SUMMARY

Obligatory cave species exhibit dramatic trait modifications such as eye reduction, loss of pigmentation and an increase in touch receptors. As molecular studies of cave adaptation have largely concentrated on vertebrate models, it is not yet possible to probe for genetic universalities underlying cave adaptation. We have therefore begun to study the strongly cave-adapted small carrion beetle Ptomaphagus hirtus. For over 100 years, this flightless signature inhabitant of Mammoth Cave, the world's largest known cave system, has been considered blind despite the presence of residual lens structures. By deep sequencing of the adult head transcriptome, we discovered the transcripts of all core members of the phototransduction protein machinery. Combined with the absence of transcripts of select structural photoreceptor and eye pigmentation genes, these data suggest a reduced but functional visual system in P. hirtus. This conclusion was corroborated by a negative phototactic response of P. hirtus in light/dark choice tests. We further detected the expression of the complete circadian clock gene network in P. hirtus, raising the possibility of a role of light sensation in the regulation of oscillating processes. We speculate that P. hirtus is representative of a large number of animal species with highly reduced but persisting visual capacities in the twilight zone of the subterranean realm. These can now be studied on a broad comparative scale given the efficiency of transcript discovery by next-generation sequencing.

INTRODUCTION

Caves represent one of the earth's most challenging environments to adapt to because they require organisms to subsist without sunlight as an energy source and aid in visual orientation. And yet, subterranean habitats harbor complex communities of highly adapted species that tend to exhibit a stereotypic suite of modified traits referred to as troglomorphy (Christiansen, 2005; Culver and Pipan, 2009). Species that are able to maintain permanent subterranean populations (eutroglophiles), for instance, are often characterized by decreased eye size (microphthalmy) and reduced body pigmentation. Even more dramatic, obligatory subterranean species (troglobionts) exhibit the complete loss of peripheral visual organs (anophthalmy) and body pigmentation (Sket, 2008). Molecular studies of cave adaptation were pioneered in and have subsequently focused on the Mexican cave tetra Astyanax mexicanus (Jeffery, 2009). Very recently, this historical vertebrate bias has been alleviated by the molecular genetic analysis of eye and pigmentation loss in the waterlouse Asellus aquaticus (Protas et al., 2011). Both of these systems constitute dramatic examples of recent and fast evolution to the troglobiont state. The genetic consequences of different degrees of long-term cave adaptation, ranging from eutroglophiles to troglobionts, remain poorly explored.

Coleoptera (beetles) constitute one of the most dominant invertebrate components of cave biota. The major beetle families found in caves are ground beetles (Carabidae: Trechinae), followed by small carrion beetles (Cholevidae or Cholevinae within Leiodidae) and rove beetles (Staphylinidae) (Moldovan, 2005) with large distributions in the palearctic and nearctic ecozones. In addition, a large diversity of aquatic troglobiotic diving beetles (Dytiscidae) has been discovered in Australia (Faille et al., 2010; Leys and Watts, 2008; Leys et al., 2003; Ribera et al., 2010). The Coleoptera are thus an ideal study group to explore the breadth of cave-adaptive states. A case in point is the small carrion beetle genus Ptomaphagus. This taxon encompasses over 50 ancestral surface-dwelling (epigean) eutroglophile and troglobiotic species in the southeast United States (Peck, 1973; Peck, 1984; Peck, 1998). One of the best-studied representatives is the small troglobiont Ptomaphagus hirtus (Tellkampf) (Packard, 1888; Peck, 1973; Peck, 1975; Tellkampf, 1844), which is endemic to the cave system in and around Mammoth Cave National Park (Fig. 1A,C,E) (Brucker, 2005). Ptomaphagus hirtus adults, which measure between 2.0 and 2.8 mm in body length, are characterized by loss of the hindwings and the near-complete reduction of the compound eye to a non-pigmented small lens patch (Fig. 1E). This contrasts with the well-developed compound eyes of eutroglophilic representatives of this genus such as Ptomaphagus cavernicola, Schwarz 1989, which shows a wide geographic distribution in the southeast United States, extending into Mexico, and has been collected both in and outside caves (Fig. 1A,B,D) (Peck, 1973).

Fig. 1.

Geographic distribution and morphology of the peripheral visual system in the small carrion beetle species Ptomaphagus hirtus and Ptomaphagus cavernicola. (A) Red area indicates documented occurrence of P. hirtus in the Mammoth cave system in Kentucky. Light blue areas identify states with frequent collection of P. cavernicola. Data based on (Peck, 1973). (B) Lateral view of P. cavernicola. (C) Lateral view of P. hirtus. (D) Lateral view of the adult head of P. cavernicola. Arrow points to compound eye. (E) Lateral view of the adult head of P. hirtus. Arrow points to residual lens structure. Inset shows a DIC image of the lens taken at high magnification from the dissected lateral head cuticle. Scale bars in A and C: 1 mm. Scale bars in D and E: 100 μm.

