Bull. Org. mond. Sante 1 1962, 27, 483-489Bull. Wid Hith Org.

The Identification of the Larvae of African Simulium*R. W. CROSSKEY 1

Identification of the larval stages of African Simulium-vectors of onchocerciasis-isbecoming increasingly important. In this paper attention is drawn to the principal morpho-logical structures that are of value for identification and illustrations are given to indicatethe various characters mentioned. The author provides an identification key to the larvaeof the different species-groups of African Simulium and to the larvae of those species ofmost importance to man, and also presents a tentative key by which a larva may be placedin the probable instar of its development.

With the growing interest in the Simuliidae as thevectors of onchocerciasis it becomes increasinglyimportant to be able to identify the larvae of theAfrican species of Simulium s.l., and to be able todecide not only to what species or species-groupany particular larva may belong, but also in whichinstar of its development it may be. Until recentlythis has appeared to be a very intractable problem(the monograph of Freeman & de Meillon (1953)deals almost solely with adult flies and pupae), andlarval identification has been regarded as almostimpossible except in those cases where the larvaewere associated with other arthropods or occurredin conspecific masses with the pupae of a singlespecies.Recent taxonomic study of the larvae of African

species (Crosskey, 1960) has, however, shown thattheir specific recognition, at least in the case of themore commonly collected species, is not alwaysespecially difficult, and that many good larvalcharacters exist for their separation into species-groups, corresponding in the main to those basedon adult and pupal characters by Freeman &de Meillon (op. cit.). There are, though, in somegroups examples of " species-pairs ", in which it isdifficult to find really good and constant larvalcriteria for species separation (e.g., S. unicornutumPomeroy and S. cervicornutum Pomeroy; S. voraxPomeroy and S. colas-belcouri Grenier & Ovazza).On the other hand, one or two individual speciesare clearly recognizable, at any rate in medium-sized or mature larvae, by certain apparently unique

* Paper presented at the Second WHO Conference onOnchocerciasis, Brazzaville, June 1961.

1 Dipterist, Commonwealth Institute of Entomology,London, England.

characters which they do not share with otherAfrican species; for instance, the setae on the prolegin S. damnosum; the combination of fan-shapedscales and simple setae on the abdominal cuticlein S. griseicolle Becker; the straight " cut-off "cephalic fans in S. copleyi Gibbins; and the extra-ordinary hypostomium in S. berneri Freeman. Somegroups are better characterized than others, and theruficorne-group may be instanced as a particularlydistinctive group: this group differs from all othersin possessing the combination of dark head-spotsand ventral abdominal papillae.The objectives of the present contribution are,

first, to draw attention to certain important larvalcharacters through the presentation of an identifica-tion key to the larvae of the different species-groupsand the most important species, and, secondly, toindicate the lines along which it appears that itmight be possible to determine the instar of anylarva. The meaning of the terms employed for larvalstructures is apparent from the accompanying figures.Most larval characters vary during larval life.

The number of rays in the cephalic fans, the numberof hooklet rows in the posterior and proleg circlets,the number of antennal segments, and the numberof secondary lobules in the anal gills all increaseduring larval development; after the attainment ofthe four-segmented condition the antennal segmentscontinue to change their relative lengths. But twocharacters, the form of the hypostomium and theshape of the postgenal cleft, appear to remain fairlyconstant during maturation of the larva, althoughthe actual size of these structures naturally increasesin successive instars as the head-capsule grows. Theshape of the postgenal cleft is perhaps the mostvaluable single character in the taxonomy of the

1182 -483

484 R. W. CROSSKEY

FIG. 1-3LARVAL STRUCTURES OF SIMULIUM

A5D0INAL

ORGAN~~~ ~ ~ ~ ~ ~ ~ ~ ~ ACSSR ETL OTRO

LEG

bSSCLERITE PAPILLA CIRCTI.

CEPHA IC PAN

NYPOSVOMIUM

POC AL CERVICAL SCLERITS OCPUT

2 3

1. Larva In left lateral view.2. Larval head, ventral view.3. Larval head, dorsal view.In Fig. 2 and 3 only one of the cephalict anal a shown.

African Simulium larvae, and may prove useful indetermining even the smallest larvae; in combinationwith hypostomial structure (and perhaps othercharacters as yet undiscovered), it may ultimatelyenable us to identify the very young larvae, at leastas far as species-group if the actual species cannotbe certainly determined. Thus intensive study ofyoung larvae should make it possible to separatethose of unimportant harmless species from thoseof the important species, as can already be donewith later-stage or fully grown larvae.

