving radiance as a function of chlorophyll concentration) and a Bio-optical Technique (using particulate backscatter along with chlorophyll concentration). The long-term evaluation of the new multi-algorithm method was performed using a multi-year MODIS dataset (2002 to 2006; during the boreal Summer-Fall periods - July to December) along the Central West Florida Shelf between 25.75°N and 28.25°N. Algorithm validation was done with in situ measurements of the abundances of K. brevis; cell counts ≥1.5×10(4) cells l(-1) defined a detectable HAB. Encouraging statistical results were derived when either or both algorithms correctly flagged known samples. The majority of the valid match-ups were correctly identified (~80% of both HABs and non-blooming conditions) and few false negatives or false positives were produced (~20% of each). Additionally, most of the HAB-positive identifications in the satellite data were indeed HAB samples (positive predictive value: ~70%) and those classified as HAB-negative were almost all non-bloom cases (negative predictive value: ~86%). These results demonstrate an excellent detection capability, on average ~10% more accurate than the individual algorithms used separately. Thus, the new Hybrid Scheme could become a powerful tool for environmental monitoring of K. brevis blooms, with valuable consequences including leading to the more rapid and efficient use of ships to make in situ measurements of HABs.     

Dietary exposure to harmful algal bloom (HAB) toxins in the endangered manatee (Trichechus manatus latirostris) and green sea turtle (Chelonia mydas) in Florida,USA

Florida is a hotspot for cyano- and microalgal harmful algal blooms (HABs) with annual red-tide events off-shore and blooms of Lyngbya spp. commonly observed in both marine and freshwater environments. This region also provides extensive foraging habitat for large populations of herbivorous green turtles (Chelonia mydas) and manatees (Trichechus manatus latirostris). The exposure of aquatic organisms to HAB toxins is not well known and whilst acute exposures are better understood, the vulnerability of aquatic animals to chronic exposure from multiple HAB toxins over prolonged periods has rarely been addressed. This study aimed to identify the presence of toxic compounds produced by HAB species commonly found in Florida (brevetoxins, okadaic acid, saxitoxins and Lyngbya toxins) in tissues and gut samples from manatee and green sea turtles that stranded in Florida, USA. Muscle, liver and alimentary tract samples were opportunistically collected from 14 manatees and 13 green turtles that stranded on the Florida shoreline between December 2003 and February 2006. Samples from each animal were assessed for the presence of brevetoxin, okadaic acid, lyngbyatoxin-A and saxitoxin. Nine (64%) manatees and 11 (85%) turtles were found to have been exposed to one or more of the HAB toxins. Okadaic acid and saxitoxin were only found in alimentary tract samples, whereas brevetoxin was more widely distributed. No lyngbyatoxin-A was observed in any tissue samples. The majority of turtles (13) stranded on the Atlantic coast between St. Johns and Monroe counties, with one on the Gulf coast at Bay County, whereas nine manatees were stranded on the Gulf coast between Levy and Lee counties, with the remaining five between Volusia and Brevard counties on the Atlantic coast. This HAB toxin screen has identified that a large proportion of a random sample of turtles and manatees that stranded in Florida in 2003–2006 were exposed to HAB toxins. Most of the concentrations measured were low, and the toxins were directly linked to the death of only three of these animals. However, the presence of these compounds, and in some cases the presence of multiple HAB toxins in individual animals, indicates that turtles and manatees in Florida are exposed to deleterious compounds at sub-lethal levels in their environment, which could ultimately compromise their health.     

