Salinity tolerance of wild Poecilia reticulata (guppy) under laboratory conditions

The present study was carried out to investigate the salinity tolerance of Poecilia reticulata (guppy) occurring in Sri Jayewardenepura canal system, in order to assess its suitability to control brackishwater breeding mosquito species such as Aedes sp. Guppies were exposed to different salinity levels directly and gradually under laboratory conditions in glass tanks. To test the direct effect of salinity, 9 different salinities (ranging from 0ppt as a control to35ppt) prepared in three replicates, each stocked with 10 fish were kept for a period of 12 weeks. To test the effect of gradual increase of salinity, three tanks containing freshwater were stocked with 10 fish in each and a gradual increase of salinity (by 5ppt once in 2 weeks up to 38ppt) was done. Mortality rates, growth (total length) and the breeding of fish were recorded in both experiments at regular time intervals. When the fish were exposed to different salinities directly, fish started dying at 10 ppt (10% mortality) after the fourth day of exposure and this mortality rate was observed in 10-20ppt salinity ranges within the 12 weeks (0% mortality was recorded in control tank).  50% survival was shown at 28ppt salinity level at the end of the 12 th week. Beyond 28ppt, there was 100% mortality. Fish showed an average of 5-7 mm growth in salinity levels up to 28ppt but they were not significantly different (p=0.886). Breeding was seen in salinity levels up to 20ppt producing 7-15 fry/female and the fry also could tolerate the salinity levels they were bred into with a mortality level of 20-30% within the study period. Contrast to the above results, the fish when exposed to gradual increasing of salinity levels could survive up to 38ppt with only a 20% mortality rate. They showed a length increase between 1-5mm, in all salinity levels but they were not significantly different (p=0.330). In gradually increasing salinity levels, fish were seen breeding even at 35 ppt (7-10 fry/female) with 10-20 % mortality rate. The results show that the guppy can tolerate and survive in salinity levels up to 28ppt when they are directly introduced from the wild and they can tolerate up to a level of 38ppt salinity if the salinity levels increase gradually. They can grow under saline conditions and breed successfully and the fry can survive well in the saline environment. This makes wild caught P. reticulata (guppy) a potential candidate that can be used as a bio-control agent in the control of species such as Aedes sp-mosquito that breed in brackish water environments of Sri Lanka.                                   KEYWORDS: Gappy,Salinity,Aedes sp,Bio-contro

Effect of selected water quality parameters on the prevalence of Poecilia reticulata (Guppy) population in Sri Jayewardenepura canal system

Present study was carried out to determine the effect of some selected water quality parameters on the prevalence of P. reticulata(Guppy)  population in Sri Jayewardenepura canal system. Fish and water samples were collected in six locations from January 2016 to December 2017. Dissolved Oxygen (DO), Biochemical Oxygen Demand (BOD), Electrical Conductivity (EC), Hardness, Alkalinity, Nitrate-Nitrogen (N-NO3-), Orthophosphate (PO4 3-) ,Temperature, pH ,Water velocity and depths in each sites were measured. Total lengths and weights of specimens (n-520) were measured and relative densities (RD) were calculated. Guppy was the most abundant species in all sites and high densities were recorded at slow moving and stagnated shallow waters.  Results revealed that all the sites were subjected to moderate or high pollution and most conspicuous were the low DO (0.83±0.12-4.84±0.76 mg/l) and high BOD (1.78±0.4-9.10±0.6 mg/l) levels. Polluted water quality of studied locations were further confirmed by high values for (N-NO3-) (3.9±0.8-11.5±1.2 mg/l), PO43- (1.9±0.4-3.9±0.3mg/l) and EC (257.6±12.8-801.6±42.0µS/cm). Other recorded water quality parameters pH (6.6±0.2–8.3±0.4), Temperature (29.9±1.1-8.3±0.4), Hardness (80.0±10.4- 113.3±13.1 mg/l) and Alkalinity (70.7±11.5-179.6±20.4 mg/l) are in suitable range of fish and aquatic life.  Despite the fact that the locations were polluted, RD of guppy (32.7±27.7-85.7±22.9) were considerable in all locations. The study showed that the RD of guppy significantly varied with some of the water quality parameters; decreasing with increasing DO, N-NO3- ,PO43- levels (P≤0.05) as well as with water depths and velocity of water and increasing with increasing pH, BOD and alkalinity of water. The study recorded that the average adult sizes of fish were smaller (♀-35 mm, ♂-25 mm) than the previous records (♀-60 mm, ♂- 40 mm) in the present study area. Thus, study concludes that, although the water quality parameters are affecting their growth rates and relative densities. KEYWORDS:     Guppy, Water quality parameters, Polluted water, Fish density &nbsp