It is especially important to be able to distinguishthe very early larvae of the S. neavei Roubaudcomplex from those of other species, since it appearsfrom the recent work of Williams, Hynes &Kershaw,' that the earliest instars in S. neavei maybe non-phoretic. Williams et al. (op. cit.) suggestthat very young larvae-those less than about one-third grown-in both the neavei-complex and the

1 Williams, T. R., Hynes, H. B. N. & Kershaw, W. E.(1961) Freshwater crabs and Simulium neavei (Unpublishedworking document AFR/Onchocerciasis (1961)/A-2).

IDENTIFICATION OF THE LARVAE OF AFRICAN SIMULIUM

FIG. 4-7THE HYPOSTOMIUM IN AFRICAN SIMULIUM

4 5*

6 7

4 & 5. The type of hypostomlum In the vast majorlty of species.6. The unique hypostomium of S. berneri.7. The type of hypostomlonm found only in S. neavel and its allies.Flg. 4, 5 and 6 are reproduced by permission of the Trustees of the British Museum (Natural History).

S. copleyi Gibbins complex (in which there is a

phoretic association with mayflies) live attached tostones; hence they could be confused with younglarvae of normal " free-living " non-phoretic species.However, from intensive study of very young larvaefrom stones or other possible substrata in known" neavei streams " it might prove possible to dis-tinguish the very young neavei-complex larvae inthe non-phoretic stages by means of the hypo-stomium. As mentioned above, this character appears

to remain more constant than most other charactersas the larva grows (see, for example, the figures ofthe hypostomium in each of the seven larval instarsof,S. damnosum given by Grenier & Feraud (1960)),and consequently might be expected to have a

similar form in the young non-phoretic neaveilarvae to that in the larger larvae after attachmentto the crabs has occurred. Later-stage larvae of theneavei-complex, i.e., those attached to crabs, allpossess a hypostomium of characteristic shape and

I

485

486 R. W. CROSSKEY

usually with a rather even row of 13 apical teeth(Fig. 7), and this is true of the smallest larvae whichI have been able to find from crabs in the BritishMuseum collection-larvae probably in the third orfourth instar (assuming, as discussed below, thatthere are six or seven instars in African simuliidlarvae). The neavei type of hypostomium is foundonly in larvae of the neavei-complex species, anddoes not occur in other African Simuliidae; thus itsfinding in a larva is diagnostic for this complex, and

should it occur in very young non-phoretic neaveilarvae would serve to separate these from otherspecies living in the same habitat.

In order that the larger and fully mature larvaeof African Simulium can be placed in their species-groups, the following key has been prepared; a keyto the larvae of the 21 species occurring in WestAfrica has been given elsewhere (Crosskey, 1960),and can be used to place most of the common EastAfrican species also.

KEY TO THE LATER-STAGE LARVAE OF THE SPECIES-GROUPSAND PRINCIPAL SPECIES OF AFRICAN SIMULIUM'

1. Hypostomium of unique form, as in Fig. 6. .......... S. berneri FreemanHypostomium not of this form . . . . . . . . . . . . . . . . . . . . 2

2. Hypostomium with a rather even row of 13 apical teeth and shaped much asin Fig. 7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S. neavei-complexHypostomium with 9 apical teeth and shaped as in Fig. 4 or 5 (slightlydifferent in some rare copleyi-complex species) . . . . . . . . . . . . . . 3

3. Thorax as well as abdomen with scales or setae . . . . . . . . . . . . . 4Thorax bare, abdomen with or without sparse scales or spines dorsally . . . 6

4. Proleg with flattened setae. Anterior abdominal segments with weak or verystrong dorso-lateral conical protuberances. Head-capsule with some strongpigmentation and spots. Length of mature larva, 5.0-6.5 mm . . . . . . . S. damnosum TheobaldProleg bare. Abdominal segments normal. Head-capsule unpigmented. Lengthof mature larva, 3.75-4.25 mm . . . . . . . . . . . . . . . . . . . . 5

5. Abdominal cuticle with fan-shaped scales and simple spine-like setae. Postgenalcleft very large and rounded, occupying most of ventral surface of head . . S. griseicolle BeckerAbdominal cuticle with one type of flattened scale-like setae. Postgenal cleftsmaller and not strongly rounded . . . . . . . . . . . . . . . . . . . S. albivirgulatum

Wanson & Henrard

6. Head-capsule with distinct pattern of dark head-spots and abdomen withventral papillae . . . . . . . . . . . . . . . . . . . . . . . . . . . S. ruficorne-groupHead-capsule with pale head-spots surrounded by variable darker pigmenta-tion, or if with dark head-spots then abdomen without ventral papillae . . . 7

7. Ventral papillae present (sometimes weak in hirsutum-group). Head-capsulewith negative pattern of pale head-spots with at least traces of dark pigmenta-tion around them. Rows of hypostomial setae lying parallel to lateral marginsof hypostomium (Fig. 5) . . . . . . . . . . . . . . . . . . . . . . .

