Amino acids in nectar enhance longevity of female Culex quinquefasciatus mosquitoes

Culex mosquitoes feed on a wide range of nectars consisting of mostly carbohydrates and amino acids, however, little is known about the utilization and effects of these different carbohydrates and their accompanying amino acids on longevity. Culex quinquefasciatus larvae were reared on low- and high-quantity food diets to produce adults that were nutritionally representative of wild-caught and laboratory-reared mosquitoes. Emerging adults reared on low- or high-quantity food diets as larvae were then provided Lantana camara nectar mimics containing mixtures of carbohydrates and amino acids to evaluate effects of nectar amino acids on longevity. Carbohydrates (with or without amino acids) were a critical component of the adult diet, and in their absence, adult mosquitoes died within 3-5 days. The nectar mimic that contained both carbohydrates and amino acids did not increase adult longevity of males originating from either poorly or well-fed larvae. However, females receiving adult diets containing both carbohydrates and amino acids lived 5% longer than females fed adult diets with only sugar.     

Pathogenesis of Bacillus sphaericus strain SSII-1 infections in Culex pipiens quinquefasciatus ( = C. pipiens fatigans) larvae

Numbers of viable bacteria in second instar Culex pipiens quinquefasciatus larvae were determined following ingestion of pathogenic strain SSII-1 and nonpathogenic Bacillus sphaericus. Numbers of nonpathogenic B. sphaericus recovered from larvae declined rapidly after cessation of feeding, as did numbers of pathogenic SSII-1 cells fed at LD₂₀ dosage. When pathogenic cells were fed at LD₇₀ dosage, the number of B. sphaericus in larvae increased following initial decline. When chloroformtreated SSII-1 cultures, in which all bacteria except spores were dead, were fed at LD₁₀ and LD₉₈ dosages, no viable B. sphaericus were recovered from larvae. In all SSII-1 treatments, other bacterial flora multiplied rapidly in larvae following onset of mortality; the role of this multiplication in the pathogenesis was not determined. It is proposed that toxic material is released when SSII-1 cells are digested and that multiplication of B. sphaericus in the larval gut is not essential in the pathogenesis. There appears to be no difference in the pathogenesis when differing numbers of B. sphaericus. i.e., LD_10–20 or LD_70–98 dosages, are ingested. Possible nature of the toxic material is discussed.     

Consequences of the introduction of the planarian Girardia anceps (Tricladida: Dugesiidae) in artificial containers with larvae of the mosquitoes Aedes aegypti and Culex pipiens (Diptera: Culicidae) from Argentina

Aedes aegypti and Culex pipiens are container-dwelling mosquito species that are vectors of important diseases to man, such as dengue and lymphatic filariasis, respectively. Predators of these pests are an interesting alternative to be incorporated to biological control measures. We tested the consequences of introducing individuals of Girardia anceps, a native freshwater flatworm species, within artificial water containers where larvae of these mosquitoes thrive. Our goals were to ascertain if mosquito species, density of larvae (high or low), type of water container (tires or ovitraps), and presence or absence of planarians affected mosquito survivorship (measured as number of individuals reaching the pupa stage) in manipulated artificial containers. Furthermore, we monitored ovitraps in the field along several months in order to explore the long-term effect of the presence of planarian on the colonization of these containers by feral mosquitoes under natural conditions. We found that the presence of planarians reduced the number of mosquitoes reaching pupation and that such reduction depends on the initial density of larvae. Reduction of populations of A. aegypti was high along the breeding season of this mosquito, being the effect less evident in C. pipiens. G. anceps could be an agent of control against container-breeding mosquitoes if its release in small water containers is complemented with other suitable management strategies.     

Olfactory responses of the antennal trichoid sensilla to chemical repellents in the mosquito, Culex quinquefasciatus

