Larvicidal potential of cell wall degrading enzymes from Trichoderma asperellum against Aedes aegypti (Diptera: Culicidae)

Aedes aegypti is a mosquito vector of arboviruses such as dengue, chikungunya, zika and yellow fever that cause important public health diseases. The incidence and gravity of these diseases justifies the search for effective measures to reduce the presence of this vector in the environment. Bioinsecticides are an effective alternative method for insect control, with added ecological benefits such as biodegradability. The current study demonstrates that a chitinolytic enzyme complex produced by the fungus Trichoderma asperellum can disrupt cuticle formation in the L3 larvae phase of A. aegypti, suggesting such biolarvicidal action could be used for mosquito control. T. asperellum was exposed to chitin from different sources. This induction of cell wall degrading enzymes, including chitinase, N-acetylglucosaminidase and β-1,3-glucanase. Groups of 20 L3 larvae of A. aegypti were exposed to varying concentrations of chitinolytic enzymes induced with commercial chitin (CWDE) and larvae cell wall degrading enzymes (L-CWDE). After 72 h of exposure to the CWDE, 100% of larvae were killed. The same percent mortality was observed after 48 h of exposure to L-CWDE at half the CWDE enzyme mixture concentration. Exoskeleton deterioration was further observed by scanning and electron microscopy. Our findings indicate that L-CWDE produced by T. asperellum reflect chitinolytic enzymes with greater specificity for L3 larval biomolecules. This specificity is characterized by the high percentage of mortality compared with CWDE treatments and also by abrupt changes in patterns of the cellular structures visualized by scanning and transmission electron microscopy. These mixtures of chitinolytic enzymes could be candidates, as adjuvant or synergistic molecules, to replace conventional chemical insecticides currently in use.     

Bionomics of Aedes aegypti subpopulations (Diptera: Culicidae) from Argentina

Differences in biological features of immature and adult Aedes aegypti, as well as variability in vector competence, seem consistent with the existence of genetic variation among subpopulations and adaptation to local conditions. This work aims to compare the bionomics of four Ae. aegypti subpopulations derived from different geographical regions reared under temperate conditions. Life statistics of three Ae. aegypti subpopulations from the provinces of Córdoba, Salta, and Misiones were studied based on horizontal life tables. The Rockefeller strain was used as a control. The development time required to complete the larva and pupa stages varied from 6.91 to 7.95 and 1.87 to 2.41 days, respectively. Significant differences were found in mean larval development time between the Córdoba and Orán subpopulations. The larva‐pupa development time was similar in all the subpopulations. However, survival values varied significantly between the Orán and San Javier subpopulations. The proportion of emergent males did not differ from females within each subpopulation nor among them. Adult longevity was similar among the subpopulations. The average number of eggs laid by each female was significantly different. The Rockefeller strain laid a significantly greater number of eggs (463.99 eggs/female) than the rest of the subpopulations. Moreover, differences in the demographic growth parameter R_o were detected among the four subpopulations. The differences obtained in larval development time, larva‐pupa survival values, and net reproductive rates among the subpopulations might reflect underlying genetic differences as a result of colonization from different regions that probably involve adaptations to local conditions.     

Synthesis and larvicidal activity of indole derivatives against Aedes aegypti (Diptera: Culicidae)

In light of the challenges to control Aedes aegypti and the critical role that it plays as arbovirus vector, it is imperative to adopt strategies that provide fast, efficient and environmentally safe control of the insect population. In the present study, we synthesized six indole derivatives (C1‐C6) and examined their larvicidal activity and persistence against Ae. aegypti larvae, as well as their toxicity towards Raw 264.7 macrophages, Vero cells, Chlorella vulgaris BR017, Scenedesmus obliquus BR003, Caenorhabditis elegans N2 and Galleria mellonella. Among the bioactive compounds (C1, C2, C4 and C5), C2 exerted the strongest larvicidal activity against Ae. aegypti, with LC50 = 1.5 μg/ml (5.88 µM) and LC90 = 2.4 μg/ml (9.50 µM), indicating that the presence of chlorine or bromine groups in the aromatic ring improved the larvicidal activity of the indole derivatives. C1, C2, C4 and C5 did not reduce viability of RAW 264.7 macrophages, Vero cells, C. elegans N2 and G. mellonella. Compounds C1, C2 and C5 did not affect the growth of C. vulgaris BR017 and S. obliquus BR003. Analysis of larvicidal persistence under laboratory conditions revealed that the effect of compounds C1, C2, C4 and C5 lasted for 30 days and caused 100% of larvae mortality within few hours. Altogether, our findings demonstrate that the indole derivatives C1, C2, C4 and C5 effectively control Ae. aegypti larvae population, without clear signs of toxicity to mammalian cells, algae, C. elegans and G. mellonella.     

