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.

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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.

     

Ultrastructure of the neurosecretory system of the Colorado potato beetle,Leptinotarsa decemlineata (Say)

Summary The ultrastructure of seven types of neurosecretory cells (NSC) in the medial and lateral groups of the protocerebrum is described. The differences among cell types established earlier by light microscopy parallel differences in size and appearance of the neurosecretory particles observed in electron micrographs. No relationship was found between the affinity for Gomori's paraldehyde fuchsin stain and the nature of the particles.The secretions of the A-, A₁-, and C-types of NSC of the medial group are characterized by electron-dense neurosecretory granules of 1250 Å dia., medium-dense granules of 2100 Å, and electron-lucent vesicles of 1700 Å, respectively. The L-type NSCof the lateral group contain smaller (1300 Å) or larger (1700 Å) neurosecretory granules. The medial B- and E-types of NSC and the lateral L_B-type contain granulated vesicles (1200 Å) of the same appearance. These cell types differ in other respects and most likely have separate functions.The author wishes to thank the Laboratory of Virology of the Agricultural University for the use of the electron microscope, Mr. J. Groenewegen and Miss J. van Rinsum for technical assistance, and Professor J. Lattin for correcting the English text. Part of the work has been done while the author was in the service of the Netherlands Organization for the Advancement of Pure Research (ZWO, grant 942-48), and the National Council for Agricultural Research (TNO).     

A proctolin-like peptide and its immunocytochemical localization in the Colorado potato beetle,Leptinotarsa decemlineata

Summary Evidence is presented that neurons in the adult Colorado potato beetle contain a proctolin-like substance. By use of immunocytochemical methods the location of immunoreactive neurons in the central and stomatogastric nervous systems is described. No such neurons were found in the proto- and deutocerebrum or optic lobe. Few immunoreactive neurons are present in the tritocerebrum and numerous proctolin-immunoreactive neurons occur in all ventral ganglia and in the frontal ganglion. Two groups of neurosecretory cells in the suboesophageal ganglion contain a proctolin-immunoreactive substance. In these cells this material is co-localized with a bovine pancreatic polypeptide/FMRF amide-like substance and with a vasopressin/vasotocin/oxytocin-like substance. Proctolin-immunoreactive axon terminals were found on the musculature of the fore- and hindgut and of the vas deferens, and on some segmental muscles. Furthermore, proctolin-immunoreactive neurosecretory axon terminals were found in the corpus cardiacum. The proctolin-like substance may therefore function both as a neurotransmitter/neuromodulator and as a neurohormone. The presence of a proctolin-like substance was also demonstrated with a sensitive bioassay. On fractionation of extracts of the nervous systems of Leptinotarsa decemlineata with high performance liquid chromatography most of the proctolin-like bioactive material comigrated with authentic proctolin. This shows that a proctolin-like substance in this insect is very similar to, if not identical with, the known pentapeptide proctolin.     

Serotonin-like immunoreactivity in the ventral nerve cord of the Colorado potato beetle,Leptinotarsa decemlineata: Identification of five different neuron classes

Summary In an immunohistochemical study of the ventral nerve cord of L. decemlineata, five distinct neuron categories were distinguished: 1) Two paired segmental twin interneurons occur in each ganglion or neuromere; their axons distribute processes over almost the entire nerve cord and run to the cerebral ganglion complex. In contrast, other axons are distributed locally. 2) Four large frontal neurosecretory neurons occur in the suboesophageal ganglion (SOG), two of which have axons that run into the mandibular nerves to form a neurohemal plexus on the surface of cerebral nerves. 3) A pair of large caudal neurons occur in the terminal ganglion and innervate the hindgut. 4) Local miniature interneurons occur in the SOG. 5) Terminal neurons are present in the last abdominal ganglion. Segmental twin interneurons appear to be grouped into 3 functional units spanning several ganglia. Their axons run to specific projection areas, which separate the functional units, and which mark the externally visible separation of condensed ganglion complexes. A possible role of the most caudal functional unit might be the synaptic control of caudal neurons innervating the hindgut.     

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.     

