Biological control potential of Turkish entomopathogenic nematodes against the Colorado potato beetle,Leptinotarsa decemlineata

Three Turkish isolates of the entomopathogenic nematodes Steinernema carpocapsae, Steinernema feltiae and Heterorhabditis bacteriophora were evaluated under laboratory conditions (in different temperatures and doses) for their biocontrol efficiency against last instar Leptinotarsa decemlineata. Herein, the effects of infective juveniles in aqueous suspension against L. decemlineata were evaluated. S. feltiae appeared to be the most pathogenic nematode among the tested species.     

Sperm utilization and post-copulatory female-guarding in the Colorado potato beetle, Leptinotarsa decemlineata

Crosses between white and black color morphs of the Colorado potato beetle, Leptinotarsa decemlineata Say, used as genetic markers, showed that male beetles ride and guard females to achieve the three copulations required to fill the spermatheca. Multiple matings are necessary for the females to realize their full reproductive potential. Sperm mixing occurs in the spermatheca providing partial sperm precedence. The data support the competitive mate searching theory.

---

Résumé Différentes combinaisons d'accouplements entre des mutants noirs et blancs du doryphore de la pomme de terre, Leptinotarsa decemlineata, servirent à étudier le comportement de guarde de la femelle par le mâle après un premier accouplement. Les résultats indiquent que le sperme de deux mâles accouplant la même femelle se mélange dans la spermathèque assurant ainsi un certain niveau de précédence du sperme du dernier mâle.La masse de sperme dans la spermathèque était inférieure à sa capacité chez les femelles ayant été accouplées une ou deux fois. Le doryphore mâle aurait donc avantage à demeurer sur la femelle après un premier accouplement afin d'obtenir le minimum de trois accouplements requis pour remplir la spermathèque avant que les autres mâles puissent accoupler cette femelle. Les autres mâles ont avantage à essayer de dérober cette femelle pour remplir eux mêmes la spermathèque ou tout au moins profiter d'une certaine précédence du sperme du dernier mâle pour fertiliser une partie des oeufs.Sans accouplements multiples, la femelle ne possède pas suffisamment de sperme pour féconder tous ses oeufs. II semble aussi que les accouplements répétés puissent augmenter sa fécondité.Les résultats supportent une stratégie reproductive de compétition plutôt qu'une simple maximisation du nombre d'accouplements.

     

Characterization of galeal chemosensilla in the adult Colorado beetle, Leptinotarsa decemlineata

ABSTRACT. Approximately fifteen gustatory sensilla are present on the galeae of adult Colorado potato beetle, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae). In the SEM, these sensilla are not distinguishable morphologically, but some of them differ physiologically. All are sensitive to sucrose, while only a few respond to gamma amino butyric acid (GABA). One, called the alpha sensillum, is significantly more sensitive to GABA than the others. The same sensory cell predominates in the alpha sensillum responses to GABA and sucrose. This example of differential specificity among sensilla, in an apparently uniform sensory field, is discussed.     

Genetic relationships of introduced Colorado potato beetle Leptinotarsa decemlineata populations in Xinjiang,China

The Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), is an infamous invasive species worldwide that aggressively attacks potato and other Solanaceae crops. CPB was first found in China in 1993 and has since spread across 2.77 × 10⁵ km² in Xinjiang Uygur Autonomous Region. To better understand genetic variation and migration patterns, we used seven polymorphic microsatellite loci to elucidate the genetic relationships and gene flow among 10 CPB populations across Xinjiang. (i) Overall low levels of genetic diversity were detected on the entire population in Xinjiang but most of the diversity was retained among populations during invasion. (ii) The mean pairwise F_ST was low (0.071 ± 0.043) among populations. The genetic differentiation was little (pairwise F_ST 0.038 ± 0.016) between the five interior populations (Wusu, Urumqi, Jimsar, Qitai and Mulei) and Tacheng population. The six populations might come from the same genetic group via Bayesian clustering and were closely related on a neighbor‐joining tree. Combining the history data, the five interior populations may have originated from Tacheng. (iii) Gene flow was frequent, especially among the five interior populations. Individuals from the interior populations could be assigned to Tacheng at higher probabilities (means 0.518 ± 0.127) than vice versa (means 0.328 ± 0.074), suggesting that the beetle population has spread from the border to the interior in Xinjiang.     

