Plasmodium hermani: Experimental transmission by Culex salinarius and comparison with other susceptible Florida mosquitoes

Culex salinarius is susceptible to Plasmodium hermani, a malarial parasite of wild turkeys in Florida. The sporogonous cycle was completed and mosquitoes with infected salivary glands transmitted the parasite by bites. Transmission was also achieved by intraperitoneal and intravenous injections of whole body slurry. This is the third species found to be susceptible to turkey malaria in Florida. A comparison of C. salinarius with two other susceptible Florida mosquitoes, Culex nigripalpus and Wyeomyia vanduzeei, revealed that C. salinarius was more susceptible to P. hermani based on oocyst counts. C. nigripalpus has previously been demonstrated as an experimental and a natural vector of P. hermani, whereas W. vanduzeei has been designated as an experimental host only. In W. vanduzeei at least 30% of the oocysts were melanized (“black bodies”) and this mosquito did not transmit the parasite via bites. Additional detailed comparisons of comparative susceptibility and transmission potentials of these three species to turkey malaria, P. hermani, have been made.     

Factor Affecting Sex Ratios of a Mermithid Parasite of Mosquitoes

The ratio of male to female Reesimermis nielseni Tsai and Grundmann, a nematode parasite of mosquito larvae, increased as the number of parasites per host increased. Hosts with a single nematode produced 9% males compared with essentially 100% males in hosts with more than 7 parasites; hosts with 3 nematodes produced about equal numbers of males and females. Males of R. nielseni generally emerged before females because of the earlier death of multiple-infected mosquitoes. The species of the host mosquito influenced the sex ratio, but the size of a specific host at the time of invasion did not. Host diet also had a noticeable influence on the sex ratio of the nematode: singly infected hosts from a starved population produced 92% males compared with 13% in the normally fed group. The importance of these factors in the mass rearing of R. nielseni is discussed.     

A laboratory evaluation of the microsporidian Vavraia culicis as an agent for mosquito control

The susceptibility of the mosquitoes Aedes aegypti, Aedes taeniorhynchus, Anopheles albimanus, Culex pipiens quinquefasciatus, Culex salinarius, and Culex tarsalis to infection by the microsporidian Vavraia culicis was determined. Using 18-hr exposures to 5 × 10³, 1 × 10⁴, 5 × 10⁴, and 1 × 10⁵ spores/ml, C. salinarius, C. tarsalis, and A. albimanus were found to be significantly more susceptible than A. aegypti. The most severe infections were observed in C. salinarius and C. tarsalis, although heavy infections of approximately 1 million spores per adult were recorded at the higher dosages in all species tested except A. aegypti. Production trials indicated that up to 5.4 × 10⁸ spores could be routinely produced in individual corn earworms, Heliothis zea. Inactivation of the spores by sunlight was measured by observing the subsequent incidence of infection and spore production in A. albimanus. These two measurements provided an LT₉₀ of 5.5 and 3.3 hr, respectively.     

The comparative activity of the Australian and united states strains of Culicinomyces clavosporus bioassayed in mosquito larvae of three different genera

Anopheles hilli, Culex quinquefasciatus, and Aedes aegypti were used as test insects to compare the activity of the Australian and United States strains of Culicinomyces clavosporus. To minimize the variability incurred by using different larval batches, both strains were bioassayed at the same time using one batch of larvae. Six pairs of assays for each of the three test species were conducted in this manner. It was found that there was no difference in potency of the two strains in any one of the three species. A between species comparison, with the data pooled for both strains, showed that A. aegypti was more susceptible to the fungus than A. hilli. The susceptibility of C. Quinquefasciatus appeared to be intermediate but the fiducial limits of the weighted mean LC₅₀ overlapped with those of the other two species. From the results of these experiments it would seem that, with regard to potency, both strains of Culicinomyces may be equally promising for the biological control of mosquitoes.     

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.     

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.     

Studies on the culicine mosquito host range of Bacillus sphaericus and Bacillus thuringiensis var. israelensis with notes on the effects of temperature and instar on bacterial efficacy

