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Dev Biol. Author manuscript; available in PMC 2011 Sep 15.
Published in final edited form as:
Dev Biol. 2010 Sep 15; 345(2): 170–179.
Published online 2010 Jul 12. doi:  10.1016/j.ydbio.2010.07.004
PMCID: PMC2927803

Developmental timing of a sensory-mediated larval surfacing behavior correlates with cessation of feeding and determination of final adult size


Controlled organismal growth to an appropriate adult size requires a regulated balance between nutrient resources, feeding behavior and growth rate. Defects can result in decreased survival and/or reproductive capability. Since Drosophila adults do not grow larger after eclosion, timing of feeding cessation during the third and final larval instar is critical to final size. We demonstrate that larval food exit is preceded by a period of increased larval surfacing behavior termed the Intermediate Surfacing Transition(IST) that correlates with the end of larval feeding. This behavioral transition occurred during the larval Terminal Growth Period (TGP), a period of constant feeding and exponential growth of the animal. IST behavior was dependent upon function of a subset of peripheral sensory neurons expressing the Degenerin/Epithelial sodium channel(DEG/ENaC) subunit, Pickpocket1(PPK1). PPK1 neuron inactivation or loss of PPK1 function caused an absence of IST behavior. Transgenic PPK1 neuron hyperactivation caused premature IST behavior with no significant change in timing of larval food exit resulting in decreased final adult size. These results suggest a peripheral sensory mechanism functioning to alter the relationship between the animal and its environment thereby contributing to the length of the larval TGP and determination of final adult size.

Keywords: Drosophila, growth, feeding behavior, developmental timing, sensory neurons


Essentially all multicellular organisms utilize both cell-autonomous and systemic physiological mechanisms to coordinate nutrient utilization, energy homeostasis and growth from embryo to final adult size(Martin and Hall, 2005; Shingleton, 2005; Stocker and Hafen, 2000; Taniguchi et al., 2006; Wullschleger et al., 2006). The species-specific uniformity of final body size reflects an intricate regulatory hierarchy capable of responding to potentially broad variations in nutrient availability associated with local environmental conditions(Nijhout, 2003; Nijhout, 2008). Most organisms are able to strike a delicate balance between feeding or fasting, nutrient utilization or storage and rapid or slow growth(Wang et al., 2006). It is generally agreed that genetic, environmental or pathological alterations of these same systems in humans contribute significantly to the increased incidence of obesity, type 2 diabetes and cardiovascular disease in modern society (Badman and Flier, 2005; O’Rahilly and Farooqi, 2008; Oswal and Yeo, 2007; Patti and Kahn, 2004; Schwartz and Porte, 2005; Smyth and Heron, 2006; Spiegel and Nabel, 2006).

Genetic approaches in model organisms such as Drosophila melanogaster have contributed to a better understanding of links between energy metabolism, feeding behavior and growth control due in part to the evolutionary conservation of cell-autonomous mechanisms controlling nutrient utilization and growth(Jacinto and Michael N. Hall 2003; Jia et al., 2004; Pan et al., 2004; Taniguchi et al., 2006). Since adult homometabolous insects such as D. melanogaster do not continue to grow after metamorphosis and eclosion, mechanisms coordinating nutrient-dependent cell growth and the developmental timing of the cessation of larval feeding behavior are critical to organismal survival. After egg hatching, Drosophila larvae progress through three instars or growth periods separated by cuticle molts to accommodate the drastic increase in size. During the first, second and early third instars, larvae exhibit foraging behavior characterized by complete immersion within the food source and constant feeding necessary to fuel the rapid growth associated with larval development. Final adult size is primarily established by the size of the larva when it stops feeding but can also be influenced by hormonal signals during post-feeding stages(Mirth and Riddiford, 2007; Okamoto et al., 2009; Slaidina et al., 2009; Stern and Emlen, 1999). The timing of feeding cessation is thought to be determined by mechanisms coordinating feeding behavior, nutrient storage and energy homeostasis to ensure the accumulation of sufficient nutritional reserves for survival through metamorphosis(Davidowitz et al., 2003; Johnston and Gallant, 2002; Mirth and Riddiford, 2007; Nijhout, 2003).

Work in both Manduca sexta and Drosophila melanogaster has described developmental checkpoints used by feeding larvae to monitor growth, regulate the length of the feeding period and determine the timing of entry into wandering stage(Davidowitz et al., 2003; Mirth and Riddiford, 2007; Nijhout, 2003). Although the precise characteristics of these checkpoints vary somewhat between species, it is clear that some form of size assessment is made early in the final instar to determine whether the animal has achieved a “critical larval size” necessary for survival through metamorphosis(Caldwell et al., 2005; Colombani et al., 2005; Davidowitz et al., 2003; Mirth et al., 2005; Nijhout, 1984). Critical size assessment in D. melanogaster is thought to occur early in the larval third instar (70–80h after egg laying, AEL) when animals are still displaying foraging behavior(Mirth et al., 2005; Mirth and Riddiford, 2007). Cessation of feeding and transition to wandering stage will not occur for ~20 hours. This period between attainment of larval critical size and the cessation of feeding and growth has been called the terminal growth period(TGP)(Shingleton et al., 2007). Since growth is proceeding at a very rapid rate during the third instar, the length of the TGP plays a key role in determination of final body size (Mirth and Riddiford, 2007; Shingleton et al., 2007). The larger question becomes just how individual larvae incorporate internal and external cues to determine when an appropriate larval size standard has been reached signaling an end to larval feeding and growth.

