Is there an ecological scenario where terrestrial insect larvae can show food choices?

Is there an ecological scenario where terrestrial insect larvae can show food choices?

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I have been going through literature on insect food choices. I plan to study the effect of prior experience on food choices in both adults and larvae of the red flour beetle, Tribolium castaneum. There are plenty of studies that focus on larval and adult experience having an effect on adult preference. I would like to look at how larval experience changes larval choice, but only if it has some ecological relevance. However, I could not find a single study that provides an ecological relevance to larvae making food choices.

This appears to be due to the fact that larvae are usually sessile, and since adult females are the ones making the oviposition choices, therefore most studies seem to assume that the choices of the larvae don't matter. (Preference Performance Hypothesis)

Larval food choice might be ecologically relevant under conditions where females make "mistakes" during oviposition, say if the environment is spatially/temporally fluctuating. But if they show limited dispersal (or slower dispersal than adults), then does their choice really matter?

Does anyone know of any examples (published or unpublished) where insect larval food choice is ecologically relevant? It would help me out a lot to design my study. Thanks in advance!

The diamondback moth (DBM), Plutella xylostella L. (Lepidoptera: Plutellidae) is one of the most important pests of cruciferous crops in the world.. A strain capable of complete development on sugar pea (Pisum sativum L. var. macrocarpon) has recently appeared. A recent study (Henniges-Janssen, Heckel & Groot (2014), "Preference of Diamondback Moth Larvae for Novel and Original Host Plant after Host Range Expansion"; link) compared the feeding preferences of L4 larvae reared during previous larval instars on cabbage and pea. Pea-reared larvae of this strain preferred peas, while cabbage-reared larvae preferred cabbage. L4 larvae of the original strain preferred cabbage irrespective of their previous diet. I don't think this example has been shown to occur in the field, but the two plants are both fed upon by these caterpillars in the wild.

There is also evidence that larvae of Cydia pomonella (codling moth), a marjo agricultural pest of orchards with a near-global distribution, learn to avoid apple flavoured with saccharine if they have previously encountered saccharine-flavoured noxious plants (Pszczolkowski & Brown, 2005); this probably meets your second criterion for ecological relevance more closely (evidence for learning to avoid odour/flavour cues encountered in a toxic plant, thereby increasing survival) but it's less relevant to a real-world field situation than the first example.

I also found an article on associative learning in insects here that may include some useful examples.

Terrestrial invertebrates in the Rhynie chert ecosystem

The Early Devonian Rhynie and Windyfield cherts remain a key locality for understanding early life and ecology on land. They host the oldest unequivocal nematode worm (Nematoda), which may also offer the earliest evidence for herbivory via plant parasitism. The trigonotarbids (Arachnida: Trigonotarbida) preserve the oldest book lungs and were probably predators that practiced liquid feeding. The oldest mites (Arachnida: Acariformes) are represented by taxa which include mycophages and predators on nematodes today. The earliest harvestman (Arachnida: Opiliones) includes the first preserved tracheae, and male and female genitalia. Myriapods are represented by a scutigeromorph centipede (Chilopoda: Scutigeromorpha), probably a cursorial predator on the substrate, and a putative millipede (Diplopoda). The oldest springtails (Hexapoda: Collembola) were probably mycophages, and another hexapod of uncertain affinities preserves a gut infill of phytodebris. The first true insects (Hexapoda: Insecta) are represented by a species known from chewing (non-carnivorous?) mandibles. Coprolites also provide insights into diet, and we challenge previous assumptions that several taxa were spore-feeders. Rhynie appears to preserve a largely intact community of terrestrial animals, although some expected groups are absent. The known fossils are (ecologically) consistent with at least part of the fauna found around modern Icelandic hot springs.

This article is part of a discussion meeting issue ‘The Rhynie cherts: our earliest terrestrial ecosystem revisited’.

1. Introduction

The Rhynie and adjacent Windyfield cherts outcrop in Aberdeenshire in Scotland. They are one of the most important windows into early life on land [1]. Fossils are preserved, often three-dimensionally, with extraordinary fidelity in the translucent chert matrix [2] and offer unparalleled insights into the external (and internal) anatomy of the animals that lived in this Early Devonian hot springs environment. Previous ecological interpretations of the Rhynie terrestrial fauna were offered by Tasch [3], Kevan et al. [4], Rolfe [5,6], Anderson & Trewin [7] and Fayers & Trewin [8]. Since these papers were published, a nematode worm [9] and several new arachnids have been described [10–13], and some previously known fossils assigned to the insects or myriapods have been reinterpreted [14,15]. Other studies focused on how the extinct trigonotarbid arachnids may have breathed [16], walked [17] and visualized their surroundings [18]. Here, we offer a modern synthesis of the terrestrial animals (primarily arthropods) known from Rhynie and their probable ecological roles. We review functional morphology, which provides evidence of where and how these animals could have lived. This is particularly important for extinct groups with no direct modern counterpart. For groups with living representatives, however, detailed comparisons of exactly how their descendants live and feed permit more accurate inferences about the Early Devonian fossils. Finally, we compare the Rhynie fauna with arthropod communities on the margins of modern hot springs.

2. Material and methods

Fossils of terrestrial animals from the Rhynie and Windyfield cherts were reviewed from the literature and compared, where possible, to closely related extant species as ecological proxies. References for individual groups are given below. The depositional setting [2] and stratigraphic age of the cherts of around 409–411 million years ([19], but see [20]) has been addressed previously in some detail. Because of the excellent preservation, it has been suggested [21] that at least the mites and springtails at Rhynie could be Recent contaminants incorporated through secondary silicification. We reject this argument (see also [22]) because: there is no known mechanism for re-mobilizing the silica in the crystalline chert both extinct and modern-looking arthropods are preserved with similar textures and arthropod material has also been recovered from drill cores well below the weathered surface which gave no opportunity for modern contaminants to enter the matrix.

3. Results

(a) Nematoda: Enoplia

Nematode worms living in water films in the terrestrial environment are effectively freshwater organisms, but a remarkable find from Rhynie is Palaeonema phyticum Poinar, Kerp & Hass, 2008 [9] (figure 1a) described from the stomatal chambers of the early land plant Aglaophyton majus Edwards, 1986. Trace fossils [23] and a putative body fossil [24] of marine Nematoda have been described from the Early Ordovician of China, but the Early Devonian P. phyticum remains the oldest unequivocal nematode body fossil. The worms were estimated at 0.1–1 mm in length, and assigned to an extinct family, Palaeonematidae [9], within the order Enoplia. The authors proposed this fossil was one of the oldest examples of a relationship between a terrestrial plant and an animal. Specifically, they interpreted P. phyticum as having reproduced within the plant tissue because eggs, juveniles and adults are present together. From the structure of the buccal cavity the authors also inferred that it was an epistrate feeder, using a ‘tear and swallow’ strategy. Poinar et al. [9] suggested that the Rhynie nematodes may have fed on the plant's cortical cells—which would also make it the oldest known herbivore—and that they could have supplemented their diet with microorganisms. In this context, it is worth noting an earlier comment by Challoner et al. [25] that some plant damage observed at Rhynie could have been induced by nematodes. We should mention that both free-living nematodes and (partially silicified) parasitic nematodes in the parenchyma cells of the plant Eleocharis have been observed in the modern Big Blue Hotspring at Yellowstone (Alan Channing 2017, personal communication).

Figure 1. Nematode worms and arachnids from Rhynie. (a) Palaeonema phyticum Poinar, Kerp & Hass, 2008 (Nematoda: Enoplia). (b,c) Palaeocharinus spp. (Arachnida: Trigonotarbida). (d,e) Two examples of mites (Arachnida: Acariformes). (f–h) Eophalangium sheari Dunlop, Anderson, Kerp & Hass, 2004 (Arachnida: Opiliones). (i) Saccogulus seldeni Dunlop, Fayers, Hass & Kerp, 2006 (Arachnida incertae sedis).

According to Bik et al. [26], the subclass Enoplia is the earliest branching clade of the Nematoda. It can be divided into a largely marine Enoplida and a terrestrial Triplonchida, the latter containing some modern plant parasites. These authors noted the paucity of nematode fossils (most come from amber), but appear to have overlooked the Rhynie species as a potential calibration point for their molecular dating. Bik et al. ([26] and references therein) also touched on the debate regarding whether nematodes originated in a marine or a terrestrial environment and—if they were marine—how often they crossed into freshwater and terrestrial habitats. Plant feeding in modern nematodes is thought to have arisen at least four times independently among the twelve currently recognized major clades [27]. Given the limited number of morphological characters, much of today's phylogenetic work is based on molecular data, and there has been no attempt to date to incorporate the extinct Rhynie family into a modern tree. If possible, this would test whether plant feeding in the fossil family is independent, or dates the origins of one of the four known transitions to plant parasitism. Bird et al. [27] reported the presence of a stylet in all the modern nematode plant parasites which the animals use to pierce plant cells, and which appears to have arisen convergently. A stylet is absent in P. phyticum [9] implying that it does not belong to one of the modern plant parasite clades, and evolved this lifestyle independently. Bird et al. [27] also noted that the modern plant parasites are often closely related to fungivorous nematodes, which suggests that (in these groups) plant-feeding evolved from fungus-feeding. Given that Rhynie has a rich fungal assemblage [28] it is interesting to speculate whether there were other nematodes in the original ecosystem which used this resource.

(b) Arachnida

(i) Trigonotarbida

Trigonotarbids are an extinct (Silurian–Permian) arachnid order. Of the five original species described from Rhynie [29]—all in the genus Palaeocharinus Hirst, 1923—only two are likely to be valid [30] the others appear to have been diagnosed on preservational artefacts. Hirst's fossils [29,31] are about 3–4 mm in body length (figure 1b). A slightly larger (ca 6 mm) and more tuberculate Palaeocharinus species was later added [12] from the adjacent Windyfield chert. Some Rhynie trigonotarbids are up to 14 mm long [5] however, these are only known in dorso–ventral section and it is difficult to assess whether they belong to the same genus as the smaller fossils. The exquisite preservation of the Rhynie trigonotarbids has contributed towards an excellent understanding of the group's morphology and palaeobiology. A key feature of the group is the presence of book lungs [32] (figure 1c) which confirm that they were unequivocally terrestrial, contra Tasch [3] who suggested that the entire Rhynie fauna may have been aquatic. These structures remain the oldest evidence for lungs in any animal group and re-investigation [16] confirmed that they are anatomically modern. For example, the air spaces between individual lamellae are separated from one another by tiny struts called trabeculae which prevent the lung from collapsing under its own weight. The presence of two pairs of lungs also phylogenetically places the trigonotarbids in the arachnid clade Pantetrapulmonata alongside spiders, whip spiders, whip scorpions and schizomids. That said, the Rhynie material also preserves some characters such as divided tergites, a locking mechanism between the prosoma and opisthosoma, and a tiny claw at the end of the pedipalp [33] which are shared with the rare and enigmatic living order Ricinulei.

Palaeocharinus has lateral eye tubercles bearing up to 15 individual lenses [18]. They appear to document a transition between a compound eye, as in horseshoe crabs and the extinct eurypterids, and the eyes of modern arachnids which have five lenses or fewer. It is not clear to what extent Palaeocharinus relied on vision—its median eyes look upwards at an angle and its lateral eyes look more to the side—and most modern arachnids rely more on tactile setae and/or vibrations as sensory input. The legs of Palaeocharinus have sparse setae, but do become more setose towards the tip. There is no evidence that Palaeocharinus had trichobothria (fine hairs used to detect air currents), but at least the metatarsus joint towards the ends of the legs bears groups of slit sense organs which in modern arachnids act as sensory strain gauges. Garwood & Dunlop [17] analysed details of the leg articulations in Palaeocharinus to constrain the degree to which the joints could bend in any given direction. From this, coupled with a digital reconstruction of the whole animal, they estimated the centre of mass and extrapolated a three-dimensional animation of a likely walking pattern based on comparisons with living arachnids.

These anatomical elements give the overall impression of Palaeocharinus as a short-legged, cursorial predator. The mouthparts consist of ‘clasp-knife’ chelicerae (figure 1c), with a curved fang articulating against a basal article bearing a row of teeth [34]. Like spiders, trigonotarbids may have pierced and restrained prey items with the fangs and then pressed the victim against the tooth row as part of the mastication process. None of the material shows evidence for an opening from a prosomal venom gland on the fang, which is otherwise only known in spiders. The Rhynie trigonotarbids also have a row of denticles on the pedipalp coxae as well as mesal projections from the anterior leg coxae which may have aided mastication. These are not seen in this form in any living arachnids, and could represent a remnant of something akin to the coxal gnathobases used to chew food in horseshoe crabs and eurypterids.

Most arachnids digest their prey pre-orally, regurgitating enzymes onto the food and sucking up the liquefied remains. Preoral digestion is likely to have been less effective in an aquatic environment, so again supports a mode of life on land. Two lines of evidence strongly support preoral digestion in Rhynie trigonotarbids. Rolfe [6, plate 1] figured an amorphous mass of cuticle held in the mouthparts of a trigonotarbid and there is a putative filtering device [34] between the labrum and the labium consisting of several downward-pointing opposing hairs or platelets. These suggest that particulate matter was filtered out, and so not ingested, and solid items would not be expected in the gut. Some authors [4] suggested that trigonotarbids may have been facultative spore feeders. Plant-eating by spiders is very rare, but not unknown [35], and in a few cases fungal spores were consumed. However, in the absence of direct evidence for spore feeding, such as a Palaeocharinus holding spores in its mouthparts or gut traces, we feel that the preserved morphology is more consistent with that of a predator.

(ii) Acariformes

The Rhynie mites remain the oldest unequivocal record of the order Acariformes. The other conventionally accepted mite order, Parasitiformes, is not known until the Cretaceous. The Rhynie fossils (figure 1d,e) were initially assigned [29] to a single species, Protacarus crani Hirst, 1923, with body lengths of ca 290–440 µm, and tentatively referred to the modern family Eupodidae. Subsequently, Dubinin [36] recognized four additional species. There has been an unfortunate tendency in the past to treat all Rhynie mites as an amorphous group of generalist detritivores. Hence we consider it important here to review the species' systematic placements, as well as the ecology of modern members of their respective groups. We should, however, caution that there has been no taxonomic revision of the mites since Dubinin's study, and that he based his interpretations on the (albeit excellent) published illustrations rather than direct observation of the fossils. The original species, P. crani, is currently placed in the clade Endeostigmata and the family Alycidae. Two further species are also considered to be endostigmatids: Protospeleorchestes pseudoprotacarus Dubinin, 1962 in the family Nanorchestidae and Pseudoprotacarus scoticus Dubinin, 1962 in the family Alicorhagiidae. The two remaining Rhynie mites are placed in a different clade, Prostigmata, specifically the group Eupodides and the family Tydeidae which contains Palaeotydeus devonicus Dubinin, 1962 and Paraprotacarus hirsti Dubinin, 1962.