Potential of crude seed extract of celery, Apium graveolens L., against the mosquito Aedes aegypti (L.) (Diptera: Culicidae).

Crude seed extract of celery, Apium graveolens, was investigated for anti-mosquito potential, including larvicidal, adulticidal, and repellent activities against Aedes aegypti, the vector of dengue haemorrhagic fever. The ethanol-extracted A. graveolens possessed larvicidal activity against fourth instar larvae of Ae. aegypti with LD50 and LD95 values of 81.0 and 176.8 mg/L, respectively. The abnormal movement observed in treated larvae indicated that the toxic effect of A. graveolens extract was probably on the nervous system. In testing for adulticidal activity, this plant extract exhibited a slightly adulticidal potency with LD50 and LD95 values of 6.6 and 66.4 mg/cm2, respectively. It showed repellency against Ae. aegypti adult females with ED50 and ED95 values of 2.03 and 28.12 mg/cm2, respectively. It also provided biting protection time of 3 h when applied at a concentration of 25 g%. Topical application of the ethanol-extracted A. graveolens did not induce dermal irritation. No adverse effects on the skin or other parts of the body of human volunteers were observed during 3 mo of the study period or in the following 3 mo, after which time observations ceased. A. graveolens, therefore, can be considered as a probable source of some biologically active compounds used in the development of mosquito control agents, particularly repellent products.     

Larvicidal effect of pepper plants on Aedes aegypti (L.) (Diptera: Culicidae).

Ethanolic extracts derived from three species of the Piperaceae (pepper) family, Piper longum L., P. ribesoides Wall., and P. sarmentosum Roxb. ex Hunt., were evaluated for efficacy against early 4th instar larvae of Aedes aegypti mosquitoes using larvicidal bioassays. The highest larvicidal efficacy was established from P. longum, followed by P. sarmentosum and P. ribesoides, with LC50 values of 2.23, 4.06, and 8.13 ppm, respectively. Observations of morphological alterations on treated 4th instar larvae revealed that most organs, except anal papillae, had a normal structural appearance that was similar to controls. Under light microscopy, the internal structures of anal papillae in the treated larvae showed shrinkage, while the external features were normal in appearance. Ultrastructural studies, however, clearly demonstrated external destruction, with extensive damage and shrunken cuticle of the anal papillae. The structural deformation of anal papillae probably led to their dysfunction, which may be intrinsically associated with the death of the larvae. This study affords some evidence regarding the action site of the pepper extracts and suggests their potential in developing new types of larvicides used for mosquito control.     

Insecticide susceptibility status of Aedes aegypti (Diptera: Culicidae) from Townsville

Insecticide susceptibility of a 1995 Townsville Aedes aegypti (L.) population was investigated in the laboratory according to World Health Organization guidelines. Using baseline data from two Townsville populations (1955 and 1989), larval bioassays detected significant increases in susceptibility to synthetic pyrethroids and malathion, and significant reductions in susceptibility to most organophosphates and propoxur. Adult bioassays detected significant resistance to bendiocarb and DDT. Comparison of larval data with the international reference ROCK strain showed substantial resistance to have developed to malathion and fenthion. Further analysis revealed the presence of distinct substrains in the baseline 1989 population, which displayed varying levels of temephos susceptibility. We concluded that susceptibility investigations should assess mosquito populations collected from many sites within an area rather than taking a single population from one site, and that the 1989 Ae. aegypti colony would be unsuitable for use as a susceptible reference population.     

Toxicity of Thiophenes from Echinops transiliensis (Asteraceae) against Aedes aegypti (Diptera: Culicidae) Larvae