Physiological mechanisms determining the developmental timing of larval feeding cessation and final adult size in insects have been shown to vary significantly between species, however, all appear to rely upon an increase in levels of circulating 20-hydroxyecdysone(referred to here as ecdysone) to end larval feeding during the final instar(Riddiford, 1993; Riddiford et al., 2003). Although sharp bursts of ecdysone release are associated with major developmental transitions such as the larval molts, recent studies suggest that control of basal ecdysone levels may have a more direct role in the regulation of larval growth rate(Colombani et al., 2005; Layalle et al., 2008; Nijhout, 2008). Recent work in Drosophila has suggested that nutrient-dependent growth of a single tissue, the prothoracic gland, mediates the increase in circulating ecdysone(Layalle et al., 2008; Mirth et al., 2005; Mirth and Riddiford, 2007; Nijhout, 2008). Increased DILP-dependent growth of the prothoracic gland was shown to increase synthesis and release of ecdysteroid hormone causing downstream activation of ecdysone-dependent gene transcription(Caldwell et al., 2005; Colombani et al., 2005; Mirth et al., 2005). TOR activity within the prothoracic gland appears to coordinate nutrition with levels of ecdsyone production to influence developmental timing(Layalle et al., 2008). In this way, Drosophila larvae are able to alter TGP length with variations in nutrient availability thereby allowing growth to near normal size even under conditions of a limited food supply(Colombani et al., 2005; Layalle et al., 2008; Nijhout, 2008).

An alternative or supplemental mechanism using peripheral sensory neurons to assess abdominal stretch or distension as a measure of overall larval size has been identified in other insect species(Nijhout, 2003; Nijhout, 2008). Results from certain species of the insect order Hemiptera, encompassing aphids, cicadas and true bugs, suggest that size assessment and progression to metamorphosis is controlled by peripheral stretch receptors in the abdomen that are activated when a particular critical size is achieved to induce larval feeding cessation and the transition to pupation(Nijhout, 2003; Nijhout, 2008). Artificial expansion of the abdomen of the milkweed bug, Oncopeltus fasciatus, by saline injection during the final larval instar caused premature metamorphosis(Nijhout, 1979). During normal growth conditions, these stretch receptor neurons are only activated once the animal reaches an appropriate critical size. In the blood-sucking Hemiptera, Rhodnius prolixus and Dipetalogaster maximus, abdominal stretch receptors are activated in response to distension of the gut following a blood meal resulting in subsequent pupariation(Chiang and Davey, 1988; Nijhout, 1984; Wigglesworth, 1934).

Such a direct role for peripheral sensory neurons in the control of larval feeding cessation has not been clearly shown for other insect species including Drosophila. However, previous studies have demonstrated that changes in Drosophila larval food-associated behavioral responses occurring during the final larval instar are dependent upon function of a small subset of peripheral sensory neurons expressing the Drosophila Degenerin/Epithelial Sodium Channel(DEG/ENaC) subunit, Pickpocket1(PPK1)(Adams et al., 1998; Ainsley et al., 2008; Ainsley et al., 2003). The DEG/ENaC family is a diverse group with members in both invertebrate and vertebrate species(Kellenberger and Schild, 2002; Welsh et al., 2002). DEG proteins were first identified as mechanosensory channels in the nematode, C. elegans(Chalfie, 2008; Goodman and Schwarz, 2003) and shown to be structurally related to ENaCs previously shown to play a crucial role in Na+ absorption in the distal part of the kidney tubule(Kellenberger and Schild, 2002). Subsequent analysis of the vertebrate acid sensing ion channels(ASICs) has led to the suggestion that members of this far reaching family of channel subunits are essential for numerous evolutionarily conserved sensory signaling processes including acid-sensation, mechanosensation and nociception(Krishtal, 2003; Lingueglia, 2007).

The midthird instar behavioral transition from foraging to wandering behavior is also reflected in altered taxis behavior in response to chemical and thermal cues as well as changes in photophobic behavior (Ainsley et al., 2008; Gong, 2009; Min and Condron, 2005; Sawin-McCormack et al., 1995; Wu et al., 2003). Loss of PPK1 or inhibition of the function of PPK1 neurons caused larvae to maintain foraging-associated behaviors throughout the wandering period(Ainsley et al., 2008; Ainsley et al., 2003). PPK1 expression is restricted to the three class IV multiple dendritic(mdIV) neurons within each hemisegment of the larval peripheral nervous system as well as two bipolar neurons innervating the posterior spiracles, the external openings to the larval tracheal system(Samakovlis et al., 1996). The mdIV neurons project a complex dendritic arbor tiling the inner surface of the larval body wall(Adams et al., 1998; Ainsley et al., 2008; Grueber and Jan, 2004; Jan and Jan, 2003). These findings suggest the possibility of a complimentary role for peripheral sensory neurons in assessment of larval size and control of larval feeding behavior in Drosophila(Ainsley et al., 2008).

