Effect of entomopathogenic fungus, Beauveria bassiana on larvae of three species of mosquitoes

Beauveria bassiana, an entomopathogenic fungus, was evaluated for its potential against second and third instar larvae of Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti. Conidiospores of this fungus were effective in causing infection leading to mortality of different larval instars. Larvae of Cx. quinquefasciatus were more susceptible to infection than An. stephensi and the second instar larvae of these two species were more susceptible than third instar larvae. Larvae of Ae. aegypti were resistant to infection by B. bassiana.     

Laboratory evaluation of Mesocyclops annulatus (Wierzejski, 1892) (Copepoda: Cyclopidea) as a predator of container-breeding mosquitoes in Argentina

In laboratory bioassays we tested the predatory capacity of the copepod Mesocyclops annulatus on Aedes aegypti and Culex pipiens larvae. A single adult female of M. annulatus caused 51.6% and 52.3% mortality of 50 first instar larvae of Ae. aegypti and Cx. pipiens respectively, in a 72 h test period. When alternative food was added to the containers, mortality rates declined to 16% and 10.3% for Ae. aegypti and Cx. pipiens respectively. When 50 first instar larvae of each of the two mosquito species tested were placed together with a single adult female of M. annulatus, mortality rates were 75.5% for Ae. aegypti larvae and 23.5% for Cx. pipiens larvae in a three day test period. Different density of adult females of M. annulatus ranged from 5 to 25 females produced mortality rates of Ae. aegypti first instar larvae from 50% to 100% respectively. When a single adult female of M. annulatus was exposed to an increasing number of first-instar Ae. aegypti larvae ranging from 10 to 100, 100% mortality was recorded from 1 to 25 larvae, then mortality declined to 30% with 100 larvae. The average larvae killed per 24 h period by a single copepod were 29.     

Biochemical and cytoimmunological evidence for the control of Aedes aegypti larval trypsin with Aea-TMOF

Trypsin and chymotrypsin-like enzymes were detected in the gut of Aedes aegypti in the four larval instar and pupal developmental stages. Although overall the amount of trypsin synthesized in the larval gut was 2-fold higher than chymotrypsin, both enzymes are important in food digestion. Feeding Aea-Trypsin Modulating Oostatic Factor (TMOF) to Ae. aegypti and Culex quinquefasciatus larvae inhibited trypsin biosynthesis in the larval gut, stunted larval growth and development, and caused mortality. Aea-TMOF induced mortality in Ae. aegypti, Cx. quinquefasciatus, Culex nigripalpus, Anopheles quadrimaculatus, and Aedes taeniorhynchus larvae, indicating that many mosquito species have a TMOF-like hormone. The differences in potency of TMOF on different mosquito species suggest that analogues in other species are similar but may differ in amino acid sequence or are transported differently through the gut. Feeding of 29 different Aea-TMOF analogues to mosquito larvae indicated that full biological activity of the hormone is achieved with the tetrapeptide YDPA. Using cytoimmunochemical analysis, intrinsic TMOF was localized to ganglia of the central nervous system in larvae and male and female Ae. aegypti adults. The subesophageal, thoracic, and abdominal ganglia of both larval and adult mosquitoes contained immunoreactive cells. Immunoreactive cells were absent in the corpus cardiacum of newly molted 4th instar larvae but were found in late 4th instar larvae. In both males and females, the intrinsic neurosecretory cells of the corpus cardiacum were filled with densely stained immunoreactive material. These results indicate that TMOF-immunoreactive material is synthesized in sugar-fed male and female adults and larvae by the central nervous system cells.     