Insecticide Susceptibility Status in Aedes albopictus, in Selected Areas in Colombo District

Aedes albopictus (Skuse) is considered as the secondary vector of dengue viral transmission in Sri Lanka. As it is dominated throughout the semi urban and rural areas, the risk of Dengue is known to be maintained by, them. In the absence of anti-viral medication and with limited availability of a commercial vaccine for public health use, vector control remains an effective means for reducing disease morbidity. Knowledge about genetic mutations associated with insecticide resistance (IR) is a prerequisite for developing rapid resistance diagnosis, and the distribution and frequency of IR conferring mutations are important information for making smart vector control decisions. A phenylalanine (F) to cysteine (C) substitution at position 1,534 within the third domain of the voltage gated sodium channel (NaV) is associated with resistance to type I pyrethroids, permethrin and type II pyrethroids, deltamethrin. The purpose of this study was to identify the pyrethroid resistance status and to detect the mutation and its frequency in A. albopictus in selected areas. Eggs were collected from two dengue high risk areas in Colombo district. Samples were reared to adulthood and their resistance status against above insecticides was determined by standard WHO susceptibility bioassays. Both populations assessed by adult bioassays were susceptible to both insecticides. Thirtyinsects from each susceptible population were then genotyped for the F1534C mutation. All the PCR products were separated on 3.5% agarose gel and visualized by UV trans-illumination. Towards both insecticides the overall susceptibility was 100% (≥98% mortality: Susceptible), in all two areas.There was no mortality among control mosquitoes. Nevertheless heterozygotes of approximately 90 and 110 bp, corresponding to alleles 1534 Phe+ and 1534 Cyskdr respectively were observed in susceptible genotypes from semi urban areas in Colombo. A slow frequencies of the 1534 Cyskdr (0.150–Maharagama permethrin susceptible population and 0.050–Rattanapitiya Deltamethrin susceptible population) were found in ourstudy, there is a selection pressure with PY favoring the homozygous kdr in the studied localities. Therefore we suggest that this mutation has been just emerged or reversed to 1534 Phe+ over generations through time.Keywords: Knock-down resistance, Voltage gated sodium channel, Pyrethroid, Aedesalbopictus, Insecticide resistanc

DIFFERENTIAL BIO·ECOLOGY OF ANOPHELINE SIBLING SPECIES: A RELEVANCE TO MALARIA CONTROL IN SRI LANKA

Human intervention by various means has altered natural ecosystems and thus paved wayfor emergence of new vectors and their propagation leading to the spread of malaria worldwide. Vector ecological studies have been undertaken in order to locate risk areas for. malaria and to formulate appropriate strategy for vector control. The prevalence andabundance of mosquito immature stages in various breeding sites reflect the ovipositionreference of females and it can be attributed at inter specific and intraspecific levels.Knowledge, especially at intraspecific level, on bio-ecology of vectors is an essentialprerequisite for formulating effective environmental management interventions for thecontrol of malaria.Morphologically more or less similar but reproductively isolated members of a taxon aretermed sibling species and the taxon are species complex. Sibling species exhibitdifferences with respect to feeding and resting preference, rate of development of resistanceto insecticide, susceptibility to parasites and seasonal prevalence.Among known malaria vectors in Sri Lanka, Anopheles culicifacies, the major vector ofmalaria, An. Subpictus and An. Annularis, the subsidiary vectors arc reported to be existingas species complex in the Indian subcontinent. Studies on the biononiics of the two siblingspecies B am! E of A/I. Culicifacies in Sri Lanka reveal that these two sympatricpopulations arc diverged in certain bio- ecological aspects which are essential forimplementing a successful vector control program. Laboratory infective studies showedthat species E could support the extrinsic cycle of Plasmodium I'ivax and P. falciparum.Studies on the age structure of species Band E revealed that species E has entered theepidemiologically dangerous age. Both siblings showed differential susceptibility toMalathion. Studies on the limnological characterization of larval breeding sites of speciesD and E showed that species E prevailed in variety of breeding habitats, an indication ofhigh level of adaptive variation of species E. Sibling species status of An. Annularis in SriLanka is yet to be revealed. A study on the sibling species of All .• Subpitus from a locality inN0I1h Western province of Sri Lanka revealed that species B is predominant in coastal am!species C in inland areas.