Results presented here identify a distinct developmentally-regulated behavior controlling larval body position within the food source that is functionally separable from final food exit. We demonstrate that PPK1 neurons mediate control of a stereotypical larval surfacing behavior necessary to shift foraging stage larvae from the full body immersion position to the food surface prior to complete food exit and entry into “wandering” behavior. Although not essential for organismal survival under laboratory culture conditions, such a behavioral transition may contribute to robust population survival under more challenging environmental conditions. Transgenic hyperactivation of PPK1 neurons caused a premature enhancement of larval surfacing behavior with a resulting decrease in final adult size. This suggests the possibility that, although evolutionarily altered, control of key developmental and behavioral transitions occurring during larval growth requires multi-layered coordination between metabolic, hormonal and peripheral sensory inputs.

Material and methods

Drosophila stocks

Flies were raised on standard cornmeal-yeast-agar medium. Crosses were performed at 25°C. UAS-ORK1ΔC, UAS-NaChBaceGFP and Df(2L)Exel6035 stocks were obtained from the Bloomington Drosophila Stock Center.

Generation of a constitutively-active PPK1[S551V] isoform

The full-length ppk1 cDNA was modified by PCR-based in vitro mutagenesis to encode a single amino acid substitution at position 551 converting the wild-type serine residue to a valine. The substitution was confirmed by DNA sequencing and the resulting ppk1[S551V] cDNA was cloned into the pUAST vector. UAS-PPK1[S551V] transgenic animals were generated by routine P-element mediated transformation (Spradling, 1986). Transposon inserts were generated and maintained in an isogenic w1118 background.

Generation of targeted ppk1 deficiency chromosomes

The ppk1 null mutant genotype was produced using two overlapping deficiency chromosomes(see Supp. Fig. 1) generated using techniques previously described for the recovery of targeted deficiencies by FLP/FRT mediated excision of mapped transposon insertions (Parks et al., 2004; Thibault et al., 2004). Df(2L)ppk1Aid(14334094–14383291) was generated using P{XP}d03030(+) and PBac{WH}f01473(−). Df(2L)ppk1MirB(14368856–14409711) was generated using PBac{WH}f07052(+) and P{XP}d02171(−). Transposon stocks were obtained from the Harvard Medical School Exelixis Collection and hsFLP stocks from the Bloomington Drosophila Stock Center. The presence, orientation and breakpoints of the final deficiencies were verified by genomic PCR using previously described protocols for hybrid and two-sided PCR (Parks et al., 2004) followed by sequencing of PCR products. For both deficiencies, the following primers were used: 5'-AATGATTCGCAGTGGAAGGCT-3' and 5'-GACGCATGATTATCTTTTACGTGAC-3'.

Correct size and position of the deficiencies were verified using a two-sided PCR technique (Parks et al., 2004) followed by sequencing. For Df(2L)ppk1Aid, the following primer pairs were used: 5'-AATGATTCGCAGTGGAAGGCT-3' + 5'-GCACCTTTTCACCACGAACT-3' and 5'-GACGCATGATTATCTTTTACGTGAC-3' + 5'-CAAGCAGCCTGAATTGATGA-3'. For Df(2L)ppk1MirB, the following primer pairs were used: 5'-GACGCATGATTATCTTTTACGTGAC-3' + 5'-TGTGGTAGTCGACGCTTCTG-3' and 5'-AATGATTCGCAGTGGAAGGCT-3' + 5'-GATAGCGCGCAAAGAAAAAC-3'. Confirmation that the Df(2L)ppk1Aid/Df(2L)ppk1MirB stock removed the ppk1 genomic region was obtained using the following primers located within the overlapping portion of the two deficiencies: 5'-GCCGAGATCAGGGAGGATG-3' + 5'-CTAGTTCTCAGATTTTTCCTCTGGT-3'.

Area Restricted Search (ARS) Behavior

Assay was performed as previously described (Ainsley et al., 2008). Staged larvae were grown at 25 °C under constant light conditions to suppress the effects of circadian rhythms. Larvae were removed from food, rinsed and allowed to acclimate to a specified temperature on a 1.5% agarose plate. Larvae were then permitted to roam freely while the number of stops and turns (separated by at least 1 peristaltic wave) were counted over 2 minutes.

