Surveillance of the mosquito Aedes aegypti and its biocontrol with the copepod Mesocyclops aspericornis in Australian wells and gold mines

Abstract. A survey of the dengue vector mosquito Aedes aegypti was undertaken using runnel traps to detect immature stages (larvae and pupae) in flooded disused mine shafts and wells in Charters Towers, Queensland, Northern Australia. The town has a history of dengue fever since 1885 when goldminers were the first recorded victims. During the latest dengue epidemic in 1993, 2% of the population had laboratory-confirmed dengue virus Type 2, despite source reduction of Ae.aegypti breeding-sites at ground level or above. This led to suspicions that dengue vector Ae.aegypti breeding-sites might be below ground level. When surveyed in March 1994, Ae.aegypti immatures were found in 9/10 wells and 1/6 mine shafts. The water in wells and mines had similar characteristics -except that turbidity was higher in the mines, which more often contained predators of mosquito immatures.
The copepod Mesocyclops aspericornis was collected from water in 1/10 wells and 2/6 mine shafts. Laboratory predation trials resulted in 95.5–100% predation by 25 copepods/1 on Ae.aegypti first-instar larvae up to 200 larvae/1. Five wells containing Ae.aegypti in the survey were inoculated with fifty indigenous M.aspericornis , and five wells (one positive and four negative in the survey) were left untreated as controls. Nine months later, in December 1994, Ae.aegypti had been eliminated from all five treated wells but all untreated control wells contained Ae.aegypti , except for one well that contained a natural population of M.aspericornis. The role of wells and mines as winter/ dry season refuges of Ae.aegypti in northern Australia is reviewed, and we recommend the use of M.aspericornis as a cost-effective, environmentally acceptable and persistent agent for the sustainable control of Ae.aegypti , especially in inaccessible breeding sites.     

The effect of predatory fish exudates on the ovipostional behaviour of three mosquito species: Culex quinquefasciatus, Aedes aegypti and Culex tarsalis

Abstract.  Three mosquito species, Culex tarsalis Coquillett, Culex quinquefasciatus Say and Aedes aegypti L. (Diptera: Culicidae), were examined in laboratory binary choice experiments to investigate whether fish exudates from the mosquitofish, Gambusia affinis (Baird & Girard) (Cyprinodontiformes: Poecilliidae), deter oviposition and whether the responses of these mosquito species to fish exudates in oviposition sites are consistent with the risk of predation from fish experienced by each species in their respective natural breeding habitats. Culex tarsalis was deterred significantly from egg laying by the presence of fish exudates in oviposition cups, consistent with high levels of predation by fish in natural breeding sites. Egg laying by Cx quinquefasciatus was slightly reduced in water with fish exudates, but was not consistently deterred by water conditioned by mosquitofish, consistent with the species' relatively low risk of fish predation in natural habitats. Oviposition by container-breeding Ae. aegypti was not deterred by the presence of fish exudates in oviposition cups, consistent with a low risk of predation by fish in natural habitats.     

Evaluations of larvicidal activity of medicinal plant extracts to Aedes aegypti （Diptera： Culicidae） and other effects on a non target fish

A preliminary study was conducted to investigate the effects of the extracts of 112 medicinal plant species, collected from the southern part of Thailand, on Aedes aegypti. Studies on larvicidal properties of plant extracts against the fourth instar larvae revealed that extracts of 14 species showed evidence of larvicidal activity. Eight out of the 14 plant species showed 100% mosquito larvae mortality. The LC50 values were less than 100μg/mL （4.1μg/ mL-89.4μg/mL）. Six plant species were comparatively more effective against the fourth instar larvae at very low concentrations. These extracts demonstrated no or very low toxicity to guppy fish （Poecilia reticulata）, which was selected to represent most common non-target organism found in habitats ofAe. aegypti, at concentrations active to mosquito larvae. Three medicinal plants with promising larvicidal activity, having LC50 and LC50 values being 4.1 and 16.4 μg/mL for Mammea siamensis, 20.2 and 34.7 μg/mL forAnethum graveolens and 67.4 and 110.3μg/mL forAnnona muricata, respectively, were used to study the impact of the extracts on the life cycle ofAe. aegypti. These plants affected pupal and adult mortality and also affected the reproductive potential of surviving adults by reducing the number of eggs laid and the percentage of egg hatchability. When each larval stage was treated with successive extracts at the LC50 value, the first instar larvae were found to be very susceptible to A. muricata and the second instar larvae were found to be susceptible to A. graveolens, while the third and fourth instar larvae were found to be susceptible to M. siamensis. These extracts delayed larval development and inhibited adult emergence and had no adverse effects on P. reticulata at LC50 and LC50 values, except for the M. siamensis extract at its LC50 value.     

