Effect of Aedes aegypti (Diptera:Culicidae) Age on Sex Ratios in Romanomermis culicivorax (Nematoda:Mermithidae)

First, second, third, early and late fourth-instar larvae, and pupae of Aedes aegypti were infected with Romanomerrais culicivorax and reared at 20, 25, and 30 C. An increase in the ratio of male to female nematodes was observed with increase in host age at the time of infection at each temperature. The number of pupal and late fourth-instar infections was low, but R. culicivorax continued to develop in adult A. aegypti. Since male nematodes were recovered from both male and female hosts infected as late fourth instars or pupae, the sex of the host did not influence the sex of the nematode.     

The effect of nematode parasitism on the osmolality and major cation concentration in the haemolymph of three larval mosquito species

Parasitism by a mermithid nematode, Romanomermis culicivorax, causes severe depletion of haemolymph carbohydrates and proteins in mosquito larvae. We undertook a study to determine if haemolymph osmolality and cation concentrations were affected also by mermithid parasitism. The haemolymph osmolality of R. culicivorax-infected and control Aedes taeniorhynchus and Culex pipiens fourth-instar larvae was not significantly different. However, the haemolymph osmolality decreased significantly in infected Anopheles quadrimaculatus. Each mosquito species demonstrated significant alterations in the haemolymph concentration of at least one cation when infected although the cation concentrations affected differed for each species. The changes observed were statistically significant but the magnitude of change was not great. Overall, despite the severe nutritional burden of the mermithid nematode, these species of mosquito larvae can continue to maintain osmoregulation.     

Observations on the biology of Octomyomermis muspratti, a nematode parasite of mosquitoes

The mosquito parasite Octomyomermis muspratti was able to infect mosquitoes in diluted sea water with conductivity readings of 4000 μmho/cm and in all dilutions of organically rich tree-hole water. In contrast, the infectivity of Romanomermis culicivorax, a more extensively studied species, was adversely affected in dilutions of sea water with conductivity of 1500 μmho/cm and no infections were observed at concentrations above 3000 μmho/cm. Also, R. culicivorax failed to infect hosts at any dilution of the tree-hole water. Diet was shown to have an effect on male-female sex ratios in developing O. muspratti, and this may be employed to enhance a better male-female ratio in laboratory cultures of this nematode. When cultures of O. muspratti were flooded every 3–4 months, they continued to produce infective-stage nematodes for up to 5 years. Also, cultures that were repeatedly flooded produced infective nematodes for more than 39 floodings.     

Distribution of breeding and control of the filariasis vector Aedes samoanus in leaf axils of Pandanus in Samoa

Water in leaf axils of the screwpine Pandanus was sampled for mosquito immature stages at seventy villages in Upolu, fifty-five in Savai'i and three in Manono, the main islands of Samoa. Ten plants in every patch of Pandanus plantation were sampled at each village. Among 23,049 mosquito larvae collected from Upolu, 77% were the filariasis vector Aedes (Finlaya) samoanus, 17.7% were Ae.(Fin.)oceanicus and 5.3% were Ae.(Fin.)tutuilae. Out of 6981 larvae taken in Savai'i, 23.2% were Ae.samoanus, 67.6%Ae.oceanicus and 9.2%Ae.tutuilae. When larval counts per plant were analysed for each district, Ae.samoanus was found to predominate in Pandanus in Upolu and Ae.oceanicus in Savai'i. However, the adult density of Ae.samoanus was higher in Savai'i and this was attributed to the large areas of forests with Freycinetia for Ae.samoanus breeding. In Pandanus in Savai'i the number of Ae.samoanus was negligible. In Upolu, with more urbanization and larger plantations, there was greater breeding of Ae.samoanus in Pandanus. Two control trials were conducted against Ae.samoanus larvae in Pandanus, one using a sand culture of the parasitic nematode Romanomermis culicivorax and the other with temephos, an organophosphate insecticide. While R.culicivorax did not adapt to the leaf axil habitat, all plants were without larvae for 5 weeks after treatment with temephos.     

Zika Virus Emergence in Mosquitoes in Southeastern Senegal, 2011

Background

Zika virus (ZIKV; genus Flavivirus, family Flaviviridae) is maintained in a zoonotic cycle between arboreal Aedes spp. mosquitoes and nonhuman primates in African and Asian forests. Spillover into humans has been documented in both regions and the virus is currently responsible for a large outbreak in French Polynesia. ZIKV amplifications are frequent in southeastern Senegal but little is known about their seasonal and spatial dynamics. The aim of this paper is to describe the spatio-temporal patterns of the 2011 ZIKV amplification in southeastern Senegal.

