Lipids and fatty acids of the southwestern corn borer,Diatraea grandiosella

The lipids of the adults and of several immature stages of the southwestern corn borer, Diatraea grandiosella, were studied after they were fed natural corn stalks or artificial diets. Linoleic acid (18:2) was the major fatty acid of the neutral lipids in both the natural and the artificial diets, but aleic acid (18:1) was the principal neutral lipid in all insect stages. Also, linoleic acid and oleic acid were the principal acids in the insect phospholipids of all stages. The content of linoleic acid in the natural diet was also high, but that in the artificial diet appeared to be much too low for insect requirements. Phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) were the major phospholipids in all growth stages. Thus, in larvae diapausing in the field, the unsaturated fatty acid content of PC was 59·3 per cent, primarily 16:1 and 18:1, and PE was 87·4 per cent, primarily 18:1, 18:2, and 18:3, and the fatty acids in the number 1- and 2-positions of PC were 53·6 and 97·2 per cent unsaturated, respectively. The haemolymph of diapausing southwestern corn borer larvae contained primarily glycerides but also had some PC and PE. Fat body from diapausing larvae contained primarily 16:0, 16:1, and 18:1 in a ratio of 1 : 1 : 2. Thus lipids of the southwestern corn borer do not reflect dietary lipids as closely as do other insects studied.     

Cold hardiness of larvae of the southwestern corn borer, Diatraea grandiosella

The cold-hardening capacity of field-collected larvae from southeast Missouri and laboratory-reared larvae of the southwestern corn borer, Diatraea grandiosella Dyar, was examined. Supercooling points of non-diapause and diapause larvae collected from maize plants grown in Missouri (36°30 N lat.) were ca.-7.0°C. The hemolymph melting points of diapause field larvae (-0.8°C) were significantly lower than those of non-diapause larvae collected in July (-0.5°C). The supercooling points of hemolymph from non-diapause and diapause field larvae ranged randomly from-10° to-18°C. Supercooling points of non-diapause laboratory larvae increased from-13° to-10°C prior to pupation, whereas those of diapause larvae increased similarly before the onset of diapause, but then decreased during diapause to ca.-17°C. No change in supercooling points or capacity to survive in the presence of ice was observed in diapause laboratory larvae acclimated at 4°C for 63 days. Laboratory and field larvae began to freeze at ca.-1.5°C in the presence of ice, but survived to several degrees below their melting points. The high supercooling points of field larvae appeared to be due to the presence of an environmental ice-nucleator. Although data for laboratory larvae indicate sufficiently low supercooling points to permit winter survival in southeastern Missouri, considerable larval mortality occurs in the field due to inoculative freezing and the presence of an ice-nucleator.     

Observations on the nutritional requirements of the southwestern corn borer, Diatraea grandiosella

The southwestern corn borer was reared on a meridic diet containing wheat germ and wheat germ oil as its only plant adjuvants. The insect's requirements for protein, a mineral mixture, and L-ascorbic acid were studied using a bioassay based on larval growth rate and adult wing condition. The principal results showed that dietary supplements of casein (5.0%), a mineral mixture with nine components (0.9%), and L-ascorbic acid (0.5%) were essential for optimum growth and development of the insect.

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Résumé Diatraea grandiosella ou Southwestern Corn Borer, a été élevé sur un aliment de type méridique et on a déterminé ses besoins quantitatifs en protéïne, en sels minéraux et en acide ascorbique. Un test biologique a été conçu, basé sur le pourcentage de chrysalides obtenues et sur les conditions de développement des ailes, cela dans chaque test et dans le groupe témoin. Un supplément alimentaire de 5% de vitamine sans caséine, a permis une croissance et un développement optima.Une modification de la teneur en caséine de 2% a un effet défavorable sur la vitesse de la croissance larvaire. L'insecte exige un mélange de sels minéraux; une concentration de 0,9% de ce mélange est optima. En l'absence de ce supplément, la croissance larvaire est significativement ralentie. L'acide L. ascorbique a aussi été reconnu indispensable, sa présence, au taux de 0,5%, donne les meileurs résultats. La plupart des larves élevées sur milieux manquants d'acide L. ascorbique meurent au cours de la mue, entre le 4ème et le 5ème stade. La comparaison entre les présents résultats et deux d'autres chercheurs sont discutés.