Acariform mites can be broadly divided into two major lineages [37]: Sarcoptiformes and Trombidiformes. Endeostigmatids are sarcoptiform mites, while eupodidids are trombidiforms. The Rhynie fossils suggest that these lineages had split from one another prior to the Early Devonian, and that some extant families can potentially be traced back over 400 million years. Sarcoptiform mites are sometimes referred to as ‘chewing mites’ since they have modified mouthparts for mastication and so are able to ingest particulate food. The most abundant sarcoptiforms are the oribatid mites, which are an important component of modern soil ecosystems. None of the Rhynie mites appear to be oribatids—a group first recorded in the Mid-Devonian—and were placed instead among the endeostigmatids, which are generally interpreted as an assemblage of early branching chewing mites [38]. Krantz & Walter [37] noted that modern endeostigmatids are often found today in extreme or dry soil habitats (e.g. deserts, microbial crusts, sea shores). We speculate that their presence at Rhynie could relate to the unusual characteristics of the hot springs environment.

With respect to modern feeding ecologies [39], some living Alycidae are predators of nematode worms a group now also known at Rhynie (see above). It has been speculated that other Alycidae are plant feeders, but these forms have elongate, needle-like mouthparts which are not seen in the Rhynie fossils. Alicorhagiidae are best considered omnivores, and belong to an assemblage of endeostigmatids known to ingest solid fragments of fungi and/or other small- and soft-bodied invertebrates. For example, some living alicorhagiids feed on fungi in the laboratory, but cultures do not prosper unless they have access to nematodes as prey items too [39]. Finally, Nanorchestidae are believed to be fluid-feeders and living species have been classified as microphytophages feeding on fungi and algae. As noted above, fungi are abundant at Rhynie as a potential food source and both green algae and cyanobacteria have been reported here as well [4].

Prostigmatid mites express a bewildering variety of lifestyles from free-living predators, to parasites, to plant feeders. At Rhynie the fossils' affinities dictate that we focus on Tydeidae, modern species of which include predators, scavengers and groups feeding on fungi or directly on plants [40]. In their catalogue of modern tydeids, Da Silva et al. [41] also listed them as phytophages, mycophages, pollenophages, insect parasites or scavengers, but further noted that the majority of today's species are scavengers or mycophages (i.e. fungi-feeders). Both of these feeding ecologies are compatible with the Rhynie palaeoenvironment. We should note in passing that Rolfe [6] identified structures which reminded him of the galls formed by eriophyoid mites (another prostigmatid group) however, gall formation and/or gall-producers at Rhynie have not been further investigated. In summary, the mite assemblage at Rhynie includes taxa which may have used a range of ecological niches.

(iii) Opiliones

The harvestman discovered at Rhynie [10] (figure 1f–h) was subsequently named Eophalangium sheari Dunlop, Anderson, Kerp & Hass, 2004 [11]. It is the oldest record of Opiliones. The material discovered suggests a compact body up to 6 mm long with elongate, slender legs. While trigonotarbids preserve lungs (see above), E. sheari has tracheal tubes [11], with a branching pattern similar to that of living harvestmen. In detail, a large tube extends into the prosoma—which would have contained the metabolically active limb musculature—and several smaller tubes serve the opisthosoma (figure 1h). These structures both represent the oldest direct evidence for tracheae in any arthropod and demonstrate unequivocally that E. sheari was a fully terrestrial animal.

Eophalangium sheari also preserves reproductive organs, allowing the different genders to be recognized, and inferences about their behaviour to be made. In modern harvestmen males either have an eversible spermatopositor (the suborder Cyphophthalmi) for depositing spermatophores on the substrate, or a penis (all other suborders) as an intromittent organ for direct sperm transfer. The Rhynie harvestman organ (figure 1g) terminates in a tapering tip, or stylus [11], and seems more likely to have been a penis used for direct copulation. The female of E. sheari preserves an annulated ovipositor (figure 1f) which, in comparison with living species, was presumably extended from the body to allow eggs to be laid in a specific place within the substrate. In its original description [11], E. sheari was assigned to the living suborder Eupnoi. However, the number of tendons in the male penis does not match the pattern seen in living eupnoids and the genital opening (or gonostome) appears to be open, rather than closed by a plate. Subsequently, Garwood et al. [42] resolved E. sheari closest to a new Carboniferous fossil harvestman based on this open gonostome and an anterior projection (or ocularium) bearing the median eyes. Together these two Palaeozoic fossils were assigned to a new extinct suborder, Tetrophthalmi, forming the sister group of Cyphophthalmi.

Unusually among arachnids, modern harvestmen are omnivorous they do not practice preoral digestion. The Rhynie harvestman does, as a result, have a visible gut trace [11], but specific gut contents cannot be identified. From an overview of feeding ecology in modern harvestmen [43], it would be misleading to claim that all harvestmen are generalists. Different modern species tend to show individual food preferences. Most harvestmen are primarily predators—be it on arthropods, molluscs or worms—but may supplement their diet by scavenging on decaying animal and/or plant material. An interesting point of note [43] is that living harvestmen often prefer soft-skinned arthropods. The collembolans at Rhynie (see below) would offer a potential source of softer prey. Given E. sheari's putative sister-group position to the suborder Cyphophthalmi, it is worth noting that modern cyphophthalmids have been observed feeding on either live or dead springtails [44]. More exotic food preferences seen in living harvestmen tend to be associated with derived clades. Thus, we would caution against inferring that E. sheari regularly consumed material like decaying vegetable matter, spores or fungi.

(iv) Arachnida incertae sedis

The final putative arachnid is Saccogulus seldeni Dunlop, Fayers, Hass & Kerp, 2006. Known only from a single specimen in thin section [13] (figure 1i), it resembles in some respects the prosoma of a spider in longitudinal section, but lacks unequivocal apomorphies of Araneae. The most remarkable feature is a long internal structure interpreted as a pharynx [13], with what looks like a dense brush of hairs or platelets forming a filtering system. Similar to Palaeocharinus above, this implies an animal using preoral digestion, but in the absence of further details about its morphology or affinities its ecology remains equivocal.

(c) Myriapoda

(i) Chilopoda

Centipedes (Chilopoda) were recorded from the Windyfield chert [7] as Crussolum sp. This genus is otherwise known from the Mid-Devonian of the USA and has been assigned to the order Scutigeromorpha, or house centipedes. The Windyfield material exhibits a distinctive cuticle type and includes isolated elements from the antennae, mouthparts and legs (figure 2a,b). A body length of about 35 mm was inferred [7]. Scutigeromorphs have a fossil record extending back to the late Silurian [45] and the presence of a forcipular apparatus in these early Palaeozoic forms, including those from Windyfield, implies biting jaws with poison glands as in living centipedes. Undheim et al. [46] noted that centipedes—and presumably also scorpions—are among the oldest terrestrial predatory arthropods to have evolved venom to subdue prey. These authors also remarked that scutigeromorph centipedes have rather delicate forcipules, largely used just to inject venom. More derived centipedes have increasingly robust mouthparts used for cutting and slicing as well. They also identified differences in venom components between scutigeromorph and scolopendromorph centipedes, which could imply a deep time split in centipede venom production and their associated feeding ecology. These Devonian fossils are in keeping with such a hypothesis.

Figure 2. Myriapods, hexapods and coprolites from Rhynie and Windyfield. (a,b) Limb elements of Crussolum sp. (Chilopoda: Scutigeromorpha). (c) Rhynimonstrum dunlopi Anderson & Trewin, 2003 (Arthropoda incertae sedis, possibly Diplopoda?). (d) Head capsule of Rhyniella preacursor Hirst & Maulik, 1926 (Hexapoda: Collembola). (e) Rhyniognatha hirsti Tillyard, 1928 (Hexapoda: affinities equivocal). (f) Leverhulmia mariae Anderson & Trewin, 2003 (probably Hexapoda). (g–i) Coprolites of the Lancifex Habgood, Hass & Kerp, 2004 type including amorphous (g) and spore-rich (h,i) contents.

Extant centipedes are invariably predators, leading to suggestions [7] that Crussolum may have been an active hunter in the Rhynie ecosystem. Scutigeromorphs can be characterized ecologically as fast-runners [47], preferentially searching for prey on the surface of the substrate. Extant scutigeromorphs retain large compound eyes while the more derived centipede orders show increasing trends towards leaf-litter and deeper soil habitats: for example shortening the legs and reducing, and finally losing, the eyes completely. Thus we can infer that Crussolum probably hunted largely on the substrate, rather than burrowing into it. Living scutigeromorphs have been reported to eat arachnids, springtails and various insects. A similar prey spectrum was available at Rhynie.

(ii) Myriapoda: Diplopoda

Rhynimonstrum dunlopi Anderson & Trewin, 2003 is based on enigmatic, but distinctive fragments of cuticle [7] which form annulate structures with specific patterns of pores (figure 2c). It was described as Arthropoda incertae sedis, but comparisons were made with the antennal articles of millipedes (Diplopoda). If this interpretation is correct it would imply an animal which was rather larger than the typical Rhynie arthropods. Millipedes today are almost always detritivores (see below), but in the absence of data confirming its affinities it is difficult to make further ecological inferences.

(d) Hexapoda

(i) Collembola

Rhynie hosts the oldest evidence for Hexapoda in the fossil record [14]. Springtails (Collembola) are represented by Rhyniella preacursor Hirst & Maulik, 1926 (figure 2d). The original descriptions [31] only recovered the head (including the mandibles) and thorax. Whalley & Jarzembowski [48] added an abdomen bearing the distinctive furcula (or spring) and the tube-like collophore on the underside. Rhyniella preacursor could thus be confirmed as an unequivocal, ca 1.5 mm long, collembolan. It had already evolved a mechanism to facilitate rapid jumps, which suggests the need to avoid predators the most likely candidates being trigonotarbids, harvestmen and centipedes (see above).

Modern collembolans largely feed on fungal hyphae and decaying plant material [49], both of which would have been abundantly available at Rhynie. A few species today are predators on animals like nematodes, rotifers and other springtails. Among previous comments on food preferences of the Rhynie species, Kevan et al. [4] suggested that the lack of a well-developed molar area in the mandibles implied dietary material that did not require mastication, such as soil microorganisms or spores. They noted plant juices as another possibility, but that R. praecursor lacks piercing mandibles that would have allowed them to create puncture wounds. However, the mandibles of R. praecursor have since been shown to possess a molar plate [50] (figure 2d). A diet requiring mastication, such as detritivory, is thus plausible although it should be mentioned that the molar regions do not directly oppose each other as would be expected for a chewing animal (Joachim Haug 2017, personal communication). Further information regarding the ecomorphology of collembolan mandibles is lacking, although some extant, carnivorous species possess asymmetrical, interlocking mandibles [49] which are absent in R. praecursor. In a discussion comment in Bernays et al. [51], Ed Jarzembowski posited through comparison with extant species that the long pretarsi of R. preacursor might have enabled locomotion on the surface of water, but this speculation has not been explored further.

Hopkin [49] commented on how similar R. preacursor is to living collembolan species. He also reviewed previous suggestions about its probable affinities, favouring the proposal of the extant family Isotomidae ([50] see also [52]). Both these studies highlighted the fact that Isotomidae possess a generalized morphology, and tend to be the dominant component within modern springtail communities. Extant members of the family are found in various litter, soil and moss habitats, and despite being more abundant in cold and damp conditions are found in a wide range of ecosystems from deserts to polar regions.

(ii) Rhyniognatha

Rhyniognatha hirsti Tillyard, 1928 is an enigmatic species [53] known from a single specimen, initially figured by Hirst & Maulik [31], preserving a pair of mandibles surrounded by harder-to-identify cuticular structures (figure 2e). Largely considered ‘insect like’ in the past, Engel & Grimaldi [14] proposed that the mouthparts were of a dicondylic nature—i.e. possessing two pivot points, a posterior condyle and anterior acetabulum—and triangular in form. From this they proposed that the animal as a whole was a pterygote (i.e. winged) insect. This tantalizing possibility has not been corroborated by any other specimens, but if correct it implies that non-insect hexapods (collembolans), apterygote insects, and some of the earliest pterygotes were all present on land by the Early Devonian. Molecular clock dating [54,55] corroborates the idea that ectognathous insects, and perhaps even pterygotes, had evolved by this point.

Irrespective of R. hirsti's affinities, the form of the mandibles—triangular, with well-differentiated incisor and molar areas—are fairly generalized and functionally comparable with those of numerous hemimetabolous insect groups (e.g. [56]), such as the Orthoptera and Blattodea. Kevan et al. [4] suggested that the mandibles are ‘reminiscent of the sharp bladed and toothed jaws of a carnivore’, while Engel & Grimaldi [14] posited that they belonged to a chewing organism, but noted that it was impossible to say whether the original diet comprised spores/pollen, leaf/stem tissue or small animals. We concur that it is impossible to reconstruct the diet of R. hirsti with any certainty, but note that the mandibles of carnivorous insects tend to have shearing cusps [57], while grasshoppers that feed on non-grass vegetation have a series of sharp pointed cusps. We tentatively suggest that R. hirsti is more like the latter condition, which hints at a non-carnivorous diet.

(iii) Leverhulmia

Leverhulmia mariae Anderson & Trewin, 2003 was initially described based on the difficult-to-study holotype specimen [7] (figure 2f), as a ca 12 mm long species assignable to Myriapoda incertae sedis. Fayers & Trewin [15] reported a new specimen, and further prepared the holotype. This revealed anterior (i.e. presumably thoracic) appendages, terminating in a pretarsus comprising a lateral pair of articulated ungues with a fixed median unguis between them. They also identified setae on the appendages as mechanoreceptors, with potential chemoreceptor structures too. In addition, they posited that the holotype represents an abdomen with a minimum of five clawless, segmented leglet pairs. Paired articulated lateral ungues imply dipluran or insect affinities, while the short median unguis is suggestive of the pretarsal arrangement of some Zygentoma (silverfish). The authors compared the abdominal leglets to the arrangement in Diplura, Zygentoma and Archaeognatha. In general, their segmented nature is suggestive of stem-, rather than crown-Hexapoda [58].

The same line of section in the holotype that makes the morphology of L. mariae challenging to interpret, however, does facilitate inferences about its ecology thanks to a significant length of gut infill [7,15]. This sinusoidal structure midway between the dorsal and ventral surface (figure 2f) contains fragments of various materials otherwise lacking in the host chert matrix, and runs the full preserved length of the fossil. The heterogenous material within this putative gut trace comprises ‘…macerated phytodebris, spores, amorphous organic matter and fragments of arthropod cuticle’ [15, p. 1121]. The authors reported that the phytodebris, while generally impossible to identify, contains rare examples of fragmented strands of xylem tissue. This is accompanied by scattered 50–80 µm embryophyte spores, fungal spore clusters found midway along the trace, and a minimum of two different types of arthropod cuticle, distinct from that of L. mariae. Of these, the first is found at one end of the gut trace, and comprises smooth, thick-walled cuticle in arcuate fragments. The other is found at the opposite end of the specimen, and consists of clustered almost-cylindrical fragments of smooth, thin-walled cuticle, one adorned by a short, socketed macroseta. Fayers & Trewin [15] identified a minimum bite size of 300 µm for this animal based on the size of the fragments, although no definitive mouthparts could be identified.

