Differential Survivorship of Invasive Mosquito Species in South Florida Cemeteries: Do Site-Specific Microclimates Explain Patterns of Coexistence and Exclusion?

Within 2 yr of the arrival of the invasive container mosquito Aedes albopictus (Skuse), the previously dominant invasive mosquito Aedes aegypti (L.) disappeared from many Florida cemeteries. At some cemeteries, however, Ae. aegypti populations seem stable despite Ae. albopictus invasion. We sought to understand this variation in the outcome (exclusion, coexistence) of this invasion, given that previous experiments show that Ae. albopictus is the superior larval competitor. We tested experimentally the hypothesis that climate-dependent egg survivorship differs between exclusion and coexistence cemeteries and that differences in invasion outcome are associated with microclimate. Viability of eggs oviposited in the laboratory and suspended in vases at six cemeteries was significantly greater for Ae. aegypti than for Ae. albopictus, and greater in 2001 than in 2006. Cemeteries differed significantly in egg survivorship of Ae. albopictus, but not of Ae. aegypti, which is consistent with the hypothesis that Ae. albopictus suffers site-specific, climate-driven egg mortality that mitigates the competitive superiority of larval Ae. albopictus. Principal component (PC) analysis of microclimate records from vases during the experiments yielded three PCs accounting for >96% of the variance in both years of experiments. Multivariate analysis of variance of the three PCs revealed significant microclimate differences among the six cemeteries and between exclusion versus coexistence cemeteries. Stepwise logistic regression of egg survivorship versus microclimate PCs yielded significant fits for both species, and twice as much variance explained for Ae. albopictus as for Ae. aegypti in both years. Higher mortalities in 2006 were associated with high average daily maximum temperatures in vases, with lethal thresholds for both species at ≈40°C. From 1990 to 2007, vase occupancy by Ae. albopictus increased and that by Ae. aegypti decreased, with increasing seasonal precipitation at one well-sampled cemetery. Results support the hypothesis that locally variable climate-driven mortality of Ae. albopictus eggs contributes to patterns of exclusion of, or coexistence with, Ae. aegypti.

Interpopulation divergence in competitive interactions of the mosquito Aedes albopictus.

Geographic variation in species interactions can have major effects on distributions. Effects of such variation can be particularly evident for invasive species, in which variation in competitive ability can influence invasive success and impacts. We tested the hypothesis that coexistence or exclusion of the resident mosquito Aedes aegypti results from variation among local populations of the invasive Aedes albopictus in competitive interactions with A. aegypti. We also examined the role of variation in fecundity-size relationships in these competitive interactions. We compared competitive abilities of nine North American populations of A. albopictus, three populations from each of three site types: extinction of A. aegypti following A. albopictus invasion, coexistence following A. albopictus invasion, and A. albopictus allopatric to A. aegypti. Competition among larvae from each A. albopictus population and a single A. aegypti population was tested in laboratory microcosms in a response surface design. We found interpopulation differences in competitive ability of A. albopictus, but no strong patterns among site types. Extinction sites had steeper average fecundity-size relationships than coexistence sites and allopatric sites, but this did not translate into superior population performance. Certain individual A. albopictus populations had exceptionally large competitive effects on A. aegypti or poor competitive responses to competition from A. aegypti, but competitive effect and response were not correlated. These results suggest that interpopulation variation in the competitive ability of A. albopictus may only partly explain the geographic pattern of coexistence with or extinction of A. aegypti. Environmental differences among regions may affect the competitive ability of A. albopictus and influence its invasion success and impact.

Differentiating the role of gamma-aminobutyric acid type A (GABAA) receptor subtypes.