Fig. 1.

Geographic distribution and morphology of the peripheral visual system in the small carrion beetle species Ptomaphagus hirtus and Ptomaphagus cavernicola. (A) Red area indicates documented occurrence of P. hirtus in the Mammoth cave system in Kentucky. Light blue areas identify states with frequent collection of P. cavernicola. Data based on (Peck, 1973). (B) Lateral view of P. cavernicola. (C) Lateral view of P. hirtus. (D) Lateral view of the adult head of P. cavernicola. Arrow points to compound eye. (E) Lateral view of the adult head of P. hirtus. Arrow points to residual lens structure. Inset shows a DIC image of the lens taken at high magnification from the dissected lateral head cuticle. Scale bars in A and C: 1 mm. Scale bars in D and E: 100 μm.

In his histological survey of North American cave animals, Packard (Packard, 1888) noted the lens-like structures in the lateral head of P. hirtus but was unable to detect anatomical evidence of an associated optic nerve or optic neuropiles. This led him to conclude that P. hirtus ‘must be blind though the eyes exist in a rudimentary state’. While cursory observations in long-term culturing experiments indicated little to the contrary (Peck, 1986), more recent insights from developmental biology recommended revisiting this issue. In Drosophila, the specification of lens-producing cells during compound eye development is contingent on the preceding differentiation of photoreceptor cells (Wolff and Ready, 1993). As the cellular dynamics of retinal differentiation is highly conserved in other insect species (for a review, see Buschbeck and Friedrich, 2008), one has to assume that the dependence of lens formation on photoreceptor differentiation is also widely conserved. The question therefore arises as to how the presence of lateral lens structures can be reconciled with the presumed absence of photoreceptors in P. hirtus. One possible explanation is the life cycle-dependent elimination of photoreceptors by programmed cell death, turning the lens into a non-functional atavistic rudiment consistent with Packard's view. Alternatively, P. hirtus might possess a functional but extremely reduced visual system.

We adopted a molecular approach to test for visual modality in P. hirtus, combining deep level RNA sequencing with gene homolog-guided transcript assembly and annotation. This strategy revealed the expression not only of insect phototransduction genes but also of the circadian clock gene network. These results suggest that P. hirtus is photosensitive and that this modality might be used to regulate seasonally or diurnally oscillating processes. Consistent with the conservation of functional vision mediated through lateral photoreceptors, P. hirtus showed negative phototaxis in light/dark choice tests. Our findings support the idea that microphthalmic inhabitants of subterranean habitats represent a distinct category of cryptozoic adaptation, which is characterized by the continued use of ambient light information (Bartkowiak et al., 1991).

MATERIALS AND METHODS

Animals

Ptomaphagus hirtus animals used for molecular and behavioral experiments were taken from a laboratory culture that was started with 15 individuals collected in autumn 2008 from the Frozen Niagara cave entrance of Mammoth Cave (Scientific Research and Collecting Permit MAC-2008-SCI-2006). Ptomaphagus cavernicola animals came from a laboratory culture that was started with 84 animals collected from Warren Cave (Florida) in the spring of 2009. Both species were cultured following previously published protocols (Peck, 1975). Animals were kept in 60 mm polystyrene Petri dishes filled with ∼0.5 mm deep layer of sieved soil obtained from the natural habitats. Culture Petri dishes were kept in Styrofoam boxes on a layer of moist tissue. To maintain saturated humidity, Petri dishes and paper towels were sprayed with water approximately every 3 days. Ptomaphagus hirtus cultures were maintained in constant dark in a coolbox incubator kept at 12°C. Ptomaphagus cavernicola cultures were kept in constant dark at ambient room temperature at 23–25°C. For feeding, cultures were regularly supplied with small amounts of dried Baker's yeast. In addition, we used Georgia 1 (GA 1) strain Tribolium castaneum animals from a culture that was maintained on yeast-enriched whole-wheat flour.

Molecular biology

Twenty-five adult P. hirtus heads were pooled for mRNA extraction. The head capsules were collected from CO2-anesthetized individuals by dissection under a binocular microscope; antennae were removed. Following dissection, each head capsule was immediately transferred into a collective 1.5 ml Eppendorf tube filled with 250 μl Trizol (Invitrogen, Carlsbad, CA, USA) and homogenized using a glass–Teflon homogenizer. The homogenized tissue was stored at –70°C until total RNA extraction following the manufacturer's protocol (Invitrogen). For library construction, mRNA was purified from 20 μg of total RNA using a mRNA purification kit (Invitrogen) and libraries were generated as described previously for sequencing with the Illumina GA II sequencing system (Daines et al., 2011).