Insect repellents are widely used to protect against insect bites and thus prevent allergic reaction and the spread of disease. To gain insight into the mosquito’s response to chemicals repellents, we investigated the interaction between the olfactory system of the mosquito Culex quinquefasciatus Say and chemical repellents using single sensillum recording. The interactions of 50 repellent chemicals with olfactory receptor neurons were measured in six different types of mosquito sensilla: long sharp trichoid (LST), short sharp trichoid (SST), short blunt trichoid I (SBT-I), short blunt trichoid II (SBT-II), short blunt trichoid-curved (SBT-C), and grooved peg (GP). A single olfactory neuron reacted to the chemical repellents in each of the sensilla except for SBT-I and SBT-II, where two neurons were involved. Other than LST and GP, which showed no or very weak responses to the repellents tested, all the sensilla showed significant excitatory responses to certain types of repellents. Terpene-derived chemicals such as eucalyptol, α-pinene, and camphor, stimulated olfactory receptor neurons in a dose-dependent manner and mosquitoes responded more strongly to terpene-derived chemical repellents than to non-terpene-derived chemicals such as dimethyl phthalate. Mosquitoes also exhibited a similar response to stereoisomers of chemicals such as (−)-β-pinene versus (+)-β-pinene, and (−)-menthone versus (+)-menthone. This study not only demonstrates the effects of chemical repellents on the mosquito olfactory system but also provides important information that will assist those screening new mosquito repellents and designing new mosquito control agents.     

Single nucleotide deletion of cqm1 gene results in the development of resistance to Bacillus sphaericus in Culex quinquefasciatus

The entomopathogen Bacillus sphaericus is one of the most effective biolarvicides used to control the Culex species of mosquito. The appearance of resistance in mosquitoes to this bacterium, however, remains a threat to its continuous use in integrated mosquito control programs. Previous work showed that the resistance to B. sphaericus in Culex colonies was associated with the absence of the 60-kDa binary toxin receptor (Cpm1/Cqm1), an alpha-glucosidase present in the larval midgut microvilli. In this work, we studied the molecular basis of the resistance developed by Culex quinquefasciatus to B. sphaericus C3-41. The cqm1 genes were cloned from susceptible (CqSL) and resistant (CqRL/C3-41) colonies, respectively. The sequence of the cDNA and genomic DNA derived from CqRL/C3-41 colony differed from that of CqSL one by a one-nucleotide deletion which resulted in a premature stop codon, leading to production of a truncated protein. Recombinant Cqm1S from the CqSL colony expressed in Escherichia coli specifically bound to the Bin toxin and had α-glucosidase activity, whereas the Cqm1R from the CqRL/C3-41 colony, with a deletion of three quarters of the receptor’s C-terminal lost its α-glucosidase activity and could not bind to the binary toxin. Immunoblotting experiments showed that Cqm1 was undetectable in CqRL/C3-41 larvae, although the gene was correctly transcribed. Thus, the cqm1R represents a new allele in C. quinquefasciatus that confers resistance to B. sphaericus.     

A systematic study on hemocyte identification and plasma prophenoloxidase from Culex pipiens quinquefasciatus at different developmental stages

Culexpipiens quinquefasciatus (C. quinquefasciatus) is an important vector that can transmit human diseases such as West Nile virus, lymphatic filariasis, Japanese encephalitis and St. Louis encephalitis. However, very limited research concerning the humoral and cellular immune defenses of C. quinquefasciatus has been done. Here we present the research on hemocyte identification and plasma including hemocyte prophenoloxidase from C. quinquefasciatus at all developmental stages in order to obtain a complete picture of C. quinquefasciatus innate immunity. We identified hemocytes into four types: prohemocytes, oenocytoids, plasmatocytes and granulocytes. Prophenoloxidase (PPO) is an essential enzyme to induce melanization after encapsulation. PPO-positive hemocytes and plasma PPO were observed at all developmental stages. As for specific hemocyte types, prophenoloxidase was found in the plasmatocytes at larval stage alone and in the smallest prohemocytes during almost all developmental stages. Moreover, the granulocytes were PPO-positive from blood-fed female mosquitoes and oenocytoids were observed PPO-positive in pupae and in adult females after blood-feeding. As for plasma, there were different patterns of PPO in C. quinquefasciatus at different developmental stages. These results are forming a basis for further studies on the function of C. quinquefasciatus hemocytes and prophenoloxidase as well as their involvement in fighting against mosquito-borne pathogens.     

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

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

The contents of adenine nucleotides and glycolytic and tricarboxylic acid cycle intermediates in activated and non-activated plerocercoids of Schistocephalus solidus (Cestoda: Pseudophyllidea)

The steady state content of adenine nucleotides, inorganic phosphate and glycolytic and tricarboxylic acid cycle intermediates were measured in freeze clamped plerocereoids of S. solidus taken from the body cavity of infected fish (non-activated plerocercoids) and the results compared with the metabolite levels in plerocercoids which had been cultured in vitro for 12 h (activated plerocercoids). Of the glycolytic intermediates, the levels of fructose-6-phosphate, fructose-1, 6-diphosphate, 3-phosphoglycerate, phosphoenolpyruvate and lactate all decreased on activation, the remainder of the glycolytic intermediates did not alter significantly. In contrast, the levels of the tricarboxylic acid cycle intermediates all increased 2–3 fold on activation, whilst the adenine nucleotides remained virtually unchanged. The differences in the steady state content in intermediary metabolites in activated and non-activated plerocereoids are discussed in relation to possible mechanisms of metabolic control.     