Host-plant discrimination and evolution of feeding preference in the Colorado potato beetle Leptinotarsa decemlineata

ABSTRACT. The behaviour of newly emerged adult Colorado potato beetles on preferred hosts follows a stereotyped pattern of sampling, feeding, grooming and rest. Reduced meal sizes on less-preferred hosts is accompanied by increased sampling and frequent interruptions in feeding. A systematic increase in pre-ingestive sampling on less-preferred foodplants indicates that beetles discriminate among closely related species within the Solanaceae. This ability may depend primarily on stimuli perceived at, and near, the leaf surface. Three geographic populations of beetles have adapted to different local host plants, but have not lost their preference for feeding on an ancestral host species. Host shifts by oligophagous insects to related plant species may evolve through selection for feeding generalists in isolated populations, and may not require genetic changes affecting the perception of a particular novel host.     

Ecdysteroids and adult diapause in the Colorado potato beetle, Leptinotarsa decemlineata

ABSTRACT. The difference in ecdysteroid activity in short-day (10-h photo-phase) and long-day (16-h photophase) Leptinotarsa decemlineata Say adults was characterized by high performance liquid chromatography, which revealed that for short-day beetles ecdysterone and ecdysone are the major constituents of the free ecdysteroids, whereas in long-day beetles products co-migrating with 2-deoxyecdysone and 2-deoxyecdysterone are just as abundant. Ecdysteroids were detected (15 ng/ml) only in the haemolymph of females. Ligation experiments showed that the induction of the diapause syndrome by inactivation of the corpora allata or the brain not only resulted in the formation of the typical diapause proteins but also in an increase of the ecdysteroid activity. It is suggested that adult diapause in L.decemlineata is regulated by a multifactorial system.     

Double-stranded RNA in exosomes: Potential systemic RNA interference pathway in the Colorado potato beetle,Leptinotarsa decemlineata

Despite extensive research during the past decade elucidating the mechanism of RNA interference (RNAi) in insects, it is not clear how ingested or injected double-stranded RNA (dsRNA) triggers RNAi response in the whole body or even its progeny, which is referred to as systemic RNAi. In the present study, we aim to understand how the dsRNA delivered into cells causes systemic RNAi using Colorado potato beetle cells (Lepd-SL1). We first tested if dsRNA treatment induces systemic RNAi in Lepd-SL1 cells. Exposure of a new batch of Lepd-SL1 cells to the conditioned medium where Lepd-SL1 cells treated with dsRNA targeting inhibitor of apoptosis were grown for 6 h induced apoptosis in these new batch of cells. We hypothesized the exosomes in the conditioned medium are responsible for RNAi-inducing effect. To test this hypothesis, we isolated exosomes from the conditioned medium from Lepd-SL1 cells that had been treated with dsGFP (dsRNA targeting gene coding for green fluorescent protein) or dsLuc (dsRNA targeting gene coding for the luciferase) were grown. RNA present in the purified exosomes was analyzed to check if long dsRNA or siRNA is accumulated in them. The results from the electrophoretic mobility shift assay clearly showed that the long dsRNAs are present in the exosomes. By knockdown of candidate genes involved in endosome recycling and generation pathways, we found that Rab4 and Rab35 are involved in exosome production and transport.     

Diet-related plasticity of the digestive proteolytic system in larvae of the Colorado potato beetle (Leptinotarsa decemlineata Say)

Quantitative and qualitative changes in digestive proteolytic activities were monitored in fourth-instar larvae of the Colorado potato beetle (Leptinotarsa decemlineata Say) subjected to three different leaf diets. Depending on the diet, the larvae exhibited variable growth rates, similar for potato (Solanum tuberosum) and eggplant (Solanum melongena) diets but lower for the tomato (Lycopersicon esculentum) diet. Interestingly, these growth rates were not associated with total protease activity in the midgut. While growth of tomato-fed insects was negligible, midgut protease activity in these insects was 1.5 and 4.2 times higher than that measured for potato- and eggplant-fed insects, respectively. As seen on gelatin-containing polyacrylamide gels, midgut extracts from insects that ingested eggplant leaves contained only a few proteinase forms, while numerous forms were observed in extracts of potato- and tomato-fed larvae. Although several forms were common to the three diets, their relative importance in the insect midgut varied. This diet-related plasticity of the digestive proteolytic system in Colorado potato beetle larvae leads one to question the potential for control approaches based on the inhibition of digestive proteases. Arch. Insect Biochem. Physiol. 36:241–250, 1997. © 1997 Wiley-Liss, Inc.     