Effects of light and hydrogen peroxide on gene expression of newly identified antioxidant enzymes in the harmful algal bloom species Chattonella marina

ABSTRACT

Antioxidant enzymes are essential proteins that maintain cell proliferation potential by protecting against oxidative stress. They are present in many organisms including harmful algal bloom (HAB) species. We previously identified the antioxidant enzyme 2-Cys peroxiredoxin (PRX) in the raphidophyte Chattonella marina. This enzyme specifically decomposes a hydrogen peroxide (H₂O₂). PRX is the only antioxidant enzyme so far identified in C. marina. This study used mRNA-seq, using Trinity assemble and blastx for annotation, to identify a further five antioxidant enzymes from C. marina: Cu Zn superoxide dismutase (Cu/Zn-SOD), glutathione peroxidase (GPX), catalase (CAT), ascorbate peroxidase (APX) and thioredoxin (TRX). In the gene expression analysis of six enzymes (Cu/Zn-SOD, GPX, CAT, APX, TRX and PRX) using light-acclimated (100 μmol photons m^?2 s^?1) C. marina cells, only PRX gene expression levels were significantly increased by strong light irradiation (1000 μmol photons m^?2 s^?1). H₂O₂ concentration and scavenging activity were also increased and significantly positively correlated with PRX gene expression levels. In dark-acclimated cells, expression levels of all antioxidant enzymes except APX were significantly increased by light irradiation (100 μmol photons m^?2 s^?1). Expression decreased the following day, with the exception of PRX expression. With the exception of CAT, gene expression of antioxidant enzymes was not significantly induced by artificial H₂O₂ treatment, although average gene expression levels were slightly increased in some enzymes. Thus, we suggest that light is the main trigger of gene expression, but the resultant oxidative stress is also a possible factor affecting the gene expression of antioxidant enzymes in C. marina.     

Effect of okadaic acid on carpet shell clam (Ruditapes decussatus) haemocytes by in vitro exposure and harmful algal bloom simulation assays

Okadaic acid (OA), produced by dinoflagellates during harmful algal blooms (HAB), belongs to the Diarrheic Shellfish Poisoning toxins that cause gastrointestinal symptoms in humans after consumption. In the present work, Ruditapes decussatus haemocytes were selected to evaluate the effect of OA on cell viability, enzymatic status and immune capacity through the measure by flow cytometry of apoptosis–cell death, non-specific esterase activity and phagocytosis. In order to compare different exposure conditions, two experiments were developed: in vitro exposure to OA and HAB simulation by feeding clams with the OA producer, Prorocentrum lima. Apoptosis was not OA dose-dependent and cell death increased in both assays. Phagocytosis of latex beads and esterase activity decreased in haemocytes incubated with OA. In contrast, esterases increased during the feeding with P. lima. Our results showed that OA and the simulated HAB caused damages on haemocyte functions and viability.     

Revisiting the 1991 algal bloom in shelf waters off Argentina: Azadinium luciferelloides sp. nov. (Amphidomataceae,Dinophyceae) as the causative species in a diverse community of other amphidomataceans

Species of the marine dinophycean genera Azadinium and Amphidoma (Amphidomataceae) mostly attract attention because of their production potential of the lipophilic polyether phycotoxin azaspiracid (AZA). The genus Azadinium probably has a very wide geographical distribution. Blooms of Azadinium from the continental shelf off Argentina have been observed back in the early 1990, but were just recently published, and the causative species, identified at that time as Azadinium cf. spinosum, could not unequivocally be determined. Here we retrospectively analyzed old archived samples of one of the South Atlantic Azadinium bloom from 1991 with electron microscopy. It turned out that the dominant nanoplanktonic dinophycean species in fact represent a new species which we describe here based on the morphology. Azadinium luciferelloides sp. nov. is a small (approximately 9–14 μm cell length) thecate dinoflagellate with the dominant plate pattern of the genus (Po, X, 4′, 3a, 6″, 6C, 5S, 6″′, 2″″), and with a small antapical spine. Azadinium luciferelloides differed from all other described species of Azadinium by the position of the ventral pore, which was located on the right ventral side in a notch of an otherwise symmetric pore plate. In addition, we recorded and documented the presence of other similar sized species of the Amphidomataceae in the samples. Our finding of Az. spinosum, Az. dalianense, Az. dexteroporum, and Amphidoma languida are the first record for the South Atlantic and thus describe an important range extension of these species. The diversity and importance of the Amphidomataceae for South Atlantic spring bloom plankton is now known and taxonomically documented, but cultures and/or analysis of AZA in field samples of the area are needed to clarify the AZA production potential of the local species and populations in order to finally evaluate the risk potential of AZA for AZA shellfish contamination in the Southwestern Atlantic region.     