Methodology/Findings

Mosquitoes were collected monthly from April to December 2011 except during July. Each evening from 18∶00 to 21∶00 hrs landing collections were performed by teams of 3 persons working simultaneously in forest (canopy and ground), savannah, agriculture, village (indoor and outdoor) and barren land cover sites. Mosquitoes were tested for virus infection by virus isolation and RT-PCR. ZIKV was detected in 31 of the 1,700 mosquito pools (11,247 mosquitoes) tested: Ae. furcifer (5), Ae. luteocephalus (5), Ae. africanus (5), Ae. vittatus (3), Ae. taylori, Ae. dalzieli, Ae. hirsutus and Ae. metallicus (2 each) and Ae. aegypti, Ae. unilinaetus, Ma. uniformis, Cx. perfuscus and An. coustani (1 pool each) collected in June (3), September (10), October (11), November (6) and December (1). ZIKV was detected from mosquitoes collected in all land cover classes except indoor locations within villages. The virus was detected in only one of the ten villages investigated.

Conclusions/Significance

This ZIKV amplification was widespread in the Kédougou area, involved several mosquito species as probable vectors, and encompassed all investigated land cover classes except indoor locations within villages. Aedes furcifer males and Aedes vittatus were found infected within a village, thus these species are probably involved in the transmission of Zika virus to humans in this environment.     

Analysis of molecular markers for metamorphic competency and their response to starvation or feeding in the mosquito, Aedes aegypti (Diptera: Culicidae)

The nutritional condition of fourth instar larvae of the yellow fever mosquito, Aedes aegypti, governs female longevity and egg production, both are key determinants of pathogen transmission. As well, nutrition provisions larval growth and development and attains its greatest pace in the last larval instar in preparation for metamorphosis to an adult. These developmental processes are regulated by a complex endocrine interplay of juvenile hormone, neuropeptides, and ecdysteroids that is nutrition sensitive. We previously determined that feeding for only 24 h post-ecdysis was sufficient for fourth instar Ae. aegypti larvae to reach critical weight and accumulate sufficient nutritional stores to commit to metamorphosis. To understand the genetic basis of metamorphic commitment in Ae. aegypti, we profiled the expression of 16 genes known to be involved in the endocrine and nutritional regulation of insect metamorphosis in two ways. The first set is a developmental profile from the beginning of the fourth instar to early pupae, and the second set is for fourth instars starved or fed for up to 36 h. By comparing the two sets, we found that seven of the genes (AaegCYP302, AaegJHE43357, AaegBrCZ4, AaegCPF1-2, AaegCPR-7, AaegPpl, and AaegSlif) were expressed during metamorphic commitment in fourth instars and in fed but not starved larvae. Based on these results, the seven genes alone or in combination may serve as molecular indicators of nutritional and metamorphic status of fourth instar Ae. aegypti larvae and possibly other mosquito species in field and laboratory studies to gauge sub-lethal effects of novel and traditional cultural or chemical controls.     

Predator–prey interactions and the cannibalism of larvae of Armigeres subalbatus (Diptera: Culicidae)

Predation and cannibalism can affect the co-existence of mosquito species or the assemblage of mosquito species (i.e., community structure). In this study, predatory feeding patterns and cannibalism of larvae of Armigeres subalbatus mosquitoes were quantified under laboratory conditions using Aedes albopictus and Culex uniformis larvae as prey organisms. Rate of consumption of prey larvae and the rate of cannibalism of 1st to 4th instar larvae of Armigeres subalbatus were reported at 24 h intervals with and without alternative food supplement. Both the 3rd and 4th instar larvae of Ar. subalbatus showed a substantial predation on the larvae of Ae. albopictus (33.6 ± 4.4) and Cx uniformis (47.3 ± 7.6). The cannibalism of the predatory larvae was strongly correlated to the larval density (r = 0.9). The predator and the prey density and the given food type were significant factors that determined the rate of pupation, death and the emergence as adults. A significant relationship was shown for the co-occurrence of predatory larvae and the prey larvae at the 85 natural breeding habitats observed at the field (χ² = 74.4, p < 0.001). Nine breeding sites (10.6%) were positive for both prey and predatory larvae. Our study showed robust information about the predatory and cannibalistic behavior of Ar. subalbatus larvae emphasizing their role in managing the population structure of mosquito communities.     

