principles of systematics and nomenclature general system and phylogeny of insects systematics of Ephemeroptera

 METHODS OF ASSOCIATING LARVAE AND ADULTS OF MAYFLIES

text from the book by Kluge 2004, with additions and corrections

Mayfly systematics is based on a combination of larval, subimaginal and imaginal characters; however, larvae and winged stages (subimago and imago) are so different, that their association represents a special problem. In literature one can find many mistakes, when such association was made wrongly, so that larvae and imagoes of different species were regarded as belonging to the same species, and vice versa, that larvae of adults of the same species were described as different species. Such confusions were made not only on the species level, but on the level of supra-species taxa (genera and families) as well.

Some features of winged stages can be found in the larval stage; this helps to associate them. As in other Pterygota, wing venation is the same in larval protopterons and adult wing (Fig.37:A; 75:A). Some authors confuse venation with tracheation; tracheation strongly changes during larval development, while venation remains constant. In the larval protopteron venation represents a system of internal hypodermal channels, which can be seen on translucent slide as light lines. For this purpose it is necessary to take such larva, which is not preparing to moult to next instar or to subimago, in other case wing hypodermis with its channels is crumpled under the larval cuticle.

Mature larvae, which are preparing to moult to subimago, can be used to extract and study subimaginal features thorax sclerotization (Fig.102:AB), caudalii, sometimes genitals (Fig.84:BC) and others. Subimaginal wings extracted from mature larva, can be spread after treating by alkali. Structure of subimaginal tarsus with claws can be studied on translucent slide of total larval leg in Canadian balsam; in shortly-moulting mayflies the same slide allows to study structure of imaginal tarsus with claws as well (Fig.98:AB).

Examination of mature larva allows one to study only selected adult characters, but not all which are necessary; particularly, in many mayfly species crumpled subimaginal genital buds inside mature larvae differ from imaginal genitals so strongly, that their comparison does not allow to associate larvae and adults correctly.

In most cases exact association of larvae and imagoes can be made by rearing only. The aim of rearing is to get such specimens, each of which has: (1) exuviae of mature larva of last instar, (2) exuviae of subimago, (3) imago. In order to do this, it is necessary to collect mature larvae, put them in a cage with water, wait until larva moults to subimago, then take its larval exuviae to alcohol and put subimago to another cage without water, than wait until subimago moults to imago and put imago and its subimaginal exuviae in alcohol together with the larval exuviae. Here are given suggestions how to do it successfully.

Some larval mayflies, especially those, which normally inhabit stagnant waters, can be kept in a simple aquarium or a can, until moulting to subimago. But larvae normally inhabiting running waters, especially mountain streams, can not live in stagnant water for a long time. For all species it is best to use special water-cages made of net and placed into natural running water (not obligatory to the same stream where these larvae inhabit).

The author uses water-cages of original construction, which can be folded and packed compactly when travelling, and can be used in all kinds of running waters, including mountain streams and greatest rivers, independently of weather and water level. Here are shown three variants of such water-cage: 

The 1st variant (Fig.1:AB) has 2 frames made of aluminium attached from outside by threads to an integral cube of polyamide (kapron) net, which has 5 equal walls 4 side walls + bottom. 

The 2nd variant (Fig.1:CD) has 2 walls made of translucent plastic and 3 walls (2 side walls + bottom) made of an integral band of polyamide net.

In the both cases the net cube (either made of the polyamide net or the polyamide net and translucent plastic) continues above by a tube made of textile. Framework (made of aluminium or translucent plastic) is supplied with 4 floats made of foamy plastic, two of which are attached to the framework, and other two are removable and serve to make the whole construction rigid when ready for work. Removable bracket with a string on its top allows closing the textile part and at the same time supporting it, in order not to allow emerged subimagoes to fly away. A plummet under the water-cage prevents overturning by wind (that is especially likely in rain when the textile above is wet and heavy). A long polyamide cord serves to tie the water-cage to something on the bank.

The 3rd variant (see photo below) has bottom made of plastic kitchen-net and walls made of textile. Its construction see here.

If there is no necessity to pack water-cages compactly for travel, the water-cage can have simpler construction. In all cases it must have following details. 