Structure? activity relationships of nine thiophenes, 2,2′: 5′,2″‐terthiophene ( 1 ), 2‐chloro‐4‐[5‐(penta‐1,3‐diyn‐1‐yl)thiophen‐2‐yl]but‐3‐yn‐1‐yl acetate ( 2 ), 4‐(2,2′‐bithiophen‐5‐yl)but‐3‐yne‐1,2‐diyl diacetate ( 3 ), 4‐[5‐(penta‐1,3‐diyn‐1‐yl)thiophen‐2‐yl]but‐3‐yne‐1,2‐diyl diacetate ( 4 ), 4‐(2,2′‐bithiophen‐5‐yl)‐2‐hydroxybut‐3‐yn‐1‐yl acetate ( 5 ), 2‐hydroxy‐4‐[5‐(penta‐1,3‐diyn‐1‐yl)thiophen‐2‐yl]but‐3‐yn‐1‐yl acetate ( 6 ), 1‐hydroxy‐4‐[5‐(penta‐1,3‐diyn‐1‐yl)thiophen‐2‐yl]but‐3‐yn‐2‐yl acetate ( 7 ), 4‐(2,2′‐bithiophen‐5‐yl)but‐3‐yne‐1,2‐diol ( 8 ), and 4‐[5‐(penta‐1,3‐diyn‐1‐yl)thiophen‐2‐yl]but‐3‐yne‐1,2‐diol ( 9 ), isolated from the roots of Echinops transiliensis, were studied as larvicides against Aedes aegypti. Structural differences among compounds 3, 5 , and 8 consisted in differing AcO and OH groups attached to C(3″) and C(4″), and resulted in variations in efficacy. Terthiophene 1 showed the highest activity (LC₅₀, 0.16 μg/ml) among compounds 1 – 9 , followed by bithiophene compounds 3 (LC₅₀, 4.22 μg/ml), 5 (LC₅₀, 7.45 μg/ml), and 8 (LC₅₀, 9.89 μg/ml), and monothiophene compounds 9 (LC₅₀, 12.45 μg/ml), 2 (LC₅₀, 14.71 μg/ml), 4 (LC₅₀, 17.95 μg/ml), 6 (LC₅₀, 18.55 μg/ml), and 7 (LC₅₀, 19.97 μg/ml). These data indicated that A. aegypti larvicidal activities of thiophenes increase with increasing number of thiophene rings, and the most important active site in the structure of thiophenes could be the tetrahydro‐thiophene moiety. In bithiophenes, 3, 5 , and 8 , A. aegypti larvicidal activity increased with increasing number of AcO groups attached to C(3″) or C(4″), indicating that AcO groups may play an important role in the larvicidal activity.     

Cyt1A from Bacillus thuringiensis Synergizes Activity of Bacillus sphaericus against Aedes aegypti (Diptera: Culicidae)

Bacillus sphaericus is a mosquitocidal bacterium recently developed as a commercial larvicide that is used worldwide to control pestiferous and vector mosquitoes. Whereas B. sphaericus is highly active against larvae of Culex and Anopheles mosquitoes, it is virtually nontoxic to Aedes aegypti, an important vector species. In the present study, we evaluated the capacity of the cytolytic protein Cyt1A from Bacillus thuringiensis subsp. israelensis to enhance the toxicity of B. sphaericus toward A. aegypti. Various combinations of these two materials were evaluated, and all were highly toxic. A ratio of 10:1 of B. sphaericus to Cyt1A was 3,600-fold more toxic to A. aegypti than B. sphaericus alone. Statistical analysis showed this high activity was due to synergism between the Cyt1A toxin and B. sphaericus. These results suggest that Cyt1A could be useful in expanding the host range of B. sphaericus.     

Cyt1A from Bacillus thuringiensis synergizes activity of Bacillus sphaericus against Aedes aegypti (Diptera: Culicidae)

Bacillus sphaericus is a mosquitocidal bacterium recently developed as a commercial larvicide that is used worldwide to control pestiferous and vector mosquitoes. Whereas B. sphaericus is highly active against larvae of Culex and Anopheles mosquitoes, it is virtually nontoxic to Aedes aegypti, an important vector species. In the present study, we evaluated the capacity of the cytolytic protein Cyt1A from Bacillus thuringiensis subsp. israelensis to enhance the toxicity of B. sphaericus toward A. aegypti. Various combinations of these two materials were evaluated, and all were highly toxic. A ratio of 10:1 of B. sphaericus to Cyt1A was 3, 600-fold more toxic to A. aegypti than B. sphaericus alone. Statistical analysis showed this high activity was due to synergism between the Cyt1A toxin and B. sphaericus. These results suggest that Cyt1A could be useful in expanding the host range of B. sphaericus.     

Chemical composition and larvicidal activity of leaf essential oil from Clausena dentata (Willd) M. Roam. (Rutaceae) against the chikungunya vector,Aedes aegypti Linn. (Diptera: Culicidae)

Larvicidal activity of essential oil and isolated compounds from Clausena dentata leaves were tested against early fourth instar Aedes aegypti larvae. GC–MS analysis of essential oil revealed the presence of fourteen compounds of which the major compounds were sabinene (21.27%), biofloratriene (19.61%), borneol (18.34%) and β-bisabolol (17.68%). The essential oil of C. dentata exhibited significant larvicidal activity, with 24 h LC₅₀ and LC₉₀ values of 140.2 and 341.6 mg/l, respectively. Larvicidal activities of the four major compounds of essential oil were also tested. The LC₅₀ values of sabinene, biofloratriene, borneol and β-bisabolol were 27.3, 47.4, 43.5 and 33.2 mg/l, respectively. Results of this study show that the leaf essential oil of C. dentata and its four major compounds may be a potent source of natural larvicides.     

