Feeding behavior of Simulium vittatum larvae in response to various suspended materials: implications for control using biological larvicides

Biologically based larvicides used to suppress nuisance populations of black flies (Diptera: Simuliidae) require that the larvae ingest the larvicide for mortality to occur. Materials present in the habitat that alter larval feeding behavior may reduce the effectiveness of the larvicide. This study was conducted to determine the effects that commonly occurring materials in larval habitats have on Simulium vittatum Zetterstedt feeding behavior. Larvae were exposed to 50 p.p.m. of kaolinite clay and 4 p.p.m. of purified diatom frustules. Larvae were also exposed to 10 000 cells or colonies per ml of viable green algae, Chlorella vulgaris Pratt and Scenedesmus quadricauda (Turp) Bréb, and the diatom Cyclotella meneghiniana Kützing to determine the effects that these materials have on larval feeding behavior. Individual larvae were recorded using a digital camcorder to determine the average number of times the larvae extended and retracted their cephalic fans per min (flick rate) in each medium. Following exposure to the selected material, neon red particles (NRP) (DayGlo) were added to the larval medium for 20 min. A spectrophotometric analysis was conducted to determine the average quantity of NRP consumed by larvae per min in each medium. The flick rate and quantity of NRP consumed by larvae was not significantly affected by clay material or viable Chlorella cells. When Scenedesmus colonies or Cyclotella cells were present in the larval medium, larval flick rate and NRP ingestion was significantly reduced. The presence of diatom frustules in the medium resulted in the most severe reduction in larval flick rate and NRP ingestion. The results suggest that larval flick rate can change depending on environmental conditions, and that suppression programs using biologically based larvicides may benefit from investigating the potential correlation between algal counts and treatment efficacy.     

Factors influencing the toxicity of xenobiotics against larval mosquitoes

In order to examine the factors influencing xenobiotic toxicity against larval mosquitoes, the larvicidal performances of two conventional insecticides (temephos and Bacillus thuringiensis var. israelensis: Bti) and a new potential phyto-insecticide (decomposed leaf litter) were compared under different conditions against three detritivorous larval mosquito types. Bioassays performed under standard conditions indicated differential tolerance levels according to the xenobiotic and the larval type. Bioassays performed under different conditions of xenobiotic dose and geometry of the water column indicated differential effects of those parameters on mortality rates. This allowed us to distinguish the performances of temephos versus those of Bti and leaf litter. These toxicological performances were examined as indicators for analysis of xenobiotic bioavailability for mosquito larvae in environmental water, and also for their comparative interest in field mosquito control.     

Ovipositional and ovicidal effects of the microbial agent Bacillus thuringiensis israelensis on Culex quinquefasciatus say (Diptera: Culicidae).

The magnitude of oviposition as well as the size, shape and the number of eggs per of egg rafts egg raft were determined after gravid Culex quinquefasciatus Say oviposited on water treated with water dispersible granules (WDG) of Bacillus thuringiensis ssp. israelensis (Bti) and on untreated water. The mean number of eggs/raft was lower in the treated than in the untreated water. Bti concentrations from 0.5 to 2.0mg/L affected the shape of egg rafts and number of eggs in each raft. As the concentration of Bti increased from 0.5 to 2.0 mg/L the shape of egg rafts became more irregular with fewer eggs in each raft. Exposure to Bti at 2- and 26-h reduced the hatching rates, and fewer eggs hatched at 26-h of exposure to Bti. As the concentration of Bti WDG increased from 0.5 to 2.0 mg/L, the hatching rate decreased. Eggs exposed for 2-h to 2.0mg/ L Bti had a hatch of 30% after 24 h, the rate increasing to 57% after 72 h. In contrast, in 26-h exposed eggs to 2.0 mg/L Bti, the hatching rate after 24 h was only 12% and this rate increased to 39% after 72 h. In larvae from eggs exposed for 2 h, the survival rate was 40% at 2.0 mg/L Bti and 87% in untreated controls. In contrast, the survival rates of larvae from 26-h exposed eggs was 91% in controls while it was 30% at 2.0 mg/L Bti. As the concentration of Bti increased from 0.5 to 2.0 mg/ 1 the survival rates of larvae decreased. The combined effects of reductions of egg rafts, low number of eggs per egg raft, and reduced hatching and survival rates could have significant cumulative effects on the yield of adult mosquitoes, and this could result in a greater control potential of this microbial agent.     