In using the key the following points should be noted: the hirsutum-group means the free-living forms and excludes theneavei and copleyi complexes; S. schoutedeni and S. mcmahoni are regarded as belonging in the cervicornutum-group and theirlarvae would key out to this group; the copleyi-complex has been omitted from this simplified key, but occurs only so far as isknown normally on mayfly nymphs.

II*NTIFICATION OF THE LARVAE OF AFRICAN SIMULIUM

Ventral papillae absent. Head-capsule with positive pattern of dark head-spots, sometimes the dark spots indistinct among general very dark pigmenta-tion (head rarely unpigmented as in S. bovis de Meillon). Rows of hypostomialsetae diverging posteriorly from lateral margins of hypostomium (Fig. 4)

8. Abdominal cuticle dorsally with large erect scales. Last abdominal segmentwith at least traces of a small accessory sclerite on either side in front ofposterior circlet.

Abdominal cuticle dorsally on the last few segments with branched fan-shapedscales or with a few simple spine-like setae. Last abdominal segment withoutaccessory sclerites.

9. Abdominal cuticle with branched fan-shaped scales

Abdominal cuticle with minute simple setae .

10. Dark brownish head-spots distinct from the creamy ground colour of the head-capsule. Abdominal cuticle bare. Postgenal cleft normally small so that thepostgenal bridge is longer than the hypostomium.Dark head-spots usually indistinct among the general dark pigmentation ofthe head-capsule (rarely head-capsule unpigmented). Abdominal cuticle withat least a few minute spines on last segments or with scattered scales (as inS. bovis). Postgenal cleft large and postgenal bridge shorter than the hypo-

S. cervicornutum-group

9

S. alcocki-group

S. hirsutum-group

S. dentulosum-group

stomium ....... medusaeforme-group

The number of larval instars in the African speciesof Simulium is in need of investigation, particularlyin S. neavei. The number is known with certaintyonly in S. damnosum, in which there are seveninstars including the so-called " mature " larva withblackened gill-spot (Grenier & Feraud, 1960). InArmenia it has been established by Terterjan (1957)that there are six instars in the larva of Simulium(Wilhelmia) paraequinum Puri, and it seems likelythat six is the usual number in most Simuliidae.According to Hinton (1958) the apparent fullymature larva is really a " pharate pupa ", havingundergone the larval-pupal moult but not havingsloughed off the last larval skin; however, for allpractical purposes the pharate pupa has to beregarded as the fully mature larva, and the stageregarded for convenience as the last larval instar.In the African species, presuming that there are

normally six or seven instars, moulting must occur

at very short intervals if the estimates of the lengthof larval life are correct (in S. damnosum, for instance,larval life is variously estimated as from 6-13 days).With six or seven instars in a period of one weeka larva must undergo an ecdysis about once a day.If the larval life is as short as is generally supposedwith tropical African species and there are normally

at least six instars, it is curious how infrequentlylarvae are observed casting their old cuticles and howinfrequently cast skins are seen, considering the largemasses in which the larvae of many species occur.The work of Terterjan (1957) and Grenier &

Feraud (1960) shows exact agreement in many ofthe more important findings, such as that the larvalantenna first attainsits four-segmented structure atthe third instar, is three-segmented in the secondinstar and two-segmented when first hatched fromthe egg (in all cases the last segment is minute andin the first instar the antenna appears superficiallylike a simple shaft). The first instar is alwaysrecognizable from the presence of the sclerotizedegg-burster on the dorsal surface of the head. Inthe later instars it is more difficult to be sure whatstage has been reached, but the pale buds on thesides of the thorax showing the developing legs,wings, halteres, and pupal respiratory organ appearto give some clue. The following key has beendrawn up in an attempt to place any larva in itscorrect instar; it is based on the presumption ofthere being at least six instars as a general rule, andmust be regarded as only a preliminary guide, whichmay require considerable modification as morebecomes known.