The inhibitory tone maintained throughout the central nervous system relies predominantly on the activity of neuronal GABAA (gamma-aminobutyric acid type A) receptors. This receptor family comprises various subtypes that have unique regional distributions, but little is known about the role played by each subtype. The majority of the receptors contain a gamma2 subunit and are sensitive to modulation by BZs (benzodiazepines), but differ with regard to alpha and beta subunits. Mutagenesis studies combined with molecular modelling have enabled a greater understanding of receptor structure and dynamics. This can now be extended to in vivo activity through translation to genetically modified mice containing these mutations. Ideally, the mutation should leave normal receptor function intact, and this is the case with mutations affecting the BZ-binding site of the GABAA receptor. We have generated mutations, which affect the BZ site of different alpha subunits, to enable discrimination of the various behavioural consequences of BZ drug action. This has aided our understanding of the roles played by individual GABAA receptor subtypes in particular behaviours. We have also used this technique to explore the role of different beta subunits in conferring the anaesthetic activity of etomidate. This technique together with the development of subtype-selective compounds facilitates our understanding of the roles played by each receptor subtype.

GABA(A) alpha 1 subunit knock-out mice do not show a hyperlocomotor response following amphetamine or cocaine treatment.

The GABA(A) receptor system provides the major inhibitory control in the CNS, with the alpha 1 beta 2 gamma 2 subunit combination being the most abundant and widely distributed form of the receptor. The alpha1 subunit knock-out (alpha1 KO) mice had a surprisingly mild overt phenotype, despite having lost approximately 60% of all GABA(A) receptors. The alpha1 KO mice had normal spontaneous locomotor activity, but were more sensitive to the sedating/ataxic effects of diazepam than wildtype (WT) mice. Pharmacological modulation of dopamine and N-methyl-D-aspartate (NMDA) receptors also produced altered responses in alpha1 KO mice compared with WT mice. As expected, the NMDA receptor antagonist MK801, amphetamine and cocaine increased locomotor activity in WT mice. Although MK801 increased locomotor activity in alpha1 KO mice, amphetamine and cocaine induced stereotypy not hyperlocomotion. Binding studies showed no gross changes in the total number of D1, D2 or NMDA receptors. Furthermore, pre-pulse inhibition of acoustic startle and the effects of cocaine in conditioned place preference were similar in both alpha1 KO and WT mice, indicating selective rather that global changes in response to dopaminergic agents. These data demonstrate subtle changes in behaviours mediated by neurotransmitters other than GABA in alpha1 KO mice and suggest that compensation may have occurred beyond the GABAergic system.

Loss of the major GABA(A) receptor subtype in the brain is not lethal in mice.

The alpha1beta2gamma2 is the most abundant subtype of the GABA(A) receptor and is localized in many regions of the brain. To gain more insight into the role of this receptor subtype in the modulation of inhibitory neurotransmission, we generated mice lacking either the alpha1 or beta2 subunit. In agreement with the reported abundance of this subtype, >50% of total GABA(A) receptors are lost in both alpha1-/- and beta2-/- mice. Surprisingly, homozygotes of both mouse lines are viable, fertile, and show no spontaneous seizures. Initially half of the alpha1-/- mice died prenatally or perinatally, but they exhibited a lower mortality rate in subsequent generations, suggesting some phenotypic drift and adaptive changes. Both adult alpha1-/- and beta2-/- mice demonstrate normal performances on the rotarod, but beta2-/- mice displayed increased locomotor activity. Purkinje cells of the cerebellum primarily express alpha1beta2gamma2 receptors, and in electrophysiological recordings from alpha1-/- mice GABA currents in these neurons are dramatically reduced, and residual currents have a benzodiazepine pharmacology characteristic of alpha2- or alpha3-containing receptors. In contrast, the cerebellar Purkinje neurons from beta2-/- mice have only a relatively small reduction of GABA currents. In beta2-/- mice expression levels of all six alpha subunits are reduced by approximately 50%, suggesting that the beta2 subunit can coassemble with alpha subunits other than just alpha1. Our data confirm that alpha1beta2gamma2 is the major GABA(A) receptor subtype in the murine brain and demonstrate that, surprisingly, the loss of this receptor subtype is not lethal.

Galanin regulates the postnatal survival of a subset of basal forebrain cholinergic neurons.

