Late Pupae Research Articles

Laboratory selection of resistance to spinosad in Culex quinquefasciatus (Diptera: Culicidae).

A southern house mosquito Culex quinquefasciatus Say colony was established from surviving late instars and pupae from a semifield evaluation on Natular XRG (a granular formulation containing 2.5% spinosad). The initial lethal levels ofNatular XRG against this colony were determined in the laboratory for the first-generation progeny (designated as F1). Selection pressure was applied at LC70-90 levels to 10,000-15,000 late third- and early fourth-instar larvae of each generation with Natular XRG. Susceptibility changes in response to selection were determined every other generation, where a gradual and steady decline in susceptibility occurred from generation F1 to F35, followed by significant decline from generations F37 to F45 For reference purposes, susceptibility of freshly collected wild populations as well as a laboratory colony of the same species was also determined concurrently, which fluctuated within a slightly wider range for the wild populations and a tighter range for the laboratory colony. By comparing with wild populations and laboratory reference colony, tolerance to spinosad was observed from generations up to F9 in the selected population. Resistance levels increased gradually from generation F11 to F35, and elevated significantly from generations F37 to F45, when resistance ratios reached 1,415.3- to 2,229.9-fold at LC50 and 9,613.1- to 17,062.6-fold at LC90. Possible mechanisms of resistance development to spinosad were discussed.

Ret rescues mitochondrial morphology and muscle degeneration of Drosophila Pink1 mutants

Parkinson's disease (PD)-associated Pink1 and Parkin proteins are believed to function in a common pathway controlling mitochondrial clearance and trafficking. Glial cell line-derived neurotrophic factor (GDNF) and its signaling receptor Ret are neuroprotective in toxin-based animal models of PD. However, the mechanism by which GDNF/Ret protects cells from degenerating remains unclear. We investigated whether the Drosophila homolog of Ret can rescue Pink1 and park mutant phenotypes. We report that a signaling active version of Ret (RetMEN2B) rescues muscle degeneration, disintegration of mitochondria and ATP content of Pink1 mutants. Interestingly, corresponding phenotypes of park mutants were not rescued, suggesting that the phenotypes of Pink1 and park mutants have partially different origins. In human neuroblastoma cells, GDNF treatment rescues morphological defects of PINK1 knockdown, without inducing mitophagy or Parkin recruitment. GDNF also rescues bioenergetic deficits of PINK knockdown cells. Furthermore, overexpression of RetMEN2B significantly improves electron transport chain complex I function in Pink1 mutant Drosophila. These results provide a novel mechanism underlying Ret-mediated cell protection in a situation relevant for human PD.

Two Rapidly Evolving Genes Contribute to Male Fitness in Drosophila

Purifying selection often results in conservation of gene sequence and function. The most functionally conserved genes are also thought to be among the most biologically essential. These observations have led to the use of sequence conservation as a proxy for functional conservation. Here we describe two genes that are exceptions to this pattern. We show that lack of sequence conservation among orthologs of CG15460 and CG15323-herein named jean-baptiste (jb) and karr, respectively-does not necessarily predict lack of functional conservation. These two Drosophila melanogaster genes are among the most rapidly evolving protein-coding genes in this species, being nearly as diverged from their D. yakuba orthologs as random sequences are. jb and karr are both expressed at an elevated level in larval males and adult testes, but they are not accessory gland proteins and their loss does not affect male fertility. Instead, knockdown of these genes in D. melanogaster via RNA interference caused male-biased viability defects. These viability effects occur prior to the third instar for jb and during late pupation for karr. We show that putative orthologs to jb and karr are also expressed strongly in the testes of other Drosophila species and have similar gene structure across species despite low levels of sequence conservation. While standard molecular evolution tests could not reject neutrality, other data hint at a role for natural selection. Together these data provide a clear case where a lack of sequence conservation does not imply a lack of conservation of expression or function.