Diffuse serotoninergic neurohemal systems associated with cerebral and suboesophageal nerves in the head of the Colorado potato beetle Leptinotarsa decemlineata

We analyzed the anatomy of two diffuse neurohemal systems for serotonin in the head of the Colorado potato beetle Leptinotarsa decemlineata by means of immunohistochemistry. One system is formed by axons from two bilateral pairs of neurons in the frontal margin of the suboesophageal ganglion that enter the ipsilateral mandibular nerve, emerge from this nerve at some distance from the suboesophageal ganglion, and cover all branches of the mandibular nerve with a dense plexus of immunoreactive axon swellings. The other system is formed by axons from two large neurons in the frontal ganglion that enter the ipsilateral frontal connectives, emerge from these connectives, and form a network of axon swellings on the labroforntal, pharyngeal, and antennal nerves and on the surface of the frontal ganglion. Immunohistochemical electron microscopy demonstrated that the axon swellings are located outside the neural sheaths of the nerves and hence in close contact with the hemolymph. We therefore suggest that these plexuses represent extensive neurohemal systems for serotonin. Most immunoreactive terminals are in direct contact with the hemolymph, and other terminals are closely associated with the muscles of the mandibles, labrum, and anterior pharynx, as well as with the salivary glands, indicating that these organs are under serotoninergic control.     

Structure-activity studies of modified citrus limonoids as antifeedants for Colorado potato beetle larvae, Leptinotarsa decemlineata

Limonin and ten structurally modified limonins were evaluated as antifeedants against 4th instar larvae of the Colorado potato beetle, Leptinotarsa decemlineata Say, in no-choice leaf dics assays. The epoxide and furan groups were shown to be essential structural requirements for high antifeedant activity.

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Résumé Le pouvoir phagodissuasif de la limonine et de 10 limonines dont la structure avait été modifiée a été évalué sur des disques de feuilles de Solanum tuberosum offerts à des larves du 4ème stade de L. decemlineata. Aucune activité dissuasive n'ayant été observée avec le tétrahydrolimonine ou le déoxylimonine, l'anneau furane et le groupe époxy sont donc nécessaires pour obtenir cette activité. Ni la réduction de la fonction 7-kéto, ni la rupture de l'anneau A de la limonine n'ont eu d'effêt sur cette activité. La réduction des fonctions 16-carbonyl et 7-kéto de la limonine ont diminué l'activité, mettant en évidence une action possible de l'anneau D-lactone dans l'activité phagodissuasive. Tandis que la déépoxydation de la limonine entraîne une perte d'activité totale, une restauration partielle de cette activité a pu être obtenu par une réduction ultérieure de la fonction 7-kéto.

     

A fine structural survey of the development of the adult fat body of Leptinotarsa decemlineata

The fat body of a female Colorado potato beetle, Leptinotarsa decemlineata, at adult ecdysis, contains a large number of protein granules which are composed of light and dark zones. Part of the light zone in some of these granules is believed to be urate. During the first two days after adult ecdysis, fat body development is not essentially different in females reared either under long- or short-day conditions. Protein granules and large vacuoles disappear and the first cell organelles are regenerated. The effect of the photoperiod on the histological structure of the fat is expressed after these events. In females reared under long-day conditions, the fat body becomes specialized for vitellogenin synthesis. Under short-day conditions, the fat body stores massive amounts of lipid until day 6 after adult ecdysis. Then the first electron-dense protein granules develop near the nucleus, and on day 10 the first autophagic vacuoles are seen. These structure changes are discussed in connection with the known biochemical properties of the adult faty body of Leptinotarsa.     

Incidence of natural enemies of the Colorado potato beetle,Leptinotarsa decemlineata [Coleoptera: Chrysomelidae] on a native host in Mexico

In central Mexico, the center of origin forLeptinotarsa decemlineata (Say) and its principal host plants,L. decemlineata is associated with a diverse complex of natural enemies. Observations during 2 years in Morelos, Mexico revealed 4 species of asopine pentatomids, 7 species of foliar searching carabids, and 2 coccinellids among the predators ofL. decemlineata. Parasitoids include 3 species of tachinids andEdovum puttleri Grissel, an eulophid egg parasitoid. These natural enemies include several predators that have not previously been reported attackingL. decemlineata. University of Maine Agriculture Experiment Station No. 1553     

Fecundity and egg fertility in the adult Colorado beetle (Leptinotarsa decemlineata) surviving larval infection by the fungus Beauveria bassiana