Risk of Aedes aegypti (Linnaeus) Development of Breeding in Polluted Drainage Systems of Urban Cities of Sri Lanka

Dengue Fever (DF) is a major public health problem at present in Sri Lanka. Drastic environmental changes due to rapid and unplanned development, extended rainfall during rainy seasons, and ambient relative humidity and temperature that favor for the breeding of Aedes aegypti (Linnaeus), the main vector of DF are the major factors contributing high incidence of dengue in the country. Dengue control programs mainly target the removal or treatment of fresh water holding artificial or natural breeding containers for controlling Ae. aegypti. Negligence of the polluted water drains during vector control programs has raised concerns and suggested the possibility of Ae. aegypti breeding. The present study was designed and conducted to evaluate the presence of Ae. aegypti in the polluted water drains in some urban areas in Sri Lanka. A preliminary entomological survey was carried out in urban areas from Chilaw to Galle to detect Ae. aegypti larvae. The drains that marked as the positive for Ae. aegypti larvae, were further surveyed for a period of three months from June to August in 2017. Ae. aegypti was recorded in drains containing polluted water in Galle city for the first time in Sri Lanka. From total of 26,949 mosquitoes, predominant species in the preliminary survey was Cx. quinquefasciatus (97.38%) followed by Cx. gelidus (0.98%), Ae. albopictus (0.62%) and Ae. aegypti (0.59%). Other species recorded were Armigeres sp., Cx. hutchinsoni and Ae. vittatus which accounted for only 0.4% of the specimens collected. Ae. aegypti was only recorded in polluted water drains with four other species during further studies carried out in the Galle district. Cx quinquefasciatus (97.2%) was predominately recorded from all the polluted drains sampled with a total of 9594 mosquitoes collected in Galle. This was followed by Ae. aegypti (1.58%) with 156 mosquitoes and Ae. vittatus (0.78%) with 77 mosquitoes. Highly deteriorated water quality characteristics were detected as low dissolved oxygen and high conductivity with visible organic pollutants at every study site. Destruction of breeding grounds during vector control programs and constant stress due to insecticidal control leads to adapt Ae. aegypti to new breeding environments. Therefore, further in-depth studies that are based on behavioral changes in the mosquitoes for selecting oviposition need to be carried out by which the control program can be well targeted to lower the disease burden.Keywords: Aedes aegypti, Polluted water drains, Insecticidal pressur

Nucleotide Sequence and Secondary Structure Variations in ITS2-rDNA Region of the Members of Anopheles culicifacies (Diptera: Culicidae) Species Complex

Anopheles culicifacies, major vector of malaria in Sri Lanka is a five member species complex. Differences of the vector competence of siblings, is still poorly delineated. Therefore, the current study was carried out to observe any relationship of the variations in vector competence to ITS2 nucleotide sequences and secondary structure characteristics of the species complex. DNA was extracted from sibling species B and E, the ITS2 region was amplified and sequenced. Sequences for A, C and D siblings were retrieved from NCBI GenBank. The complex divided into two groups, AD and BCE based on primary and secondary structure of the ITS2 sequences. Secondary structures of all species had three helices where pyrimidine-pyrimidine mismatch in Helix II and a UUUGG motif at 5‘ of Helix III were displayed only for B, C and E. Among five types of loops, interior and exterior loops were more conserved than other loop types. Results showed the major vector sibling E and poor or non vector sibling B shares identical nucleotide sequence and secondary structure. Therefore, ITS2 secondary structure is independent of the vector competence of the sibling species