Anderson & Trewin [7] suggested (and we concur) that the gut contents indicate a detritivore, because the delineation of discrete packages of arthropod cuticle, macerated plant debris, spores and amorphous organic material suggests the animal was ingesting numerous food items in various states of decay. The gut contents are also comparable to the contents of coprolites found in deposits of this age, including Rhynie (see below), which are thought to originate from detritivores. Fayers & Trewin [15] also noted that extant Archaeognatha and Zygentoma are known to be detritivores. We further observe that while the plant and arthropod elements of the gut are fragmented, presumably through mastication, the same is not true of the spores [7] presumably due to their small size. Based on the published figures, the relatively low concentration of spores suggests they were probably not a primary food source.

(e) Plant–arthropod interactions and coprolites

A potential insight into palaeoecology is arthropod-mediated plant damage and other interactions. Labandeira [59] provided a comprehensive overview, which demonstrated that from sites of equivalent age to Rhynie there is evidence of generalized palynophagy, external foliage feeding, piercing and sucking, and borings. There has been relatively little work on plant damage at Rhynie. Kevan et al. [4] reviewed and reported on surface lesions inflicted during life in Rhynie plants, and differentiated three primary type of damage. The first was suggested to be the result of metazoan penetration of plant tissues: by implication a sap-sucking arthropod, although they noted [4] this need not be unambiguously animal-mediated damage. Two further damage types could be caused by animals or fungi. Rolfe [6] concurred that the causative agent of this damage was equivocal, and could have had arthropod, fungal or other pathogenic origins. This was supported by Chaloner et al. [25, p. 178], who concluded ‘…the possibility of a sap-feeding relation involving the arthropods is probably about as far as this evidence can take us at present’. In light of this, and based on the lack of unambiguous body fossils of plant-eating arthropods (see above), we see no convincing evidence at present for arthropod-mediated herbivory in the Rhynie ecosystem.

Coprolites offer an alternative insight into palaeoecology by recording the diet of extinct organisms. Habgood et al. [60] recorded three novel coprolite ichnogenera from Rhynie. Lancifex Habgood, Hass & Kerp, 2004 was proposed for elongate structures up to about 3 mm by 1 mm (figure 2g–i). These have a diverse content, a variable degree of particle size and content degradation, are dispersed rather than clustered, and can contain mineral grains. All this points towards a detritivore. Comparisons were drawn with modern millipedes and with the similar gut contents of Leverhulmia mariae (see above), albeit then not recognized as a hexapod. Rotundafaex Habgood, Hass & Kerp, 2004 was proposed for smaller, more rounded structures, about 250 µm in diameter. Like Lancifex, these contain amorphous organic matter, fungal spores and hyphae, and plant spores. Small detritivores such as collembolans, wingless insects or small millipedes were suggested as possible producers. Finally, Bacillafaex Habgood, Hass & Kerp, 2004 was proposed for small, ca 200 µm, rod-shaped structures with amorphous organic contents. Microherbivores (possibly mites) and trigonotarbids were suggested as producers. However, the body fossils of the known mites are only about twice the size of their putative coprolites, and we should note that modern spiders (and probably trigonotarbids) are liquid-feeders and tend not to produce solid, particulate faeces. Thus we feel that the producers of Bacillafaex remain equivocal.

There is also a general hypothesis [61] that spore-feeding predated herbivory in the fossil record. Kevan et al. [4] argued that spores may have been a significant element of some Rhynie arthropods' diet, and these animals may have been central to spore dispersal. Rolfe [6] mentioned that this was not his preferred scenario for the trigonotarbids. As noted above, spores are unequivocally present in the gut of Leverhulmia mariae and in several Rhynie coprolites (figure 2h,i). In assessing their significance, we should reiterate Habgood et al.'s comments [60] that there is no distinct class of spore-rich coprolites at Rhynie as would be expected from a dedicated spore-feeder, and that where spores are found in coprolites they show little degradation. This raises questions about whether the animal actually derived any nutrients from them, or whether they were just consumed (but not processed) among the general detritus. Spore-feeding has been cited as uncommon among modern arthropods [60], and the Rhynie spores have resilient walls which could presumably resist penetration and/or digestion (Charles Wellman 2017, personal communication). We would caution against inferring spore-feeding simply because an arthropod is discovered within, or close, to a sporangium, but acknowledge that there are spore-bearing coprolites known throughout the mid to late Palaeozoic (Andrew Scott 2017, personal communication see also [62]) which could be taken as evidence for diet.

4. Discussion

Suggested ecological relationships of the terrestrial Rhynie animals recorded to date are summarized in table 1. The only potential example of true herbivory, i.e. the consumption of living plant tissue, is the nematode. Several of the mites, as well as the collembolans, are strong candidates for having been mycophages since their living relatives have been regularly reported feeding on fungi. Potential generalist detritivores include the collembolans and Leverhulmia mariae, which may be a stem-insect (or a crown-insect potentially related to bristletails or silverfish) and uniquely reveals its diet through its gut contents. Predators at Rhynie were presumably the venomous centipede, the trigonotarbid arachnids, and the harvestman the last may also have scavenged opportunistically on decaying organic matter. The mites also include groups which are known today to prey on nematodes, or at least use them to supplement their diet. These inferred food preferences are consistent with a simple, arthropod-dominated ecosystem with mycophages and/or detritivores as primary consumers—a mode of life reflected in the contents of the coprolites—and several predators at higher trophic levels. Top predators may have been the centipede and trigonotarbids, with body lengths of ca 35 and up to 14 mm, respectively. Arthropod herbivory appears to have been absent. To explain this, we might speculate that being a mycophage requires less specialization than being a herbivore because fungal tissue lacks cellulose and so is easier to digest. Furthermore, if herbivory is defined as animals biting off pieces of green plants, chewing them and then swallowing them, then it seems likely that all herbivores use a gut flora (fungi, bacteria, etc.) for digestion. For this reason it has been proposed [1,63] that herbivory effectively arose from detritivory by internalizing the decomposition process. Essentially, herbivores are merely farming their gut biota by feeding it pieces of chewed plant material.

Table 1. Summary of the terrestrial arthropod fauna of the Rhynie and Windyfield chert ecosystems, and their potential ecologies.

Rhynie thus appears viable as a terrestrial ecosystem in that the animals recorded so far all have a potential source of food (fungi, detritus, other animals). Yet as with any fossil system, we do not know if all of the original assemblage has been preserved. Missing taxa at a given time period can be inferred from dated phylogenies, but as noted by Chang & James [64] for a terrestrial example (earthworms), if there is no fossil record there is nothing to calibrate molecular clocks against and estimates of cladogenesis here are likely to be inaccurate. Are significant components of the original Rhynie fauna missing? The locality is highly unusual in its chert-based preservation via siliceous sinters deposited in a hot springs environment, and is particularly well-suited to conserving small terrestrial arthropods. It is not coincidental that Rhynie hosts the oldest records of hexapods, mites and harvestmen, and potentially the oldest nematode worms as well. These are small animals, and if other microarthropods or worms were present at Rhynie there is nothing per se to suggest that they could not be preserved. Additional groups may yet be found by processing more material. If we look at the wider picture of Devonian terrestrial arthropods there are notable absences. Scorpions are known since the Silurian, and while there is debate in the literature about whether the earliest forms were aquatic or terrestrial, they might be predicted in the Rhynie ecosystem. Another group which one would expect are millipedes. Silurian body fossils with evidence for air-breathing have been described (although new data suggest these millipedes may actually be Devonian [65] and contemporary in age with Rhynie). Millipedes are only hinted at here at Rhynie through interpretations of Rhynimonstrum dunlopi fragments and some of the coprolites.

The excellent preservation, and the presence of internal organs or gut contents in some cases, suggests the Rhynie animals were killed rather rapidly (see also [66]). Yet some arthropod fragments are disarticulated, which in turn implies a degree of post-mortem transport, or at least decay. We favour a scenario in which the terrestrial arthropod community lived on the outwash aprons of hot springs and was periodically inundated by floods of hot, silica-rich water which eventually precipitated out into the cherts. The land plants could have had a sieve-like effect, trapping arthropod remains between the in situ stems. In this scenario, we might ask whether the recovered fauna shows a bias towards smaller (slower?) animals. Were larger arthropods, perhaps a few centimetres long, more easily able to escape the inundations? Parallels could be drawn with amber where larger inclusions are a rarity and most of the arthropod fossils are quite small. However, animals like the ‘missing’ millipedes are generally quite slow-moving creatures and this hypothesis does not explain why we do not see juveniles of larger arthropods.

Trewin et al. [66] used hot springs at the Yellowstone National Park of the USA as a modern analogue to understand preservation at Rhynie. Channing & Edwards [67,68] also compared the Rhynie flora to that of Yellowstone, and to other modern and fossil hot spring communities. They argued that the Rhynie plants growing on the fringes of the hot springs were physiologically specialized (typically halophytes) and noted that Rhynie plant genera are hardly ever recorded from contemporary localities elsewhere. Among the terrestrial arthropods, only the centipede Crussolum occurs outside of Rhynie. Thus from an animal perspective, was Rhynie a typical early Devonian habitat, or a specialized ecosystem hosting a more limited spectrum of (endemic?) animals compared to the wider palaeoenvironment? Comparative studies of terrestrial arthropod communities on the margins of modern hot springs ecosystems are rare. The published literature tends to focus on aquatic species (e.g. insect larvae, crustaceans, water mites) which may be of note for being thermophilous.

One study [69] collected spiders, mites and collembolans—along with various derived insect orders—from pitfall trips set close to modern hot springs in New Zealand. Details of the taxa they found are lacking, but they noted that there were fewer invertebrates found close to the hot springs themselves, probably due to the lack of diverse habitats on the sinter terraces, and that some of the scavenging arthropods appeared to raid these terraces in search of carcasses of dead animals. A more detailed account of the soil fauna around hot springs in Iceland was provided by Tuxen [70]. Records included several taxa which would not be expected at Rhynie as they are not known elsewhere from the Devonian, e.g. parasitiform mites, several groups of derived pterygote insects and terrestrial woodlice. He also recorded soft-bodied taxa, such as earthworms and gastropods which could, theoretically, occur at Rhynie but have not been found so far. Additionally, there are several interesting parallels with Rhynie. The Icelandic hot springs margins also hosted cursorial spiders (analogous to trigonotarbids), oribatid mites (which may have evolved from endostigmatids), harvestmen, centipedes, millipedes and springtails the last including the genus Isotoma which is similar to the fossil species. Thus the terrestrial animals found at Rhynie are consistent with at least part of the fauna surrounding hot springs today for which data are available. To conclude, we would also note that Tuxen [70] found a gradation of taxa from those which occurred exclusively near hot springs through to more widely distributed taxa which happened to be more common in this habitat. That faunas can grade over quite short distances from exclusive hot springs animals to generalists in the surrounding ecosystem introduces uncertainty. How it impacts on the potential uniqueness of the Rhynie fauna—as per hypotheses about the flora (see above)—is unclear. We lack contemporary localities in the Old Red Sandstone with the same quality of preservation which could yield microarthropods and allow us to test whether Rhynie was sampling part of a more widespread Early Devonian terrestrial ecosystem.

Note added in proof

Haug & Haug (2017) recently proposed that the putative insect Rhyniognatha hirsti could in fact be a myriapod, possibly a centipede [71].

Insect Freefall: What Does It Mean for Birds?

Many people quip that they'd prefer a world without “bugs,” but as the adage goes: Be careful what you wish for. Our planet cannot function normally without insects and other invertebrates. “The little things that run the world” is what biodiversity pioneer Edward O. Wilson calls them. Insects anchor natural systems and provide invaluable natural services, as pollinators, scavengers, predators, and protein-packed prey that sustains many birds, fish, amphibians, reptiles, and mammals, in virtually all terrestrial ecosystems.

Yet insect diversity and abundance are plummeting in many places.

This leaves conservationists scrambling to find out why…and wondering what insect declines mean for other wildlife.

“We are seeing declines in abundance, diversity, and biomass of insects,” says Scott Hoffman Black, Executive Director of The Xerces Society for Invertebrate Conservation, “and by extension we have to assume that this is, and will be, impacting birds.”

Great Crested Flycatcher. Photo by Joe McDonald/Shutterstock.

The clues in this mystery include large-scale disappearance of insects, dipping bird populations, and a line-up of potential culprits including pesticides, habitat loss, and climate change. What's likely on the horizon is a choice: Do we ignore insect declines to our detriment, or change some of our most destructive day-to-day routines, which seem to be modifying our world into a more sterile place?

Vanishing Insects, Mounting Concern

Although there are not many long-term studies on insects in North America, the studies done have revealed sharp declines in certain species, including Monarch butterflies and Rusty-patched Bumble Bees, and even entire groups. “In Ohio, a study found a 33-percent reduction in abundance of butterflies over 21 years,” says Black, “and a long-term 45-year-long study with transects across California is finding declines at all sites and of all different butterfly groups.”

A study out of Germany has raised even more eyebrows, standing out for its longevity, the many sites monitored, and the focus on sampling all flying insects.

This wide-ranging, long-term investigation, published in the online journal PLOS ONE in 2017, spans 27 years of collections — and shows a more-than-75-percent decline in the flying insect biomass at 63 protected areas. The investigators used Malaise traps, tent-like contraptions that channel flying insects between fine mesh panels to a collection container. Collections made at these traps, basically masses of flying insects of many types, were weighed to gauge biomass for each reserve over different years.

In the Netherlands, rapid declines in Barn Swallows and other insect-eating farmland birds were attributed to a "depletion of the birds' food — insects." Photo by Bildagentur Zoonar GmbH/Shutterstock.

The authors write: “Our results demonstrate that recently reported declines in several taxa such as butterflies, wild bees, and moths, are in parallel with a severe loss of total aerial insect biomass, suggesting that it is not only the vulnerable species, but the flying insect community as a whole, that has been decimated over the last few decades.”

By just looking at collection jars on the shelves, the investigators could see the winnowing of insect populations. Caspar A. Hallmann, an entomologist at Radboud University Nijmegen in the Netherlands and one of the study's authors, says: “In the early 90's, you would fill a jar of one liter in about a week, requiring a more rapid replacement of jars at shorter time intervals…or they would overflow. In recent years, collectors of the Krefeld Entomological Society have used quarter-liter jars, which would not fill even after two weeks of trapping. It is really apparent.”

Silence in the Forest

Dropping insect populations impact many birds. Excepting seabirds, 96 percent of North American bird species feed insects to their young. Caterpillars alone are an important food source for at least 310 North American bird species.

Strictly insectivorous species seem to be particularly hard hit. For example, the Eastern Whip-poor-will's incessant namesake cries no longer permeate woodlands in many parts of its mapped range. After analyzing stable isotope signatures in Eastern Whip-poor-will museum specimens from Ontario, researchers wrote in the journal Frontiers in Ecology and Evolution in 2018: “For aerial insectivores, a significant change in dietary isotopes of whip-poor-wills over the past 130 years adds to the mounting evidence that population declines for many of these species may be related to changes in food supply.”