Secondary effects of the entomopathogenic hyphomycete Beauveria bassiana (Bals.) Vuill. were investigated on adults of Leptinotarsa decemlineata Say (Coleoptera:Chrysomelidae) treated as newly moulted 4th-instar larvae. Fecundity and egg hatching were monitored in surviving females mated with surviving males. When reared at 22°C, adult survivors showed a lessening of their reproductive potential during their whole life. According to the fungal inoculum dosage, the reductions of the total number of eggs laid per female and the mean number of eggs per egg mass ranged from 20% to 56% and from 18% to 46%, respectively. In contrast, at 25°C, the fecundity of survivors was not affected by the fungal infection. This variability of the secondary effects of B. bassiana according to temperature conditions might be related to the lower infection level of treated larvae at 25°C. Moreover, at both temperatures, eggs laid by females surviving fungal infection as larvae were as fertile as eggs laid by control insects. It is concluded that secondary effects of B. bassiana on the fecundity of the Colorado Potato Beetle are temperature-dependent and that they could become negligible at high temperatures under field conditions.

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Résumé Les effets secondaires de l'hyphomycète entomopathogène, Beauveria bassiana (Bals.) Vuill. sur la fécondité et la fertilité des insectes survivant à l'infection fongique ont été étudiés chez les adultes du doryphore Leptinotarsa decemlineata Say (Coleoptera:Chrysomelidae) issus de larves contaminées en début de 4ème stade. Les insectes maintenus à 22°C ont présenté une réduction de leur potentiel reproductif. Ainsi, suivant la dose d'inoculum fongique, le nombre total d'oeufs pondus par femelle et le nombre moyen d'oeufs par opplaque ont baissé de 20% à 56% et de 18% à 46%, respectivement. En revanche, à 25°C la fécondité des adultes survivants n'a pas été affectée par la maladie. Cette variabilité en fonction des conditions thermiques peut être liée à la diminution de l'effet-dose du champignon sur les larves à 25°C par rapport à 22°C. Par ailleurs, quelles que soient les conditions thermiques (22°C ou 25°C) on ne constate aucun changement significatif de la fertilité des oeufs pondus par les femelles survivantes. Les auteurs concluent que les effets secondaires de B. bassiana sur la fécondité du doryphore dépendent des conditions thermiques et qu'ils pourraient devenir négligeables à des températures élevées en plein champ.

     

Infection byBeauveria bassiana ofLeptinotarsa decemlineata larvae as a consequence of fecal contamination of the integument followingper os inoculation

A study ofper os inoculated larvae was undertaken to determine if the entomopathogenic fungus,Beauveria bassiana, is able to infect the Colorado potato beetle,Leptinotarsa decemlineata, via the alimentary tract. Surface orper os inoculated larvae which were immediately surface sterilized post inoculation did not succumb to infection, whereas those larvae not sterilized became infected. Histological studies of fed or starved, agnotobiotic (with microbial flora) and axenic larvae revealed that conidia can germinate in the gut regardless of the presence of gut microflora. However, infection via the alimentary tract was never observed in fed larvae and only noted in a single starved individual. It is concluded that infections ofper os inoclated larvae occurred after surface contamination of the integument by viable conidia contained in the frass. The rate of food passage through the gut is probably important in preventingper os infections.     

Isolation and immunochemical characterization of neuropeptides from the midgut of the Colorado potato beetleLeptinotarsa decemlineata

The midgut of the Colorado potato beetle showed endocrine cells immunopositive to monoclonals like MAC-18, MAC-3 and polyclonals to FMRF-amide and AKH-241. Extraction and HPLC-fractionation of the midgut extracts after partial purification and characterization showed immunopositive reaction to the monoclonals and also showed myotropic activity stimulating the potato beetle gut. Molecular mass of the two active peptides isolated were 22 and 26 kDa respectively. Both these peptides could be immunocytochemically demonstrated in the brain neurosecretory cells of the red cotton bug,Dysdercus cingulatus and the castor semilooper,Achaea janata as well.     

