The effect of infection with Dirofilaria immitis (dog heartworm) on fluid secretion rates in the Malpighian tubules of the mosquitoes Aedes taeniorhynchus and Anopheles quadrimaculatus

Dirofilaria immitis, the parasitic nematode causing the disease dog heartworm, is transmitted by female mosquitoes. During their development, larval nematodes reside in the cells of the Malpighian tubules of these mosquitoes for approx 13 days. We have examined the effect of the presence of these large intracellular parasites on the main physiological function of the Malpighian tubules, i.e. fluid secretion. Rates of fluid secretion were examined in vitro using both normal and infected tubules of the mosquito species Aedes taeniorhynchus and Anopheles quadrimaculatus. Tubules of A. quadrimaculatus show changes in trasport rate during the reproductive cycle. Those of A. taeniorhynchus do not. Infection with larvae of D. immitis had no effect on the rate of fluid secretion in tubules of A. quadrimaculatus. In A. taeniorhynchus by contrast, the tubules show decline in transport with time following infection. The reduction in transport capacity is proportional to the number of worms infecting the tubule. The present paper and separate ultrastructural studies demonstrate that parallel changes in microvillar ultrastructure and epithelial transport rates occur in response to infection by the parasite. In both species examined, the survival of the mosquitoes and their vector potential are determined by factors other than the transport capacities of the infected Malpighian tubules.     

Incrimination of the mosquito,Aedes taeniorhynchus,as the primary vector of heartworm,Dirofilaria immitis,in coastal Yucatan,Mexico

Mosquito collections were carried out on microfilaraemic dogs, positive for Dirofilaria sp., for 18 consecutive nights in the coastal town of Celestún, Yucatan, southeast Mexico, during the rainy season (August) of 2007. A total of 292 female mosquitoes representing 12 species of dipteran Culicidae were collected: Anopheles albimanus (Wiedemann); Anopheles crucians (Wiedemann); Anopheles pseudopunctipennis (Theobald); Culex coronator (Dyar & Knab); Culex interrogator (Dyar & Knab); Culex nigripalpus (Theobald); Culex quinquefasciatus (Say); Culex salinarius (Coquillett); Aedes aegypti (Linnaeus); Aedes scapularis (Rondani); Aedes sollicitans (Walker), and Aedes taeniorhynchus (Wiedemann). Aedes taeniorhynchus and Cx. quinquefasciatus were the species found most commonly feeding on the dogs. Filarial nematodes were observed by microscopy in nine of the mosquito species collected; however, third‐instar larvae were only observed in Ae. taeniorhynchus and An. crucians. Of 76 Ae. taeniorhynchus specimens found positive for Dirofilaria sp. by dissection, 14 were confirmed to be positive for Dirofilaria immitis (Leidy) by polymerase chain reaction (PCR). The resulting infection rate for D. immitis confirmed by PCR (6.2%) is higher than any infection rate for Ae. taeniorhynchus previously reported from the Americas.     

Inhibition of Dirofilaria immitis in gregarine-infected Aedes aegypti: preliminary observations.

This study examined some of the effects of Ascocystis culicis infections in Aedes aegypti mosquitoes and on the development of dog heartworm, Dirofilaria immitis, in A. culicis-infected A. aegypti. Infections of 100 or more gregarines had an adverse effect on mosquito larvae and the resulting pupae weighed 1 to 2 mg less than the gregarine-free stock mosquitoes. Intensity of gregarine-induced pathology of adult mosquitoes was best estimated by counting the cells of the Malpighian tubules. Larvae infected with approximately 100 gregarines per larva had at least half the number of cells in their tubules destroyed, and development of D. immitis was greatly reduced. Thus, gregarine infections may be useful in reducing populations of aedine mosquitoes and, in areas endemic for heartworm, may interrupt transmission of D. immitis.     