Toxicity tests of three strains of Bacillus sphaericus against late instars of 12 culicine mosquito species indicated a wide range of susceptibility. Culex pipiens and C. salinarius were highly susceptible (LC₅₀s < 10⁴ spores/ml) to strain 1593, and C. pipiens and C. restuans were highly susceptible to strain 2013-4. The potency of strain SSII-1 was approximately one-tenth that of strains 1593 and 2013-4 against C. pipiens. Susceptibility of Aedes species to strain 1593 was highly variable. At temperatures ≥ 20°C, A. fitchii, A. intrudens, A. stimulans, and A. vexans were moderately to highly susceptible (LC₅₀s 6 × 10³−4 × 10⁴ spores/ml), A. triseriatus was only slightly susceptible (LC₅₀ > 10⁶ spores/ml), and A. aegypti was refractory. Susceptibility of Aedes mosquitoes to strain SSII-1 was less variable, with LC₅₀s against A. aegypti, A. canadensis, A. stimulans, and A. triseriatus all being between 10⁴ and 10⁶ vegetative cells + spores/ml. All species of mosquitoes tested were, in general, highly susceptible to B. thuringiensis var. israelensis (LC₅₀s 2.3 × 10³−2.5 × 10⁴ spores/ml). In B. sphaericus toxicity tests, decreased temperatures resulted in up to a 16-fold increase in LC₅₀ and a substantial reduction in probit line slope. First-instar A. aegypti larvae were more susceptible to B. sphaericus strain SSII-1 than the three later instars, which were approximately equally susceptible; however, no significant difference was observed in the susceptibility of the four instars of A. triseriatus.     

In vivo germination and host specificity of Nosema algerae in mosquitoes.

Of five species of mosquitoes tested, Anopheles stephensi and A. albimanus were the most susceptible, A. quadrimaculatus and Culex pipiens were relatively unsusceptible and A. atroparvus was almost completely refractory to Nosema algerae. Spore germination percentages differed in the larval midguts of the five species of mosquito but not in the order which would explain the susceptibility differences. Neither midgut pH difference nor differences in passage rate through the midgut were sufficient to explain the observed differences in spore germination percentages.     

Physiological basis of host susceptibility of Florida mosquitoes to Dirofilaria immitis.

Young females of seven species of Florida mosquitoes were fed a meal of dog blood infected with Dirofilaria immitis to repletion to study the physiological mechanisms which control susceptibility and resistance in these mosquitoes. Various species of mosquitoes showed different grades of susceptibility. In all mosquitoes, microfilariae reached the midgut immediately after ingestion. Their movement from midgut to the specific host tissue—the Malpighian tubules—was either facilitated or inhibited depending on the presence or absence of anticoagulins in the salivary glands of these mosquitoes. In Anopheles quadrimaculatus, Aedes taeniorhynchus, and Aedes sollicitans, microfilariae move freely from the midgut to the Malpighian tubules, because of the presence of substantial amounts of anticoagulins in their salivary glands, and 30 to 60 mf/female developed normally to an infective stage. Very few microfilariae reached the tubules of Mansonia titillans as most of them were defaecated within a very short time after ingestion. In Aedes aegypti, Culex nigripalpus, and Culex pipiens quinquefasciatus movement of microfilariae from the midgut to the Malpighian tubules was obstructed by the coagulation of blood soon after ingestion. Coagulation of blood was followed by formation of oxyhaemoglobin crystals in C. nigripalpus and C. p. quinquefasciatus. It is suggested that secretions from symbiotic bacteria in the midgut of these mosquitoes lyse ingested red blood cells, and the released haemoglobin is oxidized to oxyhaemoglobin crystals which hinder the further movement of microfilariae and kill them.Microfilariae developed normally in A. quadrimaculatus, thus making them potentially the most susceptible mosquitoes, even though these mosquitoes did not survive to be effective potential vectors. A few microfilariae or their later developmental stages were melanized in the tubules of most A. sollicitans and A. taeniorhynchus, but the numbers of melanized stages were too few to affect the vectoring potentials of these species. In 20 per cent of A. sollicitans, 60 per cent of M. titillans, and ca. 80 per cent A. aegypti substantial numbers of the microfilariae after reaching the Malpighian tubules did not advance beyond the prelarval stage, and very few microfilariae developed successfully in the remaining mosquitoes. Very few microfilariae reached the Malpighian tubules of a small percentage of C. nigripalpus and C. p. quinquefasciatus and developed normally. The vectoring potentials of A. sollicitans, M. titillans, A. aegypti, and both Culex species were greatly hampered. These studies suggested that host-specificity of mosquitoes to D. immitis infection is controlled by the presence or absence of secondary physiological factors in their digestive tracts or in the Malpighian tubules.     

Role of mermithid nematodes in biological control of mosquitoes

Mermithid nematodes have been reported from at least 63 species of mosquitoes from all over the world, but until recently they have received little attention. Such nematodes are prime candidates as biological control agents because they have adapted to the life cycle of the host; are host specific; produce high levels of parasitism; kill the hosts; are easily handled; have a high reproductive potential; are free swimming and can be disseminated easily in the infective stage; and can be used in an inundative or inoculative manner to control mosquitoes. Nevertheless, with the exception of one mermithid species, little is known about factors that influence parasitism in mosquitoes. Also, only one species of mermithid has been successfully mass cultured to date. The exception is the mermithid Reesimermis nielseni. With this mermithid, high levels of parasitism can be obtained in natural populations of mosquitoes at reasonable dosages; preparasitic nematodes can be applied easily by using many of the standard techniques used to apply insecticides; and R. nielseni can establish itself in many of the sites in which it is released. The same potentials exist for mermithids of other aquatic insects.     