Bioinformatic analysis

Sequenced reads were aligned to the Tribolium RefSeq transcript database using blastx (Thompson et al., 1994). Reads with significant hits were sorted into putative ortholog groups based on best blastx scores. The read populations of each putative ortholog group were subjected to contig assembly with Phrap compiled with the ‘manyreads’ option (Ewing and Green, 1998). In cases of 4000 or more individual BLAST hits for a single gene (190 occurrences), 4000 sequence reads were randomly sampled from the BLAST hits for use in contig assembly. The resulting contigs were subjected to a second round of homology search against the Tribolium and Drosophila RefSeq transcript databases. The resulting database of P. hirtus transcript homologs was then searched for transcripts orthologous to Drosophila genes with functions in biological processes of interest. In cases where there was an absence of specific orthologs in the P. hirtus database, the raw sequence reads were queried by blastx with the Drosophila gene in question. To explore the possibility that low sequence conservation precluded initial inclusion in the P. hirtus transcript database, we calculated the length of protein sequence alignable between Tribolium and Drosophila orthologs as a measure of expected sequence conservation between P. hirtus and Drosophila orthologs (see Tables 1, 2, 3). All sequences of contigs discussed in this study were deposited in the Gene Expression Omnibus (accession number GSE28039). None of the over 50 open reading frame-containing contigs studied in detail contained evidence of frameshift or nonsense mutations, suggesting the absence of pseudogene transcripts.

Table 1.

Photoreceptor genes recovered from Ptomaphagus hirtus

Photoreceptor genes recovered from Ptomaphagus hirtus
Photoreceptor genes recovered from Ptomaphagus hirtus
Table 2.

Eye pigmentation genes recovered from P. hirtus

Eye pigmentation genes recovered from P. hirtus
Eye pigmentation genes recovered from P. hirtus
Table 3.

Circadian clock genes recovered from P. hirtus

Circadian clock genes recovered from P. hirtus
Circadian clock genes recovered from P. hirtus

Gene tree reconstruction

Protein sequences were aligned with PRANK using default settings (Loytynoja and Goldman, 2008). Ambiguous alignment sites were filtered with Gblocks at default setting but allowing for ‘gap positions within the final blocks’ (Castresana, 2000). The likelihood-based phylogeny estimation algorithm Tree-Puzzle was used for gene tree reconstruction, applying the Whelan–Goldman amino acid substitution model (Schmidt et al., 2002; Whelan and Goldman, 2001).

Light/dark choice tests

Single tester animals were placed in a 9 cm diameter Petri dish with a lid that was half-covered by aluminium foil, dividing the Petri dish arena into a 31.2 cm2 dark area (D-area) and an equally large light area (L-area). A commercial tungsten white light bulb (General Electric, 52 W, 120 V) was positioned perpendicularly 24 cm above the Petri dish arena. For each observation, a single animal was transferred onto the test arena with a moist brush and given 5 min for recovery and adjustment in complete darkness. Observations were started after switching on the light source, which, depending on the location of the animal in the arena, resulted in immediate exposure to light or exposure following appropriate repositioning of the lid, marking the first observation time point 0. Subsequently, the presence or absence of the animal in the L-area was recorded at 1 min intervals for a total duration of 10 min and averaged for a minimum of 10 different individuals.

The experiments with P. hirtus were performed inside the coolbox incubator at 12°C ambient temperature. A moistened paper sheet was used as the surface on the bottom of the Petri dish as preliminary observations revealed that P. hirtus showed uncoordinated walking and balancing problems on the bare plastic surface. Experiments with P. cavernicola and T. castaneum were performed at 23–25°C ambient temperature, on a moistened or dry paper surface, respectively. In all experiments, experimental animals were taken from continuously fed cultures.

RESULTS

The P. hirtus adult head transcriptome

We chose to perform RNA sequencing, which enables highly accurate qualitative and quantitative assessment of gene expression (Nagalakshmi et al., 2008; Wilhelm and Landry, 2009), on the P. hirtus adult head transcriptome to generate a database that could be probed for the conservation and expression of vision-related genes. Poly-A RNA was isolated from 25 dissected head capsules of CO2-anesthetized animals of both sexes after removal of the antennae. Using the Illumina sequencing platform, we generated 27,428,409 sequence reads of 75 bp length. Close to 20% of these (5,476,803) produced significant alignments by blastx to RefSeq protein sequences of the distantly related red flour beetle T. castaneum (Tenebrionidae) and could be sorted into 9888 putatively ortholog-specific sequence read groups. Assembly of sequence reads in each group yielded a total of 67,079 contigs with an average length of 163.3 bp. Contig orthologies were re-investigated by reciprocal BLAST against the T. castaneum and D. melanogaster RefSeq databases. This step identified 6259 and 5649 high confidence orthologs in T. castaneum and D. melanogaster, respectively. These numbers were consistent with the closer phylogenetic relationship of P. hirtus to T. castaneum. The only moderately higher number of insect-conserved loci previously identified in genome sequence comparisons (Richards et al., 2008) validated the representational depth of the P. hirtus transcript database.