Intracellular melanization of the larvae of Dirofilaria immitis in the malpighian tubules of the mosquito, Aedes sollicitans

Frequent melanization of larvae of the nematode Dirofilaria immitis parasitizing the Malpighian tubules of the mosquito, Aedes sollicitans, has been observed. Melanized and nonmelanized larvae in the Malpighian tubules were examined using light and electron microscopy. The results indicate that the pattern of melanin deposition and the ultrastructural characteristics of the pigment around the worms are identical to that observed on nematodes which have undergone humoral melanization in other dipteran insects. In the Malpighian tubules, no contact between the intracellular melanized nematodes and the hemolymph or hemocytes was observed. The results suggest that the Malpighian tubules of this species of mosquito are capable of inducing a melanotic response to invading nematode parasites. It is proposed that this is an example of “humoral” melanization at an intracellular site.     

A comparative study of flight performance and fuel utilization as a function of age in females of Florida mosquitoes

Sugar-fed females of 7 species of Florida mosquitoes were flown for 4·5 hr on a flight-mill system twice a week during their life span and analysed for flight performance (speed of flight during the first hour, the distance flown at the end of 4·5 hr flight period, and per cent reduction in flight speed of the fourth hour as compared to the first hour) and for utilization of haemolymph sugars, glycogen, and triglycerides reserves.Species-specific differences occurred in 50 per cent survival age and flight performance. Aedes taeniorhynchus, Mansonia titillans, Culex nigripalpus, Psorophora confinnis, and A. sollicitans exhibited maximum flight potential during 2 to 8 weeks after emergence, as compared with only 1 to 2 weeks in A. aegypti and Anopheles quadrimaculatus. This suggested that the former group of mosquitoes possessed a greater potential for dispersal by searching flights during their life span when compared with the latter group. Glycogen was the only energy reserve utilized during sustained tethered flight in all species. The energy requirements as calculated from depletion of glycogen during sustained flight for 4·5 hr in different species varied from 0·06 to 0·09 cal/1000 m or 16 to 32 cal/hr per g. The considerably lower values of energy expended, on the average of 16 to 22 cal/hr per g in C. nigripalpus, P. confinnis, A. sollicitans, and A. aegypti, compared with 30 to 32 cal/hr per g in A. taeniorhynchus and M. titillans, is most probably due to the substantially lower speed of the former group of mosquitoes, 1000 to 1500 m/hr, than in the latter group of mosquitoes, 1500 to 2000 m/hr. A. sollicitans and M. titillans started to show wing abrasion and distinct signs of senescence during the last 2 to 3 weeks of their life span.Non-flown females of different species maintained on sugar ab lib. achieved maximum levels of glycogen and triglycerides reserves during the second week and they maintained these levels for the major portion of their life span, after which the levels of these reserves showed a distinct decline. It is suggested that this stabilization and decline of the energy reserves control longevity and flight potential in each mosquito species.     

Further characterization of refractoriness in Aedes aegypti (L.) to infection by Dirofilaria immitis (Leidy)

Factors which control the expression of the refractory or susceptible condition to infection with Dirofilaria immitis in the mosquito. Aedes aegypti, were investigated using three protocols. (1) Microfilariae and prelarvae were injected into the hemocoel of susceptible A. aegypti. Some microfilariae and prelarvae developed to the L1 larval stage but they failed to complete development to the infective stage. (2) Enema of microfilariae and prelarvae from infected susceptible and refractory donor females were given into the midgut of uninfected susceptible and refractory recipient females. The results indicate that the conditions which inhibit the initiation of development are present in the Malpighian tubules and not in the midgut of the refractory mosquitoes. (3) Transplants of infected Malpighian tubules from susceptible and refractory donor females were made into the abdominal hemocoel of uninfected susceptible and refractory recipient females. The results showed that the refractory condition depends on the genetic makeup of the donor, not the recipient, mosquito. The above results taken as a whole indicate that the factors which control refractoriness are not present in the midgut but are present in the Malpighian tubule cells of refractory A. aegypti.     