Intermediate Surfacing Transition(IST) Behavior

Larval body exposure was quantified using digital image analysis to measure the extent of dorsal tracheal trunk visible above the food surface at a series of developmental timepoints. Six hour egg collections of the appropriate genotype were allowed to hatch and grow on standard cornmeal/yeast/sucrose media at 25°C until 70–75 hr AEL when assay vials were seeded with 50 larvae/vial. The food surface in individual vials was photographed at designated timepoints using a Nikon D40 digital camera with a Nikon AF-S Micro NIKKOR 60mm f/2.8G ED lens operated remotely with Nikon Camera Control Pro 2 software. Resulting images(see Fig. 1B–E and Fig. 3CD for examples) were scored using Adobe Photoshop to draw 1 pixel wide lines corresponding to the length of larval dorsal tracheal trunk exposed beginning at the posterior spiracles for each larva in the image(see Fig. 1E dashed line). Total tracheal trunk exposure was quantified as an esssentially nonsubjective quantitative value for surfacing behavior by scoring the total pixel length visible in each image and converting that value to cm based upon the appropriate image resolution. During the transitional period, larvae do not all surface completely but may reveal only a portion of the full body length. The mean larval body length value was used to convert the total tracheal trunk exposure values to “larval body lengths”. These dorsal tracheal trunk exposure values derived from a series of developmental timepoints were used to calculate mean and standard error for each genotype. Each data point represents n≥12 from at least three independent experiments. Resulting values were analyzed by one-way ANOVA with a Tukey posttest to establish statistical significance. Vials scored for IST were also assayed for food exit behavior by scoring the number of wandering animals (prior to spiracle eversion) on the side of the vial for each data point.

Figure 1
The Larval Intermediate Surfacing Transition(IST). (A) Plotting of larval surfacing behavior(○) relative to complete food exit(An external file that holds a picture, illustration, etc.
Object name is nihms223006ig1.jpg). Surfacing behavior was quantitated by measuring levels of dorsal tracheal trunk exposure(see Methods). Bottom panel ...
Figure 3
Premature enhanced IST behavior caused by transgenic hyperactivation of mdIV neurons. (A) Schematic representation of the constitutively active PPK1[S551V] protein indicating the two transmembrane domains (M1 and M2), the large extracellular domain and ...

Adult body weight and larval mouthhook size

Mid-second instar larvae(60 hrs AEL) of the appropriate genotypes were transferred to fresh food at a density of 50 larvae/vial and allowed to develop through metamorphosis at 25°C under constant light conditions. Freshly eclosed adults were transferred to fresh vials for 48hrs, euthanized by freezing at −70°C overnight and weighed in groups of 4 on a Mettler BasBal300. Each data point represents n ≥ 20 groups of 4 animals from at least 3 independent experiments. Since the weigth of female flies can be influenced by ovary weight, we analyzed only male flies to minimize the effect of fertility on fly weight measurements.

Larval mouthhook size was determined by image analysis using Photoshop measuring tools. Individual larvae from each appropriate genotype were staged and collected at ~70h AEL shortly following the third instar molt. Larval heads were removing using forceps and flattened under coverslips. Photomicrographs were obtained using a Nikon Eclipse E800 light microscope and the size of individual mouthhooks measured in Adobe Photoshop using the measuring/ruler tools. Each data point represents n≥12. Statistical significance was determined using a student’s t-test relative to the control ppk1GAL4/+ values.


An Intermediate Surfacing Transition precedes entry into wandering behavior

Traditional descriptions of larval behavior differentiate primarily between two overall behavioral stages, foraging(in the food) and wandering(out of the food) corresponding roughly to feeding and not feeding(Fig. 1A). Foraging larvae spend the majority of their time completely immersed in the food source such that only the posterior spiracles, the external openings to the larval tracheal system(Manning and Krasnow, 1993), are visible at the food surface(Fig. 1A–C, black arrows). Prior to wandering behavior and complete food exit, larvae begin to emerge from the food to transiently roam the food surface (Fig 1DE). This behavior, referred to here as the Intermediate Surfacing Transition (IST), is highly dynamic with larvae making frequent brief appearances at the food surface. Although any individual larva may spend only a limited time on the surface, the population of staged larvae begins to spend more and more time at the food surface during the IST. IST behavior was quantified by measuring the exposure of the dorsal tracheal trunk at the food surface at defined timepoints throughout third instar(Fig. 1AE and Methods). Tracheal trunk exposure for wild-type larvae begins to steadily increase after 90 hrs AEL and reaches its peak between 105–110 hrs AEL(Fig 1A).

IST behavior precedes the entry into wandering behavior by approximately 10 hours(Fig 1A). This corresponds temporally to the developmental transition in area restricted search(ARS)/dispersal behavior previously shown to be dependent upon function of larval peripheral sensory neurons expressing the DEG/ENaC channel subunit, PPK1(Ainsley et al., 2008). To test the possibility that these two larval behaviors may be mechanistically linked, the role of PPK1 neurons in the control of IST behavior was examined.