Physiological and morphological aspects of Aedes aegypti developing larvae: effects of the chitin synthesis inhibitor novaluron

Population control of the dengue vector mosquito, Aedes aegypti, is difficult due to many reasons, one being the development of resistance to neurotoxic insecticides employed. The biosynthesis of chitin, a major constituent of insect cuticle, is a novel target for population control. Novaluron is a benzoylphenylurea (BPU) that acts as a chitin synthesis inhibitor, already used against mosquitoes. However, information regarding BPU effects on immature mosquito stages and physiological parameters related with mosquito larval development are scarce. A set of physiological parameters were recorded in control developing larvae and novaluron was administered continuously to Ae. aegypti larvae, since early third instar. Larval instar period duration was recorded from third instar until pupation. Chitin content was measured during third and fourth instars. Fourth instars were processed histochemically at the mesothorax region, stained with hematoxylin and eosin (HE) for assessment of internal tissues, and labeled with WGA-FITC to reveal chitinized structures. In control larvae: i) there is a chitin content increase during both third and fourth instars where late third instars contain more chitin than early fourth instars; ii) thoracic organs and a continuous cuticle, closely associated with the underlying epidermis were observed; iii) chitin was continuously present throughout integument cuticle. Novaluron treatment inhibited adult emergence, induced immature mortality, altered adult sex ratio and caused delay in larval development. Moreover, novaluron: i) significantly affected chitin content during larval development; ii) induced a discontinuous and altered cuticle in some regions while epidermis was often thinner or missing; iii) rendered chitin cuticle presence discontinuous and less evident. In both control and novaluron larvae, chitin was present in the peritrophic matrix. This study showed quantitatively and qualitatively evidences of novaluron effects on Ae. aegypti larval development. To our knowledge, this is the first report describing histological alterations produced by a BPU in immature vector mosquitoes.     

A laboratory investigation of mosquito larval predation by Toxorhynchites moctezuma on Aedes aegypti

1. Functional responses of predatory Toxorhynchites moctezuma (Dyar & Knab) larvae feeding on Aedes aegypti (L.) larvae (Diptera: Culicidae) were found to be type II of Holling (1959) and Rogers (1972). 2. Estimates of searching rate were generally higher for later instar predators. The search rate of second instar predators declined as prey instar increased, but fourth instar Tx. moctezuma had the highest search rate for second instar Ae. aegypti. 3. Prey handling times were higher for early instar predators and late instar prey. 4. When presented with mixtures of two instars of Ae. aegypti, second instar Tx. moctezuma showed frequency independent selectivity for the early instars, whereas fourth instar predators showed frequency independent selectivity for the late instars of Ae. aegypti. There was no evidence of frequency dependent predation. Preferences appeared to be transitive. 5. Extended random predator equations, using parameters derived from the functional responses, did not adequately describe the outcome of predation in the prey mixture experiment, even when the possibility of optimal switching behaviour was accounted for.     

Laboratory evaluation of Alsystin, a chitin synthesis inhibitor, against Culex pipiens pipiens L. (Dip., Culicidae): effects on development and cuticle secretion

Alsystin, a benzoylphenylurea derivative, was evaluated on Culex pipiens pipiens L. Treatment was made on newly third- and fourth-instar larvae for 24 h. Mortality occurred at various developmental stages following treatment. However, death as larvae was relatively important compared with the mortality recorded for the other later developmental stages. Treatment resulted in a dose-dependent reduction in adult emergence. Moreover, treatment caused morphological aberrations depending upon the importance of larval–adult transformation and significantly increased the duration of both third and fourth larval instars. A histological study conducted on fourth instar larval integument, revealed that Alsystin delayed the ecdysis and significantly reduced the thickness of both larval and pupal cuticles secreted compared with controls. Moreover, the apolysis that occurred at day 4 in controls was not affected by treatment.     

Effects of temperature, pH and salinity on the infection of Leptolegnia chapmanii Seymour (Peronosporomycetes) in mosquito larvae