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Contribution from the Missouri Agricultural Experiment Station, Columbia, as journal series no. 7116.     

Temperature and photoperiodic regulation of diapause of the southwestern corn borer,Diatraea grandiosella

The effect of temperature and photoperiod on the onset and termination of the mature larval diapause of the southwestern corn borer, Diatraea grandiosella, was examined. The results showed that diapause induction was an extremely temperature-dependent process. Larvae reared at 23°C under short days all entered diapause whereas 90 per cent of those reared at 27°C developed continuously. A photoperiodic response was only demonstrable at 25°C, when diapause was instituted following larval exposure to daily photophases ranging from 8 to 14 hr. An examination of the sensitivity of immature larvae to low temperatures revealed that all instars showed some susceptibility to low temperature induction of diapause. No intermediate instar was found in which the developmental programme could be switched entirely from a diapause to a non-diapause one, or vice versa.Diapause development was also found to be primarily temperature regulated. The rate of termination of diapause at 30°C was significantly higher than that at 25 or 23°C. Continuous exposure to light rather than a daily photoperiod produced the highest rate of diapause development. It was also shown that a period of chilling (5°C) did not accelerate diapause development. The significance of the results in relation to the evolution and geographical distribution of the species is discussed.     

Phenological adaptations of a colonizing insect: The southwestern corn borer,Diatraea grandiosella

Summary The developmental rate, critical photoperiod, and diapause intensity were determined for three populations of the southwestern corn borer, Diatraea grandiosella, from Missouri, Mississippi and Kansas. Mississippi larvae grew at the highest rate and Missouri larvae grew at the lowest rate. The zero developmental temperatures (°C) for the Missouri population were estimated from regression lines as follows: 10.5° (eggs), 10.8° (diapausing larvae), 13.3° (non-diapausing larvae) and 11.4° (pupae). The required heat units were: 85° (eggs), 588° (diapausing larvae), 333° (non-diapausing larvae) and 149° days (pupae). However, the observed low temperature limit for larval growth under constant temperature regimes was approximately 17°C.The critical day lengths for diapause induction observed at 25°C were: 15 h 11 min (Missouri); 15 h 20 min (Mississippi); and 15 h 22 min (Kansas). The photoperiodic response of the Mississippi larvae was more or less retained at 30°C, whereas the response of the Missouri larvae was completely suppressed at this temperature. Diapause was most easily terminated in the Kansas larvae. The most intense diapause was observed in the Mississippi larvae.Model seasonal life cycles of the three geographic populations were constructed using photothermograms. Although the models showed good agreement with the field situation for the Missouri and the Kansas populations, some unknown factor(s) remains to account for an extremely long critical photoperiod in the Mississippi population.Contribution from the Missouri Agricultural Experiment Station, as journal series no. 9001     

An immunochemical study of the diapause-associated protein of the southwestern corn borer,Diatraea grandiosella