Temporal Changes Mediated by Sri Lankan Sponge Haliclona (Soestella) sp Crude Extract in in vitro Phagocytic Activity of Wistar Rats’ Peritoneal Macrophages

Temporal changes in the production of secondary metabolites by marine organisms are fully understood. Of all marine organisms, marine sponges are ranked at the top of the hierarchy due to their ability to produce structurally novel natural secondary metabolites with bioactive properties. However, variations in the abundance of bioactive compounds produced by sponges may respond to physical constraints while the habitat spongesise, competition for space, or against fouling may also cause changes in sponge secondary chemistry. Many of these biotic or abiotic factors vary between months, seasons, and years resulting strong temporal patterns in secondary metabolites. The present study thus investigated temporal changes mediated by the crude extract of a Sri Lankan marine sponge species, Haliclona (Soestella) sp, on in vitro phagocytic activity of Wistar rat peritoneal macrophages (PM). Sponge samples were harvested from Unawatuna, Galle, Sri Lanka by scuba diving in 2013 and 2015, extracted with methanol/dichloromethane, followed by filtration through Whatman No 1 filter papers and rotary evaporation. The resultant crude extract of Haliclona (Soestella) sp (HSCE) was dissolved in 5% ethanol and tested for phagocytic capacity on Wistar ratPMs at 2000, 1000, 500, 100, 10 μg/ml concentrations by ex vivo Nitro Blue Tetrazolium assay (NBT).Absorbance was obtained as a measure of NBT dye reduction by PM cells and the phagocytic capacity of these cells was calculated. The previous results, the phagocytic activity of rat PMs evaluated ex vivo was significantly higher at HSCE doses of 10, 100 and 500 μg/ml (>1; P<0.01) than at 1,000 & 2,000 μg/ml doses (<1; P<0.05), indicative of higher stimulation of PM cells by lower doses of the HSCE extracted from sponges collected in 2013. However, a dose dependent increase in phagocytic activity by PM cells was observed by HSCE extracted from sponge samples collected in 2015 (P<0.05), indicative of the higher stimulation of PM cells by higher doses of the HSCE extracted from sponges collected in 2015. Therefore, it is evident that a temporal change had occurred in the secondary metabolites responsible for phagocytic activity produced by Haliclona (Soestella) sp within a periods of two years (from 2013 -2015).Keywords: Temporal change, Haliclona (Soestella sp), Phagocytic activit

Microcystin Contamination and Potential of Microcystin Production in Major Drinking Water Bodies of Sri Lanka

Freshwater cyanobacterial blooms limit the utilization of drinking water due to decreased clarity, bad odor, anoxia in the water column and producing a diverse range of toxins; hepatotoxins, neurotoxins and cytotoxins. Hepatotoxic microcystin-LR (MC-LR) is the most intensively studied cyanotoxin and it is the most common type of cyanotoxin. The present study was based on qualitative and quantitative analysis of cyanotoxin in diverse water bodies in Sri Lanka and thirty one water bodies in different districts of Sri Lanka were selected for qualitative and quantitative analysis of microcystin-LR using PCR and ELISA techniques respectively along with assessment of some other selected physico-chemical water quality parameters. Presence of MCs producing genes, mcy A, mcy B and mcy E in field samples were analysed by PCR using specific primers and quantification of Microcystin-LR was done by Enzyme Link Immuno Sorbent Assay (ELISA) respectively. The results of the present study relvealed that most of the drinking water reservoirs were having acceptable physico-chemical parameters for drinking water and MC-LR contamination was ranged between 5.98 ppb to 0.03 ppb. The highest contamination of MC-LR was recorded in Giradurukotte Reservoir (435ppb) where moderate level of MC-LR was detected in Kurunagala lake (75.6 ppb). PCR results reveled that most of studied water bodies having MCs producing genes, mcy A, mcy B and mcy E except Labugama reservoir, Kalatuwawa reservoir, Rathkinda reservoirs, Kantale podi wewa, Minneriya wewa and Mahaweli river intake. Thus, PCR screening is a cost effective molecular biological tool to detect cyanotoxin contamination status of a water body in adavance and it may facilitate to future forcast for actual potential of cyanotoxins production and give an alarm to take nessaary treatment application for drinking water supply authorities.Keywords: Cyanotoxin, Microcystins, PCR, ELISA, Mcy gene