球形芽孢杆菌Mtx1蛋白和苏云金杆菌Cyt1Aa晶体蛋白的协同作用

采用常规的生物测定方法确定了纯化的球形芽孢杆菌(Bacillus sphaericus)的缺失信号肽的97kDa营养期杀蚊毒素(Mosquitocidal toxin 1,Mtx1)蛋白和苏云金芽孢杆菌(Bacillus thuringiensis)27.3kDa的Cyt1Aa晶体蛋白对致倦库蚊(Culex quinquefasciatus)幼虫的杀虫活性。结果表明Mtx1和Cyt1Aa不同比例的混合物对致倦库蚊的毒力比单独毒素蛋白高,经统计分析表明两毒素蛋白对目标蚊幼虫具有明显的协同作用。在LC98处理浓度下,Mtx1和Cyt1Aa按3∶1混合的混合物LT50值比单独Mtx1的提前了6.36h。表明Cyt1Aa和Mtx1对致倦库蚊具有协同毒杀作用,提高对目标蚊虫的毒力、缩短半致死时间。该结果为深入研究Mtx1和Cyt1Aa的杀蚊作用方式奠定了基础,同时为其在蚊虫防治中的应用提供了新的思路和方法。     

Combination of Mesocyclops thermocyclopoides and Bacillus thuringiensis var. israelensis: a better approach for the control of Aedes aegypti larvae in water containers.

The efficacy of a local Thai-strain of the copepod, Mesocyclops thermocyclopoides and the larvicide, Bacillus thuringiensis var. israelensis (Bti), used jointly and singly, was studied against Aedes aegypti in water containers. In a laboratory test, copepods alone produced mortality of 98-100% in 1st instar larvae of Ae. aegypti at copepod:larvae ratios ranging from 1:1 to 1:4. In an outdoor field simulated experiment that ran for 16 wk, after a single inoculation, the treatment of copepods and Bti combined yielded the better, more sustainable results than the agents used individually. Numbers of mosquito larvae per sample in the combined treatment were zero during the first 8 wk; larval numbers then increased but were maintained at a very low level for the next 4 wk after which the larval numbers increased moderately but still remained below numbers in the control. Bti alone kept the larvae at the zero level for the first 4 wk after which their numbers increased slightly and were at low levels up to 12 wk. Copepods alone maintained larval numbers at a low level as compared with those of the control. During the course of the experiment the larval numbers in the control were greater than 20 per sample. Statistically significant differences were noted among treatment means (F = 23.083, df = 3/60, P<0.01) over the total period of the study. The number of copepods in the joint treatment was significantly higher than in the copepod alone treatment for the first 8 wk (t = -4.97, df = 14, P<0.01). The density of copepods, however, for the whole 16-wk period was not significantly different in these two treatments (t = -1.51, df = 30, P>0.1).     

Salt-marsh Crustaceans, Gammarus duebeni and Palaemonetes varians as Predators of Mosquito Larvae and Their Reaction to Bacillus thuringiensis subsp. israelensis

The predatory potential of two onmnivorous crustaceans, Gammarus duebeni and Palaemonetes varians, has been examined to investigate their effect as mosquito larval predators. Mature gammarids ate 4-8 Aedes detritus larvae/24 h and palaemonids, 22-30/1 h, often killing larvae when not hungry. The crustaceans were exposed to Bacillus thuringiensis subsp. israelensis (Bti) and to mosquito larvae killed by Bti with no adverse effects, endorsing the safety of this microbial pesticide. Crustacean faecal pellets, collected post-feeding and placed in fresh marsh water, were toxic to mosquito larvae the following day. After placing the crustaceans in fresh salt-marsh water for 6 days, the fresh faecal pellets failed to kill mosquitoes apart from pellets obtained from crustaceans originally fed on the highest concentration of Bti, where there was a 50% kill after 3 days of incubation. Mosquito larvae on salt-marshes are not easy to control with ecologically undesirable toxic chemicals. Encouraging the breeding of predators such as crustaceans, or even their release, could prove to be a useful method of mosquito control to supplement the periodic inundative use of the ecologically acceptable Bti.     