The effects of temperature, pH, and NaCl concentrations on the infectivity of zoospores of Leptolegnia chapmanii (Argentine isolate) were determined for Aedes aegypti and Culex pipiens under laboratory conditions. Zoospores of L. chapmanii were infectious at temperatures between 10 and 35 degrees C but not at 5 or 40 degrees C. At the permissive temperatures, mortality rates in young instars were much higher than in older instars and larvae of Ae. aegypti were more susceptible to L. chapmanii than larvae of Cx. pipiens. At 25 degrees C, Ae. aegypti larvae challenged with L. chapmanii zoospores resulted in 100% infection at pH levels ranging from 4 to 10. Larvae of Cx. pipiens exposed to similar pH and zoospore concentrations resulted in increasing mortality rates from 62% to 99% at pH 4 to 7, respectively, and then decreased to 71% at pH 10. Aedes aegypti larvae exposed to L. chapmanii zoospores in NaCl concentrations ranging from 0 to 7 parts per thousand (ppt) at 25 degrees C resulted in 100% mortality while mortality rates for Cx. pipiens decreases from 96% in distilled water to 31.5% in water with 6 ppt NaCl. Control Cx. pipiens larvae died when exposed at a NaCl concentration of 7 ppt. Vegetative growth of L. chapmanii was negatively affected by NaCl concentrations. These results have demonstrated that the Argentinean isolate of L. chapmanii tolerated a wide range of temperatures, pH, and salinity, suggesting that it has the potential to adapt to a wide variety of mosquito habitats.     

Evaluation of some adhesives for collecting Musca domestica and Chrysomya megacephala adults or mosquito larvae in sticky traps

1. Seven types of water-insoluble adhesives were evaluated in sticky traps for collecting adults of Musca domestica L. and Chrysomya megacephala (Fabricius) or mosquito larvae (Aedes aegypti (L.) and Culex quinquefasciatus Say). 2. Adhesive viscosity affected the tackiness of the glues and this determined their trapping efficiency in air or water. 3. From the 'Hyvis' range of adhesives tested, 'Hyvis 200' was most effective for trapping adult flies. 4. With 24 h exposure to fourth instar Ae.aegypti larvae in tapwater, submerged plates coated with 'Hyvis 10', 'Hyvis 30' or 'Hyvis 200' formulations trapped the majority of larvae. In polluted water the highest rates of trapping were 17.3% of Ae.aegypti and 18.7% of Cx quinquefasciatus with 'Hyvis 200'. Floating traps were consistently less productive than submerged traps under laboratory conditions. 5. In a heavily polluted natural breeding-site of Cx quinquefasciatus, floating traps were more productive than submerged sticky traps with four of seven adhesives tested, the most efficient being 'Hyvis 200' (4.2 mosquitoes per hour) and Hyvis:polyethylene 90:10 (4.5/h). Despite the relative inefficiency of aquatic traps, emergent adults, pupae and second to fourth instars of larvae were collected quickly from the habitat.     

The effects of pH,phenol, and sodium chloride on survival and caloric,lipid, and nitrogen content of a laboratory population of chironomus attenuatus (Walk.)

Continuous flow, laboratory microcosms were used to measure the effects of pH, phenol, and NaCl on the survival of the life stages of Chironomus attenuatus, the caloric content of third and fourth instar larvae and adults, the lipid and protein-nitrogen content of fourth instar larvae, and the interaction among the various responses. The metabolism of phenol was studied using uniformly labeled phenol-C¹⁴. pH had a significant effect on the survival of all life stages of C. attenuatus. Survival was higher for all larval instars at pH 7.2 while adult emergence was higher at pH 6.2. Increasing phenol levels resulted in a nearly linear increase in caloric content. Sodium chloride affected the lipid content of fourth instar larvae. The lipid content was higher with NaCl present in the media than without NaCl. Interaction had a significant effect on all responses except survival of third and fourth instar larvae. Phenol-C¹⁴ was metabolized by bacteria, but not by C. attenuatus.     

Selective and frequency dependent predation of aquatic mosquito predator Diplonychus indicus Venkatesan & Rao (Hemiptera: Belostomatidae) on immature stages of three mosquito species

Frequency dependent mosquito larval size (II and IV instars) and species selection by the water bug Diplonychus indicus against three mosquito species Culex quinquefasciatus, Aedes aegypti and Anopheles stephensi was studied in the laboratory. The different frequencies used for each species selection were 20:30:50, 30:50:20, 50:20:30, 25:35:40, 35:40:25 and 40:25:35 of fourth instars of the respective three prey species. All nymphal water bugs (I–V instars) selected IV instar mosquito larvae and the mean proportion of late (larger) larvae eaten by the predator instars was significantly higher than the mean proportion of early (smaller) larvae eaten (F= 2.28; P < 0.001). In all six ratios used to determine the frequency dependent mosquito species selection, all the stages of the water bug selected Ae. aegypti over the other two species (F= 452.43; P < 0.001). The mean number of mosquito larvae eaten increased as its density increased based on various ratios of larvae offered. The study indicated that the predatory efficiency of D. indicus was high when Ae. aegypti was offered as prey, suggesting the utility of this mosquito predator in the control of dengue vectors.     