High Salinity Tolerance of Aedes to Breed in Brackish Waters Around the Negombo Estuary

In the midst of dengue control programs, a high occurrence of dengue and dengue hemorrhagic fever cases around the Negombo estuary is reported at the Centre for Clinical Management of Dengue & Dengue Haemorrhagic Fever, Negombo, during April/May and Sept/Oct 2014. Although preimaginal development of Aedes-dengue vector mosquito is known to exist in fresh water environment for many years, few recent findings have revealed the possibility of Aedes breeding and immature stage development in brackish water conditions. In Negombo estuary-being a partially enclosed coastal body of brackish water, we investigated the possible Aedes mosquito breeding in brackish water in and around the lagoon. Natural breeding sources were examined, and an ovitrap based study was also carried out to investigate the possible breeding, immature stage development, hatching and adult emergence in brackish water environment. The study revealed the oviposition preference (0-22 ppt), egg hatching (0-6 ppt in field conditions and 0-6 ppt in laboratory conditions for Ae.aegypti; 0-14 ppt in field conditions and 0-18 ppt in laboratory conditions for Ae. albopictus) and preimaginal development (0-6 ppt in field conditions and 0-6 ppt in laboratory conditions for Ae.aegypti; 0-14 ppt in field conditions and 0-14 ppt in laboratory conditions for Ae.albopictus) of Aedes mosquitoes. Further, the study reported the natural prevalence of preimaginal stages of Ae.albopictus in brackish water collections (2-14 ppt) around the estuary. The study confirmed the brackish water breeding of Aedes mosquitoes around the Negombo estuary. Higher salinity tolerance, compared to brackish water collections of the Northern coast, Sri Lanka was revealed. Negombo, being a fishing village and having a high population density, the study identifies the potential risk of dengue transmission in the area by providing breeding grounds for vector mosquitoes and possibly explains the high occurrence of dengue incidence around the lagoon. Further, the study suggests incorporating larvivorus fish and removing trapped receptacles in the marshy environments to intensify the control programs.Keywords: Salinity tolerance, Aedes, Negombo lagoo

Anopheles culicifacies complex: Geographical distribution of sibling species and existing methods for their identification

Meaningful epidemiological studies and effective vector control programmes depend on efficientmethods for differentiating among major vector, poor vector and non-vector of anophelinespecies complexes. Anopheles culicifacies, has a wide distribution in India, extending toEthiopia, Yemen, Iran, Afghanistan and Pakistan in the West and Bangladesh, Myanmar,Thailand, Laos and Vietnam in the East. It is also found in Nepal and Southern China to theNorth and extends to Sri Lanka in the South. This species exist as a complex of five siblingspecies provisionally designated as A, B, C, D and E. Species A and B are predominantin Northern and Southern part of India whereas species B is recorded all over the Indiansubcontinent. Species B and E are found in Sri Lanka to date. Various methods and techniqueshave been used for identifying sibling species ranging from crossing experiments cytogenetics,isoenzymes, hydrocarbon profile, DNA probes, rDNA-PCR, mt DNA-PC'R and RAPD-PCR.Studies of the suitability among different methods delineating the complex of An. culicifacies,the major malaria vector, mainly in the Indian subcontinent and in Sri Lanka have been discussedhere.Key words : Anopheles culicifacies, mosquito contro