Regulation of cuticle pigmentation in drosophila by the nutrient sensing insulin and TOR signaling pathways

Insect pigmentation is a phenotypically plastic trait that plays a role in thermoregulation, desiccation tolerance, mimicry, and sexual selection. The extent and pattern of pigmentation of the abdomen and thorax in Drosophila melanogaster is affected by environmental factors such a growth temperature and access to the substrates necessary for melanin biosynthesis. This study aimed to determine the effect of nutritional status during development on adult pigmentation and test whether nutrient sensing through the Insulin/IGF and target of rapamycin (TOR) pathways regulates the melanization of adult cuticle in Drosophila. Flies reared on low quality food exhibit decreased pigmentation, which can be phenocopied by inhibiting expression of the Insulin receptor (InR) throughout the entire fly during mid to late pupation. The loss of Insulin signaling through PI3K/Akt and FOXO in the epidermis underlying the developing adult cuticle causes a similar decrease in adult pigmentation, suggesting that Insulin signaling acts in a cell autonomous manner to regulate cuticle melanization. In addition, TOR signaling increases pigmentation in a cell autonomous manner, most likely through increased S6K activity. These results suggest that nutrient sensing through the Insulin/IGF and TOR pathways couples cuticle pigmentation of both male and female Drosophila with their nutritional status during metamorphosis.

Persistence and virulence of promising entomopathogenic fungal isolates for use in citrus orchards in South Africa

Thaumatotibia leucotreta (Meyrick) (1912) (Lepidoptera: Tortricidae) is a key economic pest of citrus in South Africa. Although a variety of control options are available, none currently registered target the soil-dwelling life stages of the pest. Three fungal isolates, two Metarhizium anisopliae sensu lato Metschnikoff (Sorokin) isolates (G 11 3 L6 and FCM Ar 23 B3) and one Beauveria bassiana sensu lato Balsamo (Vuillemin) isolate (G Ar 17 B3) have been identified as showing the greatest potential against T. leucotreta soil-dwelling life stages. Since environmental persistence is an important factor in the success of entomopathogenic fungi against soil pests, the isolates, along with two commercial isolates (B. bassiana Eco-Bb® and M. anisopliae ICIPE 69), were subjected to a six-month field persistence trial. Every month after burial of the fungal isolates, inside net bags, in orchard soil, the number of colony forming units (CFUs) per gram of soil was measured and the viability of the conidia was assessed using bioassays. All isolates investigated were capable of persisting in the soil for the duration of the trial period and in addition, were capable of initiating infection in T. leucotreta late fifth instar larvae and subsequent pupae. However, with the exception of the commercial isolates which showed some correlation, no correlation was found between the number of CFUs recorded and percentage T. leucotreta mycosis. Persistence of entomopathogenic fungi in the field is affected by a series of factors, and although the effect of these factors was not measured, the data have provided preliminary evidence towards the persistence capability of these candidate biopesticides against T. leucotreta.

Stage-specific transcription during development of Aedes aegypti.

BackgroundAedes aegypti is the most important global vector of dengue virus infection in humans. Availability of the draft genome sequence of this mosquito provides unique opportunities to study different aspects of its biology, including identification of genes and pathways relevant to the developmental processes associated with transition across individual life stages. However, detailed knowledge of gene expression patterns pertaining to developmental stages of A. aegypti is largely lacking.ResultsWe performed custom cDNA microarray analyses to examine the expression patterns among six developmental stages: early larvae, late larvae, early pupae, late pupae, and adult male and female mosquitoes. Results revealed 1,551 differentially expressed transcripts (DETs) showing significant differences in levels of expression between these life stages. The data suggests that most of the differential expression occurs in a stage specific manner in A. aegypti. Based on hierarchical clustering of expression levels, correlated expression patterns of DETs were also observed among developmental stages. Weighted gene correlation network analysis revealed modular patterns of expression among the DETs. We observed that hydrolase activity, membrane, integral to membrane, DNA binding, translation, ribosome, nucleoside-triphosphatase activity, structural constituent of ribosome, ribonucleoprotein complex and receptor activity were among the top ten ranked GO (Gene Ontology) terms associated with DETs. Significant associations of DETs were also observed with specific KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway modules. Finally, comparisons with the previously reported developmental transcriptome of the malaria vector, Anopheles gambiae, indicated that gene expression patterns during developmental processes reflect both species-specific as well as common components of the two mosquito species.ConclusionsOur study shows that genes involved in the developmental life cycle of A. aegypti are expressed in a highly stage-specific manner. This suggests that transcriptional events associated with transition through larval, pupal and adult stages are largely discrete.