Cerulean Warbler with caterpillar. Photo by Ray Hennessy/Shutterstock.

The researchers suggest that their findings could reflect the harmful effects of a decline in large night-flying moths, beetles, and other insects that left whip-poor-wills feeding at a lower trophic level, or stage in their ecosystem's food chain. The drop in prey size, diversity, and abundance may stifle whip-poor-will reproduction. More study is required to know if whip-poor-wills' and other aerial insectivores' declines are linked to pesticides, habitat loss and degradation, light pollution, or other factors.

Direct consumption aside, insects are critically important to birds in other ways. More than 85 percent of flowering plants require animal pollination in most cases this job is done by insects. “If you start to lose those pollinators,” says the Xerces Society's Black, “this also impacts many plants, and by extension birds. Many birds, for example, eat small fruits or seeds. A lot of these are from insect-pollinated plants.”

READ MORE: Chickadee Study Highlights Native Plant Benefits

Insects and spiders make up about 90 percent of the Carolina Chickadee's diet during the nesting season, and about 40 percent at other times. Researchers recently found that these nonmigratory songbirds struggled to raise their young in residential yards near and within Washington, D.C., that were packed with exotic landscaping because, due to the nonnative plants' “evolutionary novelty,” they attracted far fewer caterpillars and other insects than did native species. Biologists Desirée Narango, Douglas Tallamy, and Peter Marra wrote: “Our work demonstrates that even a common ‘urban-adapted' bird species is food-limited when nonnative plants dominate landscapes….”

“If we think about it,” says Narango, “landscaping with nonnative plants is one part of this issue of insect declines. We're making our residential areas food deserts full of plants that herbivorous insects can't eat, which means fewer insects…and less food for insectivorous birds.”

Working with homeowners as part of the citizen science program Smithsonian Neighborhood Nestwatch, Narango, Tallamy, and Marra found that areas with less than 70 percent native-plant biomass were a “dead end for insectivorous birds,” where chickadees had “lower reproductive success and unsustainable population growth…,” sometimes foregoing reproduction altogether.

A Toxic Shadow

Many scientists believe agricultural alchemy plays a big role in insect declines. Pesticides cast a broad yet invisible shadow over huge swaths of land, often well beyond areas they are meant to treat.

Pesticides and intensive farming loom large as culprits in the stark drop in France's farmland avifauna, where populations of farm-nesting species dipped by, on average, a third over the past 15 years. Partridges, Meadow Pipits, and others suffered far steeper declines in that time.

Eastern Bluebird with insect snack. Photo by Benjamin Klinger/Shutterstock.

In recent decades, 24 of 39 farmland bird species have declined in agricultural habitat encompassing 45 percent of the European Union's land. Even generalist species such as the Common Woodpigeon seem to fare better in cities than on farms, and bees as well. Farm pesticides' effects on nontarget species are suspected to be a principal cause.

Today, the world's most widely used agricultural pesticides are neonicotinoids, neurotoxins absorbed and stored in plant tissues so they repel insect pests. Neonics, as they are also known, are now banned in the European Union because of their impact on honeybees. A number of U.S. states have also introduced legislation attempting to prohibit or limit their use.

Persistent and water-soluble, neonics are highly toxic to a broad range of insects and other invertebrates. And they reach well beyond farm fields, leaching into watersheds, rising up in dust, and soaking into soil adjacent to farms, toxifying insect food plants there.

Neonics' impact on nontarget terrestrial and aquatic insects is formidable, and long-lasting as well. Depending upon soil and other factors, neonics have half-lives — the time it takes to reduce an amount of the pesticide by half — of up to 1,000 days, or nearly three years.

Although these insecticides are considered less dangerous to many vertebrates than are other pesticides, an ABC study in 2013 determined that a single neonic-coated seed can kill a bird the size of a Blue Jay.

Proving a direct link between bird declines and pesticides' impact on their insect food is difficult given other factors also at play, including habitat loss, climate change, and the direct physical effects of the chemicals on birds. But a study published in the journal Nature in 2014 was the first to “provide direct evidence that the widespread depletion of insect populations by neonicotinoids has knock-on effects on vertebrates.” In that study, in the Netherlands, rapid declines in Eurasian Skylarks, Barn Swallows, Western Yellow Wagtails, and other insect-eating farmland birds were attributed not to direct effects of the chemicals on the birds, but likely “the result of a depletion of the birds' food — insects.”

Scissor-tailed Flycatcher eating grasshopper. Photo by Brian Lasenby/Shutterstock.

Given their widespread use — for example, most corn-growing acreage in the United States is treated with neonics — these pesticides require much more research, and controls or bans. A recent review of existing research, published in Environmental Science and Pollution Research International, reports: “Correlational studies have suggested a link between neonicotinoid usage in agricultural areas and population metrics for butterflies, bees, and insectivorous birds in three different countries.”

Other potent chemical threats persist. For more than 50 years, the organophosphate chlorpyrifos has been sprayed on apples and other fruits, vegetables, nuts, and other crops. Related to sarin gas, chlorpyrifos is among the most toxic pesticides to reach aquatic ecosystems. It is a threat not only to birds that ingest it, but also to their insect food base.

ABC and other groups are calling for a ban on the use of chlorpyrifos. Environmental Protection Agency (EPA) scientists agreed that there is no way to use the pesticide safely, and the agency was on course to ban it in spring 2017. But EPA reversed course, extending its use for five years. In July, EPA rejected a challenge by a coalition of environmental and public health advocacy groups that urged the agency to ban the pesticide. ABC and others continue to advocate for legislation prohibiting chlorpyrifos' use. Meanwhile, states are taking action: California, Hawai‘i, and New York have initiated bans, and a few other states may soon follow.

This Land Is Your Land, This Land Is Wild Land

Most of the German protected areas covered in the landmark 2017 PLOS ONE study are small holdings surrounded by farmland, leading the investigators to suspect physical and chemical factors at play. “Agricultural intensification, including use of pesticides, is as far as I am concerned one of the prime suspects responsible for the insect decline,” says Hallmann.

Around the world, much of the goliath human footprint on the land comes in the form of cropland agriculture, grazing land, as well as both carefully and haphazardly logged forest. On these working lands, decisions on where habitat is cleared, whether or not it's managed with native plants, and where and how pesticides are applied will have a huge impact on insects and thus the future of birds.

Recent investigations provide a window into how, with more knowledge and the will to coexist, people can both work the land and maintain a higher diversity of insects, birds, and other wildlife. A 2019 PLOS ONE study conducted in Finland, for example, reveals that of all farm types in Europe, organic livestock farming is the only one to significantly boost populations of insectivorous and migratory birds, including swallows. The livestock and their dung draw insects rotational grazing improves habitat health and plant diversity and the semi-wild state of chemical-free pastures most closely matches untouched grassland, which is a very rare commodity in Europe these days.

Land use involving at least a partial tree canopy can be very beneficial to insects and birds, if native species are used. A study published this year in the journal Biotropica examined native canopy trees on shade-coffee farms in Nicaragua and Colombia. Researchers found that insectivorous birds favored certain species planted to provide shade for coffee plants, likely because they harbor the highest insect abundance and diversity. These preferred native trees include some in the legume family Fabaceae, such as Guanacaste and Guamo, which also benefit farmers by fixing nitrogen in the soil. Armed with this study's results, farmers can make simple choices that benefit their farms, insects, and birds.

Guanacaste tree. Photo by Raymond Pauly/Shutterstock.

Another recent study, published in the journal Wildlife Biology in January, focused on another native tree, the Andean Alder. In Colombian alder plantations, researchers found more insectivorous birds than in regenerating natural forest of about the same age (35 to 40 years). Comparing insect-eating bird abundance and diversity in both habitats, the team found that fly-catching and foliage-gleaning birds abounded in the alders, where greater sunlight penetration resulted in lush undergrowth supporting an insect bounty. The Black-capped Tyrannulet, Plushcap, and 15 other bird species were found in the alders, but not in nearby secondary forest, although with its greater diversity of trees and tree sizes, that habitat drew tree-trunk foragers not attracted to the alders, including the Powerful Woodpecker and Black-banded Woodcreeper.

In the end, the authors did not consider alder plantations replacements for natural forests, but rather complements to them. The differing habitats and their birds raise interesting questions about “green” land uses that offer significant benefits to insects and birds.

Rainforest in the Hot Seat

Climate change is very likely detrimental to insects and insectivores, including birds. Reported in Proceedings of the National Academy of Sciences in 2018, one study looked at the biomass of arthropods — invertebrates including insects, spiders, and centipedes — in Puerto Rico's largest remaining rainforest, El Luquillo.

The researchers also surveyed populations of insect-eating Anolis lizards, frogs, and birds, comparing what they found with prior survey results from the 1970s. These comparisons were made with a troubling backdrop: Over the 30-plus years between surveys at El Luquillo, the average maximum temperature in the forest there rose 3.6 degrees Fahrenheit.

The study notes a drop in arthropod biomass of between four- and eight-fold since the 1970s, with parallel declines in Anolis lizards, frogs, and birds. This includes a 90-percent drop in mist-net captures of the endemic Puerto Rican Tody — a tiny green, white, and red bird that can snap up about 40 percent of its weight in insects in a day.

Puerto Rican Tody. Photo by Falko Duesterhoeft/Shutterstock.

More study is required in El Luquillo — which was subsequently hammered by Hurricane Maria in 2017 — and other parts of the tropics, but the study's authors wrote: “Our analyses provide strong support for the hypothesis that climate warming has been a major factor driving reductions in arthropod abundances, and that these declines have in turn precipitated decreases in forest insectivores in a classic bottom-up cascade.” They added that the same scenario is likely playing out in other tropical forests experiencing significant increases in ambient temperature.

No Birds Without “Bugs”

From farm fields to alder plantations to remaining rainforest, conservationists now ponder, with a sense of urgency, human activities' unintended and intentional impacts on insects and birds.

Although many people are just now awakening to the ecological importance of insects, those who valued them all along are rolling up their sleeves to learn more and do more to conserve them.

“The authors of the German study were very fortunate to have started data collection decades ago,” says the Xerces Society's Black. “This study has got many other researchers thinking about implementing long-term monitoring.” But Black and others say that while further studies are important, so is immediate action to stem the loss of diversity and abundance of insects and other wildlife.

Exactly what insect declines mean for birds, and for us, is an emerging picture, but the todies, skylarks, and whip-poor-wills seem to tell us something we may have taken for granted before: There can't be birds without “bugs.”

Moth Biodiversity Changes Are Spatially and Taxonomically Heterogeneous

Great heterogeneity in moth trends exists geographically and taxonomically, yielding a complex picture that cautions against ambitious extrapolation and generalization. There are also methodological and statistical issues that affect the reliability of trend estimates for moths and other insects (10).

Spatially, moth trends vary at continental, regional, and even local scales, which suggests that different stressors are in play. While patterns of moth decline are prevalent across western, central, and northern European countries (17, 21 ⇓ ⇓ –24, 26), typically, there is considerable within-country heterogeneity. Conrad et al. (17) found that total abundance of macrolepidopterans decreased significantly (by 44%) in the southern half of GB over the period 1968–2002, but showed no overall change in the northern half. The median species trend at southern monitoring stations was a decrease three times greater than that at northern sites.

Regional assessments in the New World also provide evidence of spatial heterogeneity in trends. In the Missouri Ozarks, United States, a region with only modest human impacts, caterpillar numbers on oaks have fluctuated markedly over the past 20 y, but there is no signal of overall decline (27). Likewise, caterpillar collections from southeastern Arizona, United States, show great interannual fluctuation, but little evidence of decreasing abundances (data presented below). In the tropics, time-series data on moths are scarce, but two recent assessments from Central America reported greatly diminished macrolepidopteran diversity and abundance in Costa Rica (28, 29). Our data below for cloud forest caterpillars in Ecuador show no change.

Even in studies showing clear overall declines, some fraction of the moth fauna is increasing (e.g., refs. 17 and 21 and GB case study below). We are unaware of an instance where all lineages are in collapse, a signal that would implicate stressor(s) acting uniformly across families of Lepidoptera.

The assemblage-level signals described above are blind to taxonomy, lumping stenotopic species with generalists, and do not account for the colonizations of new taxa in response to land use and climate changes. Steep declines in the autochthonous fauna of a defined area could be masked by population increases of recent arrivals for example, boreal communities undergoing long-term climate warming could be particularly susceptible to such processes.

The different metrics used to measure change can contribute additional complexity to our understanding of moth biodiversity trends. A long-term study in Hungary found significant reductions in alpha and beta diversity (indicating biotic homogenization of moth communities), but no decline in total abundance (22). In GB, imputed biomass of macrolepidopterans increased, whereas abundance decreased (18, 20). Of course, temporal changes in metrics used to assess changes in biotic communities, such as species richness, biomass, and trends in abundance and occupancy, can differ for many reasons. These could be spurious, stemming from differences in measurement, scale, or time lags, or they could reflect real discrepancies driven by differential responses of individual species, with the identities of winners and losers being due to different traits, demographics, and spatial distributions, generating conflicting signals at the assemblage level (30).

Animal Diversity Web

The Himalayan water shrew ( Chimarrogale himalayica ) has a wide but sporadic distribution across the Himalayas, including: Northern India, Nepal, Laos, Myanmar, Vietnam, southern China, and Taiwan. This species is sometimes confused with other water shrew species, such as the Japanese water shrew (Chimarrogale platycephalus) and the elegant water shrew (Nectogale elegans), due to similar habitats, appearances, and taxonomy. Three subspecies have been distinguished within C. himalayica : C. h. himalayica found in the Himalayas C. h. varennei found in Myanmar, Yunnan, China, Laos, and Vietnam and C. h. leander found in Fujian, China and Taiwan. (Yuan, et al., 2013)


This semi-aquatic species is associated with clear, swift-flowing streams in forested mountainous regions. Himalayan water shrew can be found in lowland habitats at elevations of 220-250 m, and in pre-montane conifer/broad-leafed evergreen forests with an elevation range of 250-1270 m, although specimens have been collected at elevations as high as 3048 m. The depth range of this species is unknown but previous capture efforts focused on a 0.5 m deep stream. The wide distribution of Himalayan water shrew is attributed to a broad adaptive capacity and dispersal ability that is associated with an aquatic lifestyle. (He, et al., 2010 Jenkins, 2013 Lunde and Musser, 2002)

  • Habitat Regions
  • temperate
  • terrestrial
  • freshwater
  • Terrestrial Biomes
  • forest
  • mountains
  • Aquatic Biomes
  • rivers and streams
  • Range elevation 220 to 3048 m 721.78 to 10000.00 ft
  • Average depth 0.5 m 1.64 ft

Physical Description

Himalayan water shrew are small, semiaquatic mammal. Few individuals have been captured. Those few possessed a head-body length range of 111 - 132 mm, with an average of 121 mm, and a tail length of 79 - 88 mm, with an average of 85 mm. The tail is slender but densely haired. Adult Himalayan water shrews weigh between 37 - 56 g. There is no sexual dimorphism in appearance or size. Pelage is flat and dense, and grey-brown in colour with no dorsal-ventral colour boundaries. Skull and facial features include: relatively flat snout, small pinnae, and long, brown whiskers, and the brain cavity volume is large in comparison to other shrews. The greatest length of skull is between 25 – 28 millimeters, possessing 3 upper unicuspid teeth (upper incisors lack cusps). (Francis, 2008 Li and Boren, 1999 Lunde and Musser, 2002 Sterndale, 1884)

This shrew is historically placed in the subfamily Crocidurinae based on presence of white tipped teeth. However, ultraviolet rays have revealed red coloured teeth tips, and karyotyping evidence has suggested this shrew be placed in subfamily Soricinae. There are three recognized subspecies of C. himalayica , as previously mentioned, but new evidence strongly supports these subspecies as paraphyletic, and suggests they should be considered as individual species. (Mōri, et al., 1991 Yuan, et al., 2013)

Metabolic rates have not been quantified but are known to be particularly high within aquatic soricines this high metabolic rate is thought to be an adaptation for the high-energy expenditure associated with diving and foraging in cold water. (He, et al., 2010)

  • Other Physical Features
  • endothermic
  • homoiothermic
  • bilateral symmetry
  • Sexual Dimorphism
  • sexes alike
  • Range mass 37 to 56 g 1.30 to 1.97 oz
  • Range length 190 to 220 mm 7.48 to 8.66 in


Little is known about the mating systems of Himalayan water shrews.