Expression of phototransduction genes

Using a previously established compilation of conserved insect phototransduction genes as the query sequence source (Bao and Friedrich, 2009), we searched the P. hirtus adult head transcript database for orthologs of insect vision-related genes. This approach revealed the presence of transcripts of all critical components of the phototransduction protein network as characterized in Drosophila (Table 1) (for reviews, see Katz and Minke, 2009; Wang and Montell, 2007). The first step in the phototransduction signaling cascade involves the absorption of photons by the G-protein-coupled receptor complexes (GPCRs), which consist of a member of the 7-transmembrane photopigment opsin family and the G-protein subunits dGαq, Gβe and Gγe, all of which were found in the P. hirtus transcript database. Most significant was the recovery of dGαq and Gβe, which are photoreceptor specific in Drosophila (Dolph et al., 1994; Schulz et al., 1999; Scott et al., 1995; Yarfitz et al., 1991), as well as the recovery of a single opsin ortholog, which was supported by 369 single reads yielding 1008 bp of contiguous coding sequence. Gene tree analysis revealed that the P. hirtus singleton opsin was a member of the long wavelength (LW)-sensitive opsin subfamily (Fig. 2). Within this subfamily, the P. hirtus opsin was most closely related to the LW opsin of T. castaneum, as expected on taxonomic grounds. Interestingly, the selective conservation of the broadband-sensitive LW opsin in P. hirtus corresponds to the discovery of LW opsins in other species adapted to low light level ecologies such as cave crayfish (Crandall and Hillis, 1997).

Further, consistent with the preservation of functional phototransduction in P. hirtus, we recovered orthologs of key genes specifically involved in opsin deactivation and recycling, including Arrestin 1 (Arr1), Arrestin 2 (Arr2), G-protein-coupled receptorkinase 1 (Gprk1) and the Rhodopsin phosphatase retinal degeneration C (rdgC) (Table 1). We also detected the ortholog of Rab protein 6 (Rab6), which is thought to be specifically involved in the transport of rhodopsin in the Golgi complex (Shetty et al., 1998; Wang and Montell, 2007).

The second major step in Drosophila phototransduction involves the interaction of activated dGαq with the phospholipase NO RECEPTOR POTENTIAL A (NORPA) (Bähner et al., 2000; Bloomquist et al., 1988). Drosophila possess two differentially spliced isoforms of norpA, one of which, type I, is specific to the retina while the other, type II, is found in a variety of tissues outside the retina (Bloomquist et al., 1988; Kim et al., 1995; Zhu et al., 1993). Only norpA type II was detected in the P. hirtus transcript database, although both are conserved in Tribolium (Table 1). Interestingly, the Drosophila norpA type I photoresponse phenotype can be almost completely rescued by retinal misexpression of norpA type II (Kim et al., 2003). It is therefore tempting to speculate that a loss of norpA type I was compensated by norpA type II in P. hirtus.

The activation of NORPA is followed by the transient activation of the signalplex (Montell, 1998), the core of which is formed by the Ca2+ channel protein TRANSIENT RECEPTOR POTENTIAL (TRP) and the scaffolding protein INACTIVATION NO AFTERPOTENTIAL D (INAD). Orthologs of both corresponding genes were present in the P. hirtus transcript database. In addition to TRP, Drosophila photoreceptor cells express the related protein channel genes TRPL and TRPγ (Niemeyer et al., 1996; Xu et al., 2000). Only the former was represented in the P. hirtus transcript database although TRPγ is conserved in Tribolium. Also noteworthy was the recovery of the protein kinase neither inactivation nor afterpotential C (ninaC), which is specifically required for the calmodulin (CaM)-mediated localization of the signalplex in Drosophila photoreceptor cells (Porter et al., 1993).