Sustainable control of mosquito larvae in the field by the combined actions of the biological insecticide Bti and natural competitors

Integrated management of mosquitoes is becoming increasingly important, particularly in relation to avoiding recolonization of ponds after larvicide treatment. We conducted for the first time field experiments that involved exposing natural populations of the mosquito species Culex pipiens to: a) application of the biological insecticide Bacillus thuringiensis israelensis (Bti), b) the introduction of natural competitors (a crustacean community composed mainly of Daphnia spp.), or c) a combined treatment that involved both introduction of a crustacean community and the application of Bti. The treatment that involved only the introduction of crustaceans had no significant effect on mosquito larval populations, while treatment with Bti alone caused only a significant reduction in the abundance of mosquito larvae in the short‐term (within 3–10 days after treatment). In contrast, the combined treatment rapidly reduced the abundance of mosquito larvae, which remained low throughout the entire observation period of 28 days. Growth of the introduced crustacean communities was favored by the immediate reduction in the abundance of mosquito larvae following Bti administration, thus preventing recolonization of ponds by mosquito larvae at the late period (days 14–28 after treatment). Both competition and the temporal order of establishment of different species are hence important mechanisms for efficient and sustainable mosquito control.     

Relative tolerance of cirripede larval stages to acute thermal shock: A laboratory study

(1) Meroplankters drawn into once-through cooling circuits of coastal power plants are subjected to transient thermal stress. The effect of such acute thermal shock on the development of barnacle larvae was studied in the laboratory.

(2) The response of the barnacle larvae (naupliar and cyprid stages) to elevated temperature was dependent on exposure time and their stage of development.

(3) Among the stages tested, N-6 larvae showed maximum tolerance. Exposure to 37°C did not affect larval survival, but delayed development of N-2 larva to cypris by one day.

(4) Exposure at 40°C delayed, hastened or did not affect the development time of N-2 and N-4 larvae through cypris, depending on exposure time.

(5) Ten mins exposure at 43°C proved lethal to all larval stages with mortality ranging from 20 to 86%.

(6) Development success of the surviving larvae, measured in terms of cypris yield, showed no significant difference from controls, at temperatures below 40°C.

(7) Settlement activity was significantly affected in only those cyprid larvae which were exposed to 43°C for 10 min.

(8) Results of the present study indicate that thermal stress experienced in the once-through cooling system does not have significant impact on survival and development of the barnacle larvae at temperatures of 37–40°C.     

Crustacean biodiversity as an important factor for mosquito larval control

Newly established ponds, which are highly dynamic systems with changing levels of biological interactions among species, are common larval mosquito habitats. We investigated the impact of crustacean abundance and taxa diversity on mosquito oviposition and larval development. The effects of the biological larvicide Bacillus thuringiensis israelensis (Bti) on mosquito larvae were monitored according to fluctuations in crustacean communities. Populations of the mosquito Culex pipiens colonized artificial ponds that contained crustacean communities at different time points of colonization by crustaceans: 1) ‘no colonization’ (no crustaceans), 2) ‘simultaneous colonization’ by crustaceans and mosquitoes, and 3) ‘head‐start colonization’ by crustaceans (preceding colonization by mosquitoes). All types of ponds were treated with three concentrations of Bti (10, 100, or 1,000 µg/liter). Colonization of all ponds by Cx. pipiens (in terms of oviposition, larval abundance, and larval development) decreased significantly with increasing diversity of crustacean taxa. The total abundance of crustaceans had a minor effect on colonization by Cx. pipiens. The presence of crustaceans increased the sensitivity of Cx. pipiens larvae to Bti treatment by a factor of 10 and delayed the time of recolonization. This effect of Bti was relevant in the short term. In the long term, the presence of Cx. pipiens was determined by crustacean biodiversity.     