The population dynamics, reproductive strategy and life history tactics of Musculium lacusfre (Bivalvia: Pisidiidae) in Hong Kong

Hitherto uninvestigated in Hong Kong are the population dynamics, reproductive strategies and life history tactics of the Pisidiidae of which three species are known to occur locally. This study is of Musculium lacustre inhabiting a drainage ditch of the Lam Tsuen River in the New Territories of Hong Kong and extended over 15 months during 1983–84.
In Hong Kong, M. lacustre is a protandric, simultaneous hermaphrodite that matures at a length of 2 mm; the majority of each generation is, however, not mature until a length of between 4–6 mm is attained. Fertilized eggs are brooded in the inner demibranchs until they reach a length of 1.5 mm when they are released as post-foetal larvae. Musculium lacustre is univoltine. Reproduction occurs twice a year but, since M. lacustve is semelparous, the population contains, at any one time, two overstepping generations. Thus, a spring generation is recruited and grows rapidly to generate a second, smaller, autumn generation. This in turn grows rapidly and matures to produce the spring generation of the succeeding year. Adult mortality occurs in late summer and winter. A few of the late-born spring generation may overwinter to contribute to the spring generation of the succeeding year. This is not so with the autumn generation.
Such sexual and life history tactics can be related to the climate and hydrology of Hong Kong and its freshwater habitats. Reproduction takes place in spring and autumn when temperatures are moderate. Mortality is high in summer and winter when the habitat is either, respectively, hot and flushed out by summer rains or cold and dry. As M. larustre is able to overwinter but not aestivate at these latitudes (22° N), it is postulated that this holarctic species is approaching the southern limit of its asiatic range. Musculium larustre is compared with the other bivalves inhabiting fresh and brackish-water habitats in southern China.     

Occurrences of harmful algal blooms (HABs) associated with ocean environments in the South China Sea

Harmful algal blooms (HABs) occur frequently in the South China Sea (SCS), causing enormous economic losses in aquaculture. We analyzed historical HAB records during the period from 1980 to 2003 in SCS. We found that HABs-affected areas have expanded and the frequency of HABs varied during this period. The seasonal and annual variations, as well as causative algal species of HABs are different among the four regions. Areas with frequent HABs include the Pearl River Estuary (China), the Manila Bay (the Philippines), the Masinloc Bay (the Philippines), and the western coast of Sabah (Malaysia). HABs occurred frequently during March–May in the northern region of SCS, May–July in the eastern region, July in the western region, and year-round in the southern region. Among the species that cause HABs, Noctiluca scintillans dominated in the northern region, and Pyrodinium bahamense in the southern and eastern regions. Causative species also varied in different years for the entire SCS. Both P. bahamense and N. scintillans were the dominant species during 1980–2003. Some species not previously recorded formed blooms during 1991–2003, including Phaeocystis globosa, Scrippsiella trochoidea, Heterosigma akashiwo, and Mesodinium rubrum. Variations in HABs are related to various regional conditions, such as a reversed monsoon wind in the entire SCS, river discharges in the northern area, upwelling in Vietnam coastal waters during southwest winds and near Malaysia coastal waters during northeast winds, and eutrophication from coastal aquaculture in the Pearl River estuary, Manila Bay, and Masinloc Bay. Handling editor: D. Hamilton     

Group-specific component (Gc) subtypes in the Chinese population of Hong Kong

Summary The distribution of Gc subtypes in a sample of the Chinese population of Hong Kong was studied using isoelectric focusing followed by immunofixation. A sensitive modification of this technique is described. Nine distinct phenotypes were observed which appear to result from the three common alleles Gc ^IF, Gc ^IS, and Gc ², which are found in most populations. The respective gene frequencies were 0.494, 0.258, and 0.247. In addition, two rare phenotypes were observed which appear to be due to a rare allele tentatively identified as Gc ^2G2.     