The water-cage is supplied with floats in such a manner that when floating, a half of the water-cage is located in the water, and a half above the water, to allow subimagoes escape from the water. Water-cage must be not large, about 12 cm high (without the textile part) and 12 cm with: in larger water-cage it is difficult to find larval exuviae of small species, and smaller water-cage does not allow looking into it by both eyes when searching for larval exuviae and emerged adults. Walls (at least partly) are made of fine polyamide (kapron) net (cell 0.4 mm). The bottom is made of the same net, as the walls: the dust brought by water current through the walls must fall down thorough the bottom not accumulating on it. Inside the water-cage, there must be no places where larvae or their exuviae can be hidden; the framework and sutures must be outside, but not inside. No objects (stones, sand, leaves, sticks, etc.) should be putted into the water-cage; the polyamide net is a comfortable substrate for mayfly larvae, and water current brings enough food though the net for that larvae, which are not mature enough to stop feeding. The polyamide part of the water-cage is opened from above and continued by a textile tube, which has the same width as the polyamide part and subequal length. This allows to open and close the water-cage wider or narrower depending on behaviour of the emerged subimagoes, not allowing them to fly away. Cotton textile is a comfortable substrate for subimagoes. When closed, the textile part should have the form of a high roof to give more space for subimagoes, and to protect them from rain. The water-cage floating in running water should be fixed by a cord to something on the bank (tree branch, stone or something other).

Many (up to several dozens) larvae can be placed at once into the same water-cage, but it is better to use several water-cages.

It is preferable to check water-cages often, not less than three times a day, in extreme cases not less that once a day: if larval exuviae stay in water longer than one day, they are destroyed. Emerged subimagoes should be removed from the water-cage to an air-cage.

Such air-cage can be a glass tube about 10 cm length and about 2.53 cm in diameter, and is closed by cotton-wool (Fig.1:E). Inside the tube it is necessary to put a piece of paper, which is somewhat shorter than the tube and somewhat wider than its diameter; this paper must be immovably pressed to a wall of tube. It has three functions: (1) a comfortable substrate for subimago, that makes it to sit quietly and not to spend energy for flying; (2) water absorption; (3) label, on which a specimen number is written by pencil (the same number has the tube with alcohol containing larval exuviae of this specimen). It is necessary to move subimagoes from the water-cage to the air-cage, not touching them by fingers or pincer. Subimago sitting on the wall of the water-cage, can be covered by the air-cage; subimago sitting on the water surface can be moved with help of thin stick, to which it crawls if place the stick in front of subimago. It is very important to note, that glass air-cage with subimago never should be kept at direct sun light, even for a moment; it must be always in shade or in a box. Most subimagoes (apart of shortly-moulting ones) develop in about 24 hours some times less or some times more, quicker in the warm and longer in the cold.

A usual error made when rearing, is confusing of larval exuviae and adults of externally similar species when several larvae are kept together in the same water-cage. In order to avoid this, it is necessary either to use individual water-cages for each specimen (that is rather difficult), or to take out carefully all emerged subimagoes and larval exuviae each time when check the water-cage. For this purpose, it is important not to confuse exuviae of mature larvae from which subimagoes emerged, with exuviae of younger larvae, from which larvae of next instar emerged.

All stages of mayflies can be preserved in alcohol of high concentration (75% and higher). As these insects are delicate, it is better to put them into small narrow tubes full of alcohol and closed by cotton wool (see photo below), and to put these tubes into a hermetically closed glass with alcohol (see photo below): in this case tubes should not contain air bulbs, which could destroy mayflies if shaken. When you take mayflies from a cage, it is convenient to put them temporarily into large tubes (see photo below), and than move to small narrow tubes for permanent preservation. This moving material from a large tube to a small one helps to keep high concentration of alcohol. Each tube contains a label, where are written (1) place of collecting (beginning with country and such toponym which everybody can find on a map, and finishing by name of the river and village, or a distance to nearest village); (2) date (day, month, year); (3) name of the collector. Label can be written either by simple pencil (not color pencil!), or by black Indian ink (not other ink!), or on laser printer (not ink printer!). Label is a little wider than diameter of the tube and much shorter than the tube; in this case it can be putted into the tube as in photos below; at first put the label, and than insects, in order not to destroy the insect by label. 

For examination, it is useful to make slides of all parts of mayflies and their exuviae. Separated parts can be mounted in Canadian balsam; this allows to study not only cuticle, but muscles as well. Delicate translucent cuticular parts colorless wings, tergalii, subimaginal exuviae and cuticular parts treated by alkali can be mounted in glycerine or in dry condition to see better their details (in this case cover glass can be glued to the mount by Canadian balsam by sides).

cages (variants 1 and 3) and tent in pampa Yrac in Peru

cages (variant 1) close to tent near Matucana, Peru

cages (variants 1 and 3) in Amazonia

cages (variants 3 and 1) near Cayumba, Peru

male subimago of Adenophlebia sp. emerges fron larva in cage (variant 3); Rwenzori, Uganda

male subimago of boettgeri [Centroptilum] in glass tube; Rwenzori, Uganda

reared mayfly in a tube for field collecting and temporaty preservation 

reared mayfly in a tube for permanent preservation with label typed on laser printer

reared mayflies in tubes sank into a bottle with alcohol

microscopic slide in Canadian balsam with parts of one reared specimen

larval exuviae 

subimaginal exuviae

parts of imago

  

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