The neuropeptide galanin colocalizes with choline acetyltransferase, the synthetic enzyme for acetylcholine, in a subset of cholinergic neurons in the basal forebrain of rodents. Chronic intracerebroventricular infusion of nerve growth factor induces a 3- to 4-fold increase in galanin gene expression in these neurons. Here we report the loss of a third of cholinergic neurons in the medial septum and vertical limb diagonal band of the basal forebrain of adult mice carrying a targeted loss-of-function mutation in the galanin gene. These deficits are associated with a 2-fold increase in the number of apoptotic cells in the forebrain at postnatal day seven. This loss is associated with marked age-dependent deficits in stimulated acetylcholine release, performance in the Morris water maze, and induction of long-term potentiation in the CA1 region of the hippocampus. These data provide unexpected evidence that galanin plays a trophic role to regulate the development and function of a subset of septohippocampal cholinergic neurons.

Culex (Micraedes) biscaynensis n. sp. from Florida (Diptera: Culicidae).

The female, male, male genitalia, pupa, and larva of Culex biscaynensis, a new species in the Bisulcatus Group of subgenus Micraedes, are described and illustrated. The species, which is presently known only from south Florida, United States, is most similar to Culex (Micraedes) antillummagnorum Dyar.

Discovery of a bromeliad-inhabiting Culex (micraedes) sp. in south Florida.

Immatures of a Culex (Micraedes) sp. were collected from bromeliads at 6 sites in Dade County, FL. This discovery represents the first time a member of this subgenus has been detected in the continental United States. The Cx (Micraedes) sp. was found in both native and exotic bromeliads, and at some sites this Culex occurred in these plants as frequently as Wyeomyia spp.

Colonization of rock holes by Aedes albopictus in the southeastern United States.

Aedes albopictus was collected from water-holding rock holes along 3 streams in Georgia and 1 in South Carolina. To compare the occurrence of Ae. albopictus and Aedes atropalpus, rock holes were sampled for immature Aedes at 2 sites where there were numerous rock holes harboring mosquitoes. At 1 of these sites, tree holes and various types of artificial containers were also sampled for immature Aedes. At both sites, immature Ae. albopictus occurred in rock holes much less frequently than the rock-pool specialist, Aedes atropalpus. Moreover, the distribution of Ae. albopictus was limited to rock holes in less flood prone locations, whereas Ae. atropalpus was often a common mosquito even in rock holes that were among the most susceptible to flooding by rising stream levels. By contrast. Ae. albopictus was frequently found in the samples from tree holes and artificial containers. Thus, it appears that riverine rock holes that are flooded frequently may be, at best, marginal habitats for Ae. albopictus.

Spread of Aedes albopictus and decline of Ae. aegypti (Diptera: Culicidae) in Florida.

Waste tires and other types of artificial containers were sampled for immature Aedes to monitor changes in the occurrence of Aedes aegypti (L.) and Aedes albopictus (Skuse) in Florida. The initial invasion and spread of Ae. albopictus in Florida occurred in the northern part of Florida. Throughout this region, major declines in the abundance of Ae. aegypti have been associated with the expansion of Ae. albopictus in both urban and rural areas. Generally, the same results have occurred in central Florida, but at some urban locations Ae. aegypti has remained a common mosquito long after the arrival of Ae. albopictus. In southeastern Florida, Ae. aegypti is currently the dominant container-inhabiting Aedes in urban areas, whereas sites dominated by Ae. albopictus are in rural settings or in undeveloped tracts of land within urban or suburban areas. At some locations, immature Ae. albopictus were found in the same containers with another exotic mosquito, Ae. bahamensis Berlin. The persistence of thriving Ae. aegypti populations in urban areas of southern Florida indicates that Ae. albopictus might not become the dominant container Aedes in these habitats, at least not to the extent that it has in the northern part of the state.

Blood feeding and autogeny in the peridomestic mosquito Aedes bahamensis (Diptera: Culicidae).