Activity of PPK1 neurons was manipulated transgenically using the GAL4/UAS system and the previously described ppk1GAL4 transposon to drive tightly restricted expression in only the PPK1 neurons(Ainsley et al., 2008; Ainsley et al., 2003). Neurons were electrically silenced by expression of dOrk1ΔC, a constitutively active K+ leak channel that causes membrane hyperpolarization(Nitabach et al., 2002). ppk1GAL4/UAS- dOrk1ΔC larvae failed to display IST behavior prior to wandering behavior(Fig 2B). ppk1 null mutant animals were generated using two targeted overlapping deficiencies, Df(2L)ppk1Aid and Df(2L)ppk1MirB, and are indicated as DfA/DfB (see Material and methods). Loss of PPK1 activity also caused a severe disruption in appearance of the IST(Fig 2C). This behavior was rescued by transgenic expression of wild-type PPK1 in the ppk1 null mutant background(DfA/DfB) using the ppk1GAL4 driver(Fig. 2C, black circles). UAS-PPK1 and ppk1GAL4 transposons had no effect upon the ppk1 null mutant phenotype when present alone(Fig. 2C, gray and white squares).

Figure 2
Larval IST behavior requires function of mdIV sensory neurons and the PPK1 channel subunit. (A) Highly simplified depiction of a representative larval hemisegment showing the complex dendritic arbors of mdIV neurons tiling the larval body wall and their ...

Premature IST caused by hyperactivation of PPK1 neurons

The same experimental paradigm was used to determine whether hyperactivation of PPK1 neurons was sufficient to alter IST behavior. Neurons were experimentally hyperexcited by transgenic expression of two different modified ion channels capable of increasing neuronal excitability. Results from previous characterization of certain DEG/ENaC proteins in C.elegans demonstrated that mutation of a specific “DEG” residue near the second transmembrane domain could cause constitutive ion channel activity(Chalfie, 2008; Chalfie and Wolinsky, 1990). PPK1[S551V] is a constitutively-active PPK1 isoform generated by substitution of a serine residue (DEG residue) near the second transmembrane domain(Fig. 3A; also see Methods). NaChBaceGFP is a bacterial low-threshold voltage gated sodium channel previously shown to hyperactivate neuronal membranes(Luan et al., 2006; Nitabach et al., 2005).

Transgenic expression of the constitutively-active PPK1[S551V] isoform under control of ppk1GAL4 resulted in a premature and enhanced appearance of IST behavior (Fig. 3B). Transgenic larvae displayed significantly higher IST behavior at levels 3–5X normal and at timepoints 10–20 hr earlier than wild-type controls. Enhanced IST behavior caused by PPK1[S551V] expression was unaffected by a ppk1 null mutant background(DfA/DfB; ppk1GAL4:UAS-PPK1]S551V]/+)(Fig. 3B). This would be predicted since the endogenous PPK1 is replaced by PPK1[S551V].

Neuronal hyperexcitation in ppk1GAL4/UAS-NaChBaceGFP larvae(Luan et al., 2006; Nitabach et al., 2005) generated a more dramatic acceleration of IST behavior. Surfacing larvae were observed at levels >10-fold higher than wild-type and at timepoints in excess of 20 hours earlier than wild-type(Fig 3CDE). Neuronal hyperactivation resulted in 85–90% of 92h AEL transgenic larvae moving to the food surface(Fig. 3C) relative to 92h AEL wild-type control larvae(Fig. 1B) which are rarely visible on the food surface. Neuronal hyperactivation in a ppk1 null mutant background(Df/Df; ppk1GAL4:UAS-NaChBaceGFP/+) resulted in a significant suppression of enhanced IST behavior(Fig. 3F). The enhanced IST behavior was restored by transgenic expression of wild-type PPK1 protein(Df/Df; ppk1GAL4:UAS-NaChBaceGFP/ UAS-PPK1, Fig. 3F). These results are consistent with a dependence of IST behavior upon PPK1 neuron function. Increased PPK1 neuron activity is sufficient to induce premature IST behavior and loss of neuron activity causes a complete absence of the IST prior to the initiation of wandering behavior and larval food exit.

A premature developmental and behavioral transition caused by hyperactivation of PPK1 neurons

Previous studies have demonstrated a requirement for PPK1 neuron activity as a regulator of the developmentally controlled reversal in food-associated behavior observed during the midthird instar transition from foraging to wandering stage(Ainsley et al., 2008). Well-fed foraging larvae(78h AEL) faced with a shift in temperature to conditions sharply different than the food-associated temperature(25°C→20°C) will display dispersal behavior characterized by forward movement with few stops/turns(Ainsley et al., 2008). In contrast, wandering, non-feeding, larvae(108h AEL) faced with the same temperature shift showed ARS behavior, a high number of stops/turns, indicating satisfaction with their current position. This developmental reversal in thermotactic preference behavior required function of the PPK1 neurons during a critical period between 80–90h AEL(Ainsley et al., 2008).