Identification of Bioactive Compounds against Aedes aegypti (Diptera: Culicidae) by Bioassays and in Silico Assays

Most of the hematophagous insects act as disease vectors, including Aedes aegypti, responsible for transmitting some of the most critical arboviruses globally, such as Dengue. The use of repellents based on natural products is a promising alternative for personal protection compared to industrial chemical repellents. In this study, the repellent effect of essential oils extracted from Lippia thymoides, Lippia alba, Cymbopogon winterianus, and Eucalyptus globulus leaves was evaluated. Essential oils used showed repellent activity against Ae. aegypti in laboratory bioassays, obtaining protection rates above 70 % from 3.75 mg/mL and higher concentration for all analyzed oils. GC/MS identified 57 constituents, which were used in the ligand-based pharmacophore model to expose compounds with requirements for repellents that modulate mosquitoes behavior through odorant-binding protein 1 Ae. aegypti. Ligand-based pharmacophore model approach results suggested that repellent activity from C. winterianus, L. alba, and L. thymoides essential oils’ metabolites is related to Citronelal (QFIT=26.77), Citronelol (QFIT=11.29), Citronelol acetate (QFIT=52.22) and Geranil acetate (QFIT=10.28) with synergistic or individual activity. E. globulus essential oil's repellent activity is associated with Ledol (0.94 %; QFIT=41.95). Molecular docking was applied to understand the binding mode and affinity of the essential oils’ data set at the protein binding site. According to molecular docking, Citronelol (ChemPLP=60.98) and geranyl acetate (ChemPLP=60.55) were the best-classified compounds compared to the others and they can be explored to develop new repellents.     

Reappearance of Aedes aegypti (Diptera: Culicidae) in Lima, Peru

We report here the reappearance of Aedes aegypti in the Rimac district, and summarize the history of this mosquito species in Peru since its first detection in 1852. On March 17 2000 were found Ae. aegypti and Culex quinquefasciatus in Mariscal Castilla town, Flor de Amancaes, San Juan de Amancaes, El Altillo and Santa Rosa in the Rimac district, Lima Province.     

Antagonistic effects of energy status on meal size and egg-batch size of Aedes aegypti (Diptera: Culicidae).

The effect of sugar feeding on egg-batch size in Aedes aegypti was examined in a way that would distinguish between the roles of a recent sugar meal (full vs. empty crop) and of repeated sugar feeding and digestion (high vs. low energy reserves). Egg numbers of females representing the four combinations of these characteristics (full crop/high reserves, full crop/low reserves, empty crop/high reserves, empty crop/low reserves) were counted during their first gonotrophic cycle. In addition, the sizes of their replete sugar meals and human blood meals were measured to understand the interactions between them. Results demonstrated that blood-fed mosquitoes with full crops and low energy reserves produced the fewest eggs (mean = 56.2); those with empty crops and high energy reserves produced the most eggs (mean = 84.6); and those with the opposite combinations (full/high and empty/ low) had an intermediate fecundity (mean = 75.2 and 76.9, respectively). This ranking of fecundities did not correspond to blood-meal size ranks, owing to direct and indirect effects of energy reserves on meal sizes and egg number. Full-crop females with low reserves ingested the smallest blood meals (mean = 0.62 mg) and had the lowest fecundities. Full-crop females with high reserves ingested more blood (mean = 0.82 mg) and produced more eggs. But empty-crop females with low reserves ingested the largest blood meals (mean = 1.19 mg), yet produced significantly fewer eggs than their high-reserve counterparts, which took smaller blood meals (mean = 0.99 mg). These results demonstrate extremes in the reproductive penalty of crop sugar and the reproductive reward of digested sugar. Energy reserves and an empty crop are similarly valuable in promoting fecundity at the time blood is taken.     