Pathogenicity Tests on Nine Mosquito Species and Several Non-target Organisms with Strelkovimermis spiculatus (Nemata Mermithidae)

Nine species of mosquitoes and several species of non-target aquatic organisms were tested for susceptibility to the mernaithid nematode, Strelkovimermis spiculatus. All species of Anopheles, Aedes, Culex, and Toxorhynchites exposed to S. spiculatus were susceptible. Of the nine mosquito species tested, C. pipiens quinquefasciatus had the greatest tolerance to initial invasion and the highest percent infection of those that survived. High levels of infection were also achieved with Aedes taeniorhynchus and A. albopictus, but these mosquitoes were significantly less tolerant to parasitism than C. pipiens quinquefasciatus. Strelkovimermis spiculatus did not infect or develop in any of the non-target hosts tested.     

Dirofilaria immitis: effect on the longevity of Aedes trivittatus

Laboratory studies on vector mortality as related to parasite burden revealed that the mosquito Aedes trivittatus showed considerable tolerance to infection with the filarial nematode Dirofilaria immitis. Although mosquitoes exposed to a dog with a high microfilaremia (347 microfilariae/20 mm³) had a significant increase in mortality during the first 16 days postexposure, 66% of the mosquitoes lived long enough for complete parasite development to occur. Those mosquitoes exposed to a dog with a low microfilaremia (62 microfilariae/20 mm³) had no significant increase in mortality. There was a strong negative correlation between parasite burden and mosquito survival, but only mosquitoes harboring more than 15 juveniles had an increased chance of dying before D. immitis could develop to the infective stage. The retention of microfilariae within the blood clot and peritrophic membrane of A. trivittatus seems beneficial to this vector-parasite system by reducing the parasite burden and increasing mosquito longevity.     

Scalariform junctions in the malpighian tubules of the insects Rhodnius prolixus (Hemiptera: Reduviidae) and Aedes taeniorhynchus (Diptera: Culicidae)

The ultrastructure of scalariform junctions in the Malpighian tubules of the hemipteran Rhodnius prolixus and the dipteran Aedes taeniorhynchus is described. Both autocellular and intercellular scalariform junctions are illustrated. This is the first report of scalariform junctions in the Malpighian tubules of a dipteran. When combined with previous observations by other authors, the presence of scalariform junctions has now been reported in the Malpighian tubules of insects from five orders, including ametabolous, hemimetabolous, and holometabolous forms. The cell types in which scalariform junctions were found in R. prolixus and A. taeniorhynchus differ in the direction of ion and fluid transport. The cells share the capacity to transport KCl. These same cells also possess morphological features promoting close associations of mitochondria and plasma membranes in the apical region of the cell. The possible role of scalariform junctions is discussed in light of these observations.     

Early cellular responses in the Malpighian tubules of the mosquito Aedes taeniorhynchus to infection with Dirofilaria immitis (Nematoda)

Early ultrastructural changes in the Malpighian tubules of the mosquito, Aedes taeniorhynchus, were examined following infection with the nematode, Dirofilaria immitis. After ingestion by the mosquito, the microfilariae enter the cells of the Malpighian tubules, becoming intracellular. During early development, the filarial prelarvae reside in the cell cytoplasm surrounded by a clear zone without a delimiting membrane. Cells infected with prelarvae differed from uninfected cells and from cells in uninfected mosquitoes in that the volume of the apical microvilli was reduced and mitochondria were retracted from these microvilli. Morphometric analysis was used to quantify the ultrastructural consequences of infection. In infected cells, microvillar volume, the percent of microvillar volume occupied by mitochondria, and volume of mitochondria within the microvilli were significantly reduced.     

Defense reactions of mosquitoes to filarial worms: potential mechanism for avoidance of the response by Brugia pahangi microfilariae

The melanization response of Aedes trivittatus and the black-eyed Liverpool (LVP) and Rocke-feller (RKF) strains of Aedes aegypti against intrathoracically inoculated Brugia pahangi microfilariae (mff) that previously had penetrated LVP, RKF, or A. trivittatus midguts in vitro was assessed at 1, 3, and 5 days postinoculation (PI). LVP and RKF midgut-derived mff almost totally avoided the melanization response and developed normally in LVP strain A. aegypti, and although over 90% of these mff died by 5 days PI in RKF mosquitoes, the majority of these were not melanized. A. aegypti midgut-derived mff also were able to avoid the response of A. trivittatus in 33–43% of the cases. Penetration through A. trivittatus midguts, however, did not significantly affect the ability of mff to avoid the melanization response in any of the mosquitoes examined. Allogeneic and xenogeneic tissue inplants were accepted by all three mosquito species examined. Data presented support the hypothesis that mff avoid immune recognition in compatible mosquitoes by coating themselves with midgut material(s) during penetration of the midgut in their migration to the hemocoel.     

