John D. Hatle

Maximum titers of vitellogenin and total hemolymph protein occur during the canalized phase of grasshopper egg production.

Many organisms exhibit developmental plasticity only in sensitive phases and cannot respond to environmental pertubations at other times. However, we know little about the physiological events that define plastic and canalized phases. During egg production in insects, vitellogenin (Vg) accumulates first in the hemolymph and then in the eggs. In addition, storage proteins may be important resources for egg production. Therefore, we tested hypotheses on the relationships of Vg and TP (total hemolymph protein minus Vg) titers to the transition from flexible to inflexible development during egg production. In lubber grasshoppers, ∼70% of TP is contained in three proteins that range from 68 to 83 kDa. We maintained females on food treatments that produced defined plastic and canalized periods, collected hemolymph every ∼4 d, and determined the ages at which oviposition and the maximum Vg and TP titers occurred. Both Vgmax titer and especially TPmax titer were predictors of the number of eggs produced. The time from eclosion to Vgmax was significantly affected by diet, but the time from Vgmax to oviposition was not. Similarly, the time from eclosion to TPmax was significantly affected by diet, while the time from TPmax to oviposition was not. Hence, Vgmax and TPmax are physiological landmarks that occur during the canalized phase of egg production.

Juvenile hormone is a marker of the onset of reproductive canalization in lubber grasshoppers.

To meet the challenge of unpredictable environments, many animals are initially developmentally flexible (plastic) but then may become inflexible (canalized) at major developmental events. The control of reproductive output can undergo a switch from flexible to inflexible (Moehrlin, G.S., Juliano, S.A., 1998. Plasticity of insect reproduction: testing models of flexible and fixed development in response to different growth rates. Oecologia 115, 492-500), and juvenile hormone (JH) may control this switch. By manipulating food availability, we tested the hypothesis that JH is involved in the reproductive canalization that appears during oogenesis in lubber grasshoppers. We used four food treatments: (1) high (H); (2) high switched to low (HL); (3) low switched to high (LH); and (4) low (L). We collected hemolymph samples approximately every 4 days and measured the ages at which maximum JH level (JH(max)) and oviposition occurred. Diet significantly affected both age at JH(max) and age at oviposition. In contrast, diet had no significant effect on the time from JH(max) to oviposition nor on the maximum JH level observed. Our data demonstrate that, after JH(max) is reached, the time to oviposition in our grasshoppers was unresponsive to food availability. Hence, reproductive timing appears to be canalized after the JH(max). This is the first demonstration in a phytophagous insect that a particular factor (in this case, JH) can be used to mark the switch from reproductive plasticity to reproductive canalization.

Geographic variation of reproductive tactics in lubber grasshoppers

Abstract. We investigate plastic and interpopulation variation of trade-offs among reproductive tactics. There is a potential three-way trade-off among timing of reproduction, somatic storage, and investment in reproduction. We tested whether this trade-off shows latitudinal interpopulation variation. We studied populations of the lubber grasshopper, Romalea microptera, from Florida (FL; lowest latitude), Louisiana (LA; intermediate latitude), and Georgia (GA; highest latitude), each tested at three diet levels. All three populations differed in their multivariate responses of the three reproductive tactics we studied. This difference across populations was due primarily to age at first reproduction, secondarily to somatic storage, and less so to clutch mass. Age at first reproduction was least in GA, intermediate in LA, and greatest in FL grasshoppers. Somatic storage was greatest in FL and LA, and least in GA grasshoppers. Clutch mass was greatest in LA and GA, and least in FL grasshoppers. Diet levels also differed in this suite of reproductive tactics, primarily due to variation in age at reproduction. In contrast to significant, independent effects of population and diet, we find no evidence that the trade-off itself varies across populations (as indicated by the non-significant interaction of population and diet level). Thus, we show that the innate allocation of resources among reproductive tactics is different across populations, but all three populations responded similarly to a range of diet levels.