10

487

488 R. W. CROSSKEY

TENTATIVE KEY TO THE INSTARS OF A SIMULIUM LARVA

1. Egg-burster present on the head. Antenna two-segmented .. . . . . . . . . 1st instarEgg-burster absent. Antenna three- or four-segnented . . . . . . . . . . . . . . 2

2. Antenna with three segments . . . . . . . . . . . . . . . . . . . . . . . . . 2nd instarAntenna with four segments . . . . . . . . . . . . . . . . . . . . . . . . . 3

3. Thoracic buds of future legs, wings and pupal respiratory organ undeveloped . . . 3rd instarAt least traces of such buds present . . . . . . . . . . . . . . . . . . . . . . . 4

4. Buds very small and only just differentiated. Cervical sclerites not differentiated fromthe post-occiput . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4th instarBuds large and more or less easily visible, sometimes very well developed. Cervicalsclerites differentiated at the dorsal ends of the post-occiput, but not necessarily separatedoff .................................... . 5

5. " Gill-spot " whitish, individual filaments not easily distinguishable, leg and wing budswell separated from one another. Cervical sclerites not fully separated from post-occiput 5th instar" Gill-spot " slightly or much darkened, filaments discernible, leg and wing buds not wellseparated or more or less meeting one another. Cervical sclerites isolated . . . . . . 6th-7th instar

1 Grenier & Feraud (1960) regard the antenna as originally single-segmented in the first instar, two-segmented in thesecond, and three-segmented when fully formed, but in this key the very small apical cone is regarded as a segment since mostauthors consider the fully formed antenna as having four segments.

RtSUMP2

Le role joue par les Simulides africains dans la trans-mission de l'onchocercose fait sentir le besoin de disposerd'un moyen de distinguer des le stade larvaire les diff6-rentes especes et groupes d'especes. Comme le traite deFreeman et de Meillon (1953) decrit uniquement lesformes des adultes et des pupes, il n'existait pas jusqu'adate r6cente de nomenclature des larves africaines deSimulium. L'auteur du pr6sent article a propose recem-ment (1960) une systematisation des larves des especesde l'Ouest africain qui est en partie valable pour l'en-semble de la faune africaine.A l'aide de sch6mas, I'auteur met en evidence et discute

les caracteristiques morphologiques qui sont le plusutiles pour l'identification des larves. Un tableau reunitles caract6ristiques essentielles et permet de reconnaitreau stade larvaire les diff6rents groupes d'especes et lesprincipales especes africaines de simulies (reserve faitedes larves tres jeunes qu'il n'est pas toujours possible declassifier). La configuration de la fente postgenale, etdans une moindre mesure la forme de l'hypopharynx,sont parmi les caracteristiques les plus communementutilisees en taxonomie. Tout en permettant d'identifierles larves du complexe S. neavei, l'hypopharynx reste unsigne de valeur apres la fixation des larves aux crabes 'a unstade avance de leur d6veloppement; il peut contribuer

aussi a distinguer des autres larves les minuscules larves ducomplexe neavei avant cette fixation (6tant admis, commeil semble probable, que les ceufs du complexe d'especesneavei ne sont pas d6poses sur les crabes et qu'a leurspremiers stades les larves sont incapables de vivre enassociation phoretique).

Etant donne que les larves jeunes du complexe neaveine peuvent vivre en association phoretique, il est d'autantplus important d'etre en mesure de d6terminer a quelstade de leur developpement les larves de Simuliumacquierent cette propri6te. Les travaux recents conduisenta penser qu'au cours de leur vie larvaire les simuliessubissent en principe six mues successives. II n'en restepas moins vrai qu'en pratique il est loin d'etre toujoursfacile de decider A quel stade de son evolution est parve-nue une larve donnee. Pour tenter de r6soudre cette diffi-culte, l'auteur propose une table d'evaluation qui peutservir a d6terminer approximativement le stade dedeveloppement de la larve. Ce n'est d'ailleurs la qu'unprocede provisoire qui repose sur l'hypothese qu'ilexisterait normalement six mues. Ulterieurement, desmodifications devront sans doute etre apportees au furet a mesure que sera mieux connu le developpement desvarietes africaines de simulies.

IDENTIFICATION OF THE LARVAE OF AFRICAN SIMULIUM 489

REFERENCES

Crosskey, R. W. (1960) Bull. Brit. Mus. nat. Hist. (Entom.),10, 1

Freeman, P. & Meillon, B. de (1953) Simuliidae of theEthiopian Region, London, British Museum (NaturalHistory), p. 224

Grenier, P. & Feraud, L. (1960) Bull. Soc. Path. exot.,53, 563

Hinton, H. E. (1958) Ent. monthly Mag., 94, 14Terterjan, A. E. (1957) Ent. Obozr. Mosk., 36, 860