Ral mediates activity-dependent growth of postsynaptic membranes via recruitment of the exocyst

Remodelling neuronal connections by synaptic activity requires membrane trafficking. We present evidence for a signalling pathway by which synaptic activity and its consequent Ca(2+) influx activate the small GTPase Ral and thereby recruit exocyst proteins to postsynaptic zones. In accord with the ability of the exocyst to direct delivery of post-Golgi vesicles, constitutively active Ral expressed in Drosophila muscle causes the exocyst to be concentrated in the region surrounding synaptic boutons and consequently enlarges the membrane folds of the postsynaptic plasma membrane (the subsynaptic reticulum, SSR). SSR growth requires Ral and the exocyst component Sec5 and Ral-induced enlargement of these membrane folds does not occur in sec5(-/-) muscles. Chronic changes in synaptic activity influence the plastic growth of this membrane in a manner consistent with activity-dependent activation of Ral. Thus, Ral regulation of the exocyst represents a control point for postsynaptic plasticity. This pathway may also function in mammals as expression of activated RalA in hippocampal neurons increases dendritic spine density in an exocyst-dependent manner and increases Sec5 in spines.

Comparison Between Diflubenzuron and aBacillus thuringiensis israelensis– andLysinibacillus sphaericus–Based Formulation for the Control of Mosquito Larvae in Urban Catch Basins in Switzerland

A field test was conducted to evaluate a commercial biolarvicide based on Bacillus thuringiensis var. israelensis and Lysinibacillus sphaericus to control mosquitoes breeding in catch basins in southern Switzerland. The efficacy and residual activity of the microbial mosquito larvicide applied at the recommended rate of 10 g per catch basin was compared to the currently used larvicide diflubenzuron. Both products provided a very good control activity (> 97% of reduction) of late instars (3rd and 4th instars) and pupae for 4 wk. However, only the microbial formulation controlled immature stages during the whole period of the trial, with > 98% of larval reduction. A single application of the microbial larvicide applied at 10 g per catch basin significantly reduced the number of immature mosquitoes for at least 70 days. The quantity of rainfall in the 48-h period before each sampling and the water temperature did not influence the efficacy of the treatments. Under the environmental conditions encountered in southern Switzerland, the larvicide tested may be a valid alternative to diflubenzuron to control mosquitoes in urban catch basins. The long-lasting control by the microbial larvicide further reduces the number of treatments required to keep the population of mosquitoes at low levels.

The Case for a Generic Phytosanitary Irradiation Dose of 400 Gy for Lepidoptera That Infest Shipped Commodities as Pupae

The pros and cons of a generic phytosanitary irradiation dose against all Lepidoptera pupae on all commodities are discussed. The measure of efficacy is to prevent the F1 generation from hatching (F1 egg hatch) when late pupae are irradiated. More data exist for this measure than for others studied, and it is also commercially tenable (i.e., prevention of adult emergence would require a high dose not tolerated by fresh commodities). The dose required to prevent F1 egg hatch provides a liberal margin of security for various reasons. A point at issue is that correctly irradiated adults could be capable of flight and thus be found in survey traps in importing countries resulting in costly and unnecessary regulatory action. However, this possibility would be rare and should not be a barrier to the adoption of this generic treatment. The literature was thoroughly examined and only studies that could reasonably satisfy criteria of acceptable irradiation and evaluation methodology, proper age of pupae, and adequate presentation of raw data were accepted. Based on studies with 34 species in nine families, we suggest an efficacious dose of 400 Gy. However, large-scale confirmatory testing (> or = 30,000 individuals) has only been reported for one species. A dose as low as 350 Gy might suffice if results of more large-scale studies were available or the measure of efficacy were extended beyond prevention of F1 egg hatch, but data to defend measures of efficacy beyond F1 egg hatch are scarce and more would need to be generated.