Transovarial Transmission of Chikungunya Virus by Aedes albopictus Mosquitoes Ingesting Microfilariae of Dirofilaria immitis under Laboratory Conditions

Female Aedes albopictus mosquitoes of the Miki strain were experimentally fed on defibrinated sheep blood containing 5× 10⁷ PFU of chikungunya virus and 20,000 microfilariae of Dirofilaria immitis per milliliter. Fully engorged mosquitoes transmitted the virus to a small percentage of the F1 progeny, but females of the F1 generation did not transmit the virus to the F2 progeny. The control mosquitoes that ingested the virus without microfilariae did not transmit the virus to their eggs, larvae, or pupae in the F1 or F2 generations. These results showed that A. albopictus of this strain that concurrently ingested the virus and microfilariae transmitted the virus by the transovarial route under experimental conditions.     

Laboratory evaluation of lactic acid on attraction of Culex spp. (Diptera: Culicidae)

The role of lactic acid was evaluated for attraction of Culex nigripalpus, Culex quinquefasciatus, Culex tarsalis, and Aedes aegypti in the laboratory using a dual‐port olfactometer. When lactic acid was combined with chicken odor, attraction was increased for Cx. quinquefasciatus compared to chicken odor alone but not for Cx. nigripalpus, Cx. tarsalis, and Ae. aegypti. Lactic acid combined with hand odor did not change attraction of Cx. tarsalis and Ae. aegypti but decreased attraction of Cx. nigripalpus and Cx. quinquefasciatus. The addition of lactic acid to CO₂ increased attraction of Ae. aegypti and Cx. quinquefasciatus but reduced attraction of Cx. nigripalpus and Cx. tarsalis. Use of commercial lactic acid baits with CO₂ resulted in a similar trend except for Cx. nigripalpus which showed no difference. A blend of lactic acid, acetone, and dimethyl disulfide was attractive to Ae. aegypti (63.4%) but elicited low responses by all Culex spp. (1.3–26.8%). Addition of the blend to CO₂ increased attraction of Ae. aegypti and Cx. quinquefasciatus but reduced attraction of Cx. nigripalpus and Cx. tarsalis. The mixture of compounds plus CO₂ was as attractive as a hand for Cx. quinquefasciatus, Cx. tarsalis, and Ae. aegypti.     

The influence of ecological factors on mosquito abundance and occurrence in Galápagos

We sampled mosquitoes across 18 sites established at different elevations and stretching from the north to the south of Isla Santa Cruz, Galápagos. Two commonly occurring species, Ae. taeniorhynchus and Cx. quinquefasciatus, were collected along with environmental variables characteristic of the trapping sites to assess their influence on mosquito abundance and occurrence in the dry season of 2015. We captured Ae. taeniorhynchus at 14 out of 18 sites and Cx. quinquefasciatus at low and high elevation sites on Santa Cruz. We utilized two generalized linear models; the first assessed the influence of environmental variables on abundances of Ae. taeniorhynchus and the second assessed the influence of these variables on the presence of Cx. quinquefasciatus. Populations of both mosquito species declined with elevation. Rainfall data were limited, as we sampled during the dry season of 2015. Distance to mangroves and maximum humidity were significant in influencing the abundance of Ae. taeniorhynchus, while maximum humidity was found to significantly influence the presence of Cx. quinquefasciatus. Both species occurred in sites where temperature, precipitation, and humidity should allow for mosquito development as well as parasitic development of the protozoan parasites that cause avian malaria. Further research involving year‐round sampling of mosquitoes and accompanying meteorological data as well as experimental studies on vector competence are required to understand disease dynamics of parasites such as avian malaria in Galápagos.     