Mosquito larval consumption of toxic arborescent leaf-litter,and its biocontrol potential

Previously we described the mosquito larvicidal properties of decomposed leaf-litter from deciduous trees, especially the alder Alnus glutinosa (L) Gaertn., due to toxic polyphenols and other secondary compounds. To further examine the biocontrol potential of toxic leaf-litter for mosquito control, feeding rates of third-instar mosquito larvae were assessed for examples of three genera: Anopheles stephensi Liston, Aedes aegypti (L) and Culex pipiens L. (Diptera: Culicidae). When immersed in a suspension of non-toxic leaf-litter particles (approximately 0.4 mm), pre-starved larvae of all three species ingested sufficient material in 30 min to fill the anterior gut lumen (thorax plus two to three abdominal segments). Gut filling peaked after 1-2 h ingestion time, filling the intestine up to six to seven abdominal segments for Ae. aegypti, but maxima of five abdominal segments for Cx. pipiens and An. stephensi. Using three methods to quantify consumption of three materials by third-instar larvae of Ae. aegypti, the average amount of leaf-litter (non-toxic 0.4 mm particles) ingested during 3 h was determined as approximately 20 microg/larva (by dry weight and by lignin spectrophotometric assay). Consumption of humine (approximately 100 microm particles extracted from leaf-litter) during 3 h was approximately 80 microg/larva for Ae. aegypti, but only approximately 30 microg/larva for Cx. pipiens and 15 microg/larva for An. stephensi, with good concordance of determinations by dry weight and by radiometric assay. Cellulose consumption by Ae. aegypti was intermediate: approximately 40 microg/larva determined by radiometric assay. Apparent differences between the amounts of these materials ingested by Ae. aegypti larvae (humine four-fold, cellulose two-fold more than leaf-litter) may be attributed to contrasts in palatability (perhaps related to particle size or form), rather than technical discrepancies, because there was good concordance between results of both methods used to determine the amounts of humine and leaf-litter ingested. Bioassays of toxic leaf-litter (decomposed 10 months) with 4-h exposure period (ingestion time) ranked the order of sensitivity: Ae. aegypti (LC50 < 0.03 g/L) > An. stephensi (LC50 = 0.35 g/L) > Cx. pipiens (LC20 > 0.4 g/L). When immersed in the high concentration of 0.5 g/L toxic leaf-litter (0.4 mm particles), as little as 15-30 min ingestion time (exposure period) was sufficient to kill the majority of larvae of all three species, as soon as the gut lumen was filled for only the first few abdominal segments. Possibilities for mosquito larval control with toxic leaf-litter products and the need for standardized ingestion bioassays of larvicidal particles are discussed.     

Alkalization of larval mosquito midgut and the role of carbonic anhydrase in different species of mosquitoes

We have previously demonstrated the involvement of carbonic anhydrase (CA) in the alkalization mechanism of the Aedes aegypti larval midgut. In this study, we used Hansson's histochemistry to examine the distribution of the enzyme in the midgut of six different species of mosquito larvae (Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, Culex nigripalpus, Ochlerotatus taeniorhynchus, Anopheles quadrimaculatus). Additionally, we quantitated CA content in the gastric caeca, anterior and posterior midgut of fourth instar larvae from these species using the 18O isotope exchange method coupled to mass spectrometry. We also tested the effect of CA inhibitors such as methazolamide and acetazolamide in the alkalization of the midgut for these species. Our results indicate that CA is present in the larval midgut of the species studied and that it appears to be associated with the posterior midgut and gastric caeca in some species and with the anterior midgut in others. CA inhibitors appear to have a profound effect on the alkalization mechanism of the midgut with lethal consequences for most of the species tested.     