Thermal acclimation in a complex life cycle: The effects of larval and adult thermal conditions on metabolic rate and heat resistance in Culex pipiens (Diptera: Culicidae)

It has now been well established that insects can respond to variation in their environment via acclimation, yet the extent of the response varies among populations and environmental characteristics. One under-investigated theme which may contribute to this variation concerns acclimation effects across the life cycle. The present study explores how acclimation in the larval stage of Culex pipiens affects thermal relations in the adult stage. Mosquitoes were reared in a full factorial design at 18 or 26 °C as larvae and adults, then critical thermal maxima (CT_max) and metabolic rate–temperature relationships (MR–T) were determined for all 4 treatments. CT_max was positively affected by both larval and adult acclimation treatments. MR–T slope was significantly affected only by adult treatment: warm acclimated adults had on average shallower slopes and higher y-intercepts than cool acclimated ones. These results demonstrate that larval acclimation effects can alter adult phenotypes in a species whose life cycle includes two drastically different environments, an aquatic and a terrestrial stage. Studying insects with complex life cycles, especially those with aquatic or subterranean larval stages, can provide valuable information on the effects of thermal variability and predictability on phenotypic plasticity.     

Brugia malayi and B. pahangi: Cultivation in vitro of infective larvae to the fourth and fifth stages

Cultivation of fourth stage Brugia pahangi and B. malayi larvae from infective larvae (stage 3) were obtained in culture medium RPMI 1640 supplemented with 10% human AB serum and an LCC-MK₂ rhesus monkey kidney continuous cell line feeder layer. This culture system kept larvae alive in excess of 7 weeks, and served as a source for collection of the worms' secretory, excretory, and moulting antigens.     

Impact of short-term temperature challenges on the larvicidal activities of the entomopathogenic watermold Leptolegnia chapmanii against Aedes aegypti, and development on infected dead larvae

The oomycete Leptolegnia chapmanii is among the most promising entomopathogens for biological control of Aedes aegypti. This mosquito vector breeds in small water collections, where this aquatic watermold pathogen can face short-term scenarios of challenging high or low temperatures during changing ambient conditions, but it is yet not well understood how extreme temperatures might affect the virulence and recycling capacities of this pathogen. We tested the effect of short-term exposure of encysted L. chapmanii zoospores (cysts) on A. aegypti larvae killed after infection by this pathogen to stressful low or high temperatures on virulence and production of cysts and oogonia, respectively. Cysts were exposed to temperature regimes between ?12 °C and 40 °C for 4, 6 or 8 h, and then their infectivity was tested against third instar larvae (L3) at 25 °C; in addition, production of cysts and oogonia on L3 killed by infection exposed to the same temperature regimes as well as their larvicidal activity were monitored. Virulence of cysts to larvae and the degree of zoosporogenesis on dead larvae under laboratory conditions were highest at 25 °C but were hampered or even blocked after 4 up to 8 h exposure of cysts or dead larvae at both the highest (35 °C and 40 °C) and the lowest (?12 °C) temperatures followed by subsequent incubation at 25 °C. The virulence of cysts was less affected by accelerated than by slow thawing from the frozen state. The production of oogonia on dead larvae was stimulated by short-term exposure to freezing temperatures (?12 °C and 0 °C) or cool temperatures (5 °C and 10 °C) but was not detected at higher temperatures (25 °C–40 °C). These findings emphasize the susceptibility of L. chapmanii to short-term temperature stresses and underscore its interest as an agent for biocontrol of mosquitoes in the tropics and subtropics, especially A. aegypti, that breed preferentially in small volumes of water that are generally protected from direct sunlight.     