Resistance of three wild tuber-bearing potatoes to the Colorado potato beetle

The resistance of Solanum okadae Hawkes & Hjert. (PI 458367), Solanum oplocense Hawkes (PI 473368), and Solanum tarijense Hawkes (PI 414150) to the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Chrysomelidae: Chrysomelini), was studied. In replicated field trials all three accessions showed a high level of resistance to the beetle. No significant genetic variability between genotypes of the same species was found. Results from host acceptance behavior experiments, suitability for larval development tests, foliage consumption tests, and adult survival and oviposition tests supported the hypothesis that the mode of resistance differs between the three wild Solanum species. Solanum okadae and S. oplocense affected host acceptance and consumption. Because the beetle reacted differently to these two species it was hypothesized that the antifeedant chemical(s) differed in nature or quantity. S. tarijense contrasted with the other two species by affecting mostly adult colonization and oviposition.     

Genetic analysis of larval survival and larval growth of two populations of Leptinotarsa decemlineata on tomato

The genetics of adaptation to tomato in Leptinotarsa decemlineata (Say) were investigated in reciprocal F₁, F₂, and backcross populations generated from crosses between beetles from a tomato adapted population and from a population that was poorly adapted to tomato. Larvae from the parent and test populations were reared on tomato for four days, after which survivorship and larval weights were recorded. Most results indicate that differences in larval growth and survival on tomato between the parent populations are largely determined by autosomal, polygenic mechanisms, the inheritance of which involves a significant dominance component. However, results from F₂ crosses are not consistent with this conclusion. A significant difference in larval weights, but not in survival, between reciprocal F₁ populations in an analysis of combined data from four separate experiments suggests that maternal cytoplasmic effects may contribute to differences in larval performance on tomato between the adapted and unadapted populations. The unusual results obtained from F₂ crosses in this study are not atypical of results from previous studies of the genetics of adaptation to host plants by the Colorado potato beetle. Host plant adaptation by Colorado potato beetles may therefore involve unusual genetic mechanisms that are not easily assessed by classical Mendelian analysis.     

Immunocytochemical localization of neurons in the nervous system of the Colorado potato beetle with antisera against FMRFamide and bovine pancreatic polypeptide

Summary Particular neurons in the nervous system of the Colorado potato beetle, Leptinotarsa decemlineata, are recognized by antisera against bovine pancreatic polypeptide and FMRFamide. Both antisera react with the same neurons. Solid phase absorptions showed that antiserum against bovine pancreatic polypeptide cross-reacts with FMRFamide, whereas antiserum against FMRFamide cross-reacts with bovine pancreatic polypeptide. Some of the immunoreactive neurons have axons branching extensively within the neuropile, which suggests that the peptide is used as transmitter. In the corpus cardiacum, a neurohaemal organ in insects, numerous immunoreactive axon terminals are present. Here, the peptide material is presumably released as a hormone.     

Oviposition behavior of Edovum puttleri, reared on two hosts, Leptinotarsa decemlineata and L. texana

Female Edovum puttleri Grissell [Hymenoptera: Eulophidae], reared from eggs of Leptinotarsa decemlineata (Say) or Leptinotarsa texana Schaeffer [Coleoptera: Chrysomelidae], were videotaped as they attacked egg masses of L. decemlineata containing 20 host eggs. We identified 15 components of ovipositional behavior. Parasitoids reared on L. texana attacked and oviposited in significantly more host eggs than did females reared on L. decemlineata. Ethometric analyses of behavioral transitions and a clustering analysis of 34 behavioral parameters showed that females reared on L. texana attacked the host egg mass in a different manner than those reared from L. decemlineata. It was concluded that differences were associated with the host species upon which they were reared. Contrary to previous reports, mortality of unparasitized hosts was caused by an ovipositor probe of short duration, which was not related to host-feeding.     

Citrus limonoid effects on Colorado potato beetle larval survival and development

The effects of citrus limonoids, applied topically to potato (Solanum tuberosum L. var. Katahdin) foliage, on Colorado potato beetle (Leptinotarsa decemlineata) (Say) (Chrysomelidae) larval development, growth, and survival were quantified in laboratory assays and a small-plot field test. In laboratory assays, survival, development rate, and body weight decreased with increasing limonoid concentration, however these measures of larval response did not significantly differ among varying periods of limonoid exposure (three, six, or nine days). Significant limonoid application concentration and frequency effects on survival, development rate, and defoliation were observed in the field test. These results indicate the potential utility of lethal and non-lethal effects of citrus limonoids for management of the Colorado potato beetle.