Differential preservation of structural photoreceptor gene expression

Studies in Drosophila have shown that the phototransduction protein machinery is localized in the highly condensed array of microvillar extensions of the photoreceptor rhabdomere, the formation of which requires specific proteins. This includes the photoreceptor cell-specific homophilic transmembrane protein CHAOPTIC (CHP), which confers adhesion between the microvilli, and the antagonistic membrane protein PROMININ (PROM) (Zelhof et al., 2006). In addition, Drosophila photoreceptors secrete the extracellular proteoglycan protein SPACEMAKER (SPAM), also known as EYES SHUT (EYS), which results in the creation of an inter-rhabdomeral space between the Drosophila photoreceptors (Husain et al., 2006; Zelhof et al., 2006). We detected intermediate levels of chp expression in P. hirtus (Table 1), further supporting the presence of canonical insect photoreceptor cells in the adult head of this species. We also detected a low level of spam expression but found no evidence of prom in the P. hirtus transcript database (Table 1). As spam is also involved in mechanical stress protection of sensory neurons outside the visual system (Cook et al., 2008), these findings are indicative of photoreceptors in P. hirtus that are not associated with inter-rhabdomeral space formation.

Fig. 2.

Phylogenetic analysis of P. hirtus long wavelength (LW) opsin evolution. The putative P. hirtus (Phir) opsin protein sequence was aligned with the retinal opsin sequences from Drosophila melanogaster (Dmel), the honeybee Apis mellifera (Amel), the tobacco hornmoth Manduca sexta (Msex) and Tribolium castaneum (Tcas). Ambiguous regions in the resulting multiple alignment were removed, leaving 285 sites subjected to gene tree analysis. Numbers at branches indicate Tree-Puzzle branch support values.

Fig. 2.

Phylogenetic analysis of P. hirtus long wavelength (LW) opsin evolution. The putative P. hirtus (Phir) opsin protein sequence was aligned with the retinal opsin sequences from Drosophila melanogaster (Dmel), the honeybee Apis mellifera (Amel), the tobacco hornmoth Manduca sexta (Msex) and Tribolium castaneum (Tcas). Ambiguous regions in the resulting multiple alignment were removed, leaving 285 sites subjected to gene tree analysis. Numbers at branches indicate Tree-Puzzle branch support values.

Differential preservation of eye pigmentation gene expression

Insect compound eyes are usually deep brown or black in appearance because of pigment granule formation in the photoreceptors and their accessory pigment cells for optical insulation between ommatidia and the regulation of light influx within ommatidia. To investigate whether the lack of overt pigmentation in the presumptive P. hirtus lateral eyes was correlated with a reduction or loss of eye pigmentation gene transcripts, we screened the P. hirtus adult head transcriptome database for the presence of Drosophila eye pigmentation gene orthologs. While the large majority of 25 queried genes were present in P. hirtus (Table 2), we failed to detect the expression of orthologs of the ABC transporter protein genes white (w), brown (bw) and scarlet (st), which are essential and specific for membrane passage of eye pigment compounds in Drosophila (Mackenzie et al., 2000). The lack of bw corresponded to the absence of this gene in Tribolium, which expresses only one type of eye pigment (ommochrome) (Lorenzen et al., 2002). The additional lack of w and st in the P. hirtus head transcriptome suggested a gene loss-associated complete reduction of eye pigmentation in this species. This conclusion was reinforced by the absence of the eye pigmentation-associated phosphoesterase prune (pn) and kynurenine 3-monooxygenase cinnabar (cn) in the P. hirtus transcript database, both of which are conserved in Tribolium (Table 2).

Analysis of photoresponse behavior

The evidence of a functional visual system prompted the question of the significance of photic stimuli in the biology of P. hirtus. To explore the possibility of phototactic behavior in P. hirtus, we investigated dispersal behavior in a light (L) versus dark (D) choice model (Fig. 3) as detailed in Materials and methods. Strikingly, after only 2 min, less than 30% of the tested animals were recorded in the L-area. For the rest of the experiment, the proportion of animals in the L-area varied between 10 and 30%. This translated into an average phototaxis index of –0.658 or a probability of 0.17 of observing an animal in the L-area (Maria Camassa, 2001). Continuous observation of five independent animals in the same test conditions further revealed that animals tended to move faster in the L-area while resting more often in the D-area.

To evaluate how the phototactic behavior of P. hirtus compared with that of species with a fully developed visual system, we subjected the closely related eutroglophile P. cavernicola to the same L/D choice test. Ptomaphagus cavernicola animals withdrew on average twice as quickly into the D-area such that an average of less 30% were scored in the L-area 1 min after the start of the experiment. Ptomaphagus cavernicola also exhibited a stronger average negative phototaxis index (–0.889) over the subsequent 8 min of the experiment (Fig. 3A), corresponding to a probability of 0.05 of being observed in the L-area (Fig. 3B). Besides revealing a shared disposition of P. hirtus and P. cavernicola to avoid exposure to strong light, the similarity in the response of the two species was highly suggestive that P. hirtus utilized peripheral photoreception in phototaxis, which is a reasonable assumption for P. cavernicola. Continuous behavorial observation of P. cavernicola individuals revealed similar D-area-biased resting patterns to those in P. hirtus but also a faster mobility of this larger species (3–4 mm body length), contributing to the faster withdrawal into the D-area.