The temperature and humidity preferences of Haemaphysalis longicornis,ixodes holocyclus and Rhipicephalus sanguineus (ixodidae): Studies on engorged larvae

The engorged larvae of Haemaphysalis longicornis, Ixodes holocyclus and Rhipicephalus sanguineus were exposed to a range of temperatures and humidities to see whether the nature of their requirements in the laboratory were similar to the climate within the geographic range of each species. The response of H. longicornis and I. holocyclus to changes in humidity was also studied. Moulting of I. holocyclus larvae occurred from 18 to 28°C and at a saturation deficit of 4 mm Hg or less. The larvae of R. sanguineus moulted between 18 and 38°C and tolerated saturation deficits up to 35 mm Hg. The larvae of H. longicornis moulted between 15 and 38°C at saturation deficits up to 8 mm Hg. When engorged larvae of H. longicornis and I. holocyclus were exposed to very dry conditions for different periods of time and then transferred to moist conditions, the minimum pre-moult period and mortality was increased in comparison with larvae kept continuously under moist conditions. On the other hand, mortality of H. longicornis larvae was reduced in very dry conditions provided that they had been exposed to a moist environment for about 5 days previously. The larvae of I. holocyclus required at least 12 days exposure to a moist environment before any survived to moult in moderately dry conditions. The engorged larvae of both H. longicornis and I. holocyclus lost weight rapidly in dry air, whereas weight loss from R. sanguineus larvae was much slower. The rate of development was fastest in R. sanguineus and slowest in I. holocyclus.     

Effect of sulphite on adenine nucleotides of the green alga Trebouxia

The amounts of AMP, ADP and ATP were determined after sulfite incubation in photo-organotrophically cultivated cells of the green alga Trebouxia. D     

Detrimental effects of Ulva lactuca (L.) exudates and low oxygen on estuarine crab larvae

Conventional theory postulates that associations between marine macrophytes and animals are generally positive. This paper presents evidence, however, that a common species of green macroalga, Ulva lactuca (L.), is detrimental to estuarine invertebrates due to the production of toxic exudates and low oxygen tensions which occur in the seaweed beds at night. Bioassays of the responses of zoeae of five species of estuarine crabs (Callinectes sapidus Rathbun, Carcinus maenas L., Eurypanopeus depressus Smith, Neopanope texana savi Smith and Rhithropanopeus harissii Gould), using water in which Ulva lactuca was cultured for 24 h, produced 100% mortality after 22 days. No crabs survived the molt into megalopa. Hypoxic water, 0.5 ± 0.3 ppm oxygen, caused a decline in larval activity (movement), but there was no mortality over an 8-h period, Ulva-water purged to 0.4 ± 0.1 ppm oxygen caused 100% mortality in 13–40 min. These synergistic effects could be critical in estuaries where dense U. lactuca beds cause periods of low dissolved oxygen. We hypothesize larval recruitment may be limited in such systems, particularly in areas where flushing is poor.     

Resistance of field-collected populations of Culex pipiens pallens (Diptera: Culicidae) to insecticides in the Republic of Korea

Toxicities of 10 insecticides were examined against late third instars of Culex pipiens pallens, the northern house mosquito, using a direct-contact mortality bioassay. Several strains of mosquitoes were tested (insecticide-susceptible KS-CP strain and five geospatially distant field-collected strains (DG-CP, US-CP, BS-CP, GS-CP, and SG-CP)) and identified by polymerase chain reaction. Marked regional variations of insecticide susceptibility were observed. Extremely high to low levels of resistance were measured: bifenthrin, resistance ratio (RR) = 1-521; β-cyfluthrin, RR = 16-397; α-cypermethrin, RR = 9-343; deltamethrin, RR = 1-40; etofenprox, RR = 2-42; permethrin, RR = 3-46; chlorpyrifos, RR = 2-675; fenitrothion, RR = 0.5-364; and fenthion, RR = 2-360. All strains were susceptible to one or more of the insecticides examined. These results indicate that careful selection and rotational use of these insecticides may result in continued satisfactory control against field populations of northern house mosquitoes.     

Partial purification and properties of Galleria mellonella larvae proteolytic inhibitors acting on Metarhizium anisopliae toxic protease

Gel permeation, preparative isoelectric focusing, and affinity chromatography were used to purify three inhibitors of proteolytic activity from perchloric acid extracts of last instar Galleria mellonella larvae. Electrofocusing experiments revealed three isoinhibitors with different isoelectric points: inhibitor I-1 with p1 of pH 5.6, inhibitor I-2, pH 7.7, and inhibitor I-3 (of small inhibitory activity), pH 8.6. By affinity chromatography on trypsin-Sepharose 4B the I-1 was purified 9.7 ×, but 71.1% of inhibitory activity was lost. Molecular mass of the inhibitory complex was 12,600 Da. I-1 and I-2 are relatively stable to heat at several pHs with minor stability at pH 10. I-1 and I-2 inhibit serine proteases about 2.5 times as much as sulfhydryl proteases. In the same ratio protease P-1 and protease P-2 from Metarhizium anisopliae are inhibited.     

