Final Instar Larvae Research Articles

Identification and morphology of a rhyacophilid caddisfly larva from Cyprus: Rhyacophila aphrodite Malicky 1975.

Rhyacophila aphrodite Malicky 1975 is a micro-endemic species of Cyprus and the only rhyacophilid caddisfly recorded on this island. This paper describes the previously unknown larva of this species. Information on the morphology of the final larval instar is given, and the most important diagnostic features are illustrated. In the context of existing Rhyacophilidae identification keys, the larva of R. aphrodite belongs to the group fitted with one single-filament gill on each side of the meso- and metathoraces and one four-filament gill on each side of abdominal segments I to VIII. In addition, a sword process is present on each anal proleg. The distal anal claw tooth is perpendicular to the longitudinal axis of the anal claw and shorter than half of the anal claw width at the distal tooth insertion.

Silencing Taiman impairs larval development in Leptinotarsa decemlineata

An exploration of novel control strategies for Leptinotarsa decemlineata is becoming more pressing given rapid evolution of insecticide resistance and rise of production loss of potato. Dietary delivery of bacterially expressed double-stranded RNA (dsRNA) is a promising alternative for management. An important first step is to uncover possible RNA-interference (RNAi)-target genes effective against both young and old larvae. Taiman (Tai) is a basic-helix-loop-helix/Per-Arnt-Sim transcription factor that is involved in the mediation of both juvenile hormone (JH) and 20-hydroxyecdysone (20E) signaling. In the present paper, we found that continuous ingestion of dsTai for three days by third (penultimate)-instar larvae caused approximately 20% larval mortality and 80% pupation failure. The larval lethality resulted from failed cuticle and tracheae shedding, which subsequently reduced foliage consumption and nutrient absorption, and depleted lipid stores. In contrast, pupation failure derived from disturbed JH and 20E signals, and disordered nutrient homeostasis including, among others, inhibition of trehalose metabolism and reduction of chitin content. Knockdown of LdTai caused similar larval lethality and pupation impairment in second and fourth (final) larval instars. Therefore, LdTai is among the most attractive candidate genes for RNAi to control L. decemlineata larvae.

The larvae of Athripsodes longispinosus longispinosus (Martynov 1909), Athripsodes longispinosus paleochora (Malicky 1972), and Athripsodes bilineatus aegeus Malicky 1999 (Trichoptera: Leptoceridae), including a discriminatory matrix to the larvae of genus Athripsodes Billberg 1820 in Greece.

Larvae of three leptocerid caddisfly subspecies described in the present paper were sampled in Greece and the North Aegean island of Gökceada (Turkey). Information on the morphology of the final larval instar of each is given and the most important diagnostic features are illustrated. The subspecies are integrated into a synoptic discriminatory matrix including the currently known larvae of Greek species of genus Athripsodes. The species can be easily separated by head coloration; the morphology of the ventral apotome; setal and spinal patterns on the metadorsum, foretibiae, and anal prolegs; and by distribution. With respect to distribution, Athripsodes longispinosus longispinosus is known from Bulgaria, the Caucasus area, Turkey, the northern Greek mainland, and the Greek islands of Thasos, Lesbos, Andros, Ikaria, Naxos, and Rhodes. Athripsodes longispinosus paleochora is an endemic of the Greek island of Crete, and A. bilineatus aegeus has been recorded from the Peloponnese; the Greek islands of Euboea, Skiathos, Kithira, Andros; and Turkey.

Potential of South African entomopathogenic nematodes to control the leaf miner, Holocacista capensis (Lepidoptera: Heliozelidae)