Breeding occurs in May, producing a litter of five to seven offspring. Offspring are kept in a small chamber constructed in a riverbank, which usually has several openings, and one of these openings will typically be underwater. (Sterndale, 1884)

  • Key Reproductive Features
  • seasonal breeding
  • gonochoric/gonochoristic/dioecious (sexes separate)
  • sexual
  • viviparous
  • Breeding season May
  • Range number of offspring 5 to 7

Little is known about the parental investment provided by the Himalayan water shrew. As previously mentioned, there are several offspring per litter, and the young are housed in a chamber in a riverbank. (Sterndale, 1884)


Himalayan water shrews have never been kept in the captivity, and little is known about their lifespan, but they are assumed to be short-lived. (Li and Boren, 1999)


Little is known about the behavior of the Himalayan water shrew other than it is an semi-aquatic mammal associated with clear, swift-flowing streams and rivers. (Sterndale, 1884 Yuan, et al., 2013)

Home Range

Home range and territory size of individuals is unknown.

Communication and Perception

Little is known about the communication and interactions of Himalayan water shrews. The presence of long whiskers suggests reliance on tactile senses. (Sterndale, 1884)

Food Habits

Due to high metabolic demands, soricines consume as much as three times their body weight in 24 hours and can survive only a few hours without feeding.

Himalayan water shrews are insectivores. Samples of the stomach contents of C. himalayica show consumption of only insects and spiders, such as fishing spiders (Family Pisauridae). There is no evidence they consume crustaceans or fish, a common food source to other species of water shrews. (He, et al., 2010 Lunde and Musser, 2002)

  • Primary Diet
  • carnivore
    • insectivore
    • eats non-insect arthropods
    • Animal Foods
    • insects
    • terrestrial non-insect arthropods


    Little is known about predation on this species, and no predators have been identified.

    Ecosystem Roles

    C. himalayica are thought to be ecological competitors with Nectogale elegans, another water shrew of similar body size, that shares a similar habitat of swift-flowing streams in forested mountainous regions. Both are found in high altitudes (up to 3048 m), with large altitude ranges (>2000 m). These two species have never been documented to coexistence, suggesting direct ecological competition.

    Himalayan water shrews are commonly infected with the larvae of Gnathostoma nipponicum . Infection rates in the wild have been documented at >70% of the population. (Jenkins, 2013 Oyamada, et al., 1996)

    Economic Importance for Humans: Positive

    C. himalayica is harvested for local medicinal uses. (Molur, 2008)

    Economic Importance for Humans: Negative

    Although the primary habitat of this species is swift flowing streams and rivers, individuals have occasionally been found in ditches, fields, houses and barns. This presence may lead humans to perceive the Himalayan water shrew as a pest. (Sterndale, 1884)

    Conservation Status

    C. himalayica is classified by the IUCN as a species of least concern which is justified by wide geographic distribution (>2000 km), presumed large population size and the unlikeliness of a rapid population decline. Identified threats to this species include: habitat lost due to agricultural expansion and logging, harvesting for medicinal use, pest control methods, and a decline in prey species. The effects of rain forest logging on the Southeast Asian population of C. himalayica have been investigated, but these studies have been inconclusive, due to the elusiveness of this species. (Jenkins, 2013 Molur, 2008 Wells, et al., 2007)

    • IUCN Red List Least Concern
    • US Federal List No special status
    • CITES No special status
    • State of Michigan List No special status


    Kirsten Solmundson (author), University of Manitoba, Jane Waterman (editor), University of Manitoba, Tanya Dewey (editor), University of Michigan-Ann Arbor.


    living in the northern part of the Old World. In otherwords, Europe and Asia and northern Africa.

    having body symmetry such that the animal can be divided in one plane into two mirror-image halves. Animals with bilateral symmetry have dorsal and ventral sides, as well as anterior and posterior ends. Synapomorphy of the Bilateria.

    an animal that mainly eats meat

    uses smells or other chemicals to communicate

    a substance used for the diagnosis, cure, mitigation, treatment, or prevention of disease

    animals that use metabolically generated heat to regulate body temperature independently of ambient temperature. Endothermy is a synapomorphy of the Mammalia, although it may have arisen in a (now extinct) synapsid ancestor the fossil record does not distinguish these possibilities. Convergent in birds.

    forest biomes are dominated by trees, otherwise forest biomes can vary widely in amount of precipitation and seasonality.

    mainly lives in water that is not salty.

    An animal that eats mainly insects or spiders.

    having the capacity to move from one place to another.

    This terrestrial biome includes summits of high mountains, either without vegetation or covered by low, tundra-like vegetation.

    the area in which the animal is naturally found, the region in which it is endemic.

    found in the oriental region of the world. In other words, India and southeast Asia.

    breeding is confined to a particular season

    reproduction that includes combining the genetic contribution of two individuals, a male and a female

    uses touch to communicate

    that region of the Earth between 23.5 degrees North and 60 degrees North (between the Tropic of Cancer and the Arctic Circle) and between 23.5 degrees South and 60 degrees South (between the Tropic of Capricorn and the Antarctic Circle).

    reproduction in which fertilization and development take place within the female body and the developing embryo derives nourishment from the female.


    He, K., Y. Li, M. Brandley, L. Lin, Y. Wang, Y. Zhang, X. Jiang. 2010. A multi-locus phylogeny of Nectogalini shrews and influences of the paleoclimate on specialization and evolution. Molecular Phylogenetics and Evolution , 56/2: 734-746.

    Ichikawa, A., H. Nakamura, T. Yoshida. 2005. Mark-recapture analysis of the Japanese water shrew Chimarrogale platycephala in the Fujisawa Stream, a tributary of the Tenryu River, central Japan. Mammal Study , 30: 139-143.

    Jenkins, P. 2013. An account of the himalayan mountain soricid community, with the description of a new species of Crocidura (Mammalia: Soriciomorpha: Soricidae). The Raffles Bulletin of Zoology , 29: 161-175.

    Jones, G., R. Mumford. 1971. Chimarrogale from Taiwan. Journal of Mammalogy , 52/1: 228-232.

    Li, Y., J. Boren. 1999. Population Distribution, Habitat Usage, and Conservational Strategy of Himalayan water shrew (Chimarrgogale himalayica) in Taiwan . Tokay University, Republic of China: Council of Agriculture Forest Service. [Translated].

    Lunde, D., G. Musser. 2002. The capture of the Himalayan water shrew (Chimarrogale himalayica) in Vietnam. Mammal Study , 27: 137-140.

    Molur, S. 2008. "Chimarrogale himalayica" (On-line). The IUCN Red List of Threatened Species. Accessed November 01, 2015 at

    Mōri, T., S. Arai, S. Shiraishi, T. Uchida. 1991. Ultrastructural observations on spermatozoa of the soricidae, with special attention to a subfamily revision of the japanese water shrew Chimarrogale himalayica. Journal of the Mammalogical Society of Japan , 16: 1-12.

    Oyamada, T., H. Kobayashi, T. Kindou, N. Kudo, H. Yoshikawa, T. Yoshikawa. 1996. Discovery of mammalian hosts to Gnathostoma nipponicum larvae and the prevalence of the larvae in rodents and insectivores. Journal of Veterinary Medicine Science , 58/9: 839-843.

    Sterndale, R. 1884. Natural History of the Mammalia of India and Ceylon . Calcutta: Thacker, Spink. Accessed December 03, 2015 at

    Wells, K., E. Kalko, M. Lakim, M. Pfeiffer. 2007. Effects of rain forest logging on species richness and assemblage composition of small mammals in Southeast Asia. Journal of Biogeography , 34: 1087-1099.

    Yuan, S., X. Jiang, Z. Li, K. He, M. Harada, T. Oshida, L. Lin. 2013. A mitochondrial phylogeny and biogeographical scenario for asiatic water shrews of the genus Chimarrogale: implications for taxonomy and low-latidue migration routes. PLoS ONE , 8/10: 1-15.


    Phylogenetic analyses of sciomyzid DNA sequences provided strong support that the Sciomyzini, Tetanocerini and Tetanocera are monophyletic (Figure 1). We significantly estimated that (i) the ancestor of the Sciomyzidae was terrestrial (Figures 2, 3), (ii) there was a single terrestrial-to-aquatic transition early in the evolution of the Tetanocerini and, subsequently, (iii) there were at least 10 independent aquatic-to-terrestrial transitions and at least 15 transitions in feeding behaviors (Figures 2, 3, 4, Additional file 1: Figure S2). The 10:1 ratio of aquatic-to-terrestrial vs. terrestrial-to-aquatic transitions goes against the general trend observed in animals. We found that the ancestor to Tetanocera was aquatic and five Tetanocera lineages made independent aquatic-to-terrestrial transitions and seven independent transitions in feeding behaviors (Figures 2, 3, Additional file 1: Figure S2). Classifications of sciomyzids into ecological assemblages of species resulted in many non-monophyletic groupings (Figures 2, 3, 4, Additional file 1: Figure S2, Additional file 1: Figure S3) whose monophyly were rejected via phylogenetic constraint analyses (Table 2). Therefore, these findings strongly support our inferences of multiple independent transitions in feeding behaviors, habitats and prey/host usage. The damp shoreline habitat is likely a crucial transitional habitat where tetanocerine lineages that move out of the water to forage can find the same prey taxa as in the water. Once tetanocerine lineages are established on the shoreline, terrestrial molluscan taxa are available as potential food resources. From a morphological standpoint, transitioning from aquatic to terrestrial habitats is easier than the reverse, as adaptations to air-breathing just below the surface of the water are more difficult to gain than to lose. Furthermore, tetanocerine phylogenesis occurred as the Earth was going through a general drying period. These factors likely explain why so many tetanocerine lineages made secondary transitions to terrestrial environments. Finally, the results herein imply that any animal lineage that has aquatic and terrestrial members, respire the same way in both habitats and have the same type of food available in both habitats could show a similar pattern of multiple independent habitat transitions coincident with changes in behavioral and morphological traits.

    Is there an ecological scenario where terrestrial insect larvae can show food choices? - Biology

    Food webs are graphical depictions of the interconnections among species based on energy flow . Energy enters this biological web of life at the bottom of the diagram, through the photosynthetic fixation of carbon by green plants. Many food webs also gain energy inputs through the decomposition of organic matter, such as decomposing leaves on the forest floor, aided by microbes. River food webs in forested headwater streams are good examples of this.

    Energy moves from lower to higher trophic (feeding) levels by consumption: herbivores consumes plants, predators consume herbivores, and may in turn be eaten by top predators. Some species feed at more than one tropic level, hence are termed omnivores. Figure 1 provides a simplified model of such a food web.

    Generalized food web. A food web is an assemblage of organisms, including producers, consumers and decomposers, through which energy and materials may move in a community

    We can look at this food web in two ways. It can be a diagram of the flow of energy (carbon) from plants to herbivores to carnivores, and so on. We will take this approach when we examine energy flow in ecosystems. In addition, members of a food web may interact with one another via any of the four interaction types named above. An interaction between two species in one part of the web can affect species some distance away, depending on the strength and sign of the inter-connections. Often, adding a species (as when an exotic species invades a new area) or removing a species (as in a local extinction) has surprisingly far-reaching effects on many other species. This is due to the complex inter-connections of species in ecological webs.

    1. Direct effects refer to the impact of the presence (or change in abundance) of species A on species B in a two-species interaction.
    2. Indirect effects refer to the impact of the presence (or change in abundance) of species A on species C via an intermediary species (A --> B --> C).
    3. Cascading effects are those which extend across three or more trophic levels, and can be top-down (predator --> herbivore --> plant) or bottom-up (plant --> herbivore --> predator).
    4. Keystone species are those which produce strong indirect effects.

    In the rocky inter-tidal zone of Washington state, and in other, similar areas, starfish have been shown to be keystone species The entire community lives on relatively vertical rock faces in the wave-swept inter-tidal zone. The community of marine invertebrates and algae are adapted to cling or adhere to the rock face, where most fed upon the small animal life suspended in the water (plankton). A bivalve, the mussel Mytilus, is superior at attaching to rock faces, making it the competitive dominant. A starfish (Pisaster) is an effective predator of the mussels, making space available for other species, and consequently is critical to maintaining a diverse biological community.

    Instances are known where a predator so strongly suppresses its prey (herbivores), that the trophic level below (plants) benefits because it is released from the pressures of herbivory. Such “top-down” trophic cascades, where the community looks more or less ‘green’ depending on the abundance of predators, are well-known in lakes. We also know of examples where fertilizing a system, which increases plant growth, results in more predators, through the increase in abundance of herbivores. This is a “bottom-up” trophic cascade.

    Our understanding of these complex species interactions gives substance to the popular phrase, the “balance of nature”. One can also appreciate how a human-induced removal of one species (an extinction event) or the addition on one species (invasion of a community by a non-native species) could result in harm to many additional species, a topic we will consider in the second semester.

    We will gain a fuller appreciation of the complex, multi-way interactions among species as we proceed through this series of lectures. However, we can fully appreciate the complexity of these multi-way interactions, it is helpful to first understand the nuances of the various two-way interactions. We will develop our understanding of species interactions in ecological communities based on these building blocks.

    Mutualistic Interactions

    Facultative mutualisms are beneficial but not essential to survival and reproduction of either party. Obligate mutualisms are those that are essential to the life of one or both associates. We will examine an example of each.