The influence of temperature on growth rate and length of larval life of the gastropod, Crepidula plana Say

The relationship between rate of larval development and the potential to prolong larval life was examined for larvae of the marine prosobranch gastropod Crepidula plana Say. Larvae were maintained in clean glass dishes at constant temperatures ranging from 12–29°C and fed either Isochrysis galbana Parke (ISO) or a Tahitian strain of Isochrysis species (T-ISO). Under all conditions, larvae grew at constant rates, as determined by measurements of shell length and tissue biomass. Most larvae eventually underwent spontaneous metamorphosis. Regardless of temperature, faster growth was associated with a shorter planktonic stage prior to spontaneous metamorphosis. Within an experiment, higher temperatures generally accelerated growth rates and reduced the number of days from hatching to spontaneous metamorphosis. However, growth rates were independent of temperature for larvae fed ISO at 25 and 29°C and for larvae fed T-ISO at 20 and 25°C. Where growth rates were unaffected by temperature, time to spontaneous metamorphosis was similarly unaffected. Maximum durations of larval life at a given temperature were shorter for larvae of Crepidula plana than for those of the congener C. fornicata (L.), although both species grew at comparable rates. Interpretations of the ecological significance of these interspecific differences in delay capabilities will require additional data on adult distributions and larval dispersal patterns in the field.     

Haemolymph-ecdysteroid peaks occur in headless larvae of Calpodes ethlius

During the last-larval stadium of Calpodes ethlius, there is a critical period after which neck ligation no longer prevents pupation. Radioimmunoassay of haemolymph from larvae ligatured after this critical period shows that the ecdysteroid titre remains lower than normal for 3 days then rises to a prepupal peak, falls to a low level, and rises rapidly again close to the time of pupation. Hormone peaks resembling those found in normal larvae are, therefore, produced in the absence of the head. The slowly rising hormone titre seen in normal larvae prior to the prepupal peak is abolished by neck ligation, indicating that this phase of the titre retains dependence on the head after the critical period. This difference may account for the lack of intermoult wax and endocuticle secretion in neck-ligated larvae. It is concluded that peaks of haemolymph ecdysteroids can be generated at appropriate developmental stages in the absence of the head, whereas the slowly rising phase of haemolymph ecdysteroids cannot.     

The brain can eat: Establishing the existence of a central pattern generator for feeding in third instar larvae of Drosophila virilis and Drosophila melanogaster

To establish the existence of a central pattern generator for feeding in the larval central nervous system of two Drosophila species, the gross anatomy of feeding related muscles and their innervation is described, the motor units of the muscles identified and rhythmic motor output recorded from the isolated CNS. The cibarial dilator muscles that mediate food ingestion are innervated by the frontal nerve. Their motor pathway projects from the brain through the antennal nerves, the frontal connectives and the frontal nerve junction. The mouth hook elevator and depressor system is innervated by side branches of the maxillary nerve. The motor units of the two muscle groups differ in amplitude: the elevator is always activated by a small unit, the depressor by a large one. The dorsal protractors span the cephalopharyngeal skeleton and the body wall hence mediating an extension of the CPS. These muscles are innervated by the prothoracic accessory nerve. Rhythmic motor output produced by the isolated central nervous system can simultaneously be recorded from all three nerves. The temporal pattern of the identified motor units resembles the sequence of muscle contractions deduced from natural feeding behavior and is therefore considered as fictive feeding. Phase diagrams show an almost identical fictive feeding pattern is in both species.     

Histopathological effects of Bacillus thuringiensis on the midgut of tobacco hornworm larvae (Manduca sexta): Low doses compared with fasting

The cytology and ultrastructure of the midgut cells of Manduca sexta larvae are described for untreated controls, larvae which fed on a spore preparation of Bacillus thuringiensis, and larvae which were fasted for either 24 or 48 hr. New observations on the ultrastructure of midgut cells in Manduca larvae included the finding of specialized Golgi vesicles in anteriormost columnar cells and of regular arrays of expanded rough endoplasmic reticulum in goblet cells of the posterior midgut region. The present observations reveal that the columnar cells of the midgut responded cytologically in the same way to fasting as they did to exposure to the toxic spores of B. thuringiensis. The goblet cells, however, appeared unaffected by fasting but became swollen in response to feeding of B. thuringiensis spore preparation.