Unusual productivity of Aedes aegypti in septic tanks and its implications for dengue control

Increased DEN-2 virus transmission in Puerto Rico during 2005 prompted the implementation of a rapid intervention programme to suppress Aedes aegypti (L.) (Diptera: Culicidae) emergence, which in turn lead to the discovery of previously unknown breeding sites underground. Initially, the following control measures were applied in Playa/Playita (PP), a town of 1,400 households, to all areas where the number of pupae per person exceeded the expected threshold for dengue transmission; all containers likely to be aquatic habitats were turned over and containers too large to turn were treated with 1 p.p.m. methoprene. The impact of these interventions was evaluated by comparing the number of resting adult mosquitoes (by backpack aspiration and sweepnetting in bedrooms) pre-intervention, with numbers at 3 and 5 weeks post-intervention, and by evaluating pupal density at 4 weeks post-intervention in PP and in a nearby town, Coqui (CO; 1500 households), which was not treated. The pre-intervention and post-intervention densities of resting Ae. aegypti adults were significantly larger in the intervention town, although the density of pupae in surface containers was low and similar in both towns at 4 weeks post-intervention. At 3 weeks post-intervention, the density of resting adults decreased by only 18% of pre-intervention levels, but returned to pre-intervention levels 5 weeks after treatment. By contrast, the density of resting adults in CO steadily decreased to 48% and 61%, at 3 and 5 weeks after the initial surveys, respectively. Geographical Information Systems identified significant clustering of adult mosquitoes, which led to the discovery of underground aquatic habitats (septic tanks) that were producing large numbers of Ae. aegypti and Culex quinquefasciatus (Say) in the treated town. We calculated that septic tanks could produce > 18 000 Ae. aegypti and approximately 170 000 Cx quinquefasciatus adults per day. Septic tanks are likely to be common and widespread in suburban and rural Puerto Rico, where, apparently, they can contribute significantly to the maintenance of island-wide dengue virus endemicity.     

A comparison of mosquito densities,weather and infection rates of Aedes aegypti during the first epidemics of Chikungunya (2014) and Zika (2016) in areas with and without vector control in Puerto Rico

In Puerto Rico, the first records of the transmission of Chikungunya (CHIKV) and Zika (ZIKV) viruses were confirmed in May 2014 and December 2015, respectively. Transmission of CHIKV peaked in September 2014, whereas that of ZIKV peaked in August 2016. The emergence of these mosquito‐transmitted arboviruses in the context of a lack of human population immunity allowed observations of whether the outbreaks were associated with Aedes aegypti (Diptera: Culicidae) densities and weather. Mosquito density was monitored weekly in four communities using sentinel autocidal gravid ovitraps (AGO traps) during 2016 in order to provide data to be compared with the findings of a previous study carried out during the 2014 CHIKV epidemic. Findings in two communities protected against Ae. aegypti using mass AGO trapping (three traps per house in most houses) were compared with those in two nearby communities without vector control. Mosquito pools were collected to detect viral RNA of ZIKV, CHIKV and dengue virus. In areas without vector control, mosquito densities and rates of ZIKV detection in 2016 were significantly higher, similarly to those observed for CHIKV in 2014. The density of Ae. aegypti in treated sites was less than two females/trap/week, which is similar to the putative adult female threshold for CHIKV transmission. No significant differences in mosquito density or infection rates with ZIKV and CHIKV at the same sites between years were observed. Although 2016 was significantly wetter, mosquito densities were similar.     