The Cape grapevine leafminer, Holocacista capensis, a sporadic pest of economic importance, is found in South African table and wine grape vineyards. The cocoon casings, constructed by the final instar larvae, can be found attached to grape bunches, posing a phytosanitary risk for table grape export markets. The current study aimed to determine the susceptibility of leaf-mining H. capensis larvae to seven entomopathogenic nematode (EPN) species belonging to Steinernematidae and Heterorhabditidae. To determine nematode virulence occupied leaf-mining galleries were extracted from infested leaves and inoculated with 200 infective juveniles (IJs) in 50 μl of distilled water, for each of the EPNs screened. Concentration assays were conducted and and lethal dose was determined for the three most virulent species (Heterorhabditis baujardi, H. indica and H. noenieputensis), using concentrations of 0, 25, 50, 100, 200, and 400 IJs/leaf-mining larva. High mortality of leaf-mining larvae was obtained with H. baujardi (92%), H. noenieputensis (85%) and H. indica (83%). Almost double the number of H. noenieputensis (34 nematodes/insect) penetrated the insect larvae, in comparison with the other two EPNs. However, the relative potency of H. baujardi was 3.56 times higher than for H. indica, whilst that of H. indica was 2.57times higher than it was for H. noenieputensis. The results obtained in the laboratory were found to be encouraging, especially with regard to the nematodes’ ability to penetrate the leaf-mining galleries, and to infect the larvae successfully.

Functional characterization of ultraspiracle in Leptinotarsa decemlineata using RNA interference assay.

A heterodimer of ultraspiracle (USP) and ecdysone receptor (EcR) mediates 20-hydroxyecdysone (20E) signalling cascade to regulate insect moulting and metamorphosis. However, at least two questions remain to be addressed in terms of the molecular importance of USP in insect species. First, is USP involved in both regulation of ecdysteroidogenesis and mediation of 20E signalling in non-drosophilid insects, as in Drosophila melanogaster? Second, does USP play any role in larval metamorphosis except as the partner of heterodimeric receptor to activate the downstream 20E signalling genes? In this paper, we found that RNA interference (RNAi) of LdUSP in the final (fourth) instar larvae reduced the messenger RNA levels of four ecdysteroidogenesis genes (Ldspo, Ldphm, Lddib and Ldsad) and 20E titre, and repressed the expression of five 20E signal genes (EcRA, HR3, HR4, E74 and E75) in Leptinotarsa decemlineata. The LdUSP RNAi larvae remained as prepupae, with developing antennae, legs and discs of forewings and hindwings. Dietary supplement with 20E restored the expression of the five 20E signal genes, but only partially alleviated the decreased pupation rate in LdUSP RNAi beetles. Knockdown of LdUSP at the penultimate (third) instar larvae did not affect third-fourth instar moulting. However, silencing LdUSP caused similar but less severe impairments on pupation. Accordingly, we propose that USP is undoubtedly necessary for ecdysteroidogenesis, for mediation of 20E signalling and for initiation of metamorphosis in L.decemlineata.

The larva of Plectrocnemia renetta Malicky 1975 (Trichoptera, Polycentropodidae), including a discriminatory matrix to the larvae of Plectrocnemia Stephens 1836 species of Greece.

This paper describes the previously unknown larva of Plectrocnemia renetta Malicky 1975. Information on the morphology of the final instar larva is given and the most important diagnostic features are illustrated. A preliminary discriminatory matrix for the Greek larvae of Plectrocnemia Stephens 1836 is also provided. Plectrocnemia renetta and P. conspersa conspersa (Curtis 1834) belong to the group where the inner and outer dorsal secondary setae on abdominal segment IX are strongly different in length. These two species can be separated from each other by the arrangement of muscle attachment spots on the head capsule, number and length of setae on abdominal sternum IX, and by distribution patterns. With respect to zoogeography, Plectrocnemia renetta has been reported from Cyprus, Turkey, and from the Greek islands of Ikaria and Samos.

Ecdysone receptor isoforms play distinct roles in larval-pupal-adult transition in Leptinotarsa decemlineata.