To explore how the phototactic behavior of the Ptomaphagus species compared with that of non-cave-adapted Coleoptera, we also tested T. castaneum, which is characterized by crepuscular digging behavior, relatively small eyes and the lack of blue-sensitive opsin (Jackowska et al., 2007). In contrast to the Ptomaphagus species, T. castaneum demonstrated no evidence of Lor D-area preference. The proportion of animals present in the L-area decreased more slowly after onset of the experiments, never dropped below 30% and averaged 49%, translating into equal probabilities of being encountered in the Dor L-area. These findings supported the notion that both P. hirtus and P. cavernicola are characterized by cave adaptation-associated enhancement of negative phototaxis.

Conservation of circadian clock gene expression

Luminosity-based measuring of day length can play a role in the entrainment of oscillating processes through the circadian clock gene network, alone or in addition to temperature or other external zeitgeber stimuli. Remarkably, eutroglophilic and troglobiotic ground beetles exhibit day length entrainment of diurnal activity rhythms (for a review, see Lamprecht and Weber, 1992). To explore the potential for a role of vision in circadian entrainment, we also probed the P. hirtus transcript database for the presence of clock gene orthologs because the expression of clock genes is concentrated in neuronal cell clusters in the brain of insects (for a review, see Tomioka and Matsumoto, 2010). This search revealed the expression of all components of the insect clock gene core network that have been identified in Tribolium (Table 3) (Cortes et al., 2010), including the bHlH-PAS transcription factor genes period (per), cycle (cyc), tango (tgo), clock (clk) and timeless (tim) as well as interacting genes like Par-domain protein 1 (Pdp1) and vrille (vri). Previous studies reported the preservation of the photolyase chryptochrome 2 (cry2) but the likely ancestral absence of its paralog cryptochrome 1 (cry1) in Tribolium (Yuan et al., 2007; Zhu et al., 2005). Consistent with this, only cry2 was detected in the P. hirtus transcript database.

DISCUSSION

The molecular and behavioral data accumulated in this study revise the assumed blindness of P. hirtus of the past 100 years. While the anatomical localization of phototransduction in P. hirtus remains to be studied in detail, it is reasonable to assume that the lateral lens patches cover a photoreceptor cell population. This is predicted from the highly conserved developmental dependence of lens formation on photoreceptor development in insects (Friedrich et al., 2006; Friedrich et al., 1996). Also of potential significance in this context is the finding from a survey of North American Ptomaphagus that P. hirtus represents an extreme representative of a range of mildly to strongly microphthalmic species (Peck, 1973; Peck, 1984; Peck, 1986). This comparative evidence suggests that the P. hirtus lens patches were derived by gradual reduction of the compound eye. Preliminary histological analyses of the presumed lateral eyes reveal the presence of a defined, densely clustered cell population underneath a cuticular lens with remarkable similarity to the organization of the peripheral visual system in the European troglobiotic ground beetle Typhlochoromus stolzi (Bartkowiak et al., 1991) (M.F. and Elke Buschbeck, unpublished). Packard's difficulties with recognizing an optic nerve or optic neuropiles were very likely due to the correlated reduction of the central visual system (Packard, 1888). The visual ecology of its habitat, the loss of flight and the preservation of apparently only a single opsin gene suggest that P. hirtus lacks wavelength discrimination and image-forming capacity. The combination of these regressive traits is predicted to result in a similarly dramatic reduction of the central visual system as seen, for instance, in the acephalic larvae of higher dipteran species like Drosophila [for a detailed review see Friedrich (Friedrich, 2011)]. Functionality and neuroanatomical organization of the P. hirtus visual system thus represent interesting subjects of further investigation.

The transcriptome data produced further clues regarding the cellular organization of the P. hirtus visual system. The detection of CHP transcripts, for instance, is a strong indicator of peripheral photoreceptors. The lack of transcripts of all eye-specific ABC transporter genes (w, bn and st), for instance, implies the presence of unpigmented photoreceptor cells, consistent with the clear appearance of the lateral eye patches (Fig. 1E). Of note, the comparison with Tribolium suggests that the lack of bn might be ancestral for Coleoptera. Considering the heavy pigmentation of the compound eye of P. cavernicola (Fig. 1B), one has therefore to assume that, at the least, w and st were lost in the P. hirtus lineage after separation from the P. cavernicola-associated lineage due to a relaxation of selection on eye pigmentation in troglobiotic species compared with eutroglophile species. The same scenario would explain the absence of the enzymatic pigmentation genes cn and pn from the P. hirtus transcript database. The lack of cn is particularly noteworthy considering the evidence for pseudogenization of this gene in multiple cases of troglobiotic Australian diving beetles (Leys et al., 2005). The large number of enzymatic eye pigmentation genes in the P. hirtus transcriptome is best explained by functions outside the eye. This is most obviously the case for the tryptophan oxygenase vermilion (v), which has a primary function in the tryptophan metabolism pathway (Linzen, 1974; Walker et al., 1986). It is tempting to predict that the comparison of gene repertoires between epigean and troglobiotic species can be used to identify function-specific gene groups.