Influence of light and habitat on predation of Culex quinquefasciatus (Diptera: Culicidae) larvae by the waterbugs (Hemiptera: Heteroptera)

The influence of light and habitat structure on the predation of Culex quinquefasciatus larvae by the common heteropteran water bug, Diplonychus (=Sphaerodema) annulatus, D. rusticus and Anisops bouvieri was assessed in the laboratory. It was revealed that water bugs predate more in presence of light than in dark conditions. While A. bouvieri consumed more prey in structured conditions, D. annulatus and D. rusticus consumed more prey in open conditions. The selection of prey size as well as the respective numbers varied between predators and treatments significantly. Prey vulnerability (PV), an indicator of predatory efficiency, was highest for D. annulatus, moderate for D. rusticus and low for A. bouvieri. Prey consumption and PV values under different treatment conditions indicate that for belostomatid water bugs D. annulatus and D. rusticus, the order of prey consumption under different habitat and light/dark combinations is light open > dark open > light vegetated > dark vegetated. In the case of the backswimmer, A. bouvieri, the order of prey consumption appeared to be light structured > dark structured > light open > dark open. These findings were consistent with resource‐partitioning by water bug species sharing the same guild. If the observed results are extended to natural settings the efficiency of these predators in controlling mosquito populations will vary with the structural complexity of habitats and the intensity of light.     

Effect of Novaluron (Rimon 10 EC) on the mosquitoes Anopheles albimanus, Anopheles pseudopunctipennis, Aedes aegypti, Aedes albopictus and Culex quinquefasciatus from Chiapas, Mexico

Dengue fever is a serious problem in Mexico and vector control has not been effective enough at preventing outbreaks. Malaria is largely under control, but it is important that new control measures continue to be developed. Novaluron, a novel host-specific insect growth regulator and chitin synthesis inhibitor, has proved to be effective against agricultural pests, but its efficacy against larval mosquito vectors under field conditions remains unknown. In accordance with the World Health Organization Pesticide Evaluation Scheme, phase I, II and III studies were conducted to evaluate the efficacy and residual effect of Novaluron (Rimon 10 EC, Makhteshim, Beer-Sheva, Israel) on the malaria vectors Anopheles albimanus Wiedemann (Diptera: Culicidae) and Anopheles pseudopunctipennis Theobald, the dengue vectors Aedes aegypti (L) and Aedes albopictus Skuse and the nuisance mosquito Culex quinquefasciatus Say. Laboratory susceptibility tests yielded diagnostic concentrations for all five target species. Field trials to identify the optimum field dosage of Novaluron against Anopheles mosquitoes were carried out under semi-natural conditions in artificial plots and in vessels with wild mosquitoes. Efficacy was measured by monitoring mortality of larvae and pupae and the percentage of inhibition of emergence from floating cages. Dosages of Novaluron for field tests were based on pupal LC(99) (lethal concentration 99%) of An. pseudopunctipennis (0.166 mg/L) in plots and average pupal LC(99) of Ae. aegypti and Ae. albopictus (0.55 mg/L). At all dosages tested, Novaluron significantly reduced larval populations of An. albimanus, Culex coronator Dyar & Knab, Ae. albopictus and Cx. quinquefasciatus by approximately 90%, inhibited adult emergence of An. albimanus and An. pseudopunctipennis by approximately 97% for almost 4 months in experimental plots, and inhibited adult emergence of Ae. aegypti and Ae. albopictus by approximately 97% for up to 14 weeks. Recommended dosages of Novaluron for non-container breeding and container breeding mosquitoes are 0.166 mg/L and 0.55 mg/L, respectively. Overall, the residual effect was more sustained than that of temephos. The lowest dosage of Novaluron had less of an impact on non-target organisms than did temephos. Small-scale field trials in natural breeding sites treated with Novaluron at 0.6 L/ha eliminated adult emergence of An. albimanus and Cx. coronator for 8 weeks. For phase III studies, Novaluron was tested at the local and village levels, applying the optimum field rate to all natural breeding habitats within 1 km of a pair of neighbouring villages. Village-scale trials of Novaluron at 0.6 L/ha reduced An. albimanus larval populations for at least 8 weeks and, more importantly, sharply reduced the densities of adult host-seeking mosquitoes approaching houses. We conclude that Novaluron is effective and environmentally safer than temephos.     