The distribution and titre of the diapause-associated protein (DAP), which accumulates in the fat body of diapausing larvae of the southwestern corn borer, Diatraea grandiosella, were determined using an antibody raised to the purified protein. Immunoblotting and immunodiffusion showed that the highest concentration of DAP was present in the fat body. Small amounts of the protein were present in the haemolymph and even lower levels were detected in other tissues. Rocket immunoelectrophoresis showed that DAP accumulated in the fat body at the beginning of diapause, reached a plateau, and gradually declined towards the end of diapause. Similarly, the titre of DAP in last instar non-diapausing larvae treated with a JH analogue increased, reached a plateau and then declined. Double immunodiffusion and immunoblotting using the DAP-antibody and extracts of the fat body of D. grandiosella and the southern cornstalk borer, Diatraea crambidoides, and the sugarcane borer, Diatraea saccharalis, revealed the partial immunochemical identity of the DAP of D. grandiosella and a protein present in these two species. Denaturing electrophoresis with immunoblotting showed that the DAP-related proteins of D. crambidoides and D. saccharalis have apparent molecular weights of 33,000 and 36,000, respectively, as compared to a molecular weight of 35,000 for DAP of D. grandiosella. A dot blot analysis showed that no cross reaction occurred between the “diapause proteins” present in the haemolymph of adults of the Colorado potato beetle, Leptinotarsa decemlineata, and the DAP of D. grandiosella.     

Diapause of the southwestern corn borer, Diatraea grandiosella: Utilization of fat body and haemolymph reserves

The utilization of metabolic reserves of mature southwestern corn borers held in two regimes (23° 12L:12D and 30° 24L:0D) which permitted different rates of diapause development was studied. Diapause larvae which had been reared on a meridic diet or host-plant tissue were compared. Data were obtained on the fate of lipid, glycogen, protein and amino acid reserves of the fat body and haemolymph during diapause. The major results showed that lipids comprised ca. 80% of the fat body dry weight and formed the principal reserve of diapause larvae. Thin layer chromatographic analyses showed that triglycerides made up >90% of the fat body lipids. Protein, amino acids and glycogen were found to be of secondary importance during diapause since they made up <15% of the fat body dry weight. In contrast, the haemolymph had a high protein and low amino acid, lipid, and glycogen content. Haemolymph proteins supplemented the fat body lipid reserves and were drawn upon during the later stages of diapause.

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Zusammenfassung Die Art und das Schicksal der Stoffwechselreserven des Fettkörpers und der Haemolymphe diapausierender Südwest-Maiszünsler, Diatraea grandiosella, wurden untersucht. Im Labor herangezogene und im Freien gesammelte junge Diapause-Raupen wurden zwei verschiedenen Regimen unterworfen, welche signifikant verschiedene Diapause-Entwicklungsraten gewährleisteten: 23° 12L:12D und 30° 24L:0D. Als Daten wurden festgestellt: Verpuppungsraten, Gesamtkörpergewicht und der Verbrauch der Fett-, Glykogen-, Protein- und Aminosäure-Reserven des Fettkörpers und der Haemolymphe während der Diapause unter beiden Bedingungen. Obwohl die Freilandraupen im allgemeinen größer als die Labortiere waren, wurden doch wenig qualitative Unterschiede zwischen beiden Gruppen gefunden. Als Hauptergebnis zeigte sich, daß die Fette, die 80% des Fettkörper-Trockengewichts ausmachen, die hauptsächliche Stoffwechsel-Reserve darstellen. Sie bestehen überwiegend (>90%) aus Triglyceriden und wurden während der Diapause allmählich verbraucht. Eiweiß, Aminosäuren und Glykogen machen nur weniger als 15% des Fettkörper-Trockengewichts aus. Im Gegensatz dazu wies die Haemolymphe der diapausierenden Raupen einen relativ hohen Eiweiß- und geringen Aminosäure-, Fett- und Glykogen-Gehalt auf. Haemolymphe-Proteine ergänzten die Fettreserven des Fettkörpers und wurden während der späteren Stadien der Diapause abgebaut.

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Contribution from the Missouri Agricultural Experiment Station, Columbia, as journal series no. 6546.     

Influence of fertilizer on southwestern corn borer, Diatraea grandiosella, infestation and damage to field corn

Nitrogen (N) and phosphorus (P) fertilizers have differing effects on southwestern corn borer (SWCB). Diatraea grandiosella Dyar, densities and damage to field corn, Zea mays L. Nitrogen fertilization of corn is associated with an increase in number of stalks infested and girdled by corn borers, while addition of P has the opposite effect. When combinations of N and P were applied, there were no significant differences in SWCB infestation or damage from plots receiving no fertilizer. The effect of combinations of N and P on SWCB were intermediate to either element alone.