Effect of diet and feeding history on flight of Colorado potato beetle, Leptinotarsa decemlineata

We evaluated the hypothesis that Colorado potato beetle (Leptinotarsa decemlineata Say) (CPB) flight frequency is related to diet, and that it changes with duration of food unavailability or exposure to poor quality food by exposing adult overwintered and summer CPB populations to an acceptable host plant (conventional foliage), a poor host (insect resistant transgenic foliage expressing Bacillus thuringiensis tenebrionis[Btt] Cry3a toxin) and no host. Exposure to poor host and no host treatments (with or without water) decreased mean daily flight frequencies and the overall number of overwintered CPB flying, but increased the mean daily flight frequency and number of summer population CPB that flew. Overwintered CPB did not react to an absence of plants at emergence whereas summer CPB increased mean daily flight frequencies when plants and water were not available. The flight response to insect resistant foliage was similar to that for starvation treatments in both populations indicating that flight may not be triggered by Btt toxins but by starvation brought on by feeding on poor quality food. Flight was observed in all treatments for the duration of the test with two exceptions; overwintered beetles fed insect resistant foliage ceased flying after day 17 and summer beetles starved without water ceased after day 8 of a 29‐day study.     

Sequencing and characterization of a cDNA encoding a ferritin subunit of Colorado potato beetle, Leptinotarsa decemlineata

A differentially expressed cDNA fragment (P311) from Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), was identified by restriction fragment differential display-polymerase chain reaction (RFDD-PCR) technique, and showed a strong similarity to ferritin heavy chain subunits of other organisms. Based on P311, we constructed specific primers and obtained a 840-bp cDNA fragment spanning the open reading frame of CPB ferritin subunit using the rapid amplification of cDNA ends (RACE) technique. The sequence encodes 213 amino acid residues, including a 19 amino acid signal peptide. The sequence has a conserved cysteine in the N-terminus and has the seven conserved residues that comprise the ferroxidase center, which is the feature of heavy chain ferritins of vertebrates. The CPB ferritin subunit has high amino acid sequence identity with the Apriona germari (69.3%), Galleria mellonela (54.5%), Manduca sexta (54.0%), Drosophila melanogaster (53.2%), Calpodes ethlius (51.4%), and Nilaparvata lugens (47.6%) but lower identity with the Anopheles gambiae (38.7%) and Aedes aegypti (37.8%). Using Northern blot analysis, the subunit mRNA was identified from fat body and midgut of 4th instars with much higher mRNA levels found in midgut than that in fat body (2.5-fold). Nevertheless, only the levels of mRNA in fat body was induced by dexamethasone (1.5-fold).     

Quiescence in the Colorado potato beetle, Leptinotarsa decemlineata

Quiescence (death feigning) was induced in the Colorado potato beetle, Leptinotarsa decemlineata by mechanical disturbance of live and artificial plants on which beetles were walking, feeding or grooming. The response consisted of a sudden cessation of activity lasting 12 to 500 s. Recovery from this immobile state was usually preceded by either antennal or leg movement. The time taken for beetles to come out of the immobile state (i.e. recovery time) was not dependent on age or sex of beetles or on stimulus duration, but was dependent on degree of displacement and on satiation level. Repeated stimulations led to longer recovery times and to a smaller proportion of responding beetles. Stimulation within the immobile state also prolonged recovery time. The most effective frequency of mechanical disturbance for startling beetles was 30 Hz. This response is presumed to be part of the extensive defense repertoire of this beetle, but we have so far been unable to discover a natural enemy that will elicit it.     

Body movements and their role as triggers of heartbeats in pupae of the Colorado potato beetle Leptinotarsa decemlineata