Biological control of container-breeding mosquitoes, Aedes albopictus and Culex quinquefasciatus, in a Japanese island by release of Toxorhynchites splendens adults

To control container-breeding mosquitoes in the small island of Minnajima (0.56 km2), northern Okinawa, Japan, laboratory-reared adults (aged 7-10 days) of Toxorhynchites splendens (Palawan strain), a mosquito with predatory larvae, were released repeatedly during 1984, 1986 and 1987. Thirteen species of mosquitoes (Diptera: Culicidae) occurred in artificial containers, ground pools or crab-holes on the island, the predominant species being Aedes (Stegomyia) albopictus and Culex (Culex) quinquefasciatus. Predatory mosquito larvae of Culex (Lutzia) fuscanus and Cx (Lt.) halifaxii were found commonly in wet containers. In the first year of study, during a period of 54 days from 13 May to 5 July 1984, totals of 879 female and 806 male adults of Tx.splendens were released on six occasions. Similarly, between 29 April and 30 August 1986, totals of 2920 female and 2878 male adult Tx.splendens were released. In the third study year, totals of 2041 female and 1783 male Tx.splendens were released on eight occasions during 199 days from 23 April to 7 November 1987. After adult releases at two sites, the immature stages of Tx.splendens were found in 164 out of 502 traps in 1984, 421 out of 933 traps in 1986, and 151 out of 502 traps in 1987. The number of immatures of Tx.splendens present in each trap varied from 1 to 40 in 1984, 1 to 29 in 1986 and 1 to 9 in 1987. Numbers of immatures of the target species found in the traps during August-September averaged 71.9/trap/month in 1984, 114.7/trap/month in 1986 and 36.0/trap/month in 1987, significantly less in the traps with Tx.splendens than in those without them. The present field studies indicated that, in this small island, approximately 250 adult female and 200 male Tx.splendens per month should be released from April to November, and the releases should be carried out every year, in order to control effectively the target mosquitoes Ae.albopictus and Cx quinquefasciatus breeding in artificial containers in Minnajima.     

Laboratory experiments on factors affecting oviposition site selection in Toxorhynchites amboinensis (Diptera: Culicidae), with a report on the occurrence of egg cannibalism

Laboratory experiments tested the effects of water quality and the presence of conspecific and heterospecific immatures on oviposition by Toxorhynchites amboinensis (Doleschall). The females showed a highly significant preference for oak leaf infusion water over distilled water. When twenty starved third and fourth instar Tx.amboinensis larvae were present in the water, substantially fewer eggs were counted from pots containing these conspecifics, than from controls in which no larvae were present. Numbers of eggs from pots containing starved second instar larvae did not differ significantly from controls. Observations of larval behaviour while oviposition was occurring suggested that egg numbers were reduced in containers because of egg cannibalism with third and fourth instar larvae, and not because the larvae caused a deterrent effect. Subsequent experiments confirming the occurrence of substantial egg cannibalism by third and especially fourth instar larvae are described. As with larvae, the presence of Tx.amboinensis pupae in the water had little effect on oviposition. If placed in the water 24 h prior to test, pupae very slightly enhanced its attractiveness, but if introduced immediately before test there appeared to be a slight deterrent effect. With heterospecific larvae, twenty fourth instar Ae.aegypti larvae introduced into infusion water 24 h before test rendered the water slightly attractive, while water in which Ae.aegypti larvae had been reared for 48 h proved highly repellent.     

Effects of temperature on the life cycle,expansion, and dispersion of Aedes aegypti (Diptera: Culicidae) in three cities in Paraiba,Brazil

The mosquito Aedes aegypti is the primary vector of dengue and is common throughout tropical and subtropical regions. Its distribution is modulated by environmental factors, such as temperature. This study aimed to evaluate the influence of temperature on the life cycle and expansion of Ae. aegypti populations in the cities of Campina Grande, João Pessoa, and Patos. Samples of Ae. aegypti were collected in the three cities and raised in the laboratory. We assessed the life cycles of the three Ae. aegypti populations under six constant temperatures (16, 22, 28, 33, 36, and 39°C), selected on the basis of historical temperature tendencies of each city. We also used existing climate data to calculate projected temperature increases for all three areas. Our results suggest that Campina Grande, João Pessoa, and Patos will experience, respectively, maximum temperature increases of 0.030°C/year, 0.069°C/year, and 0.061°C/year, and minimum temperature increases of 0.019°C/year, ?0.047°C/year, and ?0.086°C/year. These projected increases will result in temperatures favorable to the Ae. aegypti life cycle, causing rapid population growth. Therefore, Ae. aegypti populations are likely to expand in the mesoregions represented by these cities.     