    • Gut symbionts in herbivores: mammals can't digest cellulose
    • endosymbiosis and the origin of eukaryotic cells: mitochondria, flagella, chloroplasts are thought to be derived from free-living bacteria
    • pollination systems
    • the coral polyp and its endosymbiont "alga" (actually a dinoflagellate)


    The clown fish and anemone also illustrates this point. The clown fish hides from enemies within the stinging tentacles of a sea anemone, to which the clown fish is immune. Some report this interaction as a mutualism, arguing that the clownfish drops scraps of food into the mouth of the anemone. Careful studies have failed to find much support for any benefit to the anemone, so this appears to be a commensalism.


    Species interactions within ecological webs include four main types of two-way interactions: mutualism, commensalism, competition, and predation (which includes herbivory and parasitism). Because of the many linkages among species within a food web, changes to one species can have far-reaching effects. We will next examine competition and predation, and then return to a consideration of more complicated indirect and cascading effects.


    Some of the most diverse and visually striking phenotypes seen in nature are those of camouflaged animals (Stevens & Merilaita, 2009). Background matching, or crypsis, is a common anti-predator strategy that has provided a test-bed for the theory of evolution through natural selection (Wallace, 1879 Wallace, 1889). Crypsis is selected for by visual predators such as birds (Merilaita, Lyytinen & Mappes, 2001), whereby prey that match the colour/pattern of the surrounding backgrounds survive for longer than non-matching prey (Endler, 1981 Merilaita, Scott-Samuel & Cuthill, 2017). In heterogeneous habitats, comprised of visually contrasting patches, or a gradient from one habitat type to another (Fig. 1), optimising crypsis to all of the background components presents a challenge (Merilaita, Tuomi & Jormalainen, 1999). One solution to this problem is a genetic polymorphism, which can produce two or more morphs that are specialised to different patch types (Merilaita, Lyytinen & Mappes, 2001 Surmacki, Ozarowska-Nowicka & Rosin, 2013). However, a species with a genetically fixed phenotype is restricted to camouflage on one background, or limited camouflage across varied patch colours (Fig. 1A). Therefore, in environments that change appearance across small temporal and spatial scales, detrimental phenotype-environment mismatching can occur (Cook et al., 2012 Farkas et al., 2015). In this case, selection may favour phenotypic plasticity, enabling individuals to actively change their appearance to utilise different habitat patches without compromising camouflage (Fig. 1B Stevens, 2016). An example of plasticity is colour change, which is a topic of current research interest and can be used to study the adaptive value and the physiology of camouflage (Duarte, Flores & Stevens, 2017).

    Figure 1: Possible camouflage strategies of caterpillars in response to visually heterogeneous environments.

    Rapid colour change (<2 h), as reported in fish, cephalopods, and amphibians has been widely studied (Allen et al., 2015 Buresch et al., 2011 Hanlon et al., 2009), and much is known about how chromatophores produce rapid changes in colour and pattern in these systems (Kingston et al., 2015 Mathger & Hanlon, 2007). Comparatively slower colour changes (days to months) occur in some arthropod and fish species (Llandres et al., 2013 Ryer et al., 2008). In many of these systems we still do not know whether slow colour change is adaptive, nor do we know the precise cues or biochemical processes involved. A number of potential cues have been proposed, with dietary and visual cues receiving most attention (Duarte, Flores & Stevens, 2017 Stevens & Merilaita, 2009).

    One example of a diet-induced phenotypic switch, or polyphenism, is seen in the larval stage of the moth Nemoria Arizona, which resembles inedible objects in its environment (Greene, 1989). In the spring the larvae resemble oak catkins, and in the summer they look like the branches of oak. This form of visual resemblance to inanimate objects is referred to as masquerade (Skelhorn, Rowland & Ruxton, 2010). Masquerade enables prey to avoid attack because predators misclassify these prey, rather than failing to detect them (Skelhorn et al., 2010b). The larvae of the peppered moth (Biston betularia) also masquerade as the twigs of their foodplant and change colour to match them (Noor, Parnell & Grant, 2008 Poulton, 1892). These brown and green colour morphs occur in response to the background colour on which the larvae rest (Noor, Parnell & Grant, 2008 Poulton, 1892). Changing appearance in response to background cues in the environment may be beneficial for animals that masquerade, as masquerade is often associated with polyphagy (Higginson et al., 2012). Visually hunting predators, like birds, heavily predate caterpillars that do not display warning colours (Lichter-Marck et al., 2015), and twig-mimicking caterpillars that do not match the twigs they rest on are also more likely to be predated (Skelhorn & Ruxton, 2010). Therefore, the ability to change colour could enhance masquerade in the wider range of environments these prey are likely to encounter, and consequently reduce their foraging restrictions (Ruxton, Sherratt & Speed, 2004).

    It is important to determine the exact cues eliciting colour change, as these cues initiate the colour change cascade (Duarte, Flores & Stevens, 2017), and can therefore provide information on the evolution of adaptive colour and the mechanisms of colour production (Cuthill et al., 2017). Visual stimuli exist in two forms: achromatic (luminance), and chromatic (hue/chroma). Responses to achromatic stimuli (luminance) have been reported in sand fleas, geckos, toads, and flatfish (Polo-Cavia et al., 2016 Ryer et al., 2008 Stevens et al., 2015 Vroonen et al., 2012). Tree frogs (Hyla japonica) adjust their body colour and luminance, to maximise camouflage against visually heterogeneous backgrounds, although the response to achromatic stimuli was stronger (Choi & Jang, 2014 Kang, Kim & Jang, 2016). Many of these studies propose that colour change in these animals is induced by visual cues, but the visual pathways were not explicitly studied, and additional cues such as temperature or texture were often not controlled (Lin, Lin & Huang, 2009 Polo-Cavia et al., 2016 Yamasaki, Shimizu & Fujisaki, 2009).

    To address this topic, we conducted a series of experiments to explore the type of visual cues that elicit colour change in B. betularia. The colour change in B. betularia has previously been described as a polyphenism: a switch of phenotype (Noor, Parnell & Grant, 2008). However, in the only study so far to investigate this behaviour, Noor, Parnell & Grant (2008) only provided two discrete stimuli: green vs. brown, and measured colour subjectively from a human perspective. The larvae of B. betularia are polyphagous and wind dispersed at first instar (Noor, Parnell & Grant, 2008 Tietz, 1972). The wide variety of twig colours between and within host plant species (Edmonds, 2010) presents a highly heterogeneous resting background. Therefore, it may be beneficial for individuals to change appearance on a continuous scale over time (Fig. 1D), known as a reaction norm (Woltereck, 1909). Colour reaction norms have been reported in squid, geckos, and anurans (Kang, Kim & Jang, 2016 Mathger & Hanlon, 2007 Vroonen et al., 2012), and are commonly induced by visual stimuli aquired by the animal about its environment. Reaction norms have not yet been investigated in lepidopteran larvae in this context.

    We used calibrated stimuli in order to investigate the adaptive significance of colour change in B. betularia (Stevens & Merilaita, 2009). We manipulated luminance (brightness) and colour, and evaluated the degree to which B. betularia caterpillars are able to respond to intermediate strength cues (i.e., discrete polyphenism vs. reaction norm). We also measured the response to heterogeneous twig colour environments. For the purpose of these experiments, ‘colour’ encompasses hue and chroma. Hue is defined as the direction of the colour vector, and chroma as how different a colour is from achromatic white/black (Stoddard & Prum, 2008). ‘Luminance’ is defined as achromatic intensity, or perceived brightness (Stevens, Lown & Denton, 2014 Stoddard & Prum, 2008). We modelled colour using the avian visual system which allows a more direct adaptive interpretation of larval colour change in B. betularia, compared to using human vision. We tested the following predictions: (1) larvae respond to both colour and luminance (2) larvae produce intermediate phenotypes in response to changing colour and/or luminance on a continuous scale (i.e., a reaction norm rather than a polyphenism, as suggested by Noor, Parnell & Grant (2008)) (3) when faced with a heterogeneous background, larvae adopt an intermediate colour reflecting the relative proportion of twig colours.

    Figure 2: Dowels used for luminance, colour, and heterogeneous environment experiments.


    Ethical statement

    The Institut Pasteur animal facility received accreditation from the French Ministry of Agriculture to perform experiments on live animals in compliance with the French and European regulations on the care and protection of laboratory animals. Rabbit blood draws performed in the context of this study were approved by the Institutional Animal Care and Use Committee at Institut Pasteur under protocol number 2015-0032. Mosquito collections inside Lopé National Park were conducted under permit number AR0020/14/MESR/CENAREST/CG/CST/CSAR. Mosquito collections by HLC in Gabon were performed under protocol number 0031/2014/SG/CNE approved by the National Research Ethics Committee.

    Field sampling

    Water from larval breeding sites and midguts of A. aegypti females emerging from the same larval sites were collected in Gabon in November 2014. Sylvatic collections were made inside Lopé National Park (latitude, −0.148617 longitude, 11.616715), and domestic collections were made in Lopé village (latitude, −0.099221 longitude, 11.600330) approximately 6 km from the sylvatic collection sites (Fig. 1). All of the sylvatic collections originated from rock pools, and the domestic collections were from various types of artificial containers and tires (Fig. 1B and file S5). At each larval breeding site, pupae and water were collected into a sterile 50-ml conical tube with filter-top lid using a sterile plastic pipette and brought back to the Station d’Etude des Gorilles et Chimpanzés field station. Upon arrival at the field station, 10 ml of water was transferred to a new sterile 50-ml conical tube next to the flame of a Bunsen burner and immediately stored at −20°C. The remaining water and pupae were held until the adults emerged and were visually identified as A. aegypti. The frozen water was transported back to the Centre International de Recherches Médicales de Franceville facilities in Franceville, Gabon, where the water was thawed and centrifuged at 3400 rpm for 10 min. Under a laminar flow cabinet, the supernatant was removed and replaced with 500 μl of sterile water to resuspend the bacterial pellet. The resuspended bacteria were spotted onto Whatman FTA cards (WB120401, GE Life Sciences), allowed to dry, and then wrapped in sterile foil for transport to Institut Pasteur in Paris.

    Within 12 hours of adult emergence, the midguts from A. aegypti females were dissected and stored in RNAlater (Qiagen) at +4°C. RNAlater was initially chosen to preserve the midgut tissue with the hope of being able to recover both RNA and DNA from the samples, but it was not possible to isolate a sufficient amount of RNA and DNA from each sample, so only DNA extractions were performed. Because of the lack of a laminar flow cabinet at the field station in Lopé National Park, the midguts were dissected within 50 cm of a Bunsen burner flame in an effort to maintain sterility. Before and between each dissection, the dissecting tools were disinfected with 3% bleach. Adult A. aegypti were removed from the tube in which they emerged and were cold-anesthetized. The mosquito was then surface-sterilized in 3% bleach and rinsed in sterile phosphate-buffered saline (PBS), and the midgut was dissected in a drop of sterile PBS. Because of the limited access to ethanol in the field, surface sterilization was only performed with 3% bleach. Negative controls were included in an attempt to control for potential contamination of bacteria introduced at this step (see below). The dissected midguts were placed in individual sterile tubes containing RNAlater that had been filtered through a 0.2-μm filter and aliquoted under sterile conditions. The midguts in RNAlater were stored at +4°C until being frozen at −20°C upon their arrival at Institut Pasteur in Paris until the DNA extraction was performed. Following the same procedure, the midguts from non–blood-fed, host-seeking adult females caught by HLC were also dissected. Human volunteers sat next to either the rock pools (sylvatic habitat) or the artificial containers (domestic habitat) where A. aegypti had been previously observed and caught the females as they landed and were preparing to probe.

    DNA extractions

    DNA from bacteria originating from the water samples was extracted from the Whatman FTA cards following the organic DNA extraction procedure provided by Whatman. DNA extractions were performed on two consecutive days, mixing samples each day to randomize a potential batch effect. Briefly, the filter paper was cut into small pieces using sterile scissors and soaked in 500 μl of extraction buffer [10 mM tris-HCl (pH 8.0), 10 mM EDTA, disodium salt (pH 8.0), 100 mM sodium chloride, and 2% (v/v) SDS] and 20 μl of proteinase K (20 mg/ml) prepared in sterile water overnight at 56°C with agitation. An equal volume of buffered phenol (pH 8.0) was added, vortexed briefly, and centrifuged for 10 min at maximum speed. The upper aqueous phase was transferred to a new microcentrifuge tube containing 500 μl of chloroform, vortexed thoroughly, and centrifuged for 10 min at maximum speed. The upper aqueous phase was transferred to a new tube containing 50 μl of 3 M sodium acetate (pH 5.2). Eight hundred microliters of 100% ethanol was added and vortexed. The DNA was allowed to precipitate at −20°C for at least 1.5 hours. The DNA was recovered by centrifuging for 30 min at maximum speed. The supernatant was discarded, and 1 ml of 70% ethanol was added to the pellet and centrifuged for 20 min at maximum speed. The supernatant was removed, and the pellet was air-dried for 30 min and dissolved in 50 μl of sterile water. Because of the large number of samples, the extractions were performed in two batches. To control for contamination of bacteria introduced during the DNA extraction, a negative control was made from each day of extraction by performing the extraction on a blank sample.

    Upon thawing the samples, the midguts were removed from the RNAlater and added to 300 μl of lysozyme (20 mg/ml) dissolved in Qiagen ATL buffer in a sterile tube containing grinding beads. The samples were homogenized for two rounds of 30 s at 6700 rpm (Precellys 24, Bertin Technologies). The samples were incubated at 37°C for 2 hours, after which 20 μl of proteinase K was added and vortexed briefly, followed by another incubation of 4 hours at 56°C on a shaker (300 rpm). After the incubation, 200 μl of Qiagen AL buffer and 200 μl of 100% ethanol were added to the samples and vortexed to mix. The lysate was transferred to a Qiagen DNeasy Mini Spin column (except midguts from HLC adults that were passed through Qiagen AllPrep columns), washed with buffers Qiagen AW1 and AW2 following kit instructions, and eluted in 20 μl of sterile water. To control for contamination of bacteria introduced both during the midgut dissections in the field and during the DNA extraction in the laboratory, negative controls were made by performing the same DNA extraction procedure on an aliquot of RNAlater opened at the field station in Gabon, an aliquot of sterile PBS used for midgut dissections that was opened at the field station in Gabon, and a blank midgut sample.