Rhythmic body movements and their role as triggers of intermittent heartbeats were studied in pupae of Leptinotarsa decemlineata Say. Heartbeats and body movements were recorded simultaneously by means of an optical method (infra‐red cardiography) combined with respirometry. IR‐cardiography allows heartbeats and body movements to be distinguished on the basis of their different rates (40–80 min^?1 and 4–8 min^?1, respectively) and amplitude. In the mid‐interecdysial period, abdominal movements in the pupae were always accompanied by heart activity beginning after the first 5–12 abdominal strokes. Simultaneous periods of abdominal movements and heartbeats lasted 2–5 min, while the intervening pause ranged from 40 to 72 min at 24°C. Experiments of forced heart activity showed that a slight external tactile stimulus (prodding once with a single hair), applied prior to an expected heartbeat bout, evoked abdominal movements followed soon afterwards by contractions of the dorsal vessel. Repeated prodding with a hair evoked body rotating movements (1–3 strokes) at any time with heartbeats starting at the first movement. We surmise that rhythmic body movements play an active role in the triggering of heart activity in pupae according to the principle of cardiac reflex response. This is a possible mechanism for synchronizing periods of heart activity with periods of rhythmic body movements. Haemolymph circulation in pupae is favoured when heartbeat bouts are accompanied by active body movements.     

Isolation and characterization of Locusta migratoria accessory gland myotropin I (Lom-AG-MT-I) from the brain of the Colorado potato beetle,Leptinotarsa decemlineata

A novel myotropic Colorado potato beetle peptide, active in the Locusta oviduct motility assay, was isolated from a methanolic extract of 9,000 brain complexes of adult Leptinotarsa decemlineata by means of HPLC. Its sequence is Gly-Phe-Lys-Asn-Val-Ala-Leu-Ser-Thr-Ala-Arg-Gly-Phe-NH₂. This peptide is identical to Lom-AG-MT-I, a myotropin previously isolated from the male accessory glands of Locusta migratoria, using the L. migratoria oviduct motility bioassay as a monitoring system. It strongly stimulated the frequency, amplitude, and tonus of the myogenic oviduct contractions, even at low concentrations. © 1996 Wiley-Liss, Inc.     

Intraspecific mitochondrial DNA variation in the Colorado potato beetle,Leptinotarsa decemlineata (Coleoptera: Chrysomelidae)

Restriction endonuclease analyses of mitochondrial DNA (mtDNA) were used to examine genetic variability and population structure inLeptinotarsa decemlineata (Say). A group of three enzymes, EcoRI, HpaI, and PstI, was used to reveal polymorphism both within and among some of the 10 populations tested, yielding 16 haplotypes in combination. The frequencies of these 16 haplotypes differed significantly across geographic regions, indicating some partitioning of mtDNA haplotypes. Estimates of mtDNA sequence divergence (δ) between haplotypes ranged from 0.016 to 0.135%, suggesting local differentiation of mtDNA in some populations. Analysis of these data suggests that Texas was colonized by more than one mtDNA lineage, most likely originating in Mexico. We hypothesize that a larger founder size for the initial introductions or high levels of variability in the parent population at the edge of the CPB expanding range led to the initial partitioning of haplotypes observed in samples from Texas.     

Synthetic host volatiles increase efficacy of trap cropping for management of Colorado potato beetle, Leptinotarsa decemlineata (Say)

Abstract 1 The attractiveness of pitfall traps baited with a synthetic host volatile attractant to colonizing adult Colorado potato beetle, Leptinotarsa decemlineata (Say) was evaluated in a field setting. 2 Significantly more postdiapause, colonizing adult L. decemlineata were captured in baited than unbaited pitfall traps. 3 The potential for this synthetic kairomone to enhance the efficacy of trap cropping as a management tool was evaluated by comparing conventionally managed plots with like‐sized plots bordered by either attractant‐treated trap crop or untreated trap crop. 4 More postdiapause, colonizing adults, egg masses and small larvae were present in attractant‐treated trap crops than in untreated trap crops. 5 There were no significant differences in egg mass and small larvae densities between plots bordered by attractant‐treated trap crops and conventionally managed plots, but there were significantly fewer large larvae and adult beetles in conventionally managed plots. 6 Plant canopy area of conventionally managed plots was significantly greater than in plots bordered by either type of trap crop. 7 Yields for conventionally managed plots and plots bordered by attractant‐treated trap crops did not differ, and less insecticide (44%) was applied to plots bordered by attractant‐treated trap crops.     

