Typical Insect Research Articles

Part 1: Laboratory culture of Centroptilum triangulifer (Ephemeroptera: Baetidae) using a defined diet of three diatoms

Development of methods for assessing exposure and effects of waterborne toxicants on stream invertebrate species is important to elucidate environmentally relevant information. Current protocols for freshwater invertebrate toxicity testing almost exclusively utilize cladocerans, amphipods or chironomids rather than the more typical aquatic insect taxa found in lotic systems. Centroptilum triangulifer is a parthenogenetic mayfly occurring in depositional habitats of streams and rivers of the Eastern U.S. and Canada. C. triangulifer is an ideal stream insect for toxicity testing under field and laboratory conditions because of its short life cycle, parthenogenetic mode of reproduction, and it represents a group considered sensitive to environmental stressors. In this study, a colony of C. triangulifer was reared using a defined diet of three diatoms, Mayamaea atomus var. permitis, Nitzschia cf. pusilla, and Achnanthidium minutissimum. Percent survival (⩾80%), fecundity measurements (⩾1000 eggs) and pre-egg laying weights were used as indicators of overall colony health and fitness in our laboratory water (Lab-line) and in Moderately Hard Reconstituted Water (MHRW). Lab-line reared C. triangulifer had average survival rate of 92.69% for eleven generations and 82.99% over thirteen generations. MHRW reared C. triangulifer had an average survival rate of 80.65% for four generations and three generations of fecundities greater than 1000 eggs per individual. Pre-egg laying weight and fecundity were highly correlated and a best-fit model equation was derived to estimate egg counts for future generations. Establishment of this culturing protocol provides a more ecologically relevant species for toxicity testing and aids in further stressor identification for stream bioassessments.

The complete mitochondrial genome of the Byasa alcinous (Lepidoptera: Papilionidae: Papilioninae)

The complete mitochondrial genome (mitogenome) of Byasa alcinous (Lepidoptera: Papilionidae: Papilioninae) is a circular molecule of 15,266 bp in length, containing 37 typical insect mitochondrial genes: 13 protein coding genes (PCGs), 2 rRNA genes, 22 tRNA genes and a non-coding AT-rich region. Its gene order and arrangement are identical to all other available butterfly mitogenomes. All PCGs start with a typical ATN initiation codon, except for COI, which is initiated by the CGA codon as observed in other butterfly species. Ten PCGs terminate in the complete stop codon TAA or TAG, whereas the COI, COII and ND4 genes end with single T. Ten intergenic spacers (73 bp in total), and 12 overlapping regions (28 bp in total) are dispersed throughout the whole genome. The non-coding AT-rich region is 405 bp long and contains some conserved structures similar to those found in other butterfly mitogenomes, such as the motif ATAGA followed by a 12-bp poly-T stretch and a microsatellite-like (AT)14 element preceded by the ATTTA motif. Additionally, a 11-bp poly-T sequences and a microsatellite-like (AT)7 repeated elements are detected in this region.

Invasive Prunus serotina - a new host for Yponomeuta evonymellus (Lepidoptera: Yponomeutidae)?

Introduction of non-native species of plants affects the existence and feeding preferences of herbivorous insects. The bird cherry ermine moth (Yponomeuta evonymellus ) is considered a typical monophagous insect, which feeds only on bird cherry (Prunus padus) leaves. However, in recent years, we have observed Y. evonymellus larvae feeding on leaves of the non-native (in Europe) and highly invasive black cherry (Prunus serotina). We hypothesized that this insect can feed on P. serotina leaves with no negative ef- fects on its growth and development and that the main reason why it does not accept this plant as a host is the phenological difference between the two species of cherry. Moving individuals of the three larval instars (L1, L2 and L3) from bird cherry to black cherry did not affect the percentage of adults that emerged from the pupae or the masses of the moths. In addition, in one experiment, the moths were heavier and the percentage parasitized was lower on P. serotina than on P. padus. Thus, the leaves of black cherry were at least as good a food source as P. padus for Y. evonymellus. During the feeding period, there were low concentrations of defense compounds (phenolics and condensed tannins) in the leaves of both species. However, it is likely that the low success of Y. evonymellus in infesting P. serotina is due to spring frosts and heavy rains, which are deadly for larvae in an early stage of development on black cherry. In the field these weather conditions resulted in a very high mortality of larvae in our experiment. In conclusion, the use of bird cherry as a host by Y. evonymellus is mainly determined by its phenology.