Under laboratory conditions, most colony and field-collected Aedes bahamensis Berlin females developed eggs autogenously when they had access to sugar. However, significantly fewer starved females were autogenous, and they produced smaller egg clutches. Autogenous fecundity covaried with wing length, and smaller females generally failed to express autogeny. Mating had no effect on the maturation of the initial egg clutch. Most starved, nulliparous females blood fed from a restrained host. At a south Florida field site, both parous and nulliparous Ae. bahamensis were captured with a power aspirator, but concurrent sampling with dry ice-baited, light traps collected only parous females. Host-seeking females, taken either in chicken-baited traps or as they attempted to blood feed on humans, were also parous, with a single exception. Thus, at this field site, Ae. bahamensis females normally delayed blood feeding until after their first oviposition. Whether or not Ae. bahamensis females in other south Florida populations show a similar gonotrophic pattern probably will depend upon the availability of sugar sources and conditions in the mosquito's aquatic habitat that affect adult size.

Reduced mosquito production in cemetery vases with copper liners.

Water-holding stone vases were sampled in 4 central Florida cemeteries to compare the prevalence of mosquitoes in containers with and without metallic liners. Overall, immature mosquitoes were found in more than 60% of the vases lacking liners and in more than 50% of the vases with aluminum liners. Significantly fewer vases with copper liners were positive for mosquitoes. High mortality and a lack of development were observed in a field test involving the introduction of Aedes aegypti larvae into stone vases with copper liners.

Invasion of cemeteries in Florida by Aedes albopictus.

Aedes albopictus has been found in 53 of the 67 Florida counties. The initial discoveries in 11 of these counties were made in cemeteries. At several locations, Ae. albopictus became well-established in cemeteries before appearing in nearby accumulations of waste tires. The recycling of plastic floral baskets may be aiding the spread of Ae. albopictus. Mosquitoes were commonly found in all types of flower-holding containers in cemeteries, except bronze vases. In the laboratory, most Aedes aegypti eggs laid in bronze vases hatched, but larvae subsequently died. The spread of Ae. albopictus in cemeteries seems to occur at the expense of Ae. aegypti populations. At one cemetery immature Ae. albopictus and Ae. aegypti were found in about 70% of the Aedes-positive containers at the start of a monitoring program. In subsequent collections from this site, Ae. albopictus was found in nearly all Aedes-positive containers, whereas there was a progressive decrease in containers with Ae. aegypti. This trend did not appear to be the result of any seasonal pattern because in a nearby cemetery where Ae. albopictus was absent, Ae. aegypti did not show a similar decline. Limiting flower-holding containers to those with drain holes or to bronze vases would greatly limit mosquito production.

Facultative blood-feeding in the crabhole mosquito, Deinocerites cancer.

1. Most of the female crabhole mosquitoes Deinocerites cancer Theobald entering bait traps had undeveloped ovaries. 2. Nearly half of the D. cancer females that were captured in bait traps, but denied blood, were gravid following a 1 week holding period; they developed relatively small egg clutches autogenously (mean = 7.4 eggs per gravid female). 3. Although both parous and nulliparous D. cancer females were captured exiting from crabholes, only parous females were attracted to and collected in bait traps. 4. Much larger egg clutches (mean = 41.8 eggs per female) were produced anautogenously by mosquitoes that blood-fed on chickens. 5. All D. cancer females from field collected pupae developed eggs autogenously when allowed to mate and sugar feed in the laboratory. These mosquitoes produced an initial egg clutch with a mean size equivalent to that found in blood-fed, parous mosquitoes from bait traps. 6. In the field, mating and sugar feeding rather than blood-feeding seem to stimulate production of the initial egg clutch. 7. D. cancer blood-feeding appears to be delayed until after the first egg clutch has been laid; some parous crabhole mosquitoes are facultative blood feeders and can produce an egg clutch either with or without a blood meal.

Environmental factors influencing oviposition by Culex (Culex) (Diptera: Culicidae) in two types of traps.

Seasonal and habitat influences on the egg-laying activity of four species of Culex were compared in south Florida using jar- and vat-type oviposition traps. Egg rafts of Culex nigripalpus Theobald, Cx. quinquefasciatus Say, and Cx. salinarius Coquillett were collected in every month of the year, but rafts of Cx. restuans Theobald were absent during spring and summer. Cx. nigripalpus was the dominant species in the summer and fall, and Cx. quinquefasciatus and Cx. salinarius peaked during the winter and spring. Cx. restuans oviposited most rafts in the fall (98.4%). Most Cx. nigripalpus egg rafts were laid in vats (88.5%), and most Cx. quinquefasciatus egg rafts were deposited in jars (58.7%). Fewer Cx. quinquefasciatus rafts were found in a wooded area (32.4%) with a dense undergrowth than in a more open area (67.6%), but Cx. nigripalpus showed no ovipositional preference between these two habitats. The number of rafts oviposited by all species was reduced during periods of rainfall.