Permethrin resistance variation and susceptible reference line isolation in a field population of the mosquito,Culex quinquefasciatus (Diptera: Culicidae)

This study examines the genetic variations and mechanisms involved in the development of permethrin resistance in individual mosquitoes from a field population of Culex quinquefasciatus, HAmCq^G0, and characterizes susceptible reference lines of mosquitoes with a similar genetic background to the field HAmCq^G0 strain. Six upregulated cytochrome P450 genes, CYP9M10, CYP9J34, CYP6P14, CYP9J40, CYP6AA7, and CYP4C52v1, previously identified as being upregulated in the larvae of resistant HAmCq^G8 mosquitoes were examined in the larvae of 3 strains (susceptible S‐Lab, parental HAmCq^G0 and permethrin‐selected highly resistant HAmCq^G8) and 8 HAmCq^G0 single‐egg raft colonies, covering a range of levels of susceptibility/resistance to permethrin and exhibiting different variations in the expression of A and/or T alleles at the L‐to‐F kdr locus of the sodium channel. The 2 lines with the lowest tolerance to permethrin and bearing solely the susceptible A allele at the L‐to‐F kdr locus of the sodium channels, from colonies Cx_SERC5 and Cx_SERC8, showed lower or similar levels of all 6 of the P450 genes tested compared with the S‐Lab strain, suggesting that these 2 lines could be used as the reference mosquitoes in future studies characterizing insecticide resistance in HAmCq mosquitoes. This study also provides a detailed investigation of the mechanisms involved in insecticide resistance in individuals within a population: individuals with elevated levels of resistance to permethrin all displayed one or more potential resistance mechanisms–either elevated levels of P450 gene expression, or L‐to‐F mutations in the sodium channel, or both.     

Environmental and biological factors influence the relationship between a predator fish,Gambusia holbrooki,and its main prey in rice fields of the Lower Mondego River Valley (Portugal)

We studied the relationships between a predator fish, Gambusia holbrooki, and its main food prey, within the content of a rice field food web. The influence of some environmental and biological factors on these trophic interactions, in combination with existent quantitative information, allowed us to evaluate the ecological viability of using a non-ionic surfactant, Genapol OXD-080, to control a plague caused by crayfish (Procambarus clarkii) populations in the rice fields. In the Lower Mondego River Valley, Portugal, G. holbrooki is abundant in rice fields. It feeds mainly on copepods, cladocerans and rotifers. Surface insects, such as aphids, collembolans, adult (imago) chironomids and other dipterans, are additional food. Large G. holbrooki consumed greater amounts of cladocerans and adult chironomids than other smaller size groups, while small fish prefered rotifers. Gravid females ate copepods, cladocerans, and adult chironomids and other dipterans in significantly greater amounts than immatures, males, and non-gravid females. Non-gravid females ate collembolans in significantly greater quantities than any other fish group. The population density of copepods, cladocerans, adult chironomids, and other dipterans, the area covered by aquatic vegetation, and water temperature all had significant effects on the total number of prey caught by G. holbrooki. In contrast, a negative correlation was found with rotifers, collembolans, aphids in higher densities, and of increased water volume, dissolved oxygen and pH. G. holbrooki holds a key intermediate position in the rice field food chain, feeding in large amounts of aquatic invertebrates and being eaten, in turn, by piscivores. With regard to the toxicity of Genapol OXD-080 on non-target organisms, LC₅₀ values for G. holbrooki and some of its main prey were several times lower than the concentration necessary to decrease the activity of crayfish populations in the rice fields. Thus, Genapol OXD-080 could potentially cause greater damage to the local populations of non-target species and should not be used without taking precautions not to contaminate other important biological reservoirs, such as the rice field irrigation channels. This revised version was published online in July 2006 with corrections to the Cover Date.     

Field and laboratory biology of the stem-feeding beetle Thamnurgus euphorbiae (Küster) (Col., Scolytidae) in Italy, a potential biological control candidate of leafy spurge in the USA and Canada

Thamnurgus euphorbiae overwinters as adults in dry stems of Euphorbia characias until the end of March before exiting through circular holes cut with their mandibles. Females and males appeared in the field at the same time, and the first pairs were observed on 28 March on new stems of E. characias having the open‐flower at the beginning of its development. Throughout the rest of April it was possible to find and collect the fairly abundant adults. Mating occurs on new stems of the host plant and females walk up towards the top of the stem and start to mine the centre of it in order to penetrate. Oviposition occurs in new stems and eggs are laid singly along the interior of the stem; females laid 35–85 eggs. Upon hatching, larvae started feeding on vascular bundles and the inner cortex. There are three larval instars, and pupation occurred in the stem of the host plant; this insect is univoltine. Thamnurgus euphorbiae has been accepted by the Technical Advisory Group for Biological Control Agents of Weeds for release as a biological control agent of leafy spurge in the US. Prior to this study the biology of T. euphorbiae was unknown.     