Reproductive biology of Sarotherodon mossambicus (Cichlidae) in Plover Cove Reservoir,Hong Kong

Synopsis A 16-month study (August 1972 to November 1973) of Sarotherodon mossambicus in the Plover Cove Reservoir, Hong Kong revealed a sex ratio of 1:1 for small fish (below 19 cm standard length), but above this length males predominated in the population. Monthly sex composition also showed this approximately 1:1 ratio except during the months of August and September, when again males predominated. Males and females reached M₅₀ at an average length of 17.8 cm and 16.2 cm respectively. Maturity and gonosomatic index studies showed that spawning occurred from May to October with peaks in June and August, and there were at least two spawnings each year. The relationship between fecundity and standard length of females collected during both spawnings was computed to be F = 118.49L–1150.91.     

Inter- and intraspecific differences in the life history and growth of Caridina spp. (Decapoda: Atyidae) in Hong Kong streams

1. The life‐history pattern of three populations of Caridina cantonensis (Atyidae) and one population of Caridina serrata were studied in four Hong Kong streams: Pak Ngau Shek (PNS), Kap Man Hang (KMH), Pak Tam Chung (PTC), and Lung Fu Shan (LFS). Caridina cantonensis occurred at PNS, KMH and PTC while C. serrata was present at LFS only. Monthly quantitative sampling was carried out at each study site over 2 years to investigate life‐history patterns and cohort‐specific growth rates. 2. Breeding by both species was mainly restricted to the wet season, and there was an increase in the incidence of ovigerous females as water temperatures rose at the start of the summer monsoon. The number of cohorts and the frequency of recruitment of C. cantonensis (one to three times each year) were highly site‐specific, but consistent between years. 3. Shrimp growth was size‐dependent, and decreased with increasing body size. Caridina cantonensis at PTC had the highest cohort‐specific growth rates (0.05–0.14 mg ash‐free dry weight (AFDW) mg^?1 day^?1), followed by C. serrata at LFS (6% lower), and C. cantonensis at KMH and PNS (16–39% lower). Shrimp life spans exceeded 1 year (17–22 months), depending on site and species. 4. Sexual maturity occurred at an earlier age and at a smaller size in populations with higher growth rates. Shrimps at PTC and LFS matured at approximately 4 months old; this was 3 months earlier than at KMH and 5 months earlier than at PNS. Females of C. cantonensis at PTC and C. serrata at LFS may have bred twice during each breeding season, while females in the other two populations bred once only. The number of cohorts produced by each population in each year did not vary between the years of the study. Most sexually mature individuals only survived long enough (for 10–12 months after maturation) to breed during one season. 5. The mean brood size of C. cantonensis varied among streams and was 25–28% larger at PNS and KMH than at PTC. Egg size did not differ among populations. Larger brood sizes at PNS and KMH may have been a consequence of increased availability of algal food in these unshaded stream sites. Caridina serrata had similar egg and brood sizes to C. cantonensis, but females were smaller and reproductive investment was higher. Thus, the degree of intraspecific variation in atyid breeding in Hong Kong was at least equal to, if not greater than, the extent of interspecific variation. Interspecific differences in life history may reflect the frequency of droughts and spate intensity experienced by C. serrata at LFS.     

Philasterides dicentrarchi (Ciliophora, Scuticociliatida) as the causative agent of scuticociliatosis in farmed turbot Scophthalmus maximus in Galicia (NW Spain)

Two outbreaks of scuticociliatosis affecting farmed turbot Scophthalmus maximus in Galicia are described. Moribund fish showed cutaneous ulcers, darkened skin, swimming behaviour alterations, exophthalmos, and/or abdominal distension as a result of accumulation of ascitic fluid in the body cavity. Ciliates were detected in fresh mounts of practically all organs and tissues, including the blood and ascitic fluid. Histopathological studies revealed severe encephalitis and meningitis (associated with different degrees of softening or liquefaction of the brain), necrosis of the hepatic parenchyme, severe oedema of the intestinal wall, degeneration of muscle fibres, hyperplasia of the branchial epithelium, and/or vascular and perivascular inflammation. In some cases, parasites are surrounded by abundant monocytic and lymphocytic infiltrate. We report the morphological and biometric characteristics of this ciliate, which allow identification as Philasterides dicentrarchi. We discuss possible routes of entry into the host, and environmental factors possibly facilitating infection.     