A heterodimer of two nuclear receptors, ecdysone receptor (EcR) and ultraspiracle, mediates 20‐hydroxyecdysone (20E) signaling to modulate many aspects in insect life, such as molting and metamorphosis, reproduction, diapause and innate immunity. In the present paper, we intended to determine the isoform‐specific roles of EcR during larval–pupal–adult transition in the Colorado potato beetle. Double‐stranded RNAs (dsRNAs) were prepared using the common (dsEcR) or isoform‐specific (dsEcRA, dsEcRB1) regions of EcR as templates. Ingestion of either dsEcR or dsEcRA, rather than dsEcRB1, by the penultimate (3rd) and final (4th) instar larvae caused failure of larval–pupal and pupal–adult ecdysis. The RNA interference (RNAi) larvae remained as prepupae, or became deformed pupae and adults. Determination of messenger RNA (mRNA) levels of EcR isoforms found that LdEcRA regulates the expression of LdEcRB1. Moreover, silencing the two EcR transcripts, LdEcRA or LdEcRB1 reduced the mRNA levels of Ldspo and Ldsad, and lowered 20E titer. In contrast, the expression levels of HR3, HR4, E74 and E75 were significantly decreased in the LdEcR or LdEcRA RNAi larvae, but not in LdEcRB1 depleted specimens. Dietary supplement with 20E did not restore the expression of five 20E signaling genes (USP, HR3, HR4, E74 and E75), and only partially alleviated the pupation defects in dsEcR‐ or dsEcRA‐fed beetles. These data suggest that EcR plays isoform‐specific roles in the regulation of ecdysteroidogenesis and the transduction of 20E signal in L. decemlineata.

Disruption of ecdysis in Leptinotarsa decemlineata by knockdown of chitin deacetylase 1

Abstract Chitin deacetylases (CDAs) catalyze N-deacetylation of chitin, a crucial process for chitin modification. In the present paper, LdCDA1 was identified in Leptinotarsa decemlineata. It was copiously expressed in larval foregut, hindgut and epidermis. Just before the molt in the first, second and third larval instars, the mRNA levels of LdCDA1 were high. In the fourth (final)-instar larvae, a peak occurred 4 days after ecdysis. In vivo results revealed that LdCDA1 transcriptionally responded, positively and negatively respectively, to 20-hydroxyecdysone and juvenile hormone titers. Moreover, knockdown of LdCDA1 significantly reduced foliage consumption, lengthened developing period and prevented growth in the final instar larvae. Three distinct lethal phenotypes were noted in the LdCDA1 RNAi larvae. About 30% of the RNAi larvae became moribund and finally died; approximately 50% of deformed pupae died as pharate adults; and around 20% of LdCDA1 depleted pupae finally emerged as abnormal adults and eventually died within 1 week after emergence. Furthermore, chitin content was low and the mRNA levels of five chitin biosynthesis transcripts (LdUAP1, LdUAP2, LdChSAa, LdChSAb and LdChSB) were significantly declined in the LdCDA1 RNAi larvae. In addition, glucose, trehalose and glycogen contents were increased in the LdCDA1 depleted hypomorphs, along with highly expressed genes coding for trehalose and glycogen synthesis enzymes. The findings provide a compelling piece of evidence that CDA1 is critical for chitin deposition in L. decemlineata. Moreover, LdCDA1 may be a potential target for control of the larvae.

Further studies on Melitaea arduinna (Esper, [1873]) (Lepidoptera: Nymphalidae), with observations on early stages and host-plants, from the Levant populations in Upper Galilee and the Samarian Desert, Israel

The life history and host-plants of two populations of Melitaea arduinna from Israel are described and illustrated. Their final instar larvae, adult wing morphology and wingspans are compared with those of the same species from Russia, the Caucasus and the Balkans. The taxonomic position of the Levant populations is discussed.

Developmental plasticity in metabolism but not in energy reserve accumulation in a seasonally polyphenic butterfly.

The evolution of seasonal polyphenisms (discrete phenotypes in different annual generations) associated with alternative developmental pathways of diapause (overwintering) and direct development is favoured in temperate insects. Seasonal life history polyphenisms are common and include faster growth and development under direct development than in diapause. However, the physiological underpinnings of this difference remain poorly known despite its significance for understanding the evolution of polyphenisms. We measured respiration and metabolic rates through the penultimate and final larval instars in the butterfly Pieris napi and show that directly developing larvae grew and developed faster and had a higher metabolic rate than larvae entering pupal diapause. The metabolic divergence appeared only in the final instar, that is, after induction of the developmental pathway that takes place in the penultimate instar in P. napi. The accumulation of fat reserves during the final larval instar was similar under diapause and direct development, which was unexpected as diapause is predicted to select for exaggerated reserve accumulation. This suggests that overwinter survival in diapause does not require larger energy reserves than direct development, likely because of metabolic suppression in diapause pupae. The results, nevertheless, demonstrate that physiological changes coincide with the divergence of life histories between the alternative developmental pathways, thus elucidating the proximate basis of seasonal life history polyphenisms.

Revised taxonomic status of Pseudalmenus barringtonensis Waterhouse, 1928 stat. rev. (Lepidoptera: Lycaenidae): uncovering Australia’s greatest taxonomic fraud

The Australian endemic lycaenid Pseudalmenus H.H. Druce, 1902 occupies a unique phylogenetic position within the Theclinae–Polyommatinae assemblage. Although the genus exhibits complex geographic variation, it has long been considered to be monotypic. However, evidence from adult phenotype (colour pattern), immature stages (final instar larva) and ecology (ant specificity) (total of 10 unique character states) as well as limited genetic data (mitochondrial COI) suggest that there are two species, namely, P. chlorinda (Blanchard, 1848) from Tasmania and the mainland of south-eastern Australia and P. barringtonensis Waterhouse, 1928 stat. rev., which is allopatric and narrowly restricted to montane areas in northern New South Wales. Examination of the ‘holotype’ male of P. barringtonensis in the Australian Museum showed that it is a fake, although the data label is genuine; the specimen is actually P. chlorinda chloris Waterhouse & Lyell, 1914 that has been modified with red paint to resemble P. barringtonensis. The true holotype is currently missing, but a specimen in the Australian Museum (registration No. K199026) that is part of the Colin W. Wyatt Theft Collection with a fictitious label is almost certainly the true holotype of P. barringtonensis. We discuss the history of this most unusual and bizarre circumstance and conclude that Wyatt stole the holotype sometime in 1946 before he returned to England (~72 years ago) and fabricated the fake holotype as a replacement specimen. Such a fraudulent and unprecedented act surely ranks as Australia’s greatest taxonomic fraud.

The larva of Neoneura confundens Wasscher and van't Bosch, 2013 (Odonata, Coenagrionidae) and key to the larvae of genus.

The beautifully colored damselflies included in Neoneura Selys are divided in 28 species known from North, Central and South America. Larval stage is little known, only seven species were described at this stage. We describe and illustrate the final instar larva of Neoneura confundens for the first time. Adults associated to this larva correspond to the blue form of the species and are also discussed and illustrated. The larva of N. confundens is similar to other Neoneura larvae, showing 1 premental seta and a well-marked nodus in caudal lamellae, but it can be differentiated by having fringed posterior margin in all tibiae and in middle and hind tarsi, among other characters. A key to known larvae of Neoneura and new records extending the species range in the southern cone are provided.

Effect of host plant on growth, tissue proteins and gut enzymes of pink borer Sesamia inferens

In this study, pink borer Sesamia inferens (Walker), a polyphagous insect and a major pest on millets is explored for analysing the influence of host plants on the growth, tissue proteins and midgut digestive enzymes of their larvae. The impact of feeding on different food plants on the activity of amylase, trehalase, and invertase in the midgut of the final instar larvae were assayed in vitro. The results showed that the larval feeding on the five millet hosts had significant effect on body mass, with maximum body mass being achieved when larvae fed on sorghum (0.214 g). Significantly more enzyme activity of amylase was observed in the larvae fed on finger millet. The activity of amylase, invertase and trehalase fed on pearl millet was relatively low. The results indicated that the larvae of S. inferens had more capability to utilize sorghum, finger millet and barnyard millet, suggesting that these plants could become their host plants

Methoprene-Tolerant (Met) Is Indispensable for Larval Metamorphosis and Female Reproduction in the Cotton Bollworm Helicoverpa armigera.

Juvenile hormone (JH) represses larval metamorphosis and induces adult reproduction in insects. Methoprene-tolerant (Met) is identified as an intranuclear receptor that mediates JH actions. In the present study, we characterized a Met from the severe agricultural pest, Helicoverpa armigera, namely HaMet. In the larval stage, HaMet is predominantly expressed in the epidermis and midgut, and is upregulated before each molting, whereas in adults HaMet is maximally expressed in the ovary, testis, and fat body. The immunofluorescence assay revealed that HaMet was distributed in the longitudinal and circular muscle layers of midgut in larvae, whereas in the ovary of female adults, HaMet was localized in the nucleus of the oolemma. Knockdown of HaMet in final-instar larvae shortened the time of pupation, induced abnormal pupation, and dampened pupation rate. In female adults, HaMet depletion severely suppressed the transcription of Vitellogenin (Vg) and Vitellogenin Receptor (VgR), disrupted the Vg accumulation in fat body and the yolk protein uptake in oocytes, and finally led to an impaired fecundity. Our findings therefore confirmed that HaMet acted as a nuclear receptor of JH and played an essential role in larval metamorphosis, vitellogenesis, and oocyte maturation.

The larvae of Rhyacophila tsurakiana Malicky 1984, Rhyacophila gudrunae Malicky 1972, and Rhyacophila biegelmeieri Malicky 1984, including an update for the larval key to the Greek species of genus Rhyacophila Stephens 1836 (Rhyacophilidae, Trichoptera).

Based on adults, mature pupae, and larvae sampled at the same sites on mainland Greece and on the Greek islands of Kerkyra (Corfu) and Crete, the previously unknown larval stages of Rhyacophila tsurakiana Malicky 1984, R. gudrunae Malicky 1972, and R. biegelmeieri Malicky 1984 could be associated and identified. This material enabled us to describe and illustrate the final instar larvae of these three species, and to extract reliable diagnostic characters updating the existing larval key to the Greek species of genus Rhyacophila Stephens 1836 (Karaouzas et.al. 2015). In the context of this key, R. tsurakiana (together with R. tristis Pictet 1834 and R. obtusa Klapálek 1894) lacks abdominal gills; the three species can be separated by the shape of the dorsal sclerite of abdominal segment IX and by anal proleg morphology. Rhyacophila gudrunae is the only Greek species so far where larvae are fitted with one four-filament gill on each body side of the abdomen. Finally, R. biegelmeieri has one tufted multifilament gill on each body side and can be separated from four other species sharing this gill morphology by coloration patterns and head widths; however, separation from R. nubila Zetterstedt 1840 and R. palmeni McLachlan 1879 is not yet possible. Rhyacophila tsurakiana and R. biegelmeieri are large-scale endemic caddisfly species of European Ecoregion 6 (Hellenic Western Balkan), whereas R. gudrunae is a micro-endemic species of this region and restricted to the island of Crete (Malicky 2005b; Graf et al. 2008).

Effects of larval diapause and the juvenile hormone analog, fenoxycarb, on testis development and spermatogenesis in the wax moth, Galleria mellonella (Lepidoptera: Pyralidae)

Facultative diapause in the wax moth, Galleria mellonella, occurs in the final larval instar. Application of juvenile hormone analogs (JHAs) to the larvae of this species has similar effects to diapause, in terms of prolonged development of the larval stages and the arrest in the metamorphosis of internal organs. Here, we focus on testes development and spermatogenesis at the end of larval development in G. mellonella, how they are affected by diapause induced by an environmental decrease in temperature to 18°C and the application of a JHA (fenoxycarb) to larvae. Because neither testis development nor spermatogenesis are described in detail for this species, we examined them in individuals not in diapause during the period from the last larval instar to the newly emerged adult and present a timetable of changes that occur in the development of testes in this species. These observations have increased the very limited data on the course of spermatogenesis in pyralid insects. We then used these data for comparative analysis of testes in larvae from two experimental groups: individuals in diapause and those treated with fenoxycarb. The results on the general morphology testes revealed obvious degenerative changes caused by fenoxycarb (but not by diapause), including testicular wall hypertrophy and disarrangement of testicular follicles. Moreover, treatment with fenoxycarb finally resulted in the disintegration of nearly all testicular cyst-containing germ cells at different stages of spermatogenesis, a situation never previously described in the literature. In contrast, the main effect of diapause on testes was merely the degeneration of spermatocytes in the proximal regions of the testicular follicles. Finally, the TUNEL analyses, revealed that the degenerative changes in germ cells were apoptotic in character in the testes of both individuals in diapause and fenoxycarb-treated males.

Pre-emergence growth and development in the arctic midge Trichotanypus alaskensis Brundin

Developing at low mean temperatures, arctic chironomids often have prolonged larval growth yet adult emergence is typically a brief and highly synchronous event. How does a midge population achieve synchronous emergence? Under the Absolute Spring Species Hypothesis (AbSS), adult eclosion by early-emerging species may be synchronized by overwintering as fully mature larvae. Such prepupal larvae would neither feed nor grow after spring thaw, only pupate and emerge. The podonomine Trichotanypus alaskensis Brundin is an abundant midge in tundra ponds on Alaska’s Arctic Coastal Plain, and one of the earliest-emerging species in this chironomid-dominated insect community. T. alaskensis is univoltine in these arctic ponds, with most emergence from any one pond occurring within less than a one-week span during late June, typically about three weeks after pond thaw. We evaluated T. alaskensis for conformity to the AbSS model by documenting the overwintering state of this species in a tundra pond near Barrow, Alaska, then monitoring larval growth and development of the population from spring thaw to pupation. Most T. alaskensis were immature instar IV larvae when collected in late September of both 2010 and 2011, with 10-30% still in late instar III. Immediately after pond thaw in 2011, all collected larvae had imaginal disc primordia showing early stages of instar IV development. Within the first two weeks following pond thaw, most larvae had doubled their dry mass and developed into mature (prepupal) final-instar larvae. Highly synchronized emergence by T. alaskensis is not a consequence of a population overwintering as fully-mature larvae, as per the Absolute Spring Species Hypothesis. Rather, larvae in a given tundra pond appear to develop synchronously throughout the life cycle, including a period of substantial growth and rapid prepupal development between spring thaw and early-summer emergence.

Screening for Resistance Against the Sugarbeet Root Maggot, Tetanops myopaeformis (Diptera: Ulidiidae), Using a Greenhouse Bioassay.

The sugarbeet root maggot, Tetanops myopaeformis (von Röder) (Diptera: Ulidiidae), is a major pest of sugar beet Beta vulgaris L. (Carophyllales: Amaranthaceae)in the United States and Canada. Larval feeding on roots can reduce both stand and yield. Current management practices are heavily reliant on chemical control. However, the carbamate and organophosphate insecticides that are commonly used against T. myopaeformis are being phased out of use. Host plant resistance against this pest shows promise, but difficulties with maintaining T. myopaeformis in culture have largely limited such studies to the field. A primary objective of this study was to develop protocols for rearing a laboratory colony of T. myopaeformis that would expedite assays aimed at screening for host plant resistance. Third (final) instar larvae were collected from the field and reared to the adult stage. These laboratory-reared adults laid eggs and ultimately produced a second generation of third-instar larvae in the lab. Adult flies reared from field-collected larvae were used to examine the modality of resistance of a known resistant variety by performing no-choice and paired-choice experiments alongside a susceptible variety in the greenhouse. Paired-choice tests showed no difference in oviposition rates between the two varieties, whereas no-choice tests showed significantly greater feeding damage and abundance of larvae on the susceptible variety. For the resistant variety examined here, we observed evidence of antibiosis, not antixenosis, as the putative modality of resistance. Our laboratory and greenhouse protocols can be used to expedite the process of developing varieties with resistance to this key pest of sugar beet.

The hand of man first then Santa Rosalia’s blessing: a critical examination of the supposed criticism by Samraoui (2017)

Samraoui (J Insect Conserv. https://doi.org/10.1007/s10841-017-9966-2 , 2017) claims that he shows evidence that our conservation plan of Urothemis edwardsii has failed and that natural dispersal was the only cause of the recent rapid range expansion of the species in Northeast Algeria. Here, we show that his analysis is biased, many of his arguments are erroneous and strongly contradictory, many key studies are dismissed, and the few data used as evidence to refute our conclusions rather confirm them. We also provide data to prove that our conservation plan did not cause any harm to the source population by comparing exuviae-based estimation of population size in 2012 and 2016. We discuss the need for future monitoring and management and highlight that the recommendations of Samraoui (J Insect Conserv, 2017) are misleading, and thus are unlikely to bring us closer to an effective long-term conservation of the species in the region. Beyond our criticism, we explain why we should not dismiss the direct and indirect implications of final instar larvae translocation in successful colonization of odonates in particular, which could also be applied to aquatic insects in