Biological control of strawberry tarsonemid mite Phytonemus pallidus and two-spotted spider mite Tetranychus urticae on strawberry in the UK using species of Neoseiulus (Amblyseius) (Acari: Phytoseiidae)

Two species of Neoseiulus, N. californicus and N. cucumeris, showed potential for biocontrol of phytophagous mites on strawberry. N. californicus controlled Tetranychus urticae on potted strawberry plants in a gauze-sided glasshouse at temperatures comparableto early summer in the UK (8–20°C). Both species of phytoseiid reducednumbers of the tarsonemid Phytonemus pallidus on potted strawberry plants under glasshouse conditions (15–23°C). In several experiments reductions in the range of 71–81% in numbers of tarsonemid active stages and eggs, compared to non-release plants, were obtained. The importance of establishing a suitable predator: prey ratio at an earlystage was demonstrated in an experiment where an initial ratio of 1 N. cucumeris: 10 P. pallidus gave a greater degree of controlthan 1:20 or 1:40.     

Short-distance dispersal behavior and establishment of the parasitoid Gonatocerus ashmeadi (Hymenoptera: Mymaridae) in Tahiti: Implications for its use as a biological control agent against Homalodisca vitripennis (Hemiptera: Cicadellidae)

The egg parasitoid Gonatocerus ashmeadi Girault (Hymenoptera: Mymaridae), was introduced into French Polynesia as a biological control agent to control the invasive plant feeding pest Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae). The short-distance dispersal of G. ashmeadi was monitored as part of the biological control program. G. ashmeadi showed exponential dispersal capacity with 47 m/day being a minimum estimate of its natural rate of spread at high host densities (>150 nymphs per minute of sweep net sampling) in urbanized areas at sea level, which were characterized by a high diversity of exotic ornamental plants. This rate of spread contrasted starkly with almost nonexistent establishment and dispersal where host densities were very low (<2 nymphs per minute of sweep net sampling) at high elevation (800 m) with relatively undisturbed native vegetation. Survey results across different altitudes revealed an effect of vegetative diversity and host density on the measurable mobility and establishment of G. ashmeadi. In contrast, no significant influence of wind direction was found on G. ashmeadi dispersal rate or direction. Survey results for G. ashmeadi from French Polynesia suggest that the best release establishment strategies for classical biological control of H. vitripennis are: (1) many small releases where host density is high, or (2) larger and fewer releases where host densities are low.     

Host range validation,molecular identification and release and establishment of a Chinese biotype of the Asian leaf beetle Lilioceris cheni (Coleoptera: Chrysomelidae: Criocerinae) for control of Dioscorea bulbifera L. in the southern United States

Dioscorea bulbifera, an Asian vine, is invasive in the southeastern USA. It rarely flowers but propagates from potato-like bulbils formed in leaf axils, which persist into the subsequent growing season. Lilioceris cheni Gressitt and Kimoto, a foliage-feeding beetle (Coleoptera: Chrysomelidae: Criocerinae) from Nepal, had been tested, proven to be a specialist and approved for release as a biological control agent. Regulatory delays, however, resulted in the demise of quarantine-held colonies, and acquisition of new Nepalese stock proved untenable. Searches then undertaken in southern China resulted in the collection of over 300 similar beetles. Two Chinese Lilioceris species were identified: one confirmed to be L. cheni and the other identified as Lilioceris egena (Weise). Mitochondrial analysis revealed an exact DNA match between some Chinese and one of the two Nepalese c oxidase subunit I haplotypes and all Chinese L. cheni haplotypes clustered as a single species but the comingling of the two species aroused concerns over possible hybridisation. These concerns were allayed by nuclear D2 analysis showing the absence of dual parental sequences. Nonetheless, diligence was exercised to ensure that the Chinese strains were safe to release. Abridged host testing using critical test species verified specificity. Caged releases during autumn 2011 documented the ability of adult beetles to overwinter in south Florida despite a prolonged lack of foliage. Open releases the following year produced vigorous populations that caused extensive defoliation. Preliminary observations indicate that L. cheni now contributes to the control of D. bulbifera and the bulbil-feeding L. egena should complement these effects if its host range proves appropriate.