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Résumé Nous avons étudié les corrélations entre l'utilisation d'engrais azotés et phosphatés d'une part, l'infestation et l'alimentation de D. grandiosella sur maïs en champs d'autre part. Les formules d'engrais comparées étaient: pas d'engrais, 90 et 179 kg N/ha sans P, 45 et 90 kg P/ha sans N, et les combinaisons de ces deux teneurs en N et P.La contamination était plus élevée dans les lots ayant reçu de l'azote que dans ceux traités sans N, mais la présence d'azote n'a pas réduit le décapage des tiges et la verse dus à l'alimentatin larvaire. A l'inverse, les lots n'ayant reçu que du phosphore avaient des pourcentages plus faibles de tiges contaminées par D. grandiosella et ainsi décapées et versées, que ceux qui n'avaient pas reçu P. Il n'a pas eu de différences significatives dans les contaminations ou les dégats entre les lots ayant reçu différentes combinaisons de N et P, et les effets de ces combinaisons sur l'insecte étaient intermédiaires à ceux de N et P seuls.

     

Water involvement in diapause and the resumption of morphogenesis of the southwestern corn borer, Diatraea grandiosella

The effect of water on diapause and the resumption of morphogenesis of the southwestern corn borer was examined. Diapause larvae were held under dry conditions or with access to water in various environmental regimes known to permit different rates of diapause development. Results showed that diapause development occurred at a higher rate in larvae held dry than in those continuously supplied with contact water. Larvae did not, therefore, require an exogenous water source to complete diapause development. Although the availability of water promoted the post-diapause pupal moulting cycle of larvae which had completed diapause development, those which maintained a tissue water content > ca. 55% did not require contact water to complete this cycle. The loss of water in diapause larvae held under dry conditions or with access to water along with the quantity imbibed following various periods of dehydration was estimated. The physiological significance of the results in relation to other similar data is discussed.

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Zusammenfassung Es wurde der Einfluss von Wasser auf Diapause und Wiederaufnahme der Morphogenesis bei Diatraea grandiosella untersucht. Reife Diapauselarven wurden aus Laborzuchten erhalten oder im Freiland in Südost-Missouri gesammelt. Sie wurden trocken gehalten oder hatten Zugang zu Wasser und zwar in verschiedenen Umgebungsbedingungen, bei denen bekanntermassen die Diapauseentwicklung mit verschiedener Geschwindigkeit erfolgt. Die Ergebnisse zeigten, dass die Diapauseentwicklung rascher ablief bei Larven, die trocken gehalten wurden als bei solchen mit dauerndem Kontaktwasser. Die Larven brauchen demnach keine exogene Wasserquelle zur vollständigen Diapauseentwicklung. Zwar begünstigt die Verfügbarkeit von Wasser nach der Diapause das Einsetzen der Verpuppungsphase; dennoch brauchten diejenigen Larven, deren Gewebswassergehalt bei über ca. 55% lag, überhaupt kein Kontaktwasser, um diese Phase, d.h. die Verpuppung, abzuschliessen. Der Wasserverlust von Diapauselarven bei trockenen und nassen Bedingungen wurde bestimmt und ebenso die aufgenommene Wassermenge nach Trockenperioden. Die physiologische Bedeutung der Ergebnisse in Beziehung zu anderen, ähnlichen Daten wird diskutiert.