Larvae were evaluated for timing of the developmental transition in thermotactic preference behavior by growth in standard cornmeal/yeast media at 25°C until assay at the indicated timepoint in the absence of food on an agarose sheet maintained at 20°C (Ainsley et al., 2008). Wild-type control animals normally make a major shift in thermotactic preference behavior between 92h and 108h AEL as indicated by the sharp increase in stop/turn behavior(Fig. 4A, white circles). Neuron-specific expression of PPK1[S551V] using the ppk1GAL4 driver caused a premature shift to ARS behavior by 92h AEL(Fig. 4A, gray squares) suggesting that constitutive PPK1 activation is sufficient to stimulate the behavioral transition. A similar but even more pronounced premature transition was observed in animals expressing the low-threshold voltage-gated sodium channel, NaChBaceGFP. Hyperactivation of PPK1 neurons resulting from NaChBaceGFP expression caused early third instar foraging stage(78h AEL) larvae to display ARS behavior at a timepoint approximately 30 hrs earlier than wild-type controls(Fig. 4B, black squares). These results demonstrate that the activity of PPK1 neurons is necessary and sufficient to control the progression of food-associated behaviors during third instar development.

Figure 4
Hyperactivation of mdIV neurons causes a premature larval developmental transition in food-associated behaviors. Larval food exit of wild-type(ppk1GAL4/+) larvae is indicated on the right Y-axis. The heavy-dashed line indicates the beginning of normal ...

Premature initiation of larval IST results in reduced body size

Larval IST behavior immediately precedes entry into wandering behavior and food exit suggesting that IST behavior may be a necessary preparatory step in determining the timing of entry into wandering behavior. It might be predicted that premature initiation of IST should translate into early wandering behavior. Consistent with previous results(Ainsley et al., 2008), both ppk1 null mutant(DfA/DfB) larvae and those with electrically silenced PPK1 neurons(ppk1GAL4/UAS-dOrk1ΔC) displayed a slight delay in food exit(Fig. 5A). However, food exit in animals expressing UAS-NaChBaceGFP or PPK1[S551V] in PPK1 neurons, previously demonstrated to display premature and enhanced IST behavior(Fig. 3), was not significantly different from wild-type controls(Fig 5A). This represents a clear genetic and functional distinction between the observed food surfacing behavior and final food exit.

Figure 5
Premature IST behavior results in smaller final adult size. (A) Transgenic larvae displaying premature IST behavior did not show premature larval food exit and entry in wandering behavior. (B) Length of larval mouthhooks measured shortly after third instar ...

Body size in Drosophila adults is determined by larval size at the time of feeding cessation usually associated with final food exit(Stern and Emlen, 1999). In addition, final size is dependent upon the rate of growth during the first two larval instars and the TGP(Mirth and Riddiford, 2007; Shingleton et al., 2007). Larval growth during the first two instars can be assessed by measuring the size of early third-instar mouthhooks, appendages at the anterior larval head used during feeding to direct food into the mouth. Along with the generation of a new cuticle during each molting event, larvae also produce new mouthhooks which have a characteristic morphology appropriate to each larval instar. The size of the mouthhooks produced during molting are indicative of the amount of growth during the previous instar. The length of early third instar mouthhooks in larvae with hyperexcited PPK1 neurons or loss of PPK1(DfA/DfB) were not significantly different from ppk1GAL4/+ control larvae(Fig. 5B). This demonstrates that larval feeding and growth during the first two larval instars is not affected by altered PPK1 neuron activity.

The appearance of IST behavior directly preceding larval food exit suggests a potential functional relationship between IST behavior and the end of larval feeding as a preparatory step for food exit. Premature IST behavior associated with PPK1 neuron hyperactivation could cause an early end to the TGP resulting in the generation of smaller adult animals. Results from experiments measuring the rate of larval feeding as indicated by the uptake of bromophenol blue dye in the larval gut did not show a sharp cessation of larval feeding(data not shown). However, examination of final adult size revealed a significant reduction in weight in all animals with hyperexcited PPK1 neurons as compared to wild-type animals(Fig. 5C). Expression of PPK1[S551V] or UAS-NaChBaceGFP in PPK1 neurons caused ~6% and ~11% decreases in adult male body size respectively(Fig 5C). The relative proportions of these effects are consistent with the stronger effect of NaChBaceGFP expression upon IST behavior(Figs. 3 and and4).4). Neuronal silencing of mdIV neurons by simultaneous expression of dOrk1ΔC in ppk1GAL4:UAS-NaChBacGFP animals restored adult body weight to levels comparable to wild-type animals(Fig 5C).

The production of smaller adults caused by premature IST behavior suggested that the absence of IST behavior in ppk1 null mutant larvae may conversely lead to larger final adult size due to an extended TGP. PPK1 activity was disrupted using a previously characterized ppk1RNAi transgene(Ainsley et al., 2008) to decrease PPK1 activity. When placed in trans to the Df(2L)Exel6035 deficiency chromosome, removing one copy of the endogenous ppk1 gene, the ppk1RNAi transgene caused an increase in final adult size of ~6%(Fig. 5C). ppk1 null larvae(DfA/DfB) displayed an increased final adult size of ~5%(Fig. 5B). Transgenic expression of wild-type PPK1 in DfA/DfB; ppk1GAL4/UAS-PPK1 animals lead to a decrease in final adult size although we were unable to demonstrate statistical significance of the transgenic rescue. Nonetheless, these results are consistent with a role for PPK1 neurons and the PPK1 protein in the developmental control of larval feeding behavior resulting in an effect upon larval growth and the determination of final adult size.