Susceptibility of larvae ofHeliothis zea,H. virescens,andH. armigera [lep.: Noctuidae] to 3 baculoviruses

The pattern of virulence (based on inclusion bodies) for 3 baculoviruses ofHeliothis, i.e. a unicapsid, nuclear polyhedrosis virus (HzSNPV); a multicapsid, nuclear polyhedrosis virus (HaMNPV); and a granulosis virus (HaGIV) was the same (HzSNPV>HaMNPV>HaGIV) for 3 species ofHeliothis. Based on numbers of nucleocapsids, however, the HaGIV was ca 2X more virulent than the HaMNPV for larvae ofH. virescens, (F.), and the HaMNPV was about 6X more virulent than the HaGIV for larvae ofH. armigera (Hübner). The fastest rate of larval mortality was obtained with HzSNPV. Although the mortality rate for HaGIV was faster than that of HaMNPV forH. virescens andH. armigera, it was slower than that of HaMNPV for larvae ofH. zea (Boddie). The pattern of susceptibility ofHeliothis species to HzSNPV and HaMNPV wasH. zea>H. virescens>H. armigera. Differences in susceptibility of the least susceptible species (H. armigera) and the most susceptible species (H. zea) to HzSNPV was ca. 1.6 X. Larvae ofH. zea, however, were ca. 4 to 6 X more susceptible to HaMNPV than were larvae ofH. virescens orH. armigera. A different pattern of susceptibility was recorded for HaGIV when larvae were challenged with HzSNPV and HaMNPV. Larvae ofH. virescens were ca. 20 and 35 X more susceptible to HaGIV than were larvae ofH. zea andH. armigera, respectively.     

Factors Influencing the Chlorine Susceptibility of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum

The susceptibility of representative strains of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum (the MAIS group) to chlorine was studied to identify factors related to culture conditions and growth phase that influenced susceptibility. M. avium and M. intracellulare strains were more resistant to chlorine than were strains of M. scrofulaceum. Transparent and unpigmented colony variants were more resistant to chlorine than were their isogenic opaque and pigmented variants (respectively). Depending on growth stage and growth rate, MAIS strains differed in their chlorine susceptibilities. Cells from strains of all three species growing in early log phase at the highest growth rates were more susceptible than cells in log and stationary phase. Rapidly growing cells were more susceptible to chlorine than slowly growing cells. The chlorine susceptibility of M. avium cells grown at 30°C was increased when cells were exposed to chlorine at 40°C compared to susceptibility after exposure at 30°C. Cells of M. avium grown in 6% oxygen were significantly more chlorine susceptible than cells grown in air. Chlorine-resistant MAIS strains were more hydrophobic and resistant to Tween 80, para-nitrobenzoate, hydroxylamine, and nitrite than were the chlorine-sensitive strains.     

Susceptibility of Various Species of Lepidopterous Pupae to the Entomogenous Nematode Neoaplectana carpocapsae

The susceptibility of certain species of lepidopterous pupae occurring in different ecological situations to the entomogenous nematode, Neoaplectana carpocapsae, was tested. Soil- or litter-pupating lepidopterous insects were not highly susceptible to N. carpocapsae. The most susceptible insect pupating in the soil was Spodoptera exigua with 63% pupal mortality, while Harrisinia brillians, which pupates in litter, had 55% mortality. Other soil- and litter-pupating insects had mortalities of less than 25%. Some insect species that pupate above ground were highly susceptible (> 84% mortality) to N. carpocapsae infection.     

Plasmodium berghei: mechanisms and sites of resistance to sporogonic development in different mosquitoes

An experimental study of the mechanisms and patterns of resistance to Plasmodium berghei in different mosquito species revealed a diversity of factors which prevent or inhibit sporogonic development in its different phases and in different sites in the mosquito vector. The experiments showed that Culex salinarius was a totally resistant species in which exflagellation and ookinete formation are prevented. In Aedes aegypti, ookinetes in small or moderate numbers are formed but penetration of the mosquito midgut wall is blocked and oocysts are not formed. In the three experimental vectors, Anopheles quadrimaculatus, Anopheles aztecus, and Anopheles stephensi grades of enhanced susceptibility are recognized. They are expressed in lesser numbers of abnormal and degenerative oocysts, in higher numbers of sporozoites in the salivary gland, and greater viability and invasiveness of these sporozoites. In Anopheles dureni, the natural vector of rodent malaria, one observes both in nature and under experimental conditions the highest adaptation and most pronounced grade of susceptibility to P. berghei.     

