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.     

Juvenile hormone regulation of the larval diapause of the Southwestern corn borer,Diatraea grandiosella

The hormonal mechanism which controls the larval diapause of the southwestern corn borer was examined. The onset of this facultative mature larval diapause is marked by a transition from a spotted to an immaculate larval form, and during diapause individuals may undergo one or more stationary larval ecdyses. Experiments were designed to uncover the nature of the humoral mechanism regulating this diapause state. The finding that injecting diapause larvae with 20-hydroxyecdysone only brought about a stationary larval ecdysis suggests that diapause was not maintained by the lack of ecdysone. Neck ligations performed on larvae which had just entered diapause resulted in a premature termination of diapause, and larval-pupal ecdysis occurred in the thoraco-abdominal section, suggesting that a cephalic factor was necessary for the maintenance of diapause. This finding was further supported by the discovery that injecting 20-hydroxyecdysone into the thoraco-abdominal section of previously ligated diapause larvae also resulted in a premature termination of diapause and larval-pupal ecdysis, indicating that ecdysone only initiated the pupal moulting cycle when the cephalic factor was absent.Further experiments led to the conclusion that the juvenile hormone is the cephalic factor. Topical treatment with a juvenile hormone mimic caused non-diapause mature larvae to become immaculate and enter diapause. Periodical topical application of this mimic to diapause larvae prolonged diapause and increased the number of stationary larval ecdyses. These findings suggest that the initiation and maintenance of diapause are regulated by juvenile hormone titre. Results indicate that larvae retain a high titre of juvenile hormone until the last stages of diapause. Injection of 20-hydroxyecdysone into early or middiapause larvae only caused stationary larval ecdyses, while the same injection into larvae in the late stages of diapause caused some of them to pupate. Histological studies of the neurosecretory cells, corpus cardiacum-allatum complex, and prothoracic glands showed that the endocrine system was not inactive during diapause. A new hypothesis is therefore proposed which recognizes the existence of hormonal activity during larval diapause and emphasizes the principal regulatory rôle of juvenile hormone.     

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.     

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.     

Environmental and genetic control of the larval diapause of the Southwestern corn borer, Diatraea grandiosella

ABSTRACT. The interaction of photoperiod and temperature in the regulation of the induction and termination of the larval diapause of the Southwestern corn borer, Diatraea grandiosella Dyar (Lepidoptera), was examined. A population originating from south-eastern Missouri had critical daylengths for diapause induction of about 15h 5min (ecological threshold) and llh (physiological threshold). The ecological threshold was more stable than was the physiological threshold at temperatures lower than 25°C. Above 25°C the diapause response was suppressed. The insect appears to measure photoperiods in a stationary manner since a stepwise increase or decrease in photoperiod did not affect the incidence of diapause. In the critical region of the photoperiodic response curve, a higher incidence of diapause was found among females than among males. Females entered diapause later than did males, but resumed active development earlier than males. The rate of diapause development was more temperature dependent than was the rate of diapause induction, yet it was also clearly under photoperiodic control. The temperature coefficient (Q₁₀) for this process was about 4. Several other factors including sex-linkage, age, and geographic adaptations are involved in controlling the rate of diapause development, even more so than they are in controlling diapause induction. In the laboratory, the intensity of diapause declined gradually without larvae being exposed to non-diapause inducing conditions. Incubation of field-collected larvae revealed that their sensitivity to diapause maintaining photoperiods had ended by January. Three generations of selection of a Mississippi population of D. grandiosella at 30°C and LD 12:12 led to the production of an essentially diapause-free strain and a diapause strain.     

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.

---

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.

     

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.     

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.     

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.

---

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.

---

---

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

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.     

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     

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.

---

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.

     

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.

---

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.

---

---

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

Binding of juvenile hormone,methoprene and hydroprene to haemolymph proteins of larvae of the southwestern corn borer,Diatraea grandiosella

The binding of tritiated juvenile hormone I, and the juvenile hormone mimics, methoprene and hydroprene (alkyl-3,7,11-trimethyl-2,4-dodecadienoates), to proteins of the haemolymph of larvae of the southwestern corn borer, Diatraea grandiosella, was examined in vitro. The techniques of analytical isoelectric focusing, disc electrophoresis and gel filtration were employed. Juvenile hormone was shown to bind to a high affinity low molecular weight protein in the haemolymph of diapausing larvae. This protein has an apparent dissociation constant of about $\text{3.1 × 10}{}^{\mathrm{-7}}$ M at 4°C, an isoelectric point of about 4.85, and a molecular weight of about $\text{3 × 10}{}^4$. Juvenile hormone was also bound to at least one low affinity high molecular weight protein fraction. High juvenile hormone binding activity was found in the haemolymph of newly diapaused larvae. Methoprene and hydroprene bound selectively to a different protein fraction present in the haemolymph of pre-diapausing larvae. This protein fraction had a low relative mobility when subjected to electrophoresis at pH 8.9.A preliminary study of the binding of tritiated juvenile hormone I to proteins of the haemolymph of pre-diapausing larvae of the southern cornstalk borer, Diatraea crambidoides, was also conducted in vitro. A low molecular weight binding protein with identical electrophoretic properties to that of D. grandiosella was present. The high molecular weight binding protein fraction of the two species, however, had different electrophoretic properties.     

Dietary carbohydrates: role in feeding behavior and growth of the southwestern corn borer, Diatraea grandiosella

The effect of carbohydrates on feeding behaviour and growth and development of the southwestern corn borer, Diatraea grandiosella, was examined. A feeding behavioural biossay based on establishment of first stage larvae on test or control stations within 24 hr was employed. A nutritional bioassay evaluated larval growth rates and metamorphic success of larvae reared on test or control diets. The principal results showed that glucose, fructose, sucrose, and dextrins were feeding stimulants and permitted optimum growth and development. A dietary supplement of amylopectin allowed reasonably good growth while amylose was not a utilizable carbohydrate. The pentoses, arabinose, ribose, and xylose, as well as galactose, mannose, and sorbose, were found to inhibit larval growth. Since of all these sugars only xylose acted as a feeding deterrent it was proposed that at high concentrations they or metabolic derivatives were toxic. The results are discussed in relation to those obtained with several other insect species.