Defense reactions of mosquitoes to filarial worms: comparative studies on the response of three different mosquitoes to inoculated Brugia pahangi and Dirofilaria immitis microfilariae

The melanization response of Aedes trivittatus and the Rockefeller (RKF) and black-eyed Liverpool (LVP) strains of Aedes aegypti against intrathoracically inoculated Dirofilaria immitis and Brugia pahangi microfilariae (mff) was investigated. All mff of either species were melanized in A. trivittatus following Day 2 postinoculation, and the response of this species was significantly more rapid and effective than either strain of A. aegypti. The refractory RKF strain had a significantly greater response against both D. immitis and B. pahangi than the highly susceptible LVP strain, but data suggest that the increased responsiveness was due to a physiologic incompatibility in RKF A. aegypti, thereby resulting in a greater mortality and subsequent melanization of inoculated mff. Inoculation of large numbers of mff overloaded the defense capabilities of A. aegypti (LVP), but not those of A. trivittatus. The melanization response against D. immitis mff was effectively reduced for up to 4 days in A. aegypti (LVP), but for only 1 day in A. trivittatus, when mosquitoes were maintained on a 0.3 m sucrose diet containing from 0.1 to 1.0% (w/v) phenylthiourea.     

Bioassay of three strains of Bacillus sphaericus on field-collected mosquito larvae.

Bacillus sphaericus strains 1593, 1404, and SSII-1 were assayed for infectivity against field-collected larvae of Psorophora columbiae, Culex nigripalpus, and Aedes taeniorhynchus in southwest Florida. Results indicate that all three strains are highly active against the Psorophora and Culex species. A. taeniorhynchus is also susceptible but requires higher dosages to achieve lethal responses. Tests were also conducted on the rate of infection and the differences in susceptibility of different instars to B. sphaericus. These tests indicate that nearly 75% of the mortality that occurs in the course of exposure to B. sphaericus occurs within 48 hr post-incubation with the bacteria. Furthermore, our tests indicate P. columbiae larvae decrease in susceptibility to the Bacillus with increase in larval age (instar). This investigation shows B. sphaericus to be a feasible biological control agent that warrants further study.     

Transepithelial transport of salicylate by the Malpighian tubules of insects from different orders

The organic anion salicylate is a plant secondary metabolite that protects plants against phytophagous insects. In this study, a combination of salicylate-selective microelectrodes and a radioisotope tracer technique was used to study the transepithelial transport of salicylate by the Malpighian tubules of 10 species of insects from five orders. Our results show that salicylate is transported into the lumen of the Malpighian tubules in all the species evaluated, except Rhodnius prolixus. The transepithelial transport of salicylate by the Malpighian tubules of Drosophila simulans, Drosophila erecta, Drosophila sechellia, and Acheta domesticus was saturable, Na⁺-dependent and inhibited by α-cyano-4-hydroxycinnamic acid. This transport system resembles that previously found in tubules of Drosophila melanogaster. In contrast, transepithelial transport of salicylate by Malpighian tubules of Tenebrio molitor, Plagiodera versicolora, Aedes aegypti, and Trichoplusia ni was unaffected by Na⁺-free bathing saline. The presence of both salicylate and salicylate metabolites in the secreted fluid samples from the Malpighian tubules of A. domesticus, R. prolixus, T. molitor, and T. ni indicates that insect Malpighian tubules may both transport and metabolize salicylate. The highest capacities to rid the hemolymph of salicylate were found in T. molitor, P. versicolora and Drosphila spp. Our results suggest that transport of salicylate by the Malpighian tubules might contribute to elimination of this organic anion from the hemolymph, particularly in some species that encounter high levels of organic anion in the diet.     

