Eos, t. LXII, págs. 7-22 (1986). Artificial laboratory breeding of xylophagous insect larvae and its application in cytogenetic studies 2) BY J. R. BARAGAÑO, A. NOTARIO y M. G. DE VIEDMA. INTRODUCTION HAYDAK, in 1936, managed to rear Oryzaephilus surinantensis (L.) in the la- boratory using an artificial diet. Many researchers have followed in his footsteps, so that since then, approximately 260 species of Coleoptera have been raised on nonnatural diets. Among these species there are 121 which are eminently xylophagous. They belong to seven families (Buprestidae, Elateridae, Bostrychiclae, Lyctidae, Myc- teridae, Cerambyciclae and Curculionidae). Their importance, from the economic point of view, varies widely : some of them attack living trees making them a pest ; others feed on dead or decaying wood so that they may be considered harmless or even beneficial (for example in the decomposition of tree stumps in forests) ; finally, a few cause damage to seasoned timber. Therefore, specialists in artificial breeding have been motivated by different objectives, and so have chosen the insect or insects in each case which were most suitable for obtaining specific desired results. It is clear that in the majority of cases the choice was not made at random. Generally, the insect studied was either recently established as a pest or well documented as such. •With these laboratory breeding experiments it is possible on the one hand to draw conclusions about the insects' nutritive requirements, parasitism, ethology etc ; and on the other to obtain enough specimens to try out different phytosanitary treatments with them. Both of these achievements are applicable to effectiye control of the insect problem. However, some authors, ourselves included, have followed a purer une of research towards appreciably different ends from those stated aboye. Therefore, we set aims such as: Finding the host-guest correlation in insects whose habits make their study difficult in nature. Amassing data on the morphology ami systematics of cerambycid larvae in the Iberian fauna. As DUFFY (1953) pointed out, "Whene yer the identity of a living larva is to be ascertained by rearing the adult, the necessity of individual rearing must be stressed". Individual rearing assures larvae, pupae and adults of demonstrably the same species and parenthood. Mo- reover, a guaranteed, constant supply of the different instars means that research is both easier and more reliable. 1 Research supported by a research programme CAICYT 4361/79. Hamburg, 2 Presented as a lecture at the XVII International Congress of Entomology, August 20-26, 1984. 8 J. R. BARAGASTO, A. NOTARIO, M. G. DE VIEDMA — Finding out the relationship between the immature stages and the imago. — Drawing up distribution maps. — Producing a stock of specimens on which to apply cytogenetic techniques with systematic objectives. Larval stages were chosen for our cytogenetic studies. Although our studies have not been limited to typically xylophagous forest insects (NOTARIO, 1975; BARAGASTO et al., 1982), it is true that the majority are included in this context. This study, in particular that part dealing with the rearing of the insects, deals with Coleoptera which feed as larvae on different hard and softwood trees. ARTIFICIAL DIETS FOR XYLOPHAGOUS COLEOPTERA RASMUSSEN (1956) was the first researcher who prepared an artificial diet, made up of cellulose, peptone and brewer's yeast, to rear the larvae of the cerambycid Hylotrupes bajulus (L.), although with little success. Since then, various diets have been formulated by different authors. The pioneers in rearing xylophagous Coleoptera on artificial diets in the strictest sense (NOTARIO, 1978) may be considered to be GALFORD (1969), CARLE (1969) and YEARIAN and WILKINSON (1963). GALFORD tried a mixture of products such as agar, water, cellulose, brewer's yeast, sucrose, glucose, fructose, glycine, vegetable lecithin, wheat germ, wheat germ oil, cholesterol, Wesson's salt mixture, vitamin B, Vanderzant's fortification mixture (a-tocopherol, ascorbic acid, biotin, calcium pantothenate, choline chloride, folic acid, inositol, niacinamide, pyridoxine hydrochloride, riboflavine, thiamine hydrocloride and vitamin B trituration in Mannitol), sorbic acid and methil p- hydroxybenzoate ; with this he obtained satisfactory results in ten species of Ce- rambycidae. The diet prepared by CARLE enabled him to develop the larvae of one buprestid, one mycterid, two cerambycids and four curculionids ; it consists of agar, water, cellulose, brewer's yeast, glucose, Wesson's salt mixture, ascorbic acid, benzoic acid, methyl paraben and Vanderzant's fortification mixture (the same as that used by GALFORD). To breed Ips calligraphus GERMAR (Curculionidae), YEARIAN and