Role of the CryIVD polypeptide in the overall toxicity of Bacillus thuringiensis subsp. israelensis.

The gene encoding the CryIVD protein of B. thuringiensis subsp. israelensis crystals was disrupted by in vivo recombination. The toxicity of the CryIVD protein-free inclusions was similar to that of the wild-type crystals on Anopheles stephensi larvae but was half the wild-type toxicity on Culex pipiens and Aedes aegypti larvae.     

Laboratory experiments on factors affecting oviposition site selection in Toxorhynchites amboinensis (Diptera: Culicidae), with a report on the occurrence of egg cannibalism

Laboratory experiments tested the effects of water quality and the presence of conspecific and heterospecific immatures on oviposition by Toxorhynchites amboinensis (Doleschall). The females showed a highly significant preference for oak leaf infusion water over distilled water. When twenty starved third and fourth instar Tx.amboinensis larvae were present in the water, substantially fewer eggs were counted from pots containing these conspecifics, than from controls in which no larvae were present. Numbers of eggs from pots containing starved second instar larvae did not differ significantly from controls. Observations of larval behaviour while oviposition was occurring suggested that egg numbers were reduced in containers because of egg cannibalism with third and fourth instar larvae, and not because the larvae caused a deterrent effect. Subsequent experiments confirming the occurrence of substantial egg cannibalism by third and especially fourth instar larvae are described. As with larvae, the presence of Tx.amboinensis pupae in the water had little effect on oviposition. If placed in the water 24 h prior to test, pupae very slightly enhanced its attractiveness, but if introduced immediately before test there appeared to be a slight deterrent effect. With heterospecific larvae, twenty fourth instar Ae.aegypti larvae introduced into infusion water 24 h before test rendered the water slightly attractive, while water in which Ae.aegypti larvae had been reared for 48 h proved highly repellent.     

Comparative delta-endotoxins of Bacillus thuringiensis against mosquito vectors (Aedes aegypti and Culex pipiens)

Pure crystals of seven Bacillus thuringiensis field isolates from the Lower Silesia region (Poland) were tested against larvae of Aedes aegypti L. and Culex pipiens L. (Culicidae, Diptera). The crystals of OpQ3 phylloplane isolate (belonging to the first biochemical type of B. thuringiensis subsp. japonensis, yoso, jinghongiensis) killed from 68 +/- 7% to 84 +/- 7% of the fourth instar larvae of A. aegypti. The crystals of two other strains (KpF3 and KpC1) of this group caused mortality between 3 +/- 2% and 70 +/- 7%. The LC50 ranged from 3.2 +/- 0.4 to 34.1 +/- 4.8 microg/ml. The effect of B. thulringiensis wratislaviensis H-47 crystals was the lowest with larval mortality from 0% to 17 +/- 3%. No significant (0%-37 +/- 6%) effect of B. thuringiensis crystals on the larvae of C. pipiens was observed. Our results show that the delta-endotoxins of B. thuringiensis act very specifically.     

The naturally derived insecticide spinosad is highly toxic to Aedes and Anopheles mosquito larvae

Spinosad is a naturally derived biorational insecticide with an environmentally favourable toxicity profile, so we investigated its potency against mosquito larvae (Diptera: Culicidae). By laboratory bioassays of a suspension concentrate formulation of spinosad (Tracer), the 24 h lethal concentration (LC50) against Aedes aegypti (L.) third and fourth instars was estimated at 0.025 p.p.m. following logit regression. The concentration-mortality response of third- and fourth-instar Anopheles albimanus Weidemann did not conform to a logit model. The LC50 value of spinosad in Anopheles albimanus was 0.024 p.p.m. by quadratic linear regression. A field trial in southern Mexico demonstrated that spinosad 1 p.p.m. compared with the standard temephos (Abate) 1% granules 100 g/m3 water prevented Ae. aegypti breeding in plastic containers of water for 8 weeks; at 10 p.p.m. spinosad prevented breeding for > 22 weeks. In another field trial, spinosad at 5 p.p.m. and temephos both completely eliminated reproduction of Ae. aegypti for 13 weeks. In contrast, the bacterial insecticide Bacillus thuringiensis var. israelensis (Bti, Vectobac) AS) performed poorly with just 2 weeks of complete inhibition of Ae. aegypti breeding. Spinosad also effectively prevented breeding of Culex mosquitoes and chironomids in both trials to a degree similar to that of temephos. We conclude that spinosad merits evaluation as a replacement for organophosphate or Bti treatment of domestic water tanks in Mesoamerica. We also predict that spinosad is likely to be an effective larvicide for treatment of mosquito breeding sites.     