    16S sequencing

    Libraries were made from 8 sylvatic water samples, 6 domestic water samples, 15 sylvatic midguts (9 freshly emerged and 6 HLC adults), and 11 domestic midguts (8 freshly emerged and 3 HLC adults). In addition, two technical replicates were prepared with different primer pairs used on the same water samples. Technical replicates were used to confirm the repeatability of the sequencing results. Custom-made polymerase chain reaction (PCR) primers targeting the hypervariable V5-V6 region of the bacterial 16S ribosomal RNA gene were designed following Fadrosh et al. (62). These custom primers were designed to include the necessary Illumina adapters and indexes so that only one round of PCR was needed and therefore avoid multiple rounds of PCR that could lead to a sampling bias. To overcome the issues that arise when sequencing libraries with low-diversity sequences, such as PCR amplicons, heterogeneity spacers consisting of 0 to 7 base pairs were added to the custom primers so that the sequences would be sequenced out of phase (62). A total of eight forward and eight reverse primers were designed (file S6) and used in all 8 × 8 combinations to amplify all the breeding site water and midgut samples. Four microliters of each breeding site water sample and 6 μl of each midgut sample were used to amplify the V5-V6 16S region in triplicate using Expand High-Fidelity polymerase (Sigma-Aldrich) following the manufacturer’s instructions, with the addition 0.15 μl of T4gene32 and 0.5 μl of bovine serum albumin (20 mg/ml) per reaction to improve PCR sensitivity. Water samples were amplified for 30 cycles, and midgut samples were amplified for 40 cycles. The three PCRs were pooled, and the PCR products were purified using Agencourt AMPure XP magnetic beads (Beckman Coulter). The purified PCR products were quantified by Quant-iT PicoGreen dsDNA fluorometric quantification (Thermo Fisher Scientific) and pooled for sequencing on the Illumina MiSeq platform (Illumina). The sequencing run failed multiple times with no achievable explanation except for the inability of the sequences to bind to the flow cell. The failed custom sequencing tags were replaced with sequencing tags used successfully in previous projects (57), which required performing a second round of PCR because all extracted DNA from the midgut samples had been used in the initial PCR. The second round of PCR used new custom primers containing the same V5-V6 region to rescue the samples (file S7). One microliter of each library was amplified in triplicate using Expand High-Fidelity polymerase (Sigma-Aldrich) for eight PCR cycles. The three PCRs were pooled, purified using AMPure XP magnetic beads (Beckman Coulter), and quantified using Quant-iT PicoGreen dsDNA fluorometric quantification (Invitrogen). Library quality was checked by Bioanalyzer (Aligent Technologies), and 300–base pair paired-end sequences were generated on the Illumina MiSeq platform using a V3 kit (Illumina). The raw sequence data are available at the European Nucleotide Archive under accession number PRJEB16334.

    Targeted bacterial metagenomics analysis

    Read filtering, OTU clustering, and annotation were performed with the MASQUE pipeline (, as described by Quereda et al. (63). A total of 2851 OTUs were obtained at 97% sequence identity threshold. The statistical analyses were performed with SHAMAN ( based on R software (v3.1.1) and bioconductor packages (v2.14). Because bacterial communities were expected to differ substantially between mosquito midguts and water samples, the normalization of OTU counts was performed at the OTU level by sample type (midgut or water) using the DESeq2 normalization method. All samples including the negative controls and technical replicates were included in the normalization step. The technical replicates were removed from the data set before analysis. To account for possible contamination at various steps in the sample-processing pipeline, the OTU counts were corrected with the reads from the negative controls (see above). All OTUs found in the negative control samples were removed from the normalized OTU table unless the count in a real sample was >10 times higher than the mean OTU count in the negative controls. This operation was performed with a homemade script in R (64). This normalized OTU count table with the OTUs found in the negative controls removed (file S8) was used for the richness, Shannon index, Venn diagrams, abundance heat map, and NMDS analysis. Observed richness, Shannon index, and Bray-Curtis distances were calculated with the vegan package in R (65). The effects of sample types and ecotypes on the bacterial richness were tested by fitting a generalized linear model (GLM) with a Poisson distribution. The SEs were corrected for overdispersion using a quasi-GLM model where the variance is given by the mean multiplied by the dispersion parameter. A χ 2 test was applied to compare the significance of deviance shift after adding the covariates sequentially. The effects of sample type, ecotype, and their interaction on Shannon index were tested by fitting a linear model with a normal error distribution. The response variable was power-transformed to satisfy the model assumptions. The significance of each variable was tested with an analysis of variance (ANOVA) after adding the covariates sequentially. The results of the two models were confirmed by the convergence of backward and forward selection based on the Akaike information criterion. The Bray-Curtis distances were plotted with an NMDS method constrained in two dimensions. The Spearman correlation with real distances and stress value was estimated with the vegan R package (65). Effects of habitat and sample type on β diversity were tested with the betadisper and adonis permutational multivariate ANOVA methods from the vegan R package with 999 permutations of the Bray-Curtis distance matrix derived from OTU counts.

    In SHAMAN, a GLM was fitted and vectors of contrasts were defined to determine the significance in abundance variation between sample types. The GLM included the main effect of habitat (sylvatic or domestic), the main effect of sample type (midgut or water), and their interaction. The resulting P values were adjusted for multiple testing according to the Benjamini and Hochberg procedure. All OTUs that were present in the negative controls were excluded from the final list of differentially abundant OTUs.

    To confirm the OTU-based results with an OTU-independent method, a dissimilarity matrix was generated with the SIMKA software (66). Reads with a positive match against the sequences assembled from the negative controls were removed using Bowtie v2.2.9 (67). Then, k-mers of size 32 and occurring at least greater than two times were identified with SIMKA. Bray-Curtis dissimilarity was estimated between each sample.

    Bacterial isolation

    At the same time water was removed to freeze for DNA extraction, an aliquot of the larval site water was added to 50% sterile glycerol to make 20% glycerol stocks of the larval site water. The glycerol stocks were frozen at −20°C until they were transported back to Institut Pasteur in Paris. Upon arrival in Paris, the glycerol stocks were streaked out onto agar plates made with LB medium [LBm LB with NaCl (5 mg/ml)] and PYC medium [peptone (5 g/liter), yeast extract (3 g/liter), and 6 mM calcium chloride dihydrate (CaCl2·2H2O) (pH 7.0)] plates and incubated for 3 days at 30°C. LBm and PYC were chosen for being generalist media. Individual colonies were picked from the plates and used to inoculate 3 ml of the appropriate media, which were shaken at 30°C until bacterial growth occurred and used to create new glycerol stocks of the individual isolates. The same colony was also put into 20 μl of sterile water and exposed to two rounds at 95°C for 2 min and resting on ice for 2 min. The samples were then centrifuged for 5 min at maximum speed to remove cell debris, and the supernatant was used to amplify the entire 16S region by PCR [5′-AGAGTTTGATCCTGGCTCAG-3′ (forward) and 5′-AAGGAGGTGATCCAGCCGCA-3′ (reverse)] using Expand High-Fidelity Polymerase (Sigma-Aldrich). The PCR products were purified using the QIAquick PCR Purification kit (Qiagen), quantified by NanoDrop (NanoDrop Technologies Inc.), and sequenced by Sanger sequencing. The sequences were aligned and classified at the genus level using the SILVA database ( The raw sequence data are available at the European Nucleotide Archive under accession number PRJEB16334. Individual colonies were chosen on the basis of size, color, and morphology. The purity of the colonies used in the gnotobiotic experiments was verified by restreaking the bacteria on multiple occasions.

    Gnotobiotic larvae

    Axenic larvae were created using the eighth generation of an A. aegypti laboratory colony derived from a natural population originally sampled in Thep Na Korn, Kamphaeng Phet Province, Thailand, in 2013. This mosquito strain was used to create gnotobiotic larvae as a common genetic background from a different geographical region that had, presumably, not encountered the specific bacterial isolates introduced. The rationale was to avoid potentially confounding effects of local adaptation between mosquitoes and bacterial isolates. Eggs were gently scraped off the paper they were laid on into a 50-ml conical tube. The eggs were incubated in 70% ethanol for 5 min, 3% bleach for 3 min, and 70% ethanol for 5 min. The eggs were then rinsed in sterile water three times and allowed to hatch in sterile water in a vacuum chamber. Upon hatching, the larvae were transferred to sterile 25-ml tissue-culture flasks with filter-top lids and maintained in 15 ml of sterile water. Larvae were seeded to a density of 10 to 15 larvae per flask. The larvae were maintained on 50 μl of sterile fish food every other day. The water of the larval flask was not changed for the duration of the experiment. Fish food was made sterile by resuspending ground-up fish food with water and autoclaving it for 20 min. Axenic larvae were made gnotobiotic by adding a single bacterial isolate of choice. One to 3 days before inoculating the larval flasks, the bacteria were streaked out onto agar plates with their appropriate medium. They were allowed to grow 1 to 3 days until colonies of roughly similar size were obtained. A single bacterial colony was picked and added to each 25-ml flask. The sterility of the axenic larvae, as well as efficient colonization of the gnotobiotic larvae, was verified by PCR (see below). Five third-instar larvae were collected from each gnotobiotic treatment, and 10 axenic larvae were collected from three replicate flasks at the same time (5 days after hatching). Pooled larvae from each treatment were surface-sterilized by rinsing them once in sterile water, soaking in 70% ethanol for 10 min, and rinsing three times in sterile water. The larvae were then homogenized in Qiagen ATL extraction buffer, and DNA was extracted using the Qiagen DNeasy Blood and Tissue kit. The presence or absence of bacteria was qualitatively verified by PCR using the same primers listed above for bacteria identification. Homogenates from the surface-sterilized larvae were plated to confirm that the added bacteria had colonized the larvae and that only a single morphological colony matching that of the input bacteria was present. The water in which gnotobiotic larvae developed was also plated to confirm that only the expected morphological colony was present. The axenic larval flasks were maintained for the duration of the experiment and manipulated in the same way to serve as negative controls. The amount of bacteria measured in the water of gnotobiotic treatments was not correlated to pupation rate (file S4).

    Selection of bacterial isolates for functional assays

    Because cultivable bacteria only represent a small fraction of all bacteria present, and because specific bacterial isolates do not necessarily represent OTUs, the selection of isolates for functional assays was unrelated to the 16S metagenomics data. In particular, the choice of bacterial isolates did not depend on their relative abundance or habitat of origin. Instead, it was based on an arbitrary set of selection criteria described below. The original collection of 168 bacterial isolates was narrowed down to 37 isolates to test in an initial screen of pupation rate with the hypothesis that bacterial isolates that resulted in differences in larval growth kinetics would potentially induce phenotypic differences at the adult stage. The 37 test isolates were chosen on the basis of genetic dissimilarity to other isolates (<95% genetic similarity) and previously being associated with Aedes mosquitoes in the literature. To test the pupation rate of each of the 37 initial test isolates, individual colonies were inoculated into triplicate flasks of axenic larvae, as described above. The number of pupae was counted in each flask every day for 17 days. The list of 37 isolates was further narrowed down to 16 candidate isolates based on those that reached 60% pupation. Of the 16 candidate isolates, three isolates were chosen on the basis of differences in pupation rate (file S9) and differences in the cultivable bacterial composition found in adult midguts after 4 to 6 days in the insectary (file S10). On the basis of their full-length 16S sequence, two of the three isolates were assigned to Salmonella (Ssp_ivi) and Rhizobium (Rsp_ivi) genera. The third isolate (Esp_ivi) was assigned to the Enterobacteriaceae family, but classification at the genus level was inconsistent among databases (alternatively Salmonella, Escherichia, or Shigella). Whereas Escherichia was previously found in wild A. aegypti specimens, and Shigella and Rhizobium were found in wild A. albopictus specimens, Salmonella was not previously reported to be associated with Aedes mosquitoes (20, 31). In all cases, colonies belonging to the bacterial genera that were added during the larval stage could not be recovered from the corresponding adult midguts. Even when the same bacterial genus was detected in adult midguts (file S10), the 16S sequence was distinct.

    Adult life-history traits

    After adult emergence from the different gnotobiotic treatments, 18 to 20 females were placed into triplicate 1-pint cardboard cups and maintained under standard insectary conditions (27 ± 1°C relative humidity, 75 ± 5% 12:12-hour light/dark cycle) on a sugar diet. The number of dead mosquitoes was recorded daily for 60 days until >90% of mosquitoes had died. The wings of the individual females harvested in the second replicate of the vector competence experiment (see below) were kept for later analysis. Wing length was measured from the tip (excluding the fringe) to the distal end of the allula using an ocular micrometer and a dissecting microscope. When both wings were intact, the mean of the two wing lengths was used for the statistical analysis.

    Lysozyme-like activity of hemolymph

    Antibacterial activity of the hemolymph was measured by a bacterial growth inhibition zone assay. In this assay, mosquito hemolymph was spotted onto an agar plate containing M. luteus, and the antibacterial activity of the hemolymph was measured by the area of visible bacterial clearance around the hemolymph sample. Five to 7 days after adult emergence from gnotobiotic treatments, hemolymph was collected from females and placed on agar plates seeded with M. luteus. To make the agar plate, 10 ml of agar solution [2× agar (BD BactoAgar, Becton, Dickinson and Company), freeze-dried M. luteus (5 mg/ml Sigma-Aldrich), streptomycin (0.1 mg/ml Sigma-Aldrich), and 67 mM potassium phosphate buffer (pH 6.4)] was plated, and 3-mm holes were punched in the solidified agar. Twenty females (two replicates of 10 females each) from each treatment were cold-anesthetized and stored on ice until hemolymph was collected. To collect hemolymph, 2 μl of anticoagulant solution [60% Schneider’s medium (Sigma-Aldrich), 10% fetal bovine serum (FBS), and 30% citrate buffer (pH 4.5) (98 mM NaOH, 186 mM NaCl, 1.7 mM EDTA, and 41 mM citric acid)] was injected into the thorax using a finely drawn glass capillary and a bulb dispenser (Microcaps, Drummond Scientific Co.). Ten microliters of the anticoagulant solution was then injected into the abdomen, and hemolymph was collected through capillary action by placing a capillary tube next to the injection site. The hemolymph was immediately placed on ice and then deposited in the cutout holes on the agar plates. The plates were stored at 30°C for 24 hours, and the number of individuals with detectable M. luteus growth inhibition and the size of M. luteus growth inhibition zone were calculated. The size of M. luteus growth inhibition was determined by using ImageJ ( to calculate the diameter of the clear zone. The diameter of the clear zone for the hemolymph samples was converted to lysozyme-like activity using a standard curve generated by spotting 10-fold serial dilutions of lysozyme (200 mg/ml Sigma-Aldrich) and measuring the diameter of the clear zone using ImageJ.

    Vector competence

    Following the gnotobiotic treatments, pupae were picked every day for 1 week, and adults were allowed to emerge under standard insectary conditions (27 ± 1°C relative humidity, 75 ± 5% 12:12-hour light/dark cycle). The adults were maintained in the insectary for 3 to 7 days after emergence on a standard sugar diet. Females were starved for 24 hours before the infectious blood meal. Vector competence assays were performed as previously described (68). Briefly, mosquitoes were experimentally exposed to a wild-type dengue virus serotype 1 isolate (KDH0026A) originally from Thailand (69). The isolate was passaged five times in A. albopictus C6/36 cells before its use in this study. The virus stock was diluted in cell culture medium (Leibovitz’s L-15 medium + 10% heat-inactivated FBS + nonessential amino acids + 0.1% penicillin/streptomycin + 1% sodium bicarbonate) to reach a dose of 2.4 × 10 5 FFU/ml in the first experiment and 7.15 × 10 5 FFU/ml in the second experiment. One volume of virus suspension was mixed with two volumes of freshly drawn rabbit erythrocytes washed in distilled PBS and 60 μl of 0.5 M adenosine 5′-triphosphate. After gentle mixing, 2.5 ml of the infectious blood meal was placed in each of several Hemotek membrane feeders (Hemotek Ltd.) maintained at 37°C and covered with a piece of desalted porcine intestine as a membrane. After feeding, fully engorged females were sorted into 1-pint cardboard cups and maintained under controlled conditions (28 ± 1°C relative humidity, 75 ± 5% 12:12-hour light/dark cycle) in a climatic chamber for 14 days.