The complete mitochondrial genome of the longhorn beetle, Massicus raddei

We sequenced the complete mitochondrial genome of Massicus raddei, which is the first beetle sequenced in Cerambycinae to date. The complete mitochondrial genome is 15,585 bp in length with an A + T content of 71.82%, and contains 13 protein-coding genes, 2 rRNAs, 22 tRNAs and a control region. The gene order and orientation are similar to that of typical insect species. These data will provide useful molecular information for phylogenetic relationships among the suborders of Coleoptera. By using 13 protein-coding genes as phylogenetic markers, the results support that the suborder Archostemata is a sister group to the remaining beetles and the most primitive suborder in any case; the suborder Myxophaga is sister to the suborder Adephaga.

Floral ratios in the figs of Ficus montana span the range from actively to passively pollinated fig trees

Fig trees (Ficus spp., Moraceae) and their associated obligate pollinator fig wasps (Agaonidae) are partners in what is often a pair-wise species–specific association. Their interaction centres on the unique enclosed inflorescence of Ficus species – the fig. Among dioecious fig tree species, only pollinated ovules in figs on female trees develop into seeds. On male trees, galled ovules support development of the fig wasp offspring that will transport their pollen, but no seeds develop. Some fig wasp species actively collect and disperse pollen, whereas others are typical insect pollinators in that pollen is transferred passively. Active pollination is associated with improved larval survivorship in pollinated figs. Because active pollination is much more efficient, their host figs need to contain far fewer male flowers and across numerous Ficus species anther-ovule ratios are a good predictor of pollination mode. We examined variation in inflorescence size and floral ratios among male figs of the Asian Ficus montana and its consequences for the amounts of pollen that would be available for each pollinator to collect. Inflorescence size (total flower number) was highly variable, and female pollinator offspring production was higher in figs with more female flowers. Pollinator offspring numbers and anther-ovule ratios were also highly variable, and encompassed the range typical of both actively and passively pollinated fig tree species. In combination, this variation resulted in large differences in the extent to which pollinators were competing for access to pollen, with potential fitness consequences for both partners in the mutualism.

Friction and Wear Properties of the Tergum Surface of Mole Cricket, <i>Gryllotalpa orientalis</i>

Mole cricket is typical insect living under the ground, which tergum is the main component that suffers the wear and tear from the soil particles. In order to adapt this environment, mole crickets have anatomically evolved well-suited tergum with wear-resistance property, which can protect the body from abrasion and damage. In this study, the surface morphology and structure of the tergum of mole cricket were studied and analyzed by stereomicroscope (SM) and Scanning Electron Microscope (SEM). By using a Universal Micro-Tribotester (UMT), the friction-wear properties and the performances of the tergum surface were tested along with the head direction, tail direction and lateral direction of body, respectively. The result shows that the setae have obvious influence on the friction characteristic of mole crickets tergum. By comparing with other direction, the tergum surface has better friction properties on the setae growth direction. This study can be a reference for the design of novel wear resistance surface.

The mitochondrial genome of the Russian wheat aphid Diuraphis noxia: Large repetitive sequences between trnE and trnF in aphids

To characterize aphid mitochondrial genome (mitogenome) features, we sequenced the complete mitogenome of the Russian wheat aphid, Diuraphis noxia. The 15,784-bp mitogenome with a high A+T content (84.76%) and strong C skew (−0.26) was arranged in the same gene order as that of the ancestral insect. Unlike typical insect mitogenomes, D. noxia possessed a large tandem repeat region (644bp) located between trnE and trnF. Sequencing partial mitogenome of the cotton aphid (Aphis gossypii) further confirmed the presence of the large repeat region in aphids, but with different repeat length and copy number. Another motif (58bp) tandemly repeated 2.3 times in the control region of D. noxia. All repeat units in D. noxia could be folded into stem-loop secondary structures, which could further promote an increase in copy numbers. Characterization of the D. noxia mitogenome revealed distinct mitogenome architectures, thus advancing our understanding of insect mitogenomic diversities and evolution.

An Insect Song Signal Feature Extraction Method Based on the Wavelet Packet Analysis

The wavelet packet decomposition method was used to two common insects song signal. Frequency decomposition and feature extraction were made, the feature vectors, eigenvalues and the sound quality evaluation parameter vectors were constructed, then the correlation analysis calculation were made between the eigenvalues and the sound quality evaluation parameter vectors. The results show that the correlation coefficients are good, the average correlation coefficients of cricket and grasshopper song signals are 0.8875 and 0.6942, the results of cricket is much better than grasshopper, it proved that the proposed algorithm is more suitable for cricket song signals analysis, a new and effective sound quality evaluation method for typical insect with friction sound mechanism is provided.