Sublethal effects of mosquito larvicides on swimming performance of larvivorous fish Melanotaenia duboulayi (Atheriniformes: Melanotaeniidae)

Laboratory studies were conducted to determine the sublethal effects of exposure to selected larvicides on the critical swimming speed (Ucrit) of crimson-spotted rainbowfish, Melanotaenia duboulayi (Castlenau). This native fish is common throughout southeastern Queensland, and it is increasingly being distributed as a biological control agent of mosquitoes. The selected larvicides included, two organophosphate (OP) compounds (temephos and pirimiphos-methyl), two microbial larvicides (Bacillus thuringiensis spp. israelensis [Bti] de Barjac and Bacillus sphaericus [Bs] Neide), and an insect growth regulator (IGR) (s-methoprene). Exposure to the OP temephos at 10 times the effective field concentration (EFC; 0.33 mg/liter), and OP pirimiphos-methyl at the EFC (0.50 mg/liter), resulted in a significant reduction in the Ucrit of M. duboulayi under controlled conditions. Conversely, exposure to the microbial (Bti and Bs) and IGR (s-methoprene) larvicides at 10 times the EFC had no effect on the Ucrit of M. duboulayi. Accordingly, these products are suitable for integrated pest management programs in Australia.     

Some effects of sub-lethal X-irradiation on reproduction and development in two strains of larval Taenia crassiceps

The generation times were determined for two larval strains of Taenia crassiceps. The aberrant strain (ORF) had a generation time (generation TIME = time from inoculation to initial bud production) of 23 days while for the normal (KBS) strain it was 32 days. Exposure to 10,000 R of X-irradiation of ORF larvae resulted in a significant stimulation of reproductive potential. Reproductive potential of first and second generation progeny of the irradiated ORF larvae paralleled that of non-irradiated ORF cysticerci, indicating an ephemeral effect of radiation. Doses of 10,000 R had no apparent reproductive or morphologic effect on KBS larvae.     

Upper lethal temperature limits of the common furniture beetle Anobium punctatum (Coleoptera: Anobiidae)

The susceptibility of the egg, larval and adult stages of Anobium punctatum De Geer (Coleoptera: Anobiidae) to heat (46–54°C, 25–30% RH) was investigated. The larval stage was found to be most tolerant to heat. Very short exposure (5 min) of the larvae to temperatures of 52°C and above led to 100% mortality of the larvae. Treatment for 1.5 and 1 h at 47° and 48°C, respectively, led to 100% mortality. At 46°C, longer treatment time (2.5 h) was necessary. Exposure to 45°C for 3.5 h did not lead to immediate death of the larvae. The actual mortality is assumed to be greater than the results actually reported, as most of the larvae had not resumed normal activity at the end of the two-week observation period. The egg and adult stages were more sensitive to heat than the larvae; shorter exposure times were sufficient to obtain 100% mortality.     

The effect of tubificids and chironomids on particle redistribution of lake sediment

The effects of chironomid larvae,Chironomus plumosus, and tubificid worms,Limnodrilus spp., on particle redistribution in lake sediment were investigated experimentally using pots containing sediments obtained from Lake Suwa, Japan. The chironomids and tubificids increased the water content of surface sediment. The chironomid larvae had no effect on particle size distribution, while tubificids continuously accumulated small particles on the surface sediment through their selective feeding activity. Particles larger than 0.125 mm were buried at a sediment depth of 6 cm. In Lake Suwa, long diatom frustules, large plant debris and blue-green algal flocs were found to accumulate in the deeper layer of the lake sediment inhabited by tubificids at high density.     

Laboratory and field evaluation of formulated Bacillus thuringiensis var. israelensis as a feed additive and using topical applications for control of Musca domestica (Diptera: Muscidae) larvae in caged-poultry manure

Infestations of house flies, Musca domestica L., are a continual problem around poultry establishments. Acute toxicity of two commercial Bacillus thuringiensis variety israelensis (Bti) formulations (water-dispersible granules and bran formulation) was evaluated against larvae in the laboratory and against natural populations of M. domestica larvae in the field applied in feed to chickens and as topical applications in the poultry houses. Bioassay data showed that susceptibility of M. domestica larvae increased to a given concentration of Bti as the duration of exposure increased. In the laboratory studies, the LC(50) values of Bti for the larvae ranged between 65 and 77.4 μg/ml. In the field, a concentration of 10 g Bti/kg of feed resulted in 90% reduction of larvae at 4 wk after treatment. A higher concentration (2 g/liter) of Bti in spray applications was not significantly more effective than the lower concentration of 1 g/liter. Adding Bti to chicken feed is potentially an efficient measure for the management and control of house flies in caged-poultry facilities.     