Isolation and identification of entomopathogenic fungus from Eastern Ghats of South Indian forest soil and their efficacy as biopesticide for mosquito control

The repeated usage of chemical insecticides, responsible for insecticide resistance in mosquitoes and environmental toxicity. Currently effective and environmental-safe control strategies are needed for the control disease-vector mosquitoes. Entomopathogens can be an effective alternative to chemical insecticide. Herein we isolated and tested 46 soil-borne entomopathogenic fungi belonging to six genera, namely Beauveria sp., Metarhizium sp., Fusarium sp., Aspergillus sp., Trichoderma sp., and Verticillium sp., fungi conidia were tested on Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus larvae. Bioassays results show that M. anisopliae fungal isolate causes a 100%, 98.6% and 92% mortality within six days, on Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus, respectively. M. anisopliae treated three mosquito larvae have lower lifetime with LT₅₀ values in A. stephensi, 2.931 days; A. aegypti, 2.676 days and C. quinquefasciatus, 3.254 days. 18 s rDNA sequence analysis confirmed that the isolated fungus are belonging to the genus of M. anisopliae-VKKH3, B. bassiana-VKBb03, and V. lecanii-VKPH1. Our results clearly show that M. anisopliae has good potential, as a low-cost, environmentally safe tool for the control of A. aegypti, A. stephensi, and C. quinquefasciatus mosquitoes.     

Seasonal and diel patterns of biting midges (Ceratopogonidae) and mosquitoes (Culicidae) on the Parris Island Marine Corps Recruit Depot

The Marine Corps Recruit Depot on Parris Island, SC, is surrounded by tidal salt marshes, which are breeding habitats for many pestiferous biting flies. Knowledge of biting fly behavior patterns is needed to develop effective pest management strategies in urban areas adjacent to salt marshes. We measured biting midge (Ceratopogonidae) and mosquito (Culicidae) seasonal abundance and diel activity patterns on Parris Island using CO₂‐baited suction traps from November 2001 – November 2004. Of the three biting midge species collected, Culicoides furens was most abundant (86.2% of total) and was present in high numbers from late March to November. Culicoides hollensis (12.0% of total) was present during spring and fall but absent in summer and winter; and Culicoides melleus (1.7% of total) was present in spring through fall but absent in winter. Abundance of C. furens had a positive linear correlation with air temperature and rainfall. There were nonlinear correlations between air temperature and C. hollensis and C. melleus numbers, which were most abundant at moderate temperatures. Of 18 mosquito species collected, the most abundant were Aedes taeniorhynchus (42.7% of total), Aedes sollicitans (26.3% of total), Culex salinarius (15.6% of total), Culex quinquefasciatus (7.3% of total), and Aedes vexans (5.7% of total); other species comprised <5% of collections. Aedes taeniorhynchus numbers were positively correlated with temperature and rainfall, and Cx. salinarius was correlated with soil moisture. Activity of most biting midges and mosquitoes were highest the first two hours following sunset. Species of biting flies were present in all months, suggesting that year‐round control measures are necessary to reduce exposure to potential disease vectors and nuisance biting.     

First record of West Nile Virus detection inside wild mosquitoes in Khartoum capital of Sudan using PCR

This study aimed to explore the presence of West Nile Virus (WNV) inside four species of mosquitoes: Culex univittatus (Theobald), Culex quinquefasciatus (Say) Aedes vittatus (Bigot) and Aedes vexans (Meigen). Adult wild mosquitoes were collected from different sites: Soba West, Hellat Kuku, Shambat, and Khartoum North Central Live Stock Market (KCLM). Surveys were carried out at Khartoum State during two phases: pre to the rainy season and post to the rainy season. Mosquito specimens were identified using classical keys then preserved at −80 °C freezer for two weeks till the virus examination using polymerase chain reaction (PCR) were carried out. WNV has been detected inside the three species of mosquitoes: A. vexans, C. univittatus, and C. quinquefasciatus. The species were collected from Hellat Kuku, (Shambat and Hellat Kuku), and (Shambat and KCLM) respectively. Two species of mosquitoes were positive for the virus: C. quinquefasciatus and C. univittatus. Positive results for the virus during the first phase of the study; males of C. quinquefasciatus and C. univittatus collected during the second phase of the study were also tested for the existence of the virus and they were positive. For our knowledge this study represents first record of WNV inside wild mosquitoes in Sudan. PCR technique provided reliable information because specific primer-probe sets were used for the detection of the virus. Extra studies are required to incriminate these species of mosquitoes as potential vectors of WNV.