Programmed cell death in flight muscle histolysis of the house cricket

We have characterized the process of flight muscle histolysis in the female house cricket, Acheta domesticus, through analysis of alterations of tissue wet weight, total protein content, and percent shortening of the dorsal longitudinal flight muscles (DLMs). Our objectives were to (1) define the normal course of histolysis in the cricket, (2) analyze the effects of juvenile hormone (JH) removal and replacement, (3) determine the effects of cycloheximide treatment, and (4) examine patterns of protein expression during histolysis. Our results suggest that flight muscle histolysis in the house cricket is an example of an active, developmentally regulated cell death program induced by an endocrine signal. Initial declines of total protein in DLMs indicated the JH signal that induced histolysis occurred by Day 2 and that histolysis was essentially complete by Day 3. Significant reductions in tissue weight and percent muscle shortening were observed in DLMs from Day 3 crickets. Cervical ligation of Day 1 crickets prevented histolysis but this inhibition could be reversed by continual topical treatments with methoprene (an active JH analog) although ligation of Day 2 crickets did not prevent histolysis. A requirement for active protein expression was demonstrated by analysis of synthesis block by cycloheximide and short-term incorporation of (35)S-methionine. Treatment with cycloheximide prevented histolysis. Autofluorographic imaging of DLM proteins separated by electrophoresis revealed apparent coordinated regulation of protein expression.     

From insect ovaries to sheep red blood cells: a tale of two hormones

This printed version of the Wigglesworth Lecture reviews the evidence that juvenile hormone (JH) acts on the follicular epithelium of the ovary through a membrane receptor to control access of yolk proteins to the oocyte surface. The thyroid hormones mimic this action through the same receptor. Conversely, both JH III and 3,5,3′ triiodothyronine (T3) increase the activity of Ca ATPase in isolated erythrocyte membrane preparations from sheep, apparently through the same membrane receptor. These effects are mimicked by exposure of the respective tissues to CO₂. These findings suggest that the hormones arose as biotic signals, originally using existing CO₂ receptors.     

Prominent collagen type VI expression in juvenile angiofibromas

The extracellular matrix component collagen type VI demonstrates potent growth-stimulatory effects and has been associated with aggressive tumour growth. Although, juvenile angiofibromas (JAs) often exhibit an aggressive growth pattern, the collagen type VI expression of this fibrovascular tumour has not been addressed so far. RT-PCR, Western blot analysis and immunohistochemistry were used in this study to analyse collagen type VI, type VI collagen receptor subunits (integrin α1, α2, α10, α11 and β1) and the type VI collagen receptor NG2 in JAs (N = 15) and nasal mucosa (NM, N = 8) samples. The mRNA expression of all three collagen type VI chains was found to be up-regulated significantly (P < 10⁻³–10⁻⁵, adjusted) in JAs compared to NM tissues. The Western blot analysis proved highly prominent collagen-type VI expression in JAs. The ApoTome technique revealed strong collagen-type VI signals in tumour endothelium. NG2 (P < 10⁻³, adjusted) and α11-integrin (P = 0.04, adjusted) showed a significantly higher mRNA expression levels in JAs than in NM samples. NG2, α1-, α2- and β1-intergin were located to tumour vessels, and additional stromal signals were observed for NG2 and α1-integrin in JAs. This study demonstrates a prominent collagen-type VI expression in JAs. The collagen-type VI may exert an important growth stimulus in this tumour.     

Soldier caste influences on candidate primer pheromone levels and juvenile hormone-dependent caste differentiation in workers of the termite Reticulitermes flavipes

Caste systems and the division of labor they make possible are common underlying features of all social insects. Multiple extrinsic factors have been shown to impact caste composition in social insect colonies. Primer pheromones are one type of extrinsic caste-regulatory factor; they are chemical signaling molecules produced by certain colony members to impact developmental physiology of recipient nestmates. However, only limited evidence exists regarding primer pheromones and their actions in eusocial termites. In previous research we identified two soldier-produced terpenes, γ-cadinene (CAD) and γ-cadinenal (ALD), as candidate primer pheromones of the lower termite Reticulitermes flavipes. In the present study we tested hypotheses related to CAD and ALD action in recipient individuals. We examined the influences of terminally developed soldier termites on (1) CAD and ALD levels and (2) caste differentiation in developmentally totipotent workers. Our findings show CAD and ALD (respectively) are caste stimulatory and inhibitory components of chemical blends present in soldier heads, ALD levels increase significantly (10.9×) in workers only in the presence of soldiers, and soldiers can reduce developmental-hormone response thresholds of workers, presumably via ALD action. These findings provide novel evidence supporting that CAD and ALD are authentic caste-regulatory primer pheromones in Reticulitermes.     

Shoaling in juvenile guppies: the effects of body size and shoal size

While factors affecting shoal mate choice have been examined extensively in adult guppies (Poecilia reticulata), few studies have focused on the shoaling behavior of juveniles. In this study, juvenile guppies were tested for their ability to shoal as well as their response to shoal mates of different body size and to shoals with different numbers of individuals. In dichotomous choice tests, 10-day-old guppies (mean body length=8.83 mm), 30-day-old guppies (13.17 mm) and 50-day-old guppies (18.6mm) were given the opportunity to swim near shoals of five fish or an empty chamber. In most cases, the juvenile fish demonstrated shoaling behavior, swimming near a group of fish rather than an empty chamber, regardless of the age of the stimulus shoal. When presented with two shoals, one of similar age and body size and one of dissimilar age and body size, only the 50-day-old guppies showed a significant preference for the age-matched shoal. Similarly, when choosing between a large shoal and a small shoal, only the 50-day-old guppies spent significantly more time near the larger shoal. Thus, while juveniles at each age shoaled, only 50-day-old fish demonstrated the shoal mate discrimination seen in adult fish.     