Bacterial feeding, Leishmania infection and distinct infection routes induce differential defensin expression in Lutzomyia longipalpis

BackgroundPhlebotomine insects harbor bacterial, viral and parasitic pathogens that can cause diseases of public health importance. Lutzomyia longipalpis is the main vector of visceral leishmaniasis in the New World. Insects can mount a powerful innate immune response to pathogens. Defensin peptides take part in this response and are known to be active against Gram-positive and Gram-negative bacteria, and some parasites. We studied the expression of a defensin gene from Lutzomyia longipalpis to understand its role in sand fly immune response.MethodsWe identified, sequenced and evaluated the expression of a L. longipalpis defensin gene by semi-quantitative RT-PCR. The gene sequence was compared to other vectors defensins and expression was determined along developmental stages and after exposure of adult female L. longipalpis to bacteria and Leishmania.ResultsPhylogenetic analysis showed that the L. longipalpis defensin is closely related to a defensin from the Old World sand fly Phlebotomus duboscqi. Expression was high in late L4 larvae and pupae in comparison to early larval stages and newly emerged flies. Defensin expression was modulated by oral infection with bacteria. The Gram-positive Micrococcus luteus induced early high defensin expression, whilst the Gram-negative entomopathogenic Serratia marcescens induced a later response. Bacterial injection also induced defensin expression in adult insects. Female sand flies infected orally with Leishmania mexicana showed no significant difference in defensin expression compared to blood fed insects apart from a lower defensin expression 5 days post Leishmania infection. When Leishmania was introduced into the hemolymph by injection there was no induction of defensin expression until 72 h later.ConclusionsOur results suggest that L. longipalpis modulates defensin expression upon bacterial and Leishmania infection, with patterns of expression that are distinct among bacterial species and routes of infection.

Efficacy of a mermithid nematode Romanomermis iyengari (Welch) (Nematoda: Mermithidae) in controlling tree hole-breeding mosquito Aedes albopictus (Skuse) (Diptera: Culicidae) in a rubber plantation area of Kerala, India

In rubber plantations, tree holes are one of the major types of breeding habitats of Aedes mosquitoes which transmit dengue and chikungunya. A mermithid nematode, Romanomermis iyengari, was evaluated in tree holes for its efficacy in controlling Aedes albopictus. Infection of mosquito larvae by the nematode was determined through microscopic examination on the next day of application, and evaluation of immature density of mosquito was done on the seventh day. After application of the infective stage of the nematode in a host-parasite ratio of 1:3 or 1:4, the infection rates on the different larval instars of mosquito were similar, 85.7-95.8% in first to third instars and 79.3% in fourth instar larvae or 100 and 92.9%, respectively. Parasite burden varied from 1.1 to 2.4, respectively, among first and third instar larvae applied at 1:3. At 1:4, the parasite burden was between 1.6 (fourth instar) and 4 (second instar). The increase in parasite burden due to parasite density was significant in all the larval instars (P < 0.05). High parasite burden is detrimental to parasite recycling as it can cause premature mortality of the host. Hence, the dosage of 1:3 could be considered as suitable for rubber tree hole habitats. In the nematode-applied tree holes, there was a significant level (P < 0.05) of reduction in the immature density of A. albopictus, especially late instars and pupae, confirming the efficacy of R. iyengari in infecting the mosquito and controlling pupal emergence.

Biochemical properties, expression profiles, and tissue localization of orthologous acetylcholinesterase-2 in the mosquito, Anopheles gambiae