The effect of parasitism by the mermithid Romanomermis culicivorax on the dry weight and hemolymph soluble protein content of three species of mosquitoes

The dry weight, hemolymph soluble protein composition, and content of three species of mosquitoes, Culex pipiens, Aedes taeniorhynchus, and Anopheles quadrimaculatus were examined to determine the effects of parasitism by the mermithid nematode Romanomermis culicivorax. The dry weights of infected fourth-stage larvae of all three species were significantly lower than controls. The differences in weight found between infected early and late C. pipiens and A. quadrimaculatus larvae were attributed to the weight of the parasite itself. This difference was not noticeable in A. taeniorhynchus larvae. Hemolymph proteins were severely depleted in all three mosquito species during parasitism by R. culicivorax. Analysis of protein composition by PAGE showed that these depletions were accompanied by a reduction in the number of proteins. Differences between protein composition concentrations were evident between early and late fourthstage control larvae of C. pipiens and A. quadrimaculatus. The concentration of some low-molecular-weight proteins (below 68,000) remained constant between infected and control samples of all three mosquito species.     

Matrix-Assisted Laser Desorption Ionization - Time of Flight Mass Spectrometry: An Emerging Tool for the Rapid Identification of Mosquito Vectors

Background

The identification of mosquito vectors is typically based on morphological characteristics using morphological keys of determination, which requires entomological expertise and training. The use of protein profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), which is increasingly being used for the routine identification of bacteria, has recently emerged for arthropod identification.

Methods

To investigate the usefulness of MALDI-TOF-MS as a mosquito identification tool, we tested protein extracts made from mosquito legs to create a database of reference spectra. The database included a total of 129 laboratory-reared and field-caught mosquito specimens consisting of 20 species, including 4 Aedes spp., 9 Anopheles spp., 4 Culex spp., Lutzia tigripes, Orthopodomyia reunionensis and Mansonia uniformis. For the validation study, blind tests were performed with 76 specimens consisting of 1 to 4 individuals per species. A cluster analysis was carried out using the MALDI-Biotyper and some spectra from all mosquito species tested.

Results

Biomarker mass sets containing 22 and 43 masses have been detected from 100 specimens of the Anopheles, Aedes and Culex species. By carrying out 3 blind tests, we achieved the identification of mosquito vectors at the species level, including the differentiation of An. gambiae complex, which is possible using MALDI-TOF-MS with 1.8 as the cut-off identification score. A cluster analysis performed with all available mosquito species showed that MALDI-Biotyper can distinguish between specimens at the subspecies level, as demonstrated for An gambiae M and S, but this method cannot yet be considered a reliable tool for the phylogenetic study of mosquito species.

Conclusions

We confirmed that even without any specific expertise, MALDI-TOF-MS profiling of mosquito leg protein extracts can be used for the rapid identification of mosquito vectors. Therefore, MALDI-TOF-MS is an alternative, efficient and inexpensive tool that can accurately identify mosquitoes collected in the field during entomological surveys.     

Intraguild predation of Serangium parcesetosum (Coleoptera: Coccinellidae), on whitefly Bemisia tabaci (Homoptera: Aleyrodidae) parasitized by Eretmocerus mundus (Hymenoptera: Aphelinidae)

We studied intraguild predation of Serangium parcesetosum on Eretmocerus mundus, both natural enemies of Bemisia tabaci, on eggplant under laboratory conditions. Fourth instar and adult predators attacked parasitized and unparasitized hosts equally if immature parasitoids were larvae, but avoided predating on parasitized hosts containing pupae.     

Polyamine Synthesis by the Mermithid Nematode Romanomermis culicivorax

The polyamine and amino acid composition of the mermithid nematode, Romanomermis culicivorax, and its host, Aedes aegypti, was determined. Putrescine, spermidine, spermine, cadaverine and two acetylated spermidine derivatives were present in parasitic juveniles, newly-emerged post-parasites, and eggs of R. culicivorax. Whole insect homogenates of fourth-instar A. aegypti contained the same array of polyamines, except that the putrescine:spermidine ratio was the inverse of that in parasitic R. culicivorax. Polyamines and amino acids were in greater concentrations in the nematode eggs than in other developmental stages investigated. Both the host and nematode possess the biosynthetic capacity for polyamine biosynthesis, as evidenced by measurable activities of ornithine decarboxylase in the host''s tissues and the nematode''s free-living stages.     