Laboratory and greenhouse evaluation of a synthetic host volatile attractant for Colorado potato beetle, Leptinotarsa decemlineata (Say)

Abstract 1 The attractiveness of potato plants treated with a synthetic host volatile blend [(Z)‐3‐hexenyl acetate (+/–)‐linalool, and methyl salicylate] to newly emerged and 5‐day‐old adult Colorado potato beetle, Leptinotarsa decemlineata (Say), was compared at four doses against untreated control plants and plants treated with an azadirachtin‐based antifeedant in greenhouse cage arenas. 2 Attractant‐treated plants (derived release rates of 0, 5.7, 17.1 or 57 µg/h) were significantly more attractive than untreated control plants to newly emerged and 5‐day‐old adults only at 57 µg/h. 3 Attractant‐treated plants were significantly more attractive than antifeedant‐treated plants to newly emerged and 5‐day‐old adults at the 5.7 µg/h treatment level and higher. Mean insect density on attractant‐treated plants in the attractant/antifeedant study was significantly higher than in the attractant/control study. 4 Habituation to the synthetic attractant was evaluated by exposing adult beetles to the synthetic attractant for 0, 1, 2.5, 4, 8, 12 or 16 h, before release into a wind tunnel in which an attractant‐baited plant model was placed at the upwind end. Insects exposed to the synthetic host attractant for ≤ 8 h moved to the synthetic attractant‐baited plant model whereas insects exposed to the synthetic host attractant for 12 and 16 h did not. Furthermore, beetles exposed to the synthetic attractant for 0 and 1 h moved at rates greater than, or equal to, the median whereas beetles exposed for longer time periods moved at rates significantly less than the median. 5 These results demonstrate the potential for using the synthetic plant attractant and an antifeedant as components in a stimulo‐deterrent strategy for management of the Colorado potato beetle as shown by us in another study.     

Differential responses of galeal gustatory sensilla of the adult Colorado potato beetle, Leptinotarsa decemlineata (Say), to leaf saps from host and non-host plants

ABSTRACT Responses of galeal sensilla of the Colorado potato beetle, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae), to leaf sap from Solanum tuberosum and a number of other solanaceous and non-solanaceous plants are described. A single cell responds vigorously to expressed sap from S.tuberosum , -and even to ten-fold dilutions of expressed sap. The response is characterized by a high spike frequency, low phasic activity and a tonic phase which can last for tens of seconds. At 8 s following stimulus application the primary response is typically 50% of its original activity, indicating slow adaptation to the sap stimulus. The primary response varies little among multiple stimulations of single sensilla and among sensilla on the same animal. There is a secondary response to potato leaf sap from one or more cells which have low spike amplitudes. This is usually of much lower frequency than the response from the primary cell, and it is highly variable.
The prominent and reproducible single cell response to potato sap may be an important component in signalling the presence of a host plant. This hypothesis is tested using saps from two other species of Solanum which are hosts, and several solanaceous and non-solanaceous non-hosts. Responses of galeal sensilla to the three host-plants tested were qualitatively similar and reproducible while responses to the non-hosts were highly variable and qualitatively different from responses to host saps. These results are interpreted and discussed in the context of a possible mechanism for chemosensory coding of complex stimuli in the insect/plant relationship.
Analysis of the raw electrophysiological data required extensive use of a microcomputer and several programs were developed during the course of this study. The computer analyses are briefly described in the paper and additional information and copies of the programs are available from the authors.     

Effect of an entomopathogenic nematode,Steinernema carpocapsae on haemocyte profile and phenoloxidase activity of the Colorado potato beetle,Leptinotarsa decemlineata

The Colorado potato beetle (CPB), Leptinotarsa decemlineata Say is the most destructive insect pest of potato in many areas of the world. Little is known about the haemocyte types of the CPB and its plasma phenoloxidase (PO). In this regard, we investigated the haemocyte profile and PO of CPB and its immune response to the entomopathogenic nematode, Steinernema carpocapsae. Five types of haemocytes, the plasmatocytes (~67.4%), granulocytes (~23.5%), oenocytoids (~2.4%), spherulocytes (~0.25%) and prohaemocytes (~6.5%) were identified in fourth instar CPB larvae. Total haemocyte counts (THCs) were significantly reduced in nematode-injected insects compared with control groups (P < 0.05). Nematode cellular encapsulation observed in haemolymph of nematode-injected insects may partially explain decreased THCs. Plasma PO assay showed increased PO activity in nematode-injected insects compared with control groups (P < 0.05). Plasma PO assay on native polyacrylamide gel electrophoresis (PAGE) assay with L-3, 4-dihydroxyphenylalanine as substrate showed five bands (with molecular weights of approximately 200, 118, 68.5, 62.5 and 58.75 kDa).