Fig. 3.

Behavioral analysis of phototaxis in P. hirtus. (A) Time course of average presence of experimental animals in the exposed area of the light/dark (L/D) choice test arena. X-axis indicates time points of single observations. Y-axis represents fraction of test animals present in the L-area of the test plate. (B) Phototaxis index (Y-axis) of P. hirtus and P. cavernicola calculated from data extending from the 8–10 min time points in A. Gray box background indicates maximum range over separate time intervals. Error bar represents standard deviation. Dashed line indicates neutral expectation.

Fig. 3.

Behavioral analysis of phototaxis in P. hirtus. (A) Time course of average presence of experimental animals in the exposed area of the light/dark (L/D) choice test arena. X-axis indicates time points of single observations. Y-axis represents fraction of test animals present in the L-area of the test plate. (B) Phototaxis index (Y-axis) of P. hirtus and P. cavernicola calculated from data extending from the 8–10 min time points in A. Gray box background indicates maximum range over separate time intervals. Error bar represents standard deviation. Dashed line indicates neutral expectation.

Our behavioral experiments identify avoidance of light exposure as one candidate function of the P. hirtus visual system. The strongly negative photoresponse of P. hirtus and P. cavernicola in contrast to T. castaneum is in line with the idea that negative phototaxis predisposes species to cave colonization (Langecker, 1992; Romero, 1985). Visual performance and behavior, however, need to be explored in more detail, most importantly with regard to the effects of light intensity and wavelength. The negative phototaxis of P. hirtus in our experiments is intuitively rationalized as the effect of an upper light intensity threshold, which prevents P. hirtus from leaving the cave environment or presenting itself in an area populated with visual predators. However, the phototactic response of P. hirtus may be bimodal. A complementary low-light intensity threshold level may prevent these animals from dispersing into aphotic cave zones, which are defined by the complete absence of light and extreme limitation of organic nutrients. A bimodal photoresponse may maintain P. hirtus in disphotic cave areas, which are characterized by extremely low intensities of diffuse light from cave openings and a richer organic nutrient content. Such a preference has been demonstrated in the eutroglophile ground beetle Laemostenus schreibersii (Rusdea, 1992).

In the case of P. hirtus, bimodal phototaxis may be key to maintaining a dispersal pattern coincident with that of another dominant species of the Mammoth cave system: the guano-producing cave cricket Hadenoecus subterraneus (Hubbell and Norton, 1978). This subtroglophile forms large roosting congregations at the ceiling of cave areas that are in sufficient reach of surface links for foraging excursions, which are carried out strictly at night-time and in day-spanning intervals (Poulson, 1992; Poulson et al., 1995; Studier et al., 1986). While being attracted to a diversity of decomposing organic matter, P. hirtus are known to feed frequently on the cave cricket guano (Peck, 1975), which has been rated as one of three major nutrient influx sources in the Mammoth cave system (Barr and Kuehne, 1971). A shared phototactic preference for the disphotic zone may facilitate the horizontally overlapping distribution of P. hirtus and H. subterraneus despite their vertical separation.

The preservation of circadian clock gene expression points to a second trait that characterizes P. hirtus as potentially adapted to the disphotic zone. In Drosophila, the compound eyes, the larval eyes and select brain photoreceptors cooperate in the daylight cycle-contingent entrainment of the circadian clock gene network (Helfrich-Förster, 2002; Helfrich-Förster et al., 2002; Rieger et al., 2003; Veleri et al., 2007). Intriguingly, light entrainment of locomotory activity patterns is well documented in eutroglophilic as well as troglobiotic ground beetle species with moderately to strongly reduced eyes (Lamprecht and Weber, 1975; Weber et al., 1994; Rusdea, 2000). Morphologically, however, the extreme degree of eye reduction in P. hirtus is likely to be most similar to that of the ground beetle T. stolzi (Bartkowiak et al., 1991), which exhibits only very weak light entrainment (Lamprecht and Weber, 1983; Lamprecht and Weber, 1992).