Parasitism of the mosquito Culex pipiens by the nematode Heterorhabditis bacteriophora

Various concentrations of the nematode Heterorhabditis bacteriophora were added to dishes containing second, third, and fourth larval instars of the mosquito, Culex pipiens, respectively. The infective stage nematodes were ingested by the mosquito larvae, they then penetrated through the alimentary tract in the neck region and entered the hemocoel. A melanization reaction killed many invading nematodes, but heavier concentrations overwhelmed the hosts' defense reaction and 100% mortality of third- and fourth-instar larvae was achieved using between 170 and 200 nematodes per host. Death was either due to the nematode releasing cells of the symbiotic bacterium, Xenorhabdus luminescens, into the hemocoel or to foreign bacteria (mostly Pseudomonas aeruginosa), which were introduced by the penetrating nematodes. The potential use of this nematode as a biological control agent of larval culicine mosquito is discussed.     

Larvicidal and mosquito repellent action of peppermint (Mentha piperita) oil

Oil of Mentha piperita L. (Peppermint oil), a widely used essential oil, was evaluated for larvicidal activity against different mosquito species: Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus by exposing IIIrd instar larvae of mosquitoes in enamel trays 6 × 4 inch² size filled to a depth of 3 inch with water. Of the three species tested Cx. quinquefasciatus was most susceptible followed by Ae. aegypti and An. stephensi. Application of oil at 3 ml/m² of water surface area resulted in 100% mortality within 24 h for Cx. quinquefasciatus, 90% for Ae. aegypti and 85% for An. stephensi. For Ae. aegypti 100% mortality was achieved at 3 ml/m² in 48 h or 4 ml/m² in 24 h. For An. stephensi 100% mortality was observed at 4 ml/m² in 72 h. The emergence at 3 ml/m² was also inhibited to a great extent and the few adults which emerged did not ovipost even after taking a blood meal. The oil showed strong repellent action against adult mosquitoes when applied on human skin. Percent protection obtained against An. annularis, An. culicifacies, and Cx. quinquefasciatus was 100%, 92.3% and 84.5%, respectively. The repellent action of Mentha oil was comparable to that of Mylol oil consisting of dibutyl and dimethyl phthalates.     

Interspecific discrimination and larval competition amongMicroplitis croceipes,Microplitis demolitor,Cotesia kazak (Hym.: Braconidae), andHyposoter didymator (Hym.: Ichneumonidae), parasitoids ofHeliothis virescens (Lep.: Noctuidae)

Interspecific host discrimination by adults, and larval competition among the endoparasitoidsMicroplitis croceipes (Cresson),Microplitis demolitor Wilkinson,Cotesia kazak (Telenga) andHyposoter didymator (Thunberg) were investigated usingHeliothis virescens (F.) as the host. In ovipositional choice tests, the mean number of encounters and ovipositions for unparasitized hosts was not significantly different from the mean number of encounters and ovipositions for parasitized hosts for each treatment combination (P>0.05). Thus, none of the parasitoid species discriminated between host larvae recently parasitized once by a female of another species und unparasitized hosts. However, in all but two cases, females did discriminate between unparasitized hosts and hosts in which an early first instar of the first-attacking species was developing.Cotesia kazak andH. didymator did not discriminate between unparasitized hosts and hosts parasitized by an early first instar ofM. demolitor. Larval competition among these parasitoid species was studied for three time intervals between the first and second species parasitization: 1) second species attack immediately (5–15 sec) after the first; 2) second species attack 24 h after the first; and 3) second species attack 48 h after the first. Time until egg eclosion was shortest forM. demolitor, thenC. kazak, thenM. croceipes, and longest forH. didymator. When the second parasitoid species attacked a host immediately after the first species, the species in which egg eclosion occurred first was the victor more frequently, except whenM. demolitor competed withC. kazak andH. didymator. With a 24 h delay between the first and second species to attack, the older first instar from the first parasitization usually outcompeted the younger first instar from the second attack. A first instar from the second species to attack generally outcompeted the second instar of the first species when the second parasitization had been delayed 48 h. Competiors were eliminated mainly by physical attack, butC. kazak andM. croceipes apparently also killedH. didymator eggs by physiological processes.     

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.     