WILKINSON used a diet consisting of agar, water, cellulose, brewer's yeast, sucrose, choline chloride, glycine, peanut oil, cholesterol, sorbic acid, methyl p-hydroxybenzoate and vitamin diet fortification mixture (vitamin A, vitamin D, cy-tocopherol, ascorbic acid, inositol, choline chloride, menadione, p-aminobenzoic acid, niacinamide, ri- boflavin, pyridoxine hydrochloride, thyamine hydrocloride, calcium pantothenate, biotin, folic acid and vitamin B12). Components which the three diets have in common are : agar, water, cellulose, brewer's yeast, ascorbic acid, a-tocopherol, folic acid, choline chloride, inositol, niacinamide, riboflavin, pyridoxine hydrochloride, thyamine hydrochloride, calcium pantothenate, biotin, vitamin 13 12 and the antiseptic methyl p-hydroxybenzoate. Wesson's salt mixture (calcium carbonate, copper sulphate (5E1 2 0), ferric phosphate, manganous sulphate (anhydrous), magnesium sulphate (anhydrous), potassium aluminium sulphate, potassium chloride, potassium (lihydrogen phos- phate, potassium iodide, sodium chloride, sodium fluoride, tricalcium phosphate) ARTIFICIAL LABORATORY BREEDING OF XYLOPHAGOUS 9 which appears in the diets of GALFORD and CARLE was included by YEARIAN and WILKINSON in 1965 for the rearing of Ips calligraphus GER., 1. grandicollis EICH- HOFF and 1. avulsus EICHHOFF. Components which are not common to the three diets but which are used in one or two of them are : Glucose (GALFORD, GARLE); sucrose (GALFORD, YEARIAN and WILKINSON) glycine (GALFORD, YEARIAN and WILKINSON) ; vegetable lecithin, wheat germ, wheat germ oil and vitamin B T (GALFORD) ; cholesterol (GALFORD, YEARIAN and WILKINSON) ; peanut oil, vitamin A, vitarnin D, menadione, p-aminobenzoic acid (YEARIAN and WILKINSON) ; the antiseptic sorbic acid (GALFORD, YEARTAN and WILKINSON) and the antiseptic benzoic acid (CARLE). The reasons why most of these products have been used can be summarized as follows Agar, a compound based on galactose molecules is the vital inert substance needed to bind the molecules of water. Due to the specific eating habits of the xylophages, it is clear that cellulose must be included in their diet, since, apart from supplying traces of minerals, it also plays an important part as a phagostimulant. Research undertaken on 16 Coleoptera about their nutritive requirements of the lipogenic factors choline and inositol shovved that ten of them defini- tely needed choline and six possibly needed it ; eight did not need inositol, one did (Scolytus multistriatus MARSHAM) and the others did need it, with reserves (REcHcIGL, 1977). The need for minerals is tue aspect which has been least studied in the field of insect nutrition (REcriciGL, 1977). This has been (hie to the difficulties which appear when particular inorganic ions are excluded from synthetic diets, because this exclusion considerably alters the balance of those re- maining. We can say that, in addition to potassium, magnesium and phos- phorus, other nutrients in very small quantities are probably needed by most of the insects ; it is, however, extraordinarily difficult to avoid masking of nominal suppression due to traces present in other dietary components. Therefore, vvhat is normally done is to provide the die‘tary medium with an optimum salt mixture level (and that formulated by WESSON is the most suitable). Generally, the phosphoprotein in milk, casein, is the one most commonly used to supply the insects with their amino acid requirements, although on occasions particular amino acids have been added to the medium (e. g. glycine, glutamic acid, cystine), because casein has not been able to supply them adequately. Niacinamide's biochemical importance stems from the fact that it forms part of the pyridinucleotids, which play a fundamental part as oxygen-carrying coenzymes. Assóciated with cysteamine, pantotenic acid forms pantetheine, a component of coenzyme A. The SH radical of the cysteamine activates the acetic acid and the higher fatty acids making its metabolic function very important. Thyamine is fundamental in the oxidative decarboxylation of piruvic acid (in the degradation of carbohvdrates) and of a-k-etoglutaric acid (in the citric acid cycle). 10 J. R. BARAGAÑO, A. NOTARIO, M. G. DE VIEDMA Riboflavin acts in the transfer of hydrogen to the cytochrome C or directly reduces molecular oxygen. Pyridoxine, working closely together with pyridoxarnine phosphate plays a part in the metabolism of the amino acids. The importance of folie acid lies in the incorporation of monocarbon com- ponents within other molecules. Biotin affects a large number of carboxylation and decarboxilation reactions. Vitamin B12 probably functions on the insects as a growth factor. Ascorbic acid, while of no dietetic value to the insects, is useful