    After 4 days (experiment 1) and 14 days (experiments 1 and 2), detection of dengue virus RNA was performed with a two-step reverse transcription PCR assay. Heads and bodies were separated from each other, and bodies were homogenized individually in 400 μl of RAV1 RNA extraction buffer (Macherey-Nagel) during two rounds of 30 s at 5000 rpm (Precellys 24). Total RNA was extracted using the NucleoSpin 96 Virus Core Kit following the manufacturer’s instructions (Macherey-Nagel). Total RNA was first reverse-transcribed to complementary DNA (cDNA) with random hexamers using M-MLV Reverse Transcriptase (Invitrogen). The cDNA was amplified by 45 cycles of PCR using the set of primers targeting the NS5 gene [5′-GGAAGGAGAAGGACTCCACA-3′ (forward) and 5′-ATCCTTGTATCCCATCCGGCT-3′ (reverse)]. Amplicons were visualized by electrophoresis on 2.5% agarose gels.

    In both experiments 1 and 2, the heads from infected bodies were titrated by standard focus-forming assay in C6/36 cells, as previously described (68). Briefly, heads were homogenized individually in 300 μl of Leibovitz’s L-15 medium supplemented with 2× Antibiotic-Antimycotic (Life Technologies). C6/36 cells were seeded into 96-well plates, and each well was inoculated with 40 μl of head homogenate and incubated for 1 hour at 28°C. Cells were overlaid with a 1:1 mix of carboxymethyl cellulose and Leibovitz’s L-15 medium supplemented with 0.1% penicillin (10,000 U/ml)/streptomycin (10,000 μg/ml), 1× nonessential amino acids, 2× Antibiotic-Antimycotic (Life Technologies), and 10% FBS. After 3 days of incubation, cells were fixed with 3.7% formaldehyde, washed three times in PBS, and incubated with 0.5% Triton X-100 in PBS. Cells were incubated with a mouse anti-dengue virus complex monoclonal antibody (MAB8705, Merck Millipore), washed three times with PBS, and incubated with an Alexa Fluor 488–conjugated goat anti-mouse antibody (Life Technologies). FFU were counted under a fluorescence microscope.

    Statistical analysis of mosquito phenotypes

    All statistical analyses were performed in R v3.1.2 (, unless where otherwise noted. Analysis of pupation rate was based on a three-parameter logistic model (Cumulative_proportion = K/(1 + eB(time − M) ) describing the cumulative change in pupation rate over time for each condition using least-squares nonlinear regression with the minpack.lm R package ( In this logistic model, K represents the saturation level of pupation rate (that is, the final pupation rate), B is the growth rate (that is, rate of change per unit time during the exponential phase), and M is the time at which the proportion of pupae equals 50% of the saturation level K. The extra sum-of-square F test was used to compare single parameters between two curves representing the cumulative pupation rate over time for two conditions. The P value was derived from the F test based on the F distribution and the number of degrees of freedom.

    Survival data were analyzed using a time-to-event model and Kaplan-Meier estimator in the survival R package ( Continuous variables (wing length, CFU counts, and FFU counts in the head) were analyzed using a full-factorial linear regression model and type III ANOVA, followed by verification of the normal distribution of the residuals. Binary traits (CFU prevalence, lysozyme-like activity prevalence, and vector competence binary phenotypes) were analyzed using a full-factorial logistic regression model and analysis of deviance.

    Is there an ecological scenario where terrestrial insect larvae can show food choices? - Biology

    In this course we have mainly discussed evolution within species, and evolution leading to speciation. Evolution by natural selection is caused by the interaction of populations/species with their environments.

    However, the environment of a species is always partly biotic . This brings up the possiblity that the "environment" itself may be evolving. Two or more species may in fact coevolve . And coevolution gives rise to some of the most interesting phenomena in nature.

    At its most basic, coevolution is defined as evolution in two or more evolutionary entities brought about by reciprocal selective effects between the entities . The term was invented by Paul Ehrlich and Peter Raven in 1964 in a famous article: "Butterflies and plants: a study in coevolution", in which they showed how genera and families of butterflies depended for food on particular phylogenetic groupings of plants. We have already discussed some coevolutionary phenomena:

    Coevolution might occur in any interspecific interaction. For example:

    • Interspecific competition for food or space
    • Parasite/host interactions
    • Predator/prey interactions
    • Symbiosis
    • Mutualisms
    Palatable Batesian mimics adapt to the unpalatable model by copying its pattern, but the model may not be able to escape its parasite. The first model individuals with a new, non-mimicked pattern would also lose the protection of their own species' warning pattern. Thus we can hypothesise that "coevolutionary chase" is an unlikely outcome of Batesian mimicry.

    In Müllerian mimicry the most abundant and noxious species will also be trapped by its own pattern any individuals that mimic a rarer or less noxious species will lose the protection of their own species' pattern even though, once the new mimetic pattern became common, both species would ultimately benefit. In contrast, the rarer or less noxious species always gains by mimicking the more common or noxious species, because its own species' protection is weaker than the other's. Mutual convergence is therefore unlikely because of these difficulties for the initial mimetic variants, in spite of the fact that the outcome, once achieved, is mutualistic.

    In general, there is much discussion about the likelihood of coevolution in cases where more than one species is involved in an evolutionary interactions. An "Ockham's Razor" approach to proving coevolution requires that we should first disprove the simpler hypothesis of unilateral adaptation.

    Answers to the question "How likely is coevolution?" depends what you mean by coevolution! Various types have been proposed:

    In specific coevolution, or coevolution in the narrow sense, in which one species interacts closely with another, and changes in one species induce adaptive changes in the other, and vice-versa. In some cases, this adaptation may be polygenic in other cases, there may be gene-for-gene coevolution, in which the mutual interactions are between individual loci in the two species.

    Specific coevolution may of course be short-lived, but if the interaction is very close, as in many host-parasite systems, concordant speciation or cospeciation may result where the speciation in one form causes speciation in another. Of course, cospeciation doesn't necessarily require coevolution. For example, a very unimportant but highly host-restricted parasite may always speciate whenever its host speciates, without the parasite causing any evolutionary reaction in the host.

    In diffuse coevolution, also called guild coevolution , whole groups of species interact with other groups of species, leading to changes that cannot really be identified as examples of specific, pairwise coevolution between two species. For example, a group of plant species may be fed on by a particular family of insects, which may frequently (in evolutionary time) change hosts. The plants may evolve defensive adaptations, such as defensive chemistry, or physical defenses such as spines, which work against large numbers of the species. In time, some of the insects may be able to overcome the plant's defences, leading to further evolution by the plant, and so on.

    Another related type of evolution is called escape-and-radiate coevolution. Here, an evolutionary innovation by either partner in a coevolutionary interaction enables an adaptive radiation, or speciation due to the availability of ecological opportunity. For example, it is easy to imagine that this could be a result of the diffuse kind of herbivore-plant coevolution described above.

    Phylogenies are very useful in the study of coevolution. If the phylogenies of two closely associated groups, such as host and parasite, are concordant (see overhead), this may imply:

    • That cospeciation has occurred, or
    • That one of the groups (often the parasite) has "colonized" the other (the host). Here, host shifts by the parasite may well correspond to the host phylogeny, but only because closely related hosts are similar, and liable to colonization by closely-related parasites.

    However, as we have seen, even contemporaneous cospeciation with concordant phylogenies does not prove that two lineages have coevolved. Instead, we can look at individual adaptations of the interacting species to get an idea of whether coevolution has taken place. Here are some examples:

    Defences of plants against herbivores

    Plants have many complex chemicals, called "secondary chemicals", which are not obviously used in normal metabolism. Ehrlich and Raven and others subsequently interpreted this "secondary chemistry" as an example of defensive adaptation by the plants. Many of these compounds (for instance, tannins and other phenolic compounds, alkaloids like nicotine, cocaine, opiates and THC, or cyanogenic glycosides) are highly toxic. Many animals such as insects have adapted to feeding exclusively on plants with particular defensive chemistry. If the plants evolved secondary chemistry to avoid insects, and insects evolved to handle the plant chemistry, then plant/insect coevolution has occurred.

    However, critics argue that:

    • phytophagous insects are usually rare, and therefore do not pose a threat to their host plants
    • secondary chemistry may be a byproduct of normal metabolic processes, rather than necessarily defensive

    Ant-acacias.Good evidence for insect/plant coevolution is found in the Central American plant known as "bullshorn Acacia", Acacia cornigera. This plant is similar to other members of the genus Acacia (thorn trees in the pea family), in that it has large spines which presumably protect it against mammalian herbivores (another example of coevolution, presumably against mammalian browsers). However, it lacks the cyanogenic glycosides (cyanide-producing chemicals) found in related Acacia and the thorns in this species are particularly large and hollow, and provides shelter to a species of Pseudomyrmex ant. The plant also provides proteinaceous food bodies on the tips of the leaflets, which sustain the ant colonies. These ants are particularly nasty (I can tell you from personal experience!), and are well able to deter even mammals with their wasp-like stings. It has been shown experimentally that the ants will also remove any caterpillars from the leaves that they patrol. The ants even remove vines and plants from around the base of the tree, creating a bare patch on the soil. Plants of the bullshorn Acacia which have not been occupied by ant colonies are heavily attacked by herbivores and often have vines growing in the branches.

    Related Acacia species lack hollow thorns and food bodies, and do not have specific associations with ants. They also have many cyanogenic glycosides in their leaves. This data strongly supports the idea that the bullshorn Acacia has evolved a close, mutualistic association with the ants in order to protect themselves from herbivores (and also plant competitors). It also supports the idea that the cyanogenic glycosides found in other species have a defensive role a role which has been taken over by Pseudomyrmex in the bullshorn Acacia.

    Egg mimicry in Passiflora.Similarly, we have already given examples of egg-mimicry in Passiflora, which protects plants against species of Heliconius butterflies. Female Heliconius avoid laying eggs on plants already occupied by eggs, because first instar larvae of Heliconius are highly cannibalistic the plants exploit this habit of Heliconius by creating fake yellow eggs as deciduous buds, stipule tips, or as part of the "extrafloral nectaries" on young leaves. Clearly, the plant, whose defenses of cyanogenic glycosides, alkaloids, and a host of other secondary compounts, have been breached by Heliconius, has counterevolved new defenses against this genus.

    Heliconius-egg mimicry in Passiflora Predator-prey coevolution

    Predators have obviously evolved to exploit their prey, with hunting ability being at a premium. Mammalian predators, for example, must be fast, strong and cunning enough to be able to catch their prey. It is almost as obvious that prey have evolved to protect themselves from predators. They may have a variety of defenses:

    • Large size and strength
    • Protective coverings such as shells or hard bony plates
    • Defensive weapons, such as stings or horns
    • Defensive coloration (see mimicry lecture)
    • Unpalatability and nastiness

    Two of the most famous are figs and fig-wasps, and Yucca and Yucca moths (Tegeticula).

    In both cases, the larvae are seed/flower eaters, which reduce the fertility of the flowers or inflorescences they infest.

    In both cases, the plant is completely dependent on its herbivore for pollination. The arrangement is therefore a tightly coevolved mutualism, in which the plant relies exclusively on the insect for pollination, and the insect relies exclusively on the plant for food.

    In the case of the Yucca moth the mutualism has sometimes broken down, and some clades of the moth have reverted to a parasitic mode of life -- they oviposit in the plant, but do not pollinate -- the ancestral condition for the moths.

    These examples are interesting because they represent cases where mutualisms have become so specific that they almost rival the ancient prokaryotic mutualisms of mitochondria and chloroplasts with archaebacterial cells, to produce what we now know as eukaryotes.

    Coevolutionary competitive interactions and adaptive radiation

    It is an ecological principle (Gause's principle) that related species must differ in some part of their ecology. If two species have identical or nearly identical resources, competitive exclusion will result, and the less well adapted species will go extinct.

    If this is true, and it probably is, the reverse should also occur. If a species colonizes an area where its competitors do not occur, then it may experience ecological release, and grow to very large population sizes. Not only that, the colonists may also experience disruptive selection, followed by speciation. The process can be repeated for multiple species, which evolve apart from one other to form an adaptive radiation .

    Many examples of this principle are known in island colonists. For example, we have already come across the Darwin's finches of the Galapagos islands, which have evolved into a whole range of seed-feeding and insectivorous forms. A similar, although much more diverse radiation occurs in the Hawaiian archipelago: the Hawaiian honeycreepers.

    Sometimes, the islands are " ecological islands" rather than actual islands. A number of lakes in the North temperate zone were left behind during the retreat of the ice. These lakes have in the last 10,000 years been colonized by a variety of fish. In many cases of stickleback and the trout family, multiple forms have now been produced in each lake or large fresh water body.

    Sticklebacks in Canada (Gasterosteus) often produce benthic (deep water) and limnetic (shallow water) forms (see overhead), which appear to have specialized feeding differences. These forms also keep to their own habitat, and may mate assortatively.

    Similarly, the Atlantic char (Salvelinus) in Thingvallavatn, Iceland's largest lake, have produced no less than FOUR different trophic forms similar examples are known from Norway and Ireland for other salmonids.

    Adaptations leading to ecological release, and "escape and radiate" coevolution

    As well as the colonization of new habitat, the possession of a unique adaptation may also allow adaptive radiation to colonize a new "adaptive zone" opened up as a result. There is good evidence for this:

    • A massive phylogenetic study of beetles by Brian Farrell showed that new adaptations for herbivory on flowering plants led to massive amounts of speciation. Most of the diversity of species of beetles is in the herbivorous clades (overhead).
    • The evolution of resin- or latex-bearing canals allowed plants carrying them a more rapid speciation rate than among sister taxa that lacked these adaptations. Latex and resin is a physical defence against herbivorous insects.

    Evolutionary interactions between species, and coevolution show that the complexity of genetic evolution goes on increasing, even beyond the species level. Coevolution represents an area where genetics, ecology, phylogeny all interact. To understand the evolution of life fully, the interactions between individuals and species must be explored at many levels.

    One thing is clear the majority the diversity of life and life forms is not just due to adaptation to static environments biotic interactions are probably much more important. The biotic environment is itself constantly evolving, leading to orders of magnitude more diversity possible than could be produced by evolutionary adaptation to simple physical conditions.

    Ehrlich, PR, Raven, PH 1964. Butterflies and plants: a study in coevolution. Evolution 18, 586-608.
    Farrell, BD 1998. "Inordinate fondness" explained: why are there so many beetles? Science 281, 555-559.
    Futuyma, DJ 1998. Evolutionary Biology. Chapter on coevolution.
    Thompson, JN 1994. The Coevolutionary Process. Chicago University Press.

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