Insights into the Evolution of the CSP Gene Family through the Integration of Evolutionary Analysis and Comparative Protein Modeling

Insect chemical communication and chemosensory systems rely on proteins coded by several gene families. Here, we have combined protein modeling with evolutionary analysis in order to study the evolution and structure of chemosensory proteins (CSPs) within arthropods and, more specifically, in ants by using the data available from sequenced genomes. Ants and other social insects are especially interesting model systems for the study of chemosensation, as they communicate in a highly complex social context and much of their communication relies on chemicals. Our ant protein models show how this complexity has shaped CSP evolution; the proteins are highly modifiable by their size, surface charge and binding pocket. Based on these findings, we divide ant CSPs into three groups: typical insect CSPs, an ancient 5-helical CSP and hymenopteran CSPs with a small binding pocket, and suggest that these groups likely serve different functions. The hymenopteran CSPs have duplicated repeatedly in individual ant lineages. In these CSPs, positive selection has driven surface charge changes, an observation which has possible implications for the interaction between CSPs and ligands or odorant receptors. Our phylogenetic analysis shows that within the Arthropoda the only highly conserved gene is the ancient 5-helical CSP, which is likely involved in an essential ubiquitous function rather than chemosensation. During insect evolution, the 6-helical CSPs have diverged and perform chemosensory functions among others. Our results contribute to the general knowledge of the structural differences between proteins underlying chemosensation and highlight those protein properties which have been affected by adaptive evolution.

Morphology of the male reproductive system and sperm ultrastructure of the egg parasitoid Gryon pennsylvanicum (Ashmead) (Hymenoptera, Platygastridae)

Gryon pennsylvanicum is a platygastrid hymenopteran that has lately received increasing attention in Europe due to its possible use in biological control of the conifer seed bug pest Leptoglossus occidentalis. Here the male reproductive system and the spermatogenesis of this species, along with those of Gryon muscaeformis, are examined ultrastructurally for the first time. The male genital system is formed by a pair of testes, each containing only one follicle, a pair of accessory glands and deferent ducts connected to a single ejaculatory duct. All the stages of spermatogenesis are described in detail. Characteristic features of the Gryon spp. sperm, which are 100 μm long, are the presence of a polygonal nucleus, only one mitochondrial derivative, the occurrence of the centriole adjunct and a typical insect 9 + 9 + 2 flagellar axoneme. The single derivative, however, results from a process in which one of the two mitochondria is lost during spermiogenesis. Unlike in other insects, two centrioles occur in spermatids as a consequence of the ameiotic parthenogenesis. These characteristics stand as a valuable tool for phylogenetic inferences. Furthermore this study suggests a useful strategy for laboratory mass rearing.

The gustatory sensilla on the endophytic ovipositor of Odonata

The present paper aims at describing the fine structure of coeloconic sensilla located on the cutting valves of the endophytic ovipositor of two Odonata species, the anisopteran Aeshna cyanea (Aeshnidae) and the zygopteran Ischnura elegans (Coenagrionidae), by carrying out parallel investigations under SEM and TEM. In both species these coeloconic sensilla are innervated by four unbranched neurons forming four outer dendritic segments enveloped by the dendrite sheath. One dendrite terminates at the base of the peg forming a well developed tubular body, while the other three enter the peg after interruption of the dendrite sheath. The cuticle of the peg shows an apical pore and a joint membrane. This last feature, together with the tubular body and the suspension fibers, represent the mechanosensory components of the sensillum while the pore and the dendrites entering the peg allow chemoreception. The ultrastructural organization of these coeloconic sensilla is in agreement with the one reported for insect gustatory sensilla. Our investigation describes for the first time typical insect gustatory sensilla in Odonata. Electrophysiological and behavioral studies are needed to verify the role that these structures can perform in sensing the egg-laying substrata.