Feeding behavior vs surfacing behavior vs wandering behavior

The primary job description for insect larvae could be simplistically summarized as eating and growing. This sustained activity during their first week or so of life is necessary to fulfill their most critical function as a safe container nurturing the essential imaginal disks that will eventually be transformed into adult structures during morphogenesis(Riddiford, 1993). Since maintained association with an adequate food source is key to survival, larvae display numerous food-associated behaviors in response to a variety of environmental cues. Significant changes in larval responses to these cues during the third and final larval instar result in the major transition from foraging to wandering behavior(Ainsley et al., 2008). Results presented here and elsewhere(Ainsley et al., 2008), suggest that these two major larval stages, foraging(in the food) and wandering(out of the food) actually consist of a series of separable innate behaviors meant to alter the relationship between the animal and its environment.

Midway through the third and final instar, larvae stop feeding and enter “wandering stage” when they exit the food source completely in search of an appropriate pupation site. During this transition period, larvae display striking changes in body position and the spatial relationship between larvae and the food source. The constant feeding observed in foraging stage animals is associated with maintenance of a vertical anterior mouthhooks-down larval body position with only the posterior spiracles, the external openings to the larval tracheal system(Samakovlis et al., 1996), protruding from the surface of the food. This body position allows constant contact with the food source for feeding and also maintains exposure of the posterior spiracle openings to the atmosphere to allow respiratory exchange. Full body immersion in the moist food source also prevents potential larval dessication resulting from excessive exposure to a drying external atmosphere. However, pupariation within the moist food source often results in lethality caused by suffocation due to occlusion of spiracular air exchange or by failure of adult eclosion due to inability to exit the pupal case. As a consequence, although this head-down full body immersion position is beneficial during larval foraging stages, it is essential for the animal to move to the surface of the food prior to pupariation. Results presented here show that changes in the spatial relationship between larvae and the food source during the final instar is modulated by activity of a subset of peripheral sensory neurons expressing the DEG/ENaC subunit PPK1.

Evolved differences between insect species also suggest that food surfacing behavior and complete food exit are genetically separable. Of the many Drosophila species that have been characterized, D. melanogaster is one of the few that actually fully exits the food source for pupariation. Entry into “wandering” behavior in D. melanogaster is normally associated with complete larval exit from the food and movement up the sides of culture vials in search of a moderately dry pupation site. Many closely related Drosophila species choose to simply move to the food surface where they pupate(Vandal and Shivanna, 2007). In these species, the change in body position from the vertical head-down foraging position to the food surface appears to be the key behavioral transition necessary for survival through metamorphosis. Although it is likely that this striking difference in larval behavior represents an adaptive response to environmental or food conditions, genetic and/or physiological explanations for this difference are not understood.

Behavioral manifestations of the larval foraging to wandering transition

The developmental transition in larval thermotactic preference reflected as dispersal vs ARS behavior was initiated early in the third instar correlating with the previously characterized critical period for PPK1 neuron function from 80–90h AEL(Fig. 4)(Ainsley et al., 2008). This timing coincides with the appearance of IST behavior suggesting that these two behaviors reflect the same developmental and behavioral transition preceding larval food exit. As further evidence of the functional correlation of these two intermediate behaviors, IST behavior was also dependent upon PPK1 function and activity of the PPK1 sensory neurons. IST behavior was absent in ppk1 null mutant larvae and sharply suppressed in transgenic animals with electrically silenced PPK1 neurons(Fig. 2). In addition, hyperactivation of PPK1 neurons caused a premature and enhanced appearance of IST behavior(Fig. 3) paralleling the previously observed alterations in larval thermotactic behavior(Fig. 4).

The key role of PPK1 protein for normal IST behavior suggests that the temporal timing of PPK1 expression may function as a central regulatory switch for the developmental timing of this behavioral transition. However, we have previously shown that PPK1 expression first appears in mdIV neurons in late embryos (Adams et al., 1998) and is sustained into late third instar stages (Ainsley et al., 2008; Ainsley et al., 2003). These results indicate that developmentally-regulated control of PPK1 expression is likely not a potential mechanism for control of IST behavior.

Induction of premature and enhanced IST behavior did not lead to premature final food exit but caused a significant decrease in final adult size(Fig. 5). This result is consistent with premature feeding termination caused by IST behavior. Therefore, these innate behaviors are genetically and functionally separable and the observed IST behavior is not simply the beginning step of final food exit and wandering behavior.

Both the IST and ARS behavioral transitions correlate roughly with the proposed timing of larval critical size assessment early in the third instar. As discussed earlier, the developmental period between critical size assessment and larval food exit known as the TGP is essential for growth to final maximum size. If disrupted either behaviorally or metabolically, a decrease in TGP duration should result in a decrease in final adult size. This is consistent with the observed effect of PPK1 neuron hyperactivation on final adult size(Fig. 5CD).