While it is tempting to speculate that P. hirtus measures diurnal light level changes in the disphotic cave environment, the clock gene network may be environmentally adjusted by other stimuli such as temperature (Collins et al., 2004; Yoshii et al., 2009). Consistent with this last scenario, an impact of environmental temperature oscillations on circadian rhythm regulation has been documented in a number of insects including cave-adapted carabid and catopsid beetles (for reviews, see Lamprecht and Weber, 1992; Page, 1985; Rence and Loher, 1975; Tomioka and Chiba, 1989; Weber et al., 1995; Yoshii et al., 2009). Moreover, highly sensitive temperature receptors have been found in the larval antennae of troglobiotic cholevine species (Corbiere-Tichane and Loftus, 1983; Loftus and Corbiere-Tichane, 1981; Loftus and Corbiere-Tichane, 1987). Microclimates are seasonally stable in the deeper parts of the Mammoth cave system but entrance areas, exposed to air currents, show variation in humidity and temperature (Barr and Kuehne, 1971). Moreover, there is evidence that the distribution of bottom-dwelling cave arthropods including P. hirtus is temperature and humidity dependent (Barr and Kuehne, 1971). Thus, clock gene transcript level oscillations need to be studied in relation to diurnal and seasonal light and temperature variations to elucidate the zeitgeber mechanisms in P. hirtus.

Examples of behavorial or metabolic rhythmicity in subterranean species include blind mole rats, the cavefish genera Astyanax and Nemacheilus, myriapods and crustaceans (Cooper et al., 1993; De La O-Martinez et al., 2004; Erckens and Martin, 1982; Goldman et al., 1997; Jegla and Poulson, 1968; Koilraj et al., 2000; Kumar Pati, 2001) (for a review, see Lamprecht and Weber, 1992). Curiously, while our data represent the first demonstration of the expression of the complete circadian gene network in a troglobiont, no evidence of oscillating behavior or metabolic activities has so far been reported for P. hirtus. On the contrary, previous work suggests that reproduction is season independent (Peck, 1975). Of potential significance, however, is the fact that the production of guano by H. subterraneous is seasonally variable with maximal deposition in the warmer months and close to zero production during winter (Barr and Kuehne, 1971). A study of cave cricket guano microcommunity variation demonstrated a tight correlation between climatic variations of guano richness and species composition (Poulson et al., 1995). The hypothesis that P. hirtus' life history or dispersal pattern may be seasonally adjusted therefore deserves new consideration.

In Drosophila, the biological clock controls processes as diverse as olfaction, neuronal physiology, detoxification, immune response, activity patterns and metabolism (for a review, see Allada and Chung, 2010). It is therefore difficult to speculate about the actual targets of the clock gene network in P. hirtus. We predict that transcriptomic approaches will be an efficient way not only of studying the transcriptional regulation of the clock gene network in the natural habitat but also of identifying putative target genes by virtue of correlated transcription profiles (Doherty and Kay, 2010; van der Linden et al., 2010). Our study suggests that next-generation sequencing methods will continue to serve as powerful facilitators in this endeavor. The recovery of over 5000 high confidence Drosophila and Tribolium orthologs in the adult head transcriptome of P. hirtus by RNA sequencing underlines the promise of next-generation sequencing for exploring the genetic organization of non-model organisms, by capitalizing on model species' genome sequences as a reference for assembly and annotation (Gibbons et al., 2009; Juan et al., 2010). The sensitivity of this approach is most impressively highlighted by the detection of circadian rhythm gene transcripts. In the insect species in which it has been studied, the expression of many circadian clock genes is restricted to small cell groups in the brain (Tomioka and Matsumoto, 2010). While the spatial expression characteristics of clock genes in P. hirtus remain to be determined, it is reasonable to conclude that the sensitivity of RNA sequencing has enabled the comprehensive characterization of phototransduction and clock gene expression using a very marginally cell type-enriched tissue sample. This proof-of-principle result opens the door for broad-scale comparative analyses of genetic cave adaptation.

FOOTNOTES

FUNDING

R.B. and J.C. were supported by Wayne State University graduate research enhancement funds.

Acknowledgements

Dr Rick Toomey and Dr Kurt Helf from the Mammoth Cave International Center for Science and Learning provided advice and assistance with collecting P. hirtus. David Lizdas and Bill Oldacre kindly facilitated access to Warren cave. Arianys Camacho assisted with collecting P. cavernicola. Dr Evelyn Rusdea brought the literature on circadian control in European cave beetles to our attention. The Wayne State University Scientific Computing Grid provided computational facilities for some analyses. Elke Buschbeck, Steve Cooper and two anonymous reviewers provided valuable input on the manuscript.

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