Chemical composition and larvicidal activity of edible plant‐derived essential oils against the pyrethroid‐susceptible and ‐resistant strains of Aedes aegypti (Diptera: Culicidae)

The chemical compositions and larvicidal potential against mosquito vectors of selected essential oils obtained from five edible plants were investigated in this study. Using a GC/MS, 24, 17, 20, 21, and 12 compounds were determined from essential oils of Citrus hystrix, Citrus reticulata, Zingiber zerumbet, Kaempferia galanga, and Syzygium aromaticum, respectively. The principal constituents found in peel oil of C. hystrix were β‐pinene (22.54%) and d‐limonene (22.03%), followed by terpinene‐4‐ol (17.37%). Compounds in C. reticulata peel oil consisted mostly of d‐limonene (62.39%) and γ‐terpinene (14.06%). The oils obtained from Z. zerumbet rhizome had α‐humulene (31.93%) and zerumbone (31.67%) as major components. The most abundant compounds in K. galanga rhizome oil were 2‐propeonic acid (35.54%), pentadecane (26.08%), and ethyl‐p‐methoxycinnamate (25.96%). The main component of S. aromaticum bud oil was eugenol (77.37%), with minor amounts of trans‐caryophyllene (13.66%). Assessment of larvicidal efficacy demonstrated that all essential oils were toxic against both pyrethroid‐susceptible and resistant Ae. aegypti laboratory strains at LC₅₀, LC₉₅, and LC₉₉ levels. In conclusion, we have documented the promising larvicidal potential of essential oils from edible herbs, which could be considered as a potentially alternative source for developing novel larvicides to be used in controlling vectors of mosquito‐borne disease.     

Frequency of pyrethroid resistance in Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in Thailand

Thirty‐two Aedes aegypti populations collected throughout Thailand and five populations of Aedes albopictus from southern Thailand were subjected to standard WHO contact bioassays to assess susceptibility to three commonly used synthetic pyrethroids: permethrin, deltamethrin, and lambda‐cyhalothrin. A wide degree of physiological response to permethrin was detected in Ae. aegypti, ranging from 56.5% survival (Lampang, northern Thailand) to only 4% (Kalasin in northeastern and Phuket in southern Thailand). All 32 populations of Ae. aegypti were found to have evidence of incipient resistance (62.5%) or levels of survival deemed resistant (37.5%) to permethrin. Four populations of Ae. albopictus were found with incipient resistance (97 – 80% mortality) and one with resistance (< 80%) to permethrin. The majority of Ae. aegypti populations (68.7%) was susceptible (> 98% mortality) to deltamethrin, with incipient resistance (observed 97–82% mortality) in other localities. In contrast, all populations of Ae. aegypti were completely susceptible (100% mortality) to the recommended operational dosage of lambda‐cyhalothrin. All five populations of Ae. albopictus were found completely susceptible to both deltamethrin and lambda‐cyhalothrin. Evidence of defined incipient or resistance to synthetic pyrethroids mandates appropriate response and countermeasures to mitigate further development and spread of resistance. In light of these findings, we conclude that routine and comprehensive susceptibility monitoring of dengue mosquito vectors to synthetic pyrethroids should be a required component of resistance management policies and disease control activities.     

State‐wide survey of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in Florida

Aedes aegypti and Aedes albopictus are invasive mosquito species with geographic ranges that have oscillated within Florida since their presence was first documented. Local transmission of dengue, chikungunya, and Zika viruses serves as evidence of the public health importance of these two species. It is important to have detailed knowledge of their distribution to aid in mosquito control efforts and understand the risk of arbovirus transmission to humans. Through a partnership involving the University of Florida Institute of Food and Agricultural Sciences Cooperative Extension Service and the Florida Medical Entomology Laboratory; the Florida Department of Health; and mosquito control agencies throughout Florida, a container mosquito surveillance program involving all life stages was launched in the summer of 2016 to detect the presence of Ae. aegypti and Ae. albopictus. Results from this survey were mapped to provide a picture of the current known distribution of Ae. aegypti and Ae. albopictus in Florida. Aedes aegypti and/or Ae. albopictus were detected in the 56 counties that were part of the survey. Only Aedes albopictus was detected in 26 counties, primarily in the panhandle region of Florida. The results of this work underscore the importance of maintaining container mosquito surveillance in a state where chikungunya, dengue, and Zika viruses are present and where there is continued risk for exotic arbovirus introductions.     

Larvicidal and repellent potential of Albizzia amara Boivin and Ocimum basilicum Linn against dengue vector, Aedes aegypti (Insecta:Diptera:Culicidae)

Investigations were made to test the larval toxicity and smoke repellent potential of Albizzia amara and Ocimum basilicum at different concentration (2%, 4%, 6%, 8% and 10%) against the different instar (I, II, III and IV) larvae and pupae of Aedes aegypti. The LC50 values of A. amara and O. basilicum for I instar larvae was 5.412 and 3.734, II instar 6.480 and 4.154, III instar 7.106 and 4.664, IV instar 7.515 and 5.124, respectively. The LC50 and LC90 values of pupae were 6.792%, 5.449% and 16.925%, 15.474%. The smoke toxicity of A. amara was more effective against A. aegypti than the O. basilicum.