     

Juvenile hormone and a protein associated with the larval diapause of the southwestern corn borer,Diatraea grandiosella

Pre-diapausing larvae of the southwestern corn borer, Diatraea grandiosella, were found to store an electrophoretically discrete protein fraction which makes up about 20% of the soluble proteins of the fat body of newly diapaused individuals. This fraction appears to be restricted to the larval fat body and haemolymph. It accumulates in the fat body of pre-diapausing larvae and reaches a maximum titre in newly diapaused individuals. The protein is utilized gradually during diapause so that only traces of it remain after diapause has terminated.The protein fraction was found to accumulate in the fat body of non-diapausing larvae which had been treated with a juvenile hormone (JH) mimic. This treatment induces a diapause-like state. The involvement of JH in the accumulation and utilization of the protein was investigated by examining the proteins of the fat body after larvae had been allatectomized, or had received a JH mimic application, or had received both treatments. The results showed that non-diapausing larvae began to accumulate the protein fraction within 8 days of receiving a single application of JH mimic. Allatectomy of pre-diapausing larvae was shown to suppress the accumulation of the protein. This suppression was partially reversed by a topical application of JH mimic. JH mimic treatments and allatectomy of diapausing larvae were also shown to cause a decrease in the titre of most of the proteins of the fat body including the diapause-associated protein.The diapause-associated protein has a molecular weight of about 41,000, and contains 15–17 different amino acid residues, of which aspartate, leucine, lysine, and glutamate predominate, comprising about 43, 37, 35 and 32 residues per molecule, respectively. We suggest that diapauselin is a suitable name for this protein. Our findings provide the necessary framework for undertaking a more detailed analysis of the hormonal regulation of diapauselin synthesis and utilization.     

Juvenile hormone titer determinations in the southwestern corn borer, Diatraea grandiosella, by electron capture-gas chromatography.

A procedure is described for analysis of the three known juvenile hormones (JHs) at physiological levels in 5–10 gram samples of insect hemolymph or whole bodies. The method employs conversion of the oxirane ring of the JHs to their respective 11-methoxy-10-pentafluorophenoxyacetate esters, allowing use of gas-liquid chromatography/electron capture detection for qualitative and quantitative analysis at picogram levels. The method is used to determine the JH titers of the southwestern corn borer, $\text{Diatraea}$$\text{grandiosella}$, as a function of developmental stage. The results show that diapausing larvae contain significant titers of all three known juvenile hormones, thereby supporting the theory that larval diapause in this species is under the control of the corpus allatum.     

Synthesis and release of lipophorin in larvae of the southwestern corn borer,Diatraea grandiosella: An in vitro study

The source of the lipophorin present in the larval haemolymph of the southwestern corn borer, Diatraea grandiosella, was examined in vitro. Although lipophorin was shown to be one of several proteins released from cultured fat body and midgut, only fat body was shown to synthesize lipophorin. Fat body, incubated in a medium containing [³H]leucine, was shown to release radiolabelled lipophorin using immunoprecipitation. Similar studies using midguts incubated in a medium containing [³H]leucine did not reveal any synthesis of lipophorin. Lipophorin was isolated by density-gradient ultracentrifugation from media in which the fat bodies of about 600 diapausing larvae had been incubated for 4 hr. The isolated lipophorin had a peak density of 1.11 g/ml, and contained various lipids including diacylglycerol, triacylglycerol, sterol, hydrocarbon, free fatty acid, phosphatidyl choline, phosphatidyl ethanolamine and sphingomyelin.     

Juvenile hormone and the induction of larval polymorphism and the diapause of the southwestern corn borer, Diatraea grandiosella

The southwestern corn borer, Diatraea grandiosella, enters diapause as an immaculate mature larva which is a polymorphic variant of the spotted non-diapause larva. Because dormant immaculate larvae could be obtained by treating last stage non-diapause larvae with a juvenile hormone mimic (JHM), experiments were conducted to determine whether these hormonally induced immaculate larvae (HIL) were physiologically comparable to the normal environmentally induced immaculate diapause larvae (EIL). Comparative data obtained about pupation rates, response to JHM, metabolic reserves, oxygen consumption, and the state of spermatogenesis of HIL and EIL led to the conclusion that the HIL were physiologically similar to the EIL. The results demonstrate that the developmental programme of non-diapause larvae could be switched and ‘diapause’ induced solely by the topical application of JHM. We believe that the data further support our hypothesis that the larval diapause of D. grandiosella is initiated and maintained by the juvenile hormone.     