Non-larvicidal effects of Bacillus thuringiensis israelensis and Bacillus sphaericus on oviposition and adult mortality of Culex quinquefasciatus Say (Diptera: Culicidae).

Two microbial mosquito larvicides, Bacillus thuringiensis ssp. israelensis (Bti) and Bacillus sphaericus (Bsph), have been shown to be highly effective in controlling mosquito larvae and have been used in larvicidal programs for many years. In exploring other modes of action of these agents, we studied the ovipositional response of Bsph susceptible and resistant Culex quinquefasciatus to aqueous suspensions of Bti and Bsph water dispersible granules (WDG). We quantified the level of mortality of adult mosquitoes caused by exposure to Bti and Bsph suspensions during oviposition. Significantly lower numbers of egg rafts were laid and collected from the treatments than the control regimen. There was an inverse relationship between Bacillus product concentrations and oviposition. As the concentration of Bti or Bsph increased from 0.0 to 2.0 mg/L, treated waters received progressively fewer egg rafts. In addition to the negative effects of Bacillus on oviposition, both male and female adult mosquitoes suffered high mortality on landing and imbibing on Bti and Bsph suspensions, the extent of mortality directly proportional to concentration. These two microbial agents used solely as mosquito larvicides thus have the additional benefits of reducing mosquito oviposition and killing adult mosquitoes, especially gravid females that come in contact with the treated water either for oviposition or drinking. Reducing the number of gravid females may also result in reduced transmission rates of pathogens. The combined effects of reduced oviposition and adult mortality could result in higher control potential of these microbial agents.     

Bioevaluation of Subabul (Leucaena leucocephala) proteinase inhibitors on Helicoverpa armigera

Helicoverpa armigera, a highly polyphagous pest, has a broad host spectrum, causes significant levels of yield loss in many agriculturally important crops. Serine primarily responsible for most of the proteolytic activity in the larval gut of lepidopteron insects. Neonate larvae were reared on artificial diet and chickpea seeds smeared with Subabul Trypsin Inhibitor. Larvae fed with artificial diet showed reduction in larval weight up to 21% (HSTI) and 43% (LSTI). However, larvae fed on seeds showed significant reduction in weight, 52.4% (HSTI) and 60.3% (LSTI), suggesting that the diet also plays a vital role on the effectiveness of the inhibitors on larval growth and development. HSTI and LSTI inhibited the gut proteinases from larvae fed on artificial diet significantly (41.40% and 64.36%) compared to the gut proteinases (27.80% and 38.90%) from larvae fed on chickpea seeds. Seeds smeared with 10,000 TIU resulted in complete mortality of larvae while there was no mortality observed in artificial diet. The results reveal that LSTI is a stronger inhibitor of insect gut proteinases and for larvae fed with poor nutrition in the natural ecosystems, low level expression of inhibitor would be enough to affect the growth and development. Handling editor: Chen-Zhu Wang     

松墨天牛表皮蛋白基因的克隆及表达分析

【目的】本研究旨在探索松墨天牛Monochamus alternatus Hope中昆虫表皮蛋白（ICP）基因在幼虫各组织中和不同发育阶段的时空表达模式。【方法】用cDNA末端快速扩增方法(rapid amplification of cDNA ends, RACE) 克隆了松墨天牛表皮蛋白基因,用实时荧光定量PCR分析了该基因在幼虫体内和不同虫态中的表达。【结果】克隆获得的松墨天牛幼虫表皮蛋白基命名为MoalICP（GenBank 登录号：AGX00998.1）,开放阅读框长408 bp,编码135个氨基酸,推测得到的蛋白的分子量为14.51 kDa。由MoalICP推导的蛋白与桑天牛Apriona germari ICP (AAM66718.1)氨基酸序列一致性为79%;与丽蝇蛹集金小蜂Nasonia vitripennis ICP (NP_001161297.1)、果蝇Drosophila mojavensis ICP (XP_002005461.1)、玉带凤蝶Papilio polytes ICP (BAM18876.1)等19种昆虫表皮蛋白的氨基酸序列一致性在35%~45%之间;在第10-26位氨基酸位处含有一个跨膜片段。MoalICP在幼虫头、体壁、脂肪体、血细胞、中肠和马氏管均有表达;在蛹和成虫中的表达量分别是在幼虫中的44%和161%。【结论】MoalICP与其他昆虫有较高的氨基酸序列一致性。MoalICP在松墨天牛幼虫内广泛表达;在各个发育阶段中,以成虫中的表达量最高。本文为进一步研究松墨天牛表皮蛋白基因的生理功能和松墨天牛的表皮化学奠定基础。     