Metagenomic sequencing reveals viral abundance and diversity in mosquitoes from the Shaanxi-Gansu-Ningxia region,China

BackgroundMosquitoes host and transmit numerous arthropod-borne viruses (arboviruses) that cause disease in both humans and animals. Effective surveillance of virome profiles in mosquitoes is vital to the prevention and control of mosquito-borne diseases in northwestern China, where epidemics occur frequently.MethodsMosquitoes were collected in the Shaanxi-Gansu-Ningxia region (Shaanxi Province, Gansu Province, and Ningxia Hui Autonomous Region) of China from June to August 2019. Morphological methods were used for taxonomic identification of mosquito species. High-throughput sequencing and metagenomic analysis were used to characterize mosquito viromes.ResultsA total of 22,959 mosquitoes were collected, including Culex pipiens (45.7%), Culex tritaeniorhynchus (40.6%), Anopheles sinensis (8.4%), Aedes (5.2%), and Armigeres subalbatus (0.1%). In total, 3,014,183 (0.95% of clean reads) viral sequences were identified and assigned to 116 viral species (including pathogens such as Japanese encephalitis virus and Getah virus) in 31 viral families, including Flaviviridae, Togaviridae, Phasmaviridae, Phenuiviridae, and some unclassified viruses. Mosquitoes collected in July (86 species in 26 families) showed greater viral diversity than those from June and August. Culex pipiens (69 species in 25 families) and Culex tritaeniorhynchus (73 species in 24 families) carried more viral species than Anopheles sinensis (50 species in 19 families) or Aedes (38 species in 20 families) mosquitoes.ConclusionViral diversity and abundance were affected by mosquito species and collection time. The present study elucidates the virome compositions of various mosquito species in northwestern China, improving the understanding of virus transmission dynamics for comparison with those of disease outbreaks.     

Comparative morphology of the pyloric armature of adult mosquitoes (Diptera: Culicidae)

The structure of the pyloric armature, hypothesized to aid in blood-meal digestion or parasite resistance, was compared quantitatively among the following 8 species in 5 genera of adult mosquitoes from the southeastern United States: Aedes albopictus, Aedes japonicus, Aedes triseriatus, Anopheles punctipennis, Culex pipiens s.l., Culex restuans, Orthopodomyia signifera, and Toxorhynchites rutilus. Females differed significantly among species in the structure of spines composing the armature, with Aedes spp. forming one general group, Culex spp. another, and An. punctipennis and Or. signifera a third. Relationships of species based on structural characters of the armature were consistent with recent culicid phylogenies. Although pyloric armature has been noted in mosquitoes and other insects, this is the first quantitative investigation of the mosquito pyloric armature.     

Differential susceptibility of lepidopterous pupae to infection by the nematode Neoaplectana carpocapsae

Prepupae of Galleria mellonella, Spodoptera exigua, and Pseudaletia unipuncta were highly susceptible to infection by the nematode Neoaplectana carpocapsae and its associated bacterium, Xenorhabdus nematophilus. On the other hand, pupae of the three species were significantly different in their susceptibility to the nematode. G. mellonella pupae were highly susceptible (100% mortality), S. exigua pupae were moderately susceptible (ca. 75% mortality), and P. unipuncta pupae were least susceptible (ca. 54% mortality). In the latter two species, many pupae died without any nematode development, but the typical signs associated with a nematode infection were evident. Age of the pupae and increasing the dosage of the nematode did not significantly affect mortality of S. exigua or P. unipuncta.     

Relative susceptibility of several noctuid species to a nuclear polyhedrosis virus from Heliothis armiger

Several alternate hosts were tested for their relative susceptibility to an isolate of Galleria mellonella nuclear polyhedrosis virus. Neonate Trichoplusia ni, Heliothis zea, and Manduca sexta were all susceptible to per oral administration of purified polyhedra. Of the three alternate species tested, T. ni was the most susceptible, and exhibited the most variable mortality response over the dose range tested, while M. sexta was the least susceptible. We believe this represents the first report of a lethal virus infection in a sphingid species, and useful parameters for the successful inoculation of alternate hosts are discussed.     

Two Species of the Family Incurvariidae (Lepidoptera) New to Korea

Two species of the family Incurvariidae, Vespina nielseni Kozlov and Paraclemensia monospina Nielsen, are reported for the first time from Korea. The morphological characteristics of adults and the female genitalia of the two species are redescribed briefly, with illustrations.     

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.