Identification, characterization, and developmental regulation of two storage proteins in the bamboo borer Omphisa fuscidentalis

Two insect storage proteins, OfSP1 (75 kDa) and OfSP2 (72 kDa), were purified using three different chromatographies from the hemolymph of Omphisa fuscidentalis larvae during diapause, and their genes were cloned. OfSP1 and OfSP2 concentrations in the hemolymph were high during diapause. During pupation, OfSP1 levels decreased in the male hemolymph and disappeared from the female hemolymph. OfSP1 and OfSP2 mRNA levels in the fat bodies were low during the third instar, but increased greatly during the fourth and fifth larval instars. During diapause, mRNA expression continued at a lower level than during the feeding period. The injection of 20-hydroxyecdysone (20E) into diapausing larvae caused an increase in OfSP1 and OfSP2 mRNA levels 2-3 days post-injection, followed by a decrease in expression until pupation, which occurred 2-4 days thereafter. When larvae were treated with juvenile-hormone analog (JHA), OfSP1 and OfSP2 mRNA levels gradually decreased until the onset of pupation. In Omphisa, OfSP1 and OfSP2 proteins are produced and released by the larval fat bodies in the fourth and fifth-instar larvae, and the proteins accumulate in the hemolymph until the insects enter diapause. OfSP1 may be reabsorbed by the fat bodies at the end of diapause for subsequent re-use during pupation.     

Molting in workers of the Formosan subterranean termite Coptotermes formosanus

The Formosan subterranean termite, Coptotermes formosanus, with its huge colonies, is a major urban pest in several southern states and Hawaii as well as in South Asia. Because of their cryptic nature (underground habitat) and very long life cycle, not much is known about molting in termite workers. In C. formosanus, the workers stop foraging and lose their gut fauna, respectively, approximately 10 and 5 days prior to ecdysis. In any given colony an average of 1.01% (range 0.6-1.8) of the workers were found to molt each day under laboratory conditions. Workers destined to molt become sluggish and their head capsules develop a mottled texture one day prior to ecdysis. Ecdysis was generally accomplished with the assistance of other workers, which also fed on the exuviae. Immediately after molting worker mandibles were light pink in color and became fully melanized approximately two days later. Gut fauna were acquired on the fourth day after molting. Flagellates were transferred as small encysted cells from other workers through proctodeal feeding. Juvenile hormone III titer ranged between 30-41 pg/mg bodyweight in all stages except in workers sampled 6 days prior to ecdysis. In these workers the titer was 80.5 pg/mg. The high juvenile hormones (JH) titer may also be involved in causing defaunation. Ecdysteroid titer increased from 2.1 pg/mg in non-molting workers to 359.5 and 332.4 pg/mg one and two days following defaunation, respectively. There was a second smaller peak two days prior to ecdysis.     

Localization of the bacterial agent of juvenile oyster disease (Roseovarius crassostreae) within affected eastern oysters (Crassostrea virginica)

The bacterium Roseovarius crassostreae causes seasonal mortalities among commercially produced eastern oysters (Crassostrea virginica) grown in the Northeastern United States. Phylogenetically, the species belongs to a major lineage of marine bacteria (the Roseobacter clade), within which Roseovarius crassostreae is the only known pathogen to be isolated in laboratory culture. The objective of the current study was to determine the location and nature of R. crassostreae interactions with oysters affected by juvenile oyster disease (JOD). Scanning electron microscopy of diseased individuals revealed abundant colonization of the inner shell surfaces by bacteria which were morphologically similar to R. crassostreae. The same types of cells were also observed on and within layers of host-derived conchiolin on the inner valves. Most bacterial cells were alive as determined by the use of a fluorescent viability stain. Further, most were clearly attached at the cell poles, which is consistent with the ability of R. crassostreae to express polar fimbriae. When material from the pallial fluid, soft tissue and inner valve surfaces was cultured, the highest numbers of R. crassostreae were recovered from the inner valves. These samples also contained the greatest abundance of R. crassostreae as a percentage of total colonies. Cloning and sequencing of 16S rRNA genes provided culture-independent evidence of the numerical dominance of R. crassostreae among the bacterial consortia associated with the inner shell surfaces of JOD-affected animals. The ability of R. crassostreae to colonize shell and conchiolin is consistent with the described JOD-pathology and may aid the bacteria in avoiding hemocyte-mediated killing.     

Effect of exogenous JHI on imaginal determination in Aeshna cyanea

Last instar larvae of Aeshna cyanea injected with 8 μg or more JHI before the end of the fourth ocular stage (day 7 or 8 of the instar) became perfect supernumerary larvae which indicates that adult determination does not occur before this time. When JHI is applied later (days 9–13) larval-adult intermediates are formed. It seems, therefore, that imaginal programming takes place at the end of the fourth ocular stage and might be correlated with the first ecdysone peak which occurs at the same time.