Quality control of Bacillus thuringiensis ssp. israelensis products based on toxin quantification with monoclonal antibodies

Quality control of Bacillus thuringiensis ssp. israelensis (Bti) products is currently based on international toxic units (ITUs). The potency of products is related to the activity of a standard (IPS-82, Institute Pasteur, Paris) assessed in bioassays using Aedes aegypti as a target host. The procedure is time consuming and costly, often producing variable results. The activity of Bti is based on four different insecticidal crystal proteins (ICPs): Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa. Monoclonal antibodies were produced using IPS-82 for immunisation and an enzyme-linked immunosorbent assay (ELISA) was developed. Antibodies were selected with specificity against Cry11A and Cyt1A. Cry4 specific antibodies could not distinguish between Cry4A and Cry4B. Within five replicate assessments of the three ICPs (in µg mg^-1 ICP protein), an error between 3 and 8% was recorded, whereas a 14% error was obtained comparing seven samples of the same production batch for ITUs mg^-1. The toxicity against A. aegypti expressed in ITUs correlated well with the results of the ELISA (correlation coefficient r Cyt 1=0.79; Cry 11=0.87; Cry 4=0.91) also when related to the sum of all ICPs (r=0.87). The ELISA can reduce efforts to determine Bti quality compared with the labour-intensive and variable ITU bioassay.     

Caging experiment to examine mortality during metamorphosis of coral reef fish larvae

All previous attempts to estimate early postsettlement mortality of coral reef fishes using either caging experiments or disappearance of new recruits have examined fish that had already settled, and therefore did not include the metamorphosis process. Crest nets capture unharmed transparent larvae during their migration from the open ocean to lagoon reefs before metamorphosis. We released these presettlement larvae at night into cages surrounding patch reefs and measured larval survivorship after two nights. This caging experiment involved cages enclosing the natural resident fish fauna, including predators, and others cleared of fish before releasing the larvae. The analyses of variance showed that (1) there was no difference in survivorship between the seven trials, (2) there was a significant difference between cleared and uncleared cages, and (3) there were significant differences between larval species tested. For the seven species that had a significant difference in survivorship between cleared and uncleared cages, average mortality of the larvae was 14% (range 0-26%) in cleared cages and 67% (range 29-76%) in cages with predators. The difference in mortality between species was related to the size of the larvae, as larger species exhibited reduced mortality compared to smaller species. Mortality was related to the abundance of resident fish that could act as predators or competitors. Predation can have a significant impact on the survival of metamorphosing fish larvae on coral reefs.     

A field study of potential ecological costs of resistance by ‘stem ducking’ in tall goldenrod,Solidago altissima

Culex quinquefasciatus Say (Diptera: Culicidae) is an abundant urban mosquito that is the vector of filariasis. Breeding in septic tanks, where there are very high levels of bacterial food, it is likely to have a different reaction to crowding compared with other mosquitoes. To test for the presence and type of crowding effects, four larval densities of C. quinquefasciatus varying from 0.4 to 3.2 larvae ml^?1 of water were reared in tubes. Mortality was found to greatly increase at densities above 0.8 larvae, whereas larval duration increased even above 0.4 larvae ml^?1. Changing the water in the tubes daily gave a small (but significant) response in reducing mortality and larval duration. However, when larvae kept at a low density shared the same water with larvae at high density, there was no chemical influence on their growth rate and mortality. The effect of crowding was primarily due to physical disturbances between larvae. When larvae were kept at a high density in the same volume of water, but in shallow trays with a large surface area and therefore much less contact between them, mortality was the same as for the lowest density. There was still, however, a significant increase in larval duration from 8.6 days at 0.4 larvae ml^?1 to 12.1 days at 3.2 larvae ml^?1. It is therefore concluded that the larvae respond to physical rather than chemical factors by prolonging larval development and having some increase in mortality.