Evolution of insect arylalkylamine N -acetyltransferases: Structural evidence from the yellow fever mosquito, Aedes aegypti

Arylalkylamine N-acetyltransferase (aaNAT) catalyzes the transacetylation from acetyl-CoA to arylalkylamines. aaNATs are involved in sclerotization and neurotransmitter inactivation in insects. Phyletic distribution analysis confirms three clusters of aaNAT-like sequences in insects: typical insect aaNAT, polyamine NAT-like aaNAT, and mosquito unique putative aaNAT (paaNAT). Here we studied three proteins: aaNAT2, aaNAT5b, and paaNAT7, each from a different cluster. aaNAT2, a protein from the typical insect aaNAT cluster, uses histamine as a substrate as well as the previously identified arylalkylamines. aaNAT5b, a protein from polyamine NAT -like aaNAT cluster, uses hydrazine and histamine as substrates. The crystal structure of aaNAT2 was determined using single-wavelength anomalous dispersion methods, and that of native aaNAT2, aaNAT5b and paaNAT7 was detected using molecular replacement techniques. All three aaNAT structures have a common fold core of GCN5-related N-acetyltransferase superfamily proteins, along with a unique structural feature: helix/helices between β3 and β4 strands. Our data provide a start toward a more comprehensive understanding of the structure-function relationship and physiology of aaNATs from the mosquito Aedes aegypti and serve as a reference for studying the aaNAT family of proteins from other insect species. The structures of three different types of aaNATs may provide targets for designing insecticides for use in mosquito control.

Molecular Characterization, cDNA Cloning and Phylogenetic Analysis of Cecropin Gene Isolated from Bacterial-challenged Cotton Leafworm, Spodoptera littoralis

Experiments were designed and conducted to isolate and characterize the full length cDNA of cecropin gene from the cotton leafworm, Spodoptera littoralis. In addition, sequence and phylogenetic analyses of this gene were studied. Using differential display technique, a typical cationic insect cecropin gene (SpliCec) was isolated from bacterial-challenged S. littoralis haemolymph. Five differentially displayed bands were sequenced. Based on sequence analyses of the data, specific primers for the full length of cecropin were designed and successfully amplified 388 bp amplicon. The integration of the results revealed that the 388 bp-PCR product has one open reading frame (orf) of 186 bp long, including both start codon (AUG) and stop codon (UGA). The deduced amino acid sequence of SpliCec indicated that the full length prepropeptide consists of a 22-residue signal peptide, a dipeptide prosequence and a 38-residue mature peptide. The SpliCec sequence created significant similarity with many insect cecropin sequences, especially those of M. sexta and A. convolvuli.

An FXPRLamide neuropeptide induces seasonal reproductive polyphenism underlying a life-history tradeoff in the tussock moth.

The white spotted tussock moth, Orgyia thyellina, is a typical insect that exhibits seasonal polyphenisms in morphological, physiological, and behavioral traits, including a life-history tradeoff known as oogenesis-flight syndrome. However, the developmental processes and molecular mechanisms that mediate developmental plasticity, including life-history tradeoff, remain largely unknown. To analyze the molecular mechanisms involved in reproductive polyphenism, including the diapause induction, we first cloned and characterized the diapause hormone-pheromone biosynthesis activating neuropeptide (DH-PBAN) cDNA encoding the five Phe-X-Pro-Arg-Leu-NH2 (FXPRLa) neuropeptides: DH, PBAN, and α-, β-, and γ-SGNPs (subesophageal ganglion neuropeptides). This gene is expressed in neurosecretory cells within the subesophageal ganglion whose axonal projections reach the neurohemal organ, the corpus cardiacum, suggesting that the DH neuroendocrine system is conserved in Lepidoptera. By injection of chemically synthetic DH and anti-FXPRLa antibody into female pupae, we revealed that not only does the Orgyia DH induce embryonic diapause, but also that this neuropeptide induces seasonal polyphenism, participating in the hypertrophy of follicles and ovaries. In addition, the other four FXPRLa also induced embryonic diapause in O. thyellina, but not in Bombyx mori. This is the first study showing that a neuropeptide has a pleiotropic effect in seasonal reproductive polyphenism to accomplish seasonal adaptation. We also show that a novel factor (i.e., the DH neuropeptide) acts as an important inducer of seasonal polyphenism underlying a life-history tradeoff. Furthermore, we speculate that there must be evolutionary conservation and diversification in the neuroendocrine systems of two lepidopteran genera, Orgyia and Bombyx, in order to facilitate the evolution of coregulated life-history traits and tradeoffs.