Plasticity and Canalization in the Control of Reproduction in the Lubber Grasshopper

Abstract The ability to change reproductive tactics during adult development in response to environmental variation is predicted to enhance fitness. Many organisms show phenotypic plasticity early in non-embryonic development, but later exhibit phases of developmental inflexibility (=canalization). Therefore, we studied reproduction-related hormones and proteins and their relationships to plasticity in the Eastern lubber grasshopper. Diet-switching experiments demonstrated plasticity early in the egg production cycle, but a switch to canalization late in the cycle. We measured developmental titers of 4 hemolymph compounds from single individuals from adult molt until first oviposition. These 4 compounds were the egg-yolk precursor protein vitellogenin, juvenile hormone (the central regulator of insect reproduction), major hemolymph proteins, and ecdysteroids (the arthropod molting hormone that ultimately is stored in the egg). Using diet manipulations, we investigated how these developmental titers relate to the switch from plastic to canalized egg production. All 4 hemolymph compounds reached their peak levels during the canalized phase, about 12 day before oviposition. Diet switches after these peak levels did not affect the timing to oviposition. Therefore, these peak titers were physiological events that occurred after the individual committed to laying. We compared these patterns in reproduction to the development toward adult molt, another major life-history event in insects. We observed an extended canalized phase before the adult molt. This canalized phase always included a peak of ecdysteroids. The similar patterns in the physiology of these life-history events suggested that common limitations may exist in major developmental processes of insects that are directed by hormones.

Survival advantage of sluggish individuals in aggregations of aposematic prey, during encounters with ambush predators

Movement is an important element of prey defense ensembles. The adaptive advantages of either remaining motionless or rapid escape are clear. In contrast, putative benefits are unclear for sluggish movement of aposematic prey that are neither fleeing nor avoiding detection of predators. Nonetheless, sluggish movement is common in aposematic insects. Our central hypothesis is that sluggish movement evolved in part by motion-oriented predators culling the fastest-moving insects from an aggregation. This would be particularly likely in chemically defended prey, which would deter continued predation. We test predictions of our hypothesis with feeding experiments using the sluggish, gregarious Eastern lubber grasshopper and a motion-oriented predator, the Northern leopard frog. Sluggish-moving (i.e., control) grasshoppers were significantly less likely to be eaten than fast-moving (i.e., motion-induced) grasshoppers (p = 0.0098). Next, non-moving grasshoppers were used as extreme sluggish-moving prey. Non-moving prey were significantly less likely to be eaten than sluggish-moving grasshoppers (p = 0.05). In addition, and most importantly, sluggish-moving grasshoppers in an aggregation were significantly less likely to be attacked than fast-moving grasshoppers in the aggregation (p = 0.0156). Finally, the survivorship of sluggish-moving grasshoppers in pairings vs. aggregations was not significantly different (p = 0.33). Our results demonstrate that the fastest-moving individual in an aggregation of aposematic insects is more likely than sluggish cohorts to be attacked by motion-oriented predators. This survival disadvantage for fast-moving, gregarious prey could create a selection pressure for the evolution of sluggish movement as a defense mechanism in aposematic, gregarious prey.

Tests of potential adipokinetic hormone precursor related peptide (APRP) functions: lack of responses.

The adipokinetic hormone (AKH) precursor related peptides (APRPs) are end products of the synthesis of the well-conserved AKHs. The large amount of metabolic energy devoted to APRP synthesis suggests they have an important function(s) in the insects. Several functions have been proposed, but currently none are known. We tested whether the APRPs stimulate hyperlipemia, hypertrehalosemia, fat body glycogen phosphorylase activation, Malpighian tubule secretion, and hindgut myotropia. Surprisingly, none of these responses were stimulated by APRPs isolated from the lubber grasshopper, Romalea microptera (= guttata). In addition, the APRPs delivered in concert with AKHs did not significantly increase hyperlipemia, hypertrehalosemia, or phosphorylase activation over the AKHs alone. Our data discount several proposed functions for the APRPs. Arch.

Reproductive Responses to Photoperiod by a South Florida Population of the Grasshopper Romalea microptera (Orthoptera: Romaleidae)