Modification of histidine (B10) is the causative agent for a superactive form of insulin.

The site of modification that is responsible for the formation of superactive insulin (ILM) was determined. The insulin derivative was prepared by treatment of insulin-Sepharose with ammonium bicarbonate. It was found that the insulin was bound to the resin through histidine B10, His (B10), and its ammonium bicarbonate-mediated release resulted in an insulin analog in which His (B10) was modified on the imidazole ring. This modification was reversible upon storage, resulting in normal levels of insulin activity. Amino acid analysis of a peptide containing this modified histidine revealed some aspartic acid. Since Asp (B10) insulin is also superactive, the observed superactivity may thus stem from either modification of the histidine or its conversion to aspartic acid.     

Ultra-rapid real-time PCR for the detection of Paenibacillus larvae, the causative agent of American Foulbrood (AFB)

A novel micro-PCR-based detection method, termed ultra-rapid real-time PCR, was applied to the development of a rapid detection for Paenibacillus larvae (P. larvae) which is the causative agent of American Foulbrood (AFB). This method was designed to detect the 16S rRNA gene ofP. larvae with a micro-scale chip-based real-time PCR system, GenSpector^® TMC-1000, which has uncommonly fast heating and cooling rates (10 °C per second) and small reaction volume (6 μl). In the application of ultra-rapid real-time PCR detection to an AFB-infected larva, the minimum detection time was 7 min and 54 s total reaction time (30 cycles), including the melting temperature analysis. To the best of our knowledge, this novel detection method is one of the most rapid real-time PCR-based detection tools.     

中国香港细蜉属三新种记述(蜉蝣目:细蜉科)

记述了采自中国香港的细蜉科Caenidae,细蜉属Caenis3新种：点刻细蜉Caenis aspera sp.nov.、双突细蜉Caenis bicornis sp.nov.和光滑细蜉Caenis lubrica sp.nov.。模式标本均保存在中国广州华南农业大学昆虫学系昆虫标本室。     

Heterologous expression of green fluorescent protein in Paenibacillus larvae, the causative agent of American Foulbrood of honey bees

Aims: We aimed at expressing heterologous proteins in Paenibacillus larvae, the causative agent of American Foulbrood of honey bees, as a prerequisite for future studies on the molecular pathogenesis of P. larvae infections. Methods and Results: For this purpose, we established a protocol for the transformation of the plasmid pAD43‐25 carrying a functional GFP gene sequence (gfpmut3a) into different P. larvae strains representing the two most relevant P. larvae genotypes ERIC I and ERIC II. We determined the optimal field strength for electroporation and the optimal regeneration time after transformation. Stable GFP expression could be detected in the mutants during their entire life cycles and even after sporulation and re‐germination. Conclusions: This method is suitable not only for the expression of GFP in P. larvae but also for the expression of heterologous proteins or GFP‐tagged proteins in P. larvae. Mutants can be used for infection assays because GFP expression remained stable after sporulation and re‐germination. Significance and Impact of the Study: This method provides the first true molecular tool for P. larvae and, therefore, is an immense advancement from what we had previously at our hands for the study of P. larvae pathogenesis.     

The ecology of the marine rove beetle,Bryothinusa spp. (Coleoptera: Staphylinidae) in Hong Kong

Bryothinusa spp. are common marine insect in Hong Kong. They occur in the intertidal zone of sandy shores between 0.6 and 1.2 m tide level. They emerge when the tide recedes, possibly to mate and feed, then burrow again at the advance of the incoming tide.Bryothinusa has a special respiratory apparatus for long submersions and therefore is able to inhabit the changeable intertidal zone where the competition with other living organisms is comparatively low.