Low Concentrations of Salicylic Acid Stimulate Insect Elicitor Responses in Zea mays Seedlings

Salicylic acid (SA) generally is thought to suppress jasmonic acid (JA) related signaling events. However, when we treated the roots of corn seedlings overnight with low physiological concentrations of SA (50 μM), we found a priming effect of this pretreatment on typical insect elicitor (IE)-induced responses in the leaves of these plants. IE-induced JA was more than 2-fold up regulated in SA-pretreated plants. Consequently, IE-induced volatile organic compounds (VOC) release also was significantly increased. In contrast, when corn seedlings were treated with SA overnight and then mechanically damaged, we found no significant differences in JA accumulation. We also found that the application of even lower concentrations of SA (5 μM) had no significant effect on IE-induced responses, while higher concentrations (500 μM) inhibited IE-induced JA accumulation. Likewise, shorter exposure to SA did not affect subsequent JA accumulation induced by IE or mechanical wounding. These results provide evidence for the existence of non-compatible defense priming by signaling molecules that usually are involved in a conflictive defense signaling pathway and suggests common elements in the regulation of priming plant defense responses.

Identification of kinin-related peptides in the disease vector, Rhodnius prolixus

We have used an in silico approach to identify a gene from the blood-gorging vector, Rhodnius prolixus, that is predicted to produce an insect kinin prepropeptide. The prepropeptide is 398 amino acids in length and can potentially produce a large number of kinin-related peptides following post-translational processing. A comparison with other insect kinin precursor sequences demonstrates greatest conservation at the C-terminal region of the kinin peptides. Multiple peptides predicted from the kinin gene are phenotypically expressed in R. prolixus, as revealed by MALDI-TOF MS MS, including 12 kinins and one kinin precursor peptide (KPP). Six of these peptides are characterized by the typical insect kinin C-terminal motif FX 1X 2WGamide and five of these are also found as truncated forms. Five peptides were identified with an atypical, though similar, FX 1X 2WAamide C-terminus. There is also peptide with a C-terminal DDNGamide motif and a number of non-amidated peptides.

Insectopedia (review)

Reviewed by: Insectopedia Diane M. Rodgers Hugh Raffles , Insectopedia. New York: Pantheon Books, 2010, 465 pp. This is not your typical insect encyclopedia. The A through Z compendium does not start off predictably with cataloging the life cycle of A-ants, B-bees, and C-crickets respectively—although all of these are mentioned in the book. Rather, we begin with A-air, B-beauty, and C-Chernobyl. And it is this that makes Hugh Raffles' Insectopedia refreshing to read, as the associations he makes with insects are always surprising. Raffles implores us to begin to see the small world of insects within the very first entries on Air and Beauty. But this is just as much a book about humans and their culture as it is about insects, beginning with the chapter on Chernobyl, while taking up the topic of insects mutated by radiation, human social issues are explored. Controversies about the scientific determination of safe exposure to radiation levels are discussed, along with Cornelia Hesse-Honegger's project of recording the effects of radiation on insects by painting the mutations. We learn of her unique artistic method as well as her trials with the nuclear industry and some members of the scientific community. [End Page 701] Art, politics, and the "conjoined fate" of humans and other species are all interwoven into one complex piece. A similar chapter, "The Sound of Global Warming," introduces us to David Dunn's sound recordings of bark beetles and their connection to the larger issue of climate change. Where the reader might have found crickets under C, instead there is an extensive exploration of crickets in China under G for "Generosity (the Happy Times)." Here is a good example of the explicit connection of insects to human culture that Raffles has in mind throughout the book. Insects are not categories, and they are "not merely the opportunity for culture but its co-authors" (100). While Raffles' explanation of the world of cricket fighting attains the thick description of Clifford Geertz's anthropological look at cock fighting in Balinese culture, he is after something other than an ethnography of cultural practices. This chapter, as is Raffles' intent for the book overall, is meant to impel readers to consider "relationships with other beings that accepts that they are both like and unlike ourselves, not in some generalized abstract way, but in quite particular respects that provide grounds for connections and empathies as well as points of utter disconnection" (114). The individuals, groups, and topics Raffles' features in the book all serve to reinforce this point. Two of the entomologists of whom he devotes significant space, Karl von Frisch (Language) and Jean-Henri Fabre (Evolution), are portrayed as champions of science; however, there is also something a bit more subjective that guides their insect studies. Although many of the scientists and non-scientists he features in the book clearly strive to understand insects, what Raffles seems to continuously conclude is that these creatures are a wonderful mystery, one that humans should not ignore, but may never understand. This attitude also saves Raffles from falling into the unreflective use of anthropomorphic terms. Although referring to the way that insects are often described scientifically and in human culture generally, Raffles also questions the accuracy of attributing human motive to insect behavior. As he recounts the scientific descriptions of the mating habits of the Empididae, in particular the gifting behavior of male balloon flies, what appears to be an exchange of sex for prey wrapped in silk is termed a "nuptial gift." When certain male balloon flies obtain sex with the female by presenting an empty silken husk or cotton ball provided by researchers, then the males are considered to be "cheating" and the female viewed as "tricked into sex." Raffles questions this evolutionary analysis: "What if we assume that the willingness [End Page 702] of many female flies to accept cotton balls indicates that rather than being 'worthless,' the objects have qualities unknown to us? Is it too obvious to point out (again) the hazards of presuming what an object is and what it does for beings whose ways of being are so different from our own?" (296). Tellingly...