Aedes bahamensis: its invasion of south Florida and association with Aedes aegypti.

The exotic mosquito, Aedes bahamensis, is now well-established in south Florida, where it is widely distributed throughout Dade and southern Broward Counties in both urban and rural areas east of the Everglades. When discarded automobile tires were sampled in areas near human habitation, larvae and pupae of Ae. bahamensis were frequently found in association with immature Ae. aegypti. Elsewhere, however, Ae. bahamensis generally occurred in the absence of Ae. aegypti. The persistence of Ae. bahamensis populations at specific sites was documented in egg collections from ovitraps and in larval samples from the water retained in discarded tires.

Solubilisation of bovine rumen and decolorisation of bovine blood by enzymic hydrolysis with alcalase.

Enzymic hydrolysis has potential for upgrading a range of waste materials from the meat industry. The effects of the reaction variables pH, time, temperature, enzyme concentration and rumen concentration on the solubilisation of rumen by Alcalase have been determined. Reaction progress curves showed rapid initial rates followed by a gradual decline in rate. The optimum reaction pH was 8·5. Optimum reaction temperatures for hydrolysis and solubilisation were about 55°C and 60°C, respectively, for a 3-h reaction. Solubilisation and degree of hydrolysis both increased with increasing enzyme concentration and decreased with increasing substrate concentration.

Male-dependent stenogamy in Aedes taeniorhynchus.

A male-dependent stenogamous condition was found in a strain of Aedes taeniorhynchus that was derived from a field collection taken near Puerto Peñasco, Mexico. Mating occurred readily in this strain, even when the adults were confined in 8-dram glass shell vials. Under such conditions, mating also occurred in interstrain crosses involving Puerto Peñasco males with females from Florida strains of Ae. taeniorhynchus, but not in reciprocal crosses. For males of the Puerto Peñasco strain, flight did not appear to be a prerequisite for mating.

Kinetics of the hydrolysis of lean meat protein by alcalase: Derivation of two alternative rate equations and their fit to experimental data.

Two rate equations have been developed to model the hydrolysis of ground lean meat protein by Alcalase. The first equation was based on classical Michaelis-Menten kinetics and the second on the adsorption of enzyme to the protein prior to reaction. It was assumed that this adsorption could be modelled by a Langmuir-type adsorption isotherm. Each equation considered the enzyme to be competitively inhibited by reaction product, and considered enzyme inactivation to be first order. Both rate equations have been fitted to experimental data obtained from the hydrolysis of meat protein by Alcalase. Initial rate data indicated that the adsorption model was more appropriate. However, both equations gave satisfactory fits to 11 reaction progress curves determined over a wide range of enzyme and substrate concentrations.

Hydrolysis of the sarcoplasmic, myofibrillar and connective tissue proteins of lean beef by alcalase and its relationship to whole meat hydrolysis.

The enzymic hydrolysis and solubilisation of the three meat protein fractions have been investigated in order to elucidate some aspects of the hydrolysis of whole beef protein. Much of the water-soluble sarcoplasmic protein fraction was insolubilised on heating for 25 min at 60°C. While most of this insoluble protein was easily resolubilised enzymically, some of it remained insoluble after 5 hours' hydrolysis. Solubilisation of connective tissue by Alcalase after a 3 h reaction increased markedly from 23% at 55°C to 99% at 60°C. This explains the high optimum temperature for solubilisation of whole beef by Alcalase. A significant portion of myofibrillar tissue remained insoluble after 3 hours' reaction. indicating that the majority of the insoluble solids remaining after whole meat hydrolysis at 60°C derived from myofibrillar tissue. There was good agreement between an experimental reaction progress curve for whole beef hydrolysis and one estimated from progress curves for hydrolysis of the three meat protein fractions.