Host range of the parasite Strelkovimermis spiculatus (Nematoda: Mermithidae) in Argentina mosquitoes

Strelkovimermis spiculatus ( Poinar and Camino 1986 ) is a nematode parasite of mosquitoes isolated from the Neotropical region. We investigated the host range of this parasite in mosquito populations for a better understanding of the dynamics of temporary breeding sites. Five grassy‐pool habitats filled by rainwater were sampled from the summer 2007 through the fall 2008. Eight mosquito species were collected: Anopheles albitarsis, Culex chidesteri, Culex dolosus, Culex maxi, Aedes albifasciatus, Psorophora ciliata, Psorophora cyanescens, and Psorophora albigenu. Six of these species were parasitized: Cx. chidesteri, Cx. dolosus, Cx. maxi, Ae. albifasciatus, Ps. ciliata, and Ps. cyanescens. The occurrence of this mermithid in natural mosquito populations was increased from the end of winter to the end of the spring. Prevalence ranged from 11% to 100%. High levels of infections were registered only in Ae. albifasciatus larvae, the most abundant mosquito species (95%), followed by Cx. dolosus (2.7%). Strelkovimermis spiculatus completed its development in all infected mosquito larvae. The presence of S. spiculatus in six natural mosquito populations increases the number of susceptible species to 24.     

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.     

No-choice host range tests for Allotropa sp. near mecrida,a parasitoid of the pink hibiscus mealybug,Maconellicoccus hirsutus (Hemiptera: Pseudococcidae)

Host range studies of the parasitoid Allotropa sp. near mecrida (Walker) (Hymenoptera: Platygastridae) were conducted prior to applying for a permit to release it against the pink hibiscus mealybug, Maconellicoccus hirsutus (Green) (Hemiptera: Pseudococcidae), in southern California. Using M. hirsutus as a control, four mealybug species were tested as potential hosts. Allotropa sp. near mecrida did not successfully reproduce in any of the mealybug species tested, except for the target species, M. hirsutus. The parasitoid was found to host feed upon one of the non-target species within the confined test environment. Subsequently a USDA-APHIS importation and release permit was granted.     

Susceptibility of ten species of mosquito larvae to the parasitic nematode Romanomermis iyengari and its development

Ten species of mosquitoes (Diptera: Culicidae) from five genera were exposed to preparasites of the tropical mermithid nematode species Romanomermis iyengari (Welch) (Nematoda: Mermithidae), a strain isolated in 1978 from Pondicherry. By exposing mosquito larvae during the second instar, nematode infection was invariably lethal, the rate being highest in Culex sitiens Wiedemann (95%) followed by Cx. quinquefasciatus Say (90%), Aedes aegypti (L.) (79%), Anopheles subpictus Grassi (64%), Ae. albopictus (Skuse) and Armigeres subalbatus Coquillett (62%), Cx. tritaeniorhynchus Giles (57%), Mansonia annulifera (Theobald) (46%), An. stephensi Liston (40%) and An. culicifacies Giles (36%). When fourth-instar larvae were exposed, the infection was highest in Ar. subalbatus (66%), followed by An. stephensi (52%), Cx. quinquefasciatus (47%), Ae. aegypti and An. subpictus (42%), Ae. albopictus (30%), An. culicifacies (29%), Cx. sitiens (24%), Cx. tritaeniorhynchus (19%) and Ma. annulifera (8%), with 2-45% of infected culicines surviving to adulthood. The parasitic phase of the nematode lasted 5-7 days in all the host species, yielding 1.1-3.2 parasites per II instar and 1.1-2.5 parasites per IV instar. The overall output of parasites per 100 mosquito larvae (infected + uninfected) was highest for Ae. aegypti when mosquitoes were exposed during II instar (2.53 parasites/larva) and for Ar. subalbatus when mosquitoes were exposed during IV instar (1.65/larva), and lowest for Ma. annulifera exposed during IV instar (0.09/larva). For routine laboratory culture of R. iyengari it is convenient to employ Cx. quinquefasciatus as the host yielding 90-190 parasites/100 larva.     

Carbohydrate-mediated Recognition of Larval Mosquito Hosts by Romanomermis culicivorax

Proteases, glycosidases, and lectins were tested and the results supported a role in host recognition for glycoproteins containing β-glucose and α-mannose on the cuticular surface of host and parasite. Carbohydrates containing α-glucose, galactose, fucose, or N-acetylglucosamine residues apparently are not involved in nematode attachment. Chitin or a related N-acetylglucosamine polymer was found in R. culicivorax preparasites. Treatment of preparasites with neuraminidase, which hydrolyzes sialic acids, increased nematode attachment to Anopheles freeborni larvae.