Community and Species-Specific Responses of Wild Bees to Insect Pest Control Programs Applied to a Pollinator-Dependent Crop

Wild bee conservation is regarded as essential for sustainable production of pollinator-dependent crops, yet little is known about the effects on wild bee communities of typical insect pest management programs used postbloom. We developed an insecticide program risk (IPR) index to quantify the relative risk to wild bees of insecticide programs applied to blueberry fields. This was used to determine the relationship between IPR and the abundance, diversity, and richness of wild bee communities sampled during three successive flowering seasons. In 2 of 3 yr, bee abundance and species richness declined with increasing IPR. Bee diversity declined with IPR in one of 3 yr. These results indicate that wild bee communities are negatively affected by increasingly intensive chemical pest management activities in crop fields and that interyear variability in bee populations has the potential to mask such effects in short-term studies. When several wild bee species were analyzed separately, two of three solitary and one of three social blueberry-foraging species declined with increasing IPR values, suggesting that different life histories and nesting habits may help some bee populations escape the negative effects of insecticides applied after bloom. Pollinator conservation programs aimed strictly at reducing insecticide use may have varying success, depending on the biology of the target bee species. The IPR index provides a standard method to compare pest management programs for their potential effect on wild bee communities, with broad application for use in other agricultural systems.

The complete mitochondrial genome of the leafminer Liriomyza trifolii (Diptera: Agromyzidae)

Liriomyza trifolii (Diptera: Agromyzidae) is one of the most economically significant pests in the world. In this paper we present sequence data for the complete mitochondrial genome of L. trifolii. The circular genome is 16,141 bp long and contains one encoding region including 37 genes and one non-coding A+T-rich region. Gene numbers and organization is similar to that of the typical insect mitochondrial genomes except that two additional tRNA genes are found in the A+T-rich region (tRNA(Thr) and tRNA(Leu(UUR))). All of the protein initiation codons are ATN, except ND1 which begins with GTG and COI which is initiated by the quadruplet ATCA. The 22 tRNA anticodons of L. trifolii match those observed in Drosophila yakuba, and all of tRNAs form the typical cloverleaf structure except for tRNA(Ser(AGN)), which has lost the DHU-arm. The A+T-rich region of L. trifolii also contains two previously noted Diperan features--a highly conserved polyT stretch and a (TA)(n) stretch.

A biologically inspired approach to feasible gait learning for a hexapod robot

A biologically inspired approach to feasible gait learning for a hexapod robotThe objective of this paper is to develop feasible gait patterns that could be used to control a real hexapod walking robot. These gaits should enable the fastest movement that is possible with the given robot's mechanics and drives on a flat terrain. Biological inspirations are commonly used in the design of walking robots and their control algorithms. However, legged robots differ significantly from their biological counterparts. Hence we believe that gait patterns should be learned using the robot or its simulation model rather than copied from insect behaviour. However, as we have foundtahula rasalearning ineffective in this case due to the large and complicated search space, we adopt a different strategy: in a series of simulations we show how a progressive reduction of the permissible search space for the leg movements leads to the evolution of effective gait patterns. This strategy enables the evolutionary algorithm to discover proper leg co-ordination rules for a hexapod robot, using only simple dependencies between the states of the legs and a simple fitness function. The dependencies used are inspired by typical insect behaviour, although we show that all the introduced rules emerge also naturally in the evolved gait patterns. Finally, the gaits evolved in simulations are shown to be effective in experiments on a real walking robot.