Synthesis and release of proteins from cultured larval fat body of the southwestern corn borer,Diatraea grandiosella

The larval fat body of the southwestern corn borer, Diatraea grandiosella, was cultured in vitro to examine the relationship between proteins present in the fat body, those released into the medium, and those present in the haemolymph. While the incorporation of [³H]leucine into fat body proteins was high in last instar pre-diapausing and non-diapausing larvae, it fell in early diapausing larvae to about 11% of that found in prediapausing larvae. Incorporation of [³H]leucine into the diapause-associated protein of the fat body increased gradually in pre-diapausing larvae and reached a maximum in newly-diapaused larvae at a time when the incorporation of [³H]leucine into other proteins of the fat body had declined. The proteins released from the cultured fat body showed identical electrophoretic properties and close immunochemical relationships to most of those present in the haemolymph. Small amounts of the diapause-associated protein were released in vitro from the fat body of larvae of different ages in diapause. Lipophorin was also released in vitro from the fat body of non-diapausing and diapausing larvae, and shown to be immunochemically identical to the lipophorin present in the haemolymph.     

Effects of 25-azasteroids on development and reproduction of the southwestern corn borer Diatraea grandiosella Dyar

25-Azacholesterol and 25-azacoprostane significantly increased the larval period of southwestern corn borer in a dose dependent fashion from 1 to 300 ppm in the diet. Male larvae were much more resistant than female larvae to the growth-inhibiting effects of the azasteroids. Both azasteroids caused melanization of larvae and inhibited moulting, but did not produce larval-pupal intermediates. The azasteroids caused a reduction in pupal and adult weight at higher concentrations, more so in females than in males. Pupal periods and adult longevity were shortened, and adult wing malformation occurred with the highest azasteroid concentrations. Egg production and hatchability decreased with increasing azasteroid concentrations in larval diets.     

Assembly and secretion of lipophorin by the larval fat body of the southwestern corn borer,Diatraea grandiosella: An in vitro study

The synthesis, processing, and secretion of lipophorin by the larval fat body of the southwestern corn borer, Diatraea grandiosella, was examined using in vitro techniques. Pulse-labeling of lipophorin with [³⁵S]methionine showed that apolipophorin-I and -II were each synthesized and secreted from the fat body into Grace's medium with an intracellular transit time of about 45 min. Secretion of the apolipoproteins from the fat body became insensitive to the presence of monensin, which disrupts protein processing in the Golgi complex, at 30 min, indicating that most of the pulse-labeled apolipoprotein has transited the Golgi complex by this time. Three inhibitors of protein processing, carbonylcyanide m-chlorophenyl hydrazone, monensin, and brefeldin A, inhibited secretion of lipophorin into medium. Puromycin treatment did not appear to result in the secretion into the medium of lipophorin particles containing incomplete translation products of apolipophorin-I or -II. Incubation of fat bodies with [³H]oleate resulted in the secretion of lipophorin containing [³H]glycerides, a process that was inhibited by cycloheximide, puromycin, and monensin, indicating that apolipoprotein synthesis is required for secretion of [³H]glyceride on nascent lipophorin particles. In contrast, suramin, which has been shown to block the binding of lipophorin to plasma membrane receptors, inhibited the synthesis and secretion of lipophorin, but it did not appear to inhibit the transfer of [³H]lipid from the fat body to lipophorin. Inhibitors of protein synthesis and processing, therefore, can be used to distinguish between secretion of lipophorin-associated lipids and secretion of lipids mediated by the lipid-transfer particle outside the plasma membrane of the fat body.