Effects of Toxorhynchites moctezuma larval predation on Aedes aegypti populations: experimental evaluation

Abstract. Predatory larvae of the mosquito Toxorhynchites moctezuma were used experimentally to control a standing crop of larvae of the dengue vector mosquito Ae.aegypti. Each week, fifty Ae.aegypti first instar larvae were introduced to each of five water-filled drums (220 litres) of the type commonly used for domestic water storage in Caribbean dwellings. At the beginning of the fourth week, a certain number (0, 1, 2, 5 or 10) of first instar Tx.moctezuma larvae were introduced to each drum and the daily yield of Ae.aegypti adults from each drum was monitored thereafter. The experiment was repeated three times. With only one or two Tx.moctezuma larvae, predation on Ae.aegypti larvae stopped the output of Ae.aegypti adults for 1 week. Five or ten Tx. moctezuma prevented any Ae.aegypti emergence for up to 16 weeks. Cannibalism among Tx. moctezuma larvae was seldom observed and appeared not to be a hindrance in using this species against Ae.aegypti. Thus Tx.moctezuma is regarded as a good candidate for the biological control of Ae.aegypti by augmentative releases.     

Intraspecific competition amongst larval Aedes aegypti: food exploitation or chemical interference?

Abstract. 1. Competition within and between the larval instars of the yellow fever mosquito, Aedes aegypti , can be measured by its effect on stage duration. In a series of laboratory experiments the relative importance of chemical interference and food exploitation in mediating competition between first and fourth instar larvae was investigated.
2. In contrast to the results of three previous studies, I found no evidence that a chemical growth retardant played any part in greatly prolonging the stage durations of larvae in both age classes.
3. When competition between the two age classes became important, the relative increase in stage duration wasgreater for first instar larvae than for fourth instar larvae. This result can be reproduced with Gilpin & McClelland's (1979) model of competition by food exploitation alone, providing the range of available food particle size is assumed to be an increasing function of age. An additional, but less important, refinement makes the conversion efficiency of food into larval biomass a decreasing function of age.     

Laboratory evaluation of three mosquito pathogenic strains of Bacillus sphaericus isolated in Egypt

Three strains of Bacillus sphaericus H-5a5b designated Ghar. 1 & 10, Ghar. 2 & 20, and Ghar. 3 & 30 were tested for growth, virulence, and larvicidal activity against Culex pipiens in the laboratory. Incubation temperature was positively correlated (r = 0.91) to the rate of bacterial growth (strain Ghar. 2 & 20). All three strains retained their virulence through 25 successive transfers on nutrient agar. Acetone powder preparations showed high larvicidal activity against C. pipiens, although second instar larvae were more susceptible than fourth instars to all three strains. The most active strain was Ghar. 2 & 20 with LC50 values of 0.51 mg/liter (second instar) and 1.62 mg/liter (fourth instar) after 48 hr of exposure. Mortality rates in fourth instar larvae exposed to an acetone powder form of strain Ghar. 2 & 20 were significantly greater at higher than at lower temperatures.     

Larvicidal activity of Tagetes patula essential oil against three mosquito species

Larvicidal activity of Tagetes patula essential oil was tested against the fourth instar larvae of Aedes aegypti, Anopheles stephensi, and Culex quinquefaciatus. Five different concentrations of essential oil were studied and the results were compared with that of synthetic insecticide, malathion. A. aegypti (LC(50) 13.57, LC(90) 37.91) was most susceptible followed by An. stephensi (LC(50) 12.08, LC(90) 57.62) and C. quinquefaciatus (LC(50) 22.33, LC(90) 71.89).