Abstract Reproductive tactics of some insects are affected by photoperiod because daylength is a reliable cue indicating seasonal change in environmental suitability. We tested the hypothesis that late-season (short-day) photoperiod affects the length of the oviposition cycle of the eastern lubber grasshopper, which is from a subtropical area with a winter dry season. We predicted that oviposition would occur sooner in short-day (11.5:12.5 [L:D] h) than in long-day (13.75:10.25 [L:D] h) individuals, and that costs associated with earlier oviposition would be expressed as smaller eggs or fewer eggs per female under the short-day photoperiod. Two diets (low and high food quantity) were tested at each photoperiod. Although MANOVA yielded no significant main effect of photoperiod on the timing of oviposition, number of eggs, and mean egg dry mass, there was a significant multivariate interaction between photoperiod and food for these variables. In the low food treatment, the short-day animals produced fewer, smaller eggs than did the long-day animals, but this multivariate difference between short-day and long-day animals was absent in the high-food treatment. Photoperiod and its interaction with food did not affect timing of oviposition in either multivariate or univariate analyses. The absence of the predicted change in timing of reproduction in an autumn photoperiod suggests that seasonal constraints on reproduction do not exert a strong influence on reproductive timing of this population. Our alternative hypothesis is that the combination of short days and low food availability serve as a cue to lubber grasshoppers of an oncoming dry season with potentially limiting food, and that they respond to this cue by reducing the mass of reproductive output but not by accelerating reproductive timing. Thus, we propose that these subtropical grasshoppers adjust reproductive investment in response to seasonal changes in resource availability.

Plasticity and canalization of insect reproduction: Testing alternative models of life history transitions

Life histories may show phases of both plasticity and canalization in response to feeding rate. Models for life history canalization and plasticity postulate a threshold for initiation of canalized developmental events. Some models postulate adaptive plasticity, whereas others postulate nonadaptive plasticity that results from environmental modulation of fixed development. These models have been tested by changing feeding rate at various times and determining when timing of life history events becomes unresponsive to those changes. This approach has been criticized because putative thresholds are usually not known. We use an alternative experimental design to test models of reproductive plasticity and canalization, and to estimate thresholds, in the grasshopper Romalea microptera. We develop mathematical models for published verbal models that predict how life history timing changes with feeding rate. Alternative models predict distinct relationships of time to oviposition vs. mean food intake that we test via experimental manipulation of food intake and nonlinear regressions. Regressions yield estimates of both the threshold and the duration of post-threshold development. A model postulating a fixed threshold and canalized post-threshold development provides the best, most parsimonious fit to data for this grasshopper. Thus, the simplest model, postulating no adaptive variation in development, is supported, a result that is consistent with previous experiments on this system using changing feeding rates. We use the estimate of the threshold (in units of food eaten) and measurements of hemolymph protein content to estimate the threshold in units of physiologically relevant storage. These results elucidate the structure of reproductive plasticity in this system and how this alternative experimental approach can provide testable predictions of developmental thresholds for further experiments on life history plasticity and canalization.

Sluggish Movement and Repugnant Odor Are Positively Interacting Insect Defensive Traits in Encounters with Frogs

Sluggish movement is common in chemically defended insects. We have recently shown that sluggish movement can be beneficial to prey when it fails to release the attack response of an ambush (=motion-oriented) predator. Here, we test the hypothesis that sluggish movement and chemical defense (i.e., repugnant odor) together are more defensive than either alone. We manipulated the movement and odor of lubber grasshoppers to produce four prey types: (1) sluggish-moving and high odor, (2) sluggish-moving and low odor, (3) fast-moving and high odor, and (4) fast-moving and low odor. We then offered these prey to frogs. In two independent experiments, frogs attacked prey type 1 (i.e., sluggish-moving and high-odor prey) significantly later than they attacked the other prey types. Hence, the defenses of sluggish movement and repugnant odor can act together to produce a prey that is better defended than prey with either defense alone. This may help explain why these two traits commonly cooccur in insects.

Inter-individual variation in sequestration (as measured by energy dispersive spectroscopy) predicts efficacy of defensive secretion in lubber grasshoppers

Summary. We tested whether inter-individual variation in concentration of sequestered compounds predicts defensive efficacy of the defensive secretion of onion-fed Eastern lubber grasshoppers. When fed onion, lubbers have the ability to sequester sulfur compounds into their defensive secretion, and the secretion from onion-fed lubbers is more deterrent of ants than the secretion from lubbers fed other diets (Jones et al. 1989). To test three hypotheses, we developed a method for measuring total sequestered sulfur in the secretion using energy dispersive spectroscopy (EDS). First, we found that lubbers fed an ad libitum, monophagous diet of onion produce more secretion and have higher concentrations of total sulfur in their secretions than lubbers fed other diets. These sulfur concentrations varied three-fold; we believe that this variation in the composition of defensive secretions stems from physiological variation. Second, our method was sensitive enough to detect sulfur in the secretion of some lubbers that had been fed onion only once. Third, the sulfur concentration of a secretion sample significantly predicted its ability to deter ants from foraging. Hence, our results suggest that inter-individual variation in defensive chemistry in insects offered the same diet may be ecologically important.