Peripheral sensory control of behavior

Expression of the PPK1 DEG/ENaC subunit is tightly restricted to the mdIV neurons in the larval body wall(Fig. 2A) and two bipolar neurons innervating the posterior spiracles(ps)(Adams et al., 1998; Ainsley et al., 2008). Although possible functional relationships between mdIV neurons and ps neurons are not clear, both morphological and anatomical differences suggest distinct physiological functions. The md/da sensory neurons within the larval body wall have been spatially and morphologically divided into four subclasses based upon complexity of their dendritic arbors and their relative location within the larval PNS(Grueber et al., 2002; Grueber and Jan, 2004). The mdIV neurons are just one subclass of the larger collection of md/da neurons present within the larval body wall. Although experimental characterization of the md/da neurons has been extremely useful in studies aimed at understanding the development of dendritic fields (Corty et al., 2009; Matthews et al., 2007; Zlatic et al., 2009), the physiological functions and/or any interactions between the md/da subgroups remain poorly understood.

The mdIV neurons have been implicated as nociceptive neurons involved in activating a writhing escape response when exposed to noxious heat(Hwang et al., 2007; Tracey et al., 2003). This escape response has been functionally attributed to the necessity for Drosophila larvae to protect themselves from parasitoid wasps(Hwang et al., 2007). More recently, the mdIV neurons and PPK1 were shown to be required for a larval nocifensive response to noxious mechanical stimuli(Zhong et al., 2010). The relevance of these responses to harsh thermal or mechanical stimulation within the normal endogenous larval environment remains uncertain. In experimental conditions reported here, hyperactivation of the mdIV neurons using either the constitutively-active PPK1[S511V] isoform or the low-threshold voltage-gated sodium channel, NaChBaceGFP, did not result in induction of the previously reported “nocifensive” response(Zhong et al., 2010).

PPK1 neurons appear to play a much more prominent role in the normal larval response to its environment than simply a response to harsh stimuli. This supports the possibility of a polymodal role for this class of sensory neurons that may depend upon more than one source of activation signal. It must also be noted that the ppk1GAL4 driver transposon used in our studies and in previous studies demonstrating a nociceptive mdIV function(Hwang et al., 2007; Tracey et al., 2003; Zhong et al., 2010) does not distinguish between the mdIV neurons and the single PPK1-expressing bipolar neuron innervating each posterior spiracle(ps)(Ainsley et al., 2008). In addition, there are distinct morphological differences between the three mdIV neurons(ddaC, v’ada and vdaB; see Fig. 2A) within each larval hemisegment. The mdIV ddaC neuron displays a more extensive and symmetrical dendritic arbor than the v’ada and vdaB neurons. Their relative dorsal/ventral locations within the body wall may also expose each mdIV subtype to a different range of stimuli whether mechanical or chemical. Therefore, results produced using the ppk1GAL4 transposon would not detect any differences in the relative contributions of mdIV subtypes or the ps neurons to any of the proposed functional roles.

Feeding larvae detect and respond to multiple sensory inputs providing information about food resources and their immediate environment. Responses can be separated into distinct innate behaviors meant to coordinate internal growth signals with changing environmental conditions. Our findings demonstrate that the PPK1 neurons contribute to the regulation of larval developmental timing and feeding behavior transitions within normal environmental parameters. Results presented here have focused upon the larval PPK1-expressing neurons and their role in larval feeding behavior, however, other recent work has also described a role for PPK1-expressing neurons in regulation of adult feeding behavior(Ribeiro and Dickson, 2010). Although a clear functional connection between the physiological roles of larval and adult PPK1 neurons is not yet apparent, both appear to contribute in some way to monitoring of external sensory information from food sources as input to help modulate internal nutrient homeostasis.

Supplementary Material


Supplementary Figure 1:

Generation of targeted overlapping ppk1 deficiency chromosomes. Schematic representation of 35B1 genomic region containing ppk1 (white thick arrow) and flanking genes as indicated (colored thick arrows). Df(2L)35B1Aid (black) and Df(2L)35B1Mirb (red) were generated by FLP/FRT mediated excision of existing transposon insert pairs P{XP}d03030:PBac{WH}f01473 and PBac{WH}f07052:P{XP}d02171 respectively with approximate mapped insertion sites indicated at top. Resulting deficiencies were analyzed by genomic PCR and breakpoints confirmed by sequence analysis. Combination of the two lethal overlapping Df(2L)35B1Aid and Df(2L)35B1Mirb chromosomes resulted in a viable ppk1 null mutant stock. The region of overlap removing only ppk1 is indicated by the yellow box.


The authors would like to thank Sophie Krajewski, Chelsea Bilskemper, Justin Crader and Emily Pacholski for excellent technical assistance. In addition, we thank the laboratory of Michael Welsh for assistance in generation of the PPK1[S551V] isoform. This work was supported by NIH Grant NS0707197 to WAJ. JAA was supported by a AHA predoctoral fellowship.


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