Acetylcholinesterases (AChEs) catalyze the hydrolysis of acetylcholine, a neurotransmitter for cholinergic neurotransmission in animals. Most insects studied so far possess two AChE genes: ace-1 paralogous and ace-2 orthologous to Drosophila melanogaster ace. We characterized the catalytic domain of Anopheles gambiae AChE1 in a previous study (Jiang et al., 2009) and report here biochemical properties of A. gambiae AChE2 expressed in Sf9 cells. An unknown protease in the expression system cleaved the recombinant AChE2 next to Arg110, yielding two non-covalently associated polypeptides. A mixture of the intact and cleaved AChE2 had a specific activity of 72.3 U/mg, much lower than that of A. gambiae AChE1 (523 U/mg). The order of Vmax/KM values for the model substrates was acetylthiocholine > propionylthiocholine ≈ acetyl-(β-methyl)thiocholine > butyrylthiocholine. The IC50's for eserine, carbaryl, BW284C51, paraoxon and malaoxon were 1.32, 13.6, 26.8, 192 and 294 nM, respectively. A. gambiae AChE2 bound eserine and carbaryl stronger than paraoxon and malaoxon, whereas eserine and malaoxon modified the active site Ser232 faster than carbaryl or paraoxon did. Consequently, the ki's were 1.173, 0.245, 0.029 and 0.018 μM−1min−1 for eserine, carbaryl, paraoxon and malaoxon, respectively. Quantitative polymerase chain reactions showed a similar pattern of ace-1 and ace-2 expression. Their mRNAs were abundant in early embryos, greatly decreased in late embryos, larvae, pupae, and pharate adult, and became abundant again in adults. Both transcripts were higher in head and abdomen than thorax of adults and higher in male than female mosquitoes. Transcript levels of ace-1 were 1.9- to 361.8-fold higher than those of ace-2, depending on developmental stages and body parts. Cross-reacting polyclonal antibodies detected AChEs in adult brains, thoracic ganglia, and genital/rectal area. Activity assays, immunoblotting, and tandem mass spectrometric analysis indicated that A. gambiae AChE1 is responsible for most of acetylthiocholine hydrolysis in the head extracts. Taken together, these data indicate that A. gambiae AChE2 may play a less significant role than AChE1 does in the mosquito nervous system.

Irradiation for Quarantine Control of the Invasive Light Brown Apple Moth (Lepidoptera: Tortricidae) and a Generic Dose for Tortricid Eggs and Larvae

The effects of irradiation on egg, larval, and pupal development, and adult reproduction in light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae),were examined. Eggs, neonates, third instars, fifth instars, and early stage pupae were irradiated at target doses of 60, 90, 120, or 150 Gy or left untreated as controls in replicated factorial experiments and survival to the adult stage was recorded. Tolerance to radiation generally increased with increasing age and developmental stage. A radiation dose of 120 Gy applied to eggs and neonates prevented adult emergence. A dose of 150 Gy prevented adult emergence in larvae at all stages. In large-scale validation tests, a radiation dose of 150 Gy applied to fifth instars in diet, apples or peppers resulted in no survival to the adult stage in 37,947 treated individuals. Pupae were more radio tolerant than larvae, and late stage pupae were more tolerant than early stage pupae. Radiation treatment of late pupae at 350 and 400 Gy resulted in three and one fertile eggs in 4,962 and 4,205 total eggs laid by 148 and 289 mating pairs, respectively. For most commodities, the fifth instar is the most radio tolerant life stage likely to occur with the commodity; a minimum radiation dose of 150 Gy will prevent adult emergence from this stage and meets the zero tolerance requirement for market access. For traded commodities such as table grapes that may contain E. postvittana pupae, a radiation dose > 400 Gy may be necessary to completely sterilize emerging adults. After review of the literature, a generic radiation treatment of 250 Gy is proposed for tortricid eggs and larvae in regulated commodities.

Partial Life Tables From Three Generations of &lt;I&gt;Enaphalodes rufulus&lt;/I&gt; (Coleoptera: Cerambycidae)

We used life table analyses to investigate age specific mortality and to better understand the population dynamics of the red oak borer, Enaphalodes rufulus (Haldeman) (Coleoptera: Cerambycidae). We continually sampled populations within 177 trees at primarily two sites in the Ozark National Forest in Arkansas throughout three (2-yr) generations. The first cohort (adults emerged in 2003) was sampled during a severe population outbreak, whereas the second and third (2005 and 2007) were sampled during the population crash that followed. Generation mortality was 94% in 2003 and 99% in both 2005 and 2007. Estimates of apparent mortality indicated that the E. rufulus population crash likely occurred during or before the first overwintering period (2003-2004) of the generation that emerged as adults in 2005. We found limited evidence for density dependent mortality, which suggest that intraspecific competition after the first active feeding period was apparently not an important mortality factor during E. rufulus development. Life tables revealed that E. rufulus larvae generally experienced the greatest apparent mortality during the second summer of active feeding (80-94%) when larvae were feeding in, and moving between phloem and sapwood. The least apparent mortality was incurred during the following spring and early summer (26-67%) when late stage larvae and pupae were deepest and most protected within sapwood or heartwood tunnels. We found very little evidence for mortality from associated species. Scarring of vascular tissue in response to E. rufulus feeding occurred during early life stages and may be an important tree resistance mechanism and E. rufulus mortality factor.

Community Ecology of Container Mosquitoes (Diptera: Culicidae) in Virginia Following Invasion by &lt;I&gt;Aedes japonicus&lt;/I&gt;

The success of an invasive species in a new region depends on its interactions with ecologically similar resident species. Invasions by disease vector mosquitoes are important as they may have ecological and epidemiological consequences. Potential interactions of a recent invasive mosquito, Aedes japonicus Theobald, with resident species in Virginia were evaluated by sampling larvae from containers and trapping adults. Distinct species compositions were observed for artificial containers and rock pools, with Ae. albopictus most abundant in the former and Ae. japonicus in the latter. However, these two species were found to co-occur in 21.2% of containers sampled. Among the six mosquito species most common in containers from May through September, 2006, only interspecific associations of Ae. japonicus with Aedes albopictus (Skuse) and Aedes triseriatus (Say) were significant, and both were negative. In addition to differences in habitat preference, mean crowding estimates suggest that interspecific repulsion may contribute to the significant negative associations observed between these species. High relative abundances of late instars and pupae of Ae. japonicus seem to provide this species with a mechanism of evading competition with Ae. albopictus, facilitating their coexistence in artificial containers. Although annual fluctuations were observed, trends in adult populations over a 6-yr period provide no evidence of declines. In summary, this survey of diverse container types and all life stages provided only limited evidence for competitive displacements or reductions of resident container species by Ae. japonicus, as observed elsewhere in its invasive range.

Resistance Development in Culex quinquefasciatus to Spinosad: A Preliminary Report

A Culex quinquefasciatus colony was established from surviving late instars and pupae from a semifield microcosm test with Natular XRG (a granular formulation containing 2.5% spinosad) applied at 14.2 kg/ha. The initial lethal concentrations for 50% and 90% of the population (LC50 and LC90) against Natular XRG were determined in the laboratory for the 1st generation progeny (F1). Selection was applied at LC70-90 levels to 10,000-15,000 of late 3rd and early 4th instars each generation. Susceptibility changes were determined every other generation and referenced to a susceptible laboratory colony as well as freshly collected wild populations of the same species concurrently. Tolerance to spinosad (resistance ratio < 5.0 fold) was observed up to F8 generation in the selected population. Thereafter, resistance ratios increased significantly from F10 to F16 generations, being 7.26- to 20.52-fold at the LC50 and 7.48- to 20.08-fold at the LC90 level.

Effect of Controlled Atmosphere Storage on Adults and Immature Stages of Rhizopertha dominica (Fab.)

Effect of controlled atmosphere parameters like carbon dioxide, oxygen concentration and period of storage were studied with 40, 50 and 60% concentrations of carbon dioxide; 16, 12, 8, and 4% oxygen, and the remaining as nitrogen. A setup was developed for production of gas mixtures for controlled atmosphere storage studies. The gases were metered separately, and mixed together in a gas mixing chamber generating the desired gas composition. The containers were purged with gas mixture after introducing counted number of adults or immature stages Rhizopertha dominica (Fab.), and were kept at 30oC in a temperature controlled chamber. Complete mortality of adults of was obtained in 5 days with 40% carbon dioxide at 16% oxygen level. With the reduced oxygen level of 8%, cent percent mortality of adults was achieved in 3 days. Complete mortality of eggs and early instar larvae were obtained with 40% carbon dioxide concentration in 9 days. Late instar larvae and pupae required 50% carbon dioxide concentration with 9 and 12 day treatment, respectively, for complete control. The control of all the life stages of R.dominica required a treatment with 50% carbon dioxide concentration for 12 days.

Variable host phenology does not pose a barrier to invasive weevils in a northern hardwood forest

1 A suite of invasive weevils has established in hardwood forests of the North American Great Lakes Region. We quantified patterns of host availability and the capacity of adults to succeed in a system with high host variability both within and between seasons in Michigan, U.S.A. 2 We quantified phenological development of foliage on three host species [sugar maple, Acer saccharum Marshall; ironwood, Ostrya virginiana (Mill.) K. Koch; and raspberry, Rubus spp.]. We estimated adult abundance using emergence traps and sweep net sampling over 3 years, and compared field host associations with laboratory choice assays. 3 Host plant phenology varied among species, between years, and in their interactions. The four most common weevils, Phyllobius oblongus (L.), Polydrusus sericeus (Schaller), Barypeithes pellucidus (Boheman) and Sciaphilus asperatus (Bonsdorff), emerged in early to mid-June, in approximately that order. After emergence, each species showed evidence of host preference based on their abundances on foliage. Overall, P. oblongus and B. pellucidus were most prevalent on sugar maple, P. sericeus was most prevalent on ironwood, and S. asperatus was relatively evenly distributed. Laboratory choice tests with P. oblongus and P. sericeus confirmed these preferences. 4 These four invasive species comprised over 99% of all 12 845 weevils obtained, suggesting displacement of native species. The optimal sampling methods varied among weevil species. 5 These invasive weevils contend with the highly variable conditions of their environment, and also potential phenological asynchrony, via relatively late emergence, even at the cost of lower host quality. Annual variation is greater for numbers of adults than larvae, suggesting that mortality of late instars or pupae is particularly important.

Transcription Profiling Associated With Life Cycle of Anopheles gambiae

Complex biological events occur during the developmental process of the mosquito Anopheles gambiae (Giles). Using cDNA expression microarrays, the expression patterns of 13,440 clones representing 8,664 unique transcripts were revealed from six different developmental stages: early larvae (late third instar/early fourth instar), late larvae (late fourth instar), early pupae (< 30 min after pupation), late pupae (after tanning), and adult female and male mosquitoes (24 h postemergence). After microarray analysis, 560 unique transcripts were identified to show at least a fourfold up- or down-regulation in at least one developmental stage. Based on the expression patterns, these gene products were clustered into 13 groups. In total, eight genes were analyzed by quantitative real-time polymerase chain reaction to validate microarray results. Among 560 unique transcripts, 446 contigs were assigned to respective genes from the An. gambiae genome. The expression patterns and annotations of the genes in the 13 groups are discussed in the context of development including metabolism, transport, protein synthesis and degradation, cellular processes, cellular communication, intra- or extra-cellular architecture maintenance, response to stress or immune-related defense, and spermatogenesis.

Interaction Between the Relay Loop and the SH1-SH2 Helix Region in Drosophila Muscle Myosin is Essential for Normal Motor Function, Myofibril Stability and Muscle Contraction

Molecular modeling of Drosophila muscle myosin reveals that relay domain residue E499 interacts with SH1-SH2 residue R714 in a charge-dependent manner. To explore the significance of this interaction, we generated transgenic lines expressing myosin with a mutation in the relay loop domain (E499R) or the SH1-SH2 helix (R714E). Both mutations yield ∼75% reductions in CaATPase as well as basal or actin-activated MgATPase activity. Actin-sliding velocity of E499R is reduced by 65% and R714E shows no motility. Indirect flight muscles in late pupae of each mutant display disrupted myofibril assembly. Two-hour- and two-day-old adults have severely abnormal myofibril morphology and poor sarcomere organization, with no flight ability. Therefore, the putative interaction of the relay with SH1-SH2 is indispensable for normal motor function, myofibril stability and locomotion. We next constructed a putative compensatory mutant designed to restore function of the E499R or R714E mutant by generating a transgenic line that expresses both E499R and R714E. Interestingly, calcium, basal, and actin-stimulated ATPase values are restored to 65–70% of wild type and actin-sliding velocity is at 40%. The compensatory mutation suppresses the assembly defects in pupal muscle that are seen in both E499R and R714E and reduces disrupted myofibril morphology in two-hour old indirect flight muscles. However, the E499R-R714E mutant myosin was unable to maintain sarcomere organization in two-day-old flies or to restore flight ability. Overall, our results reveal that interaction of residues at the relay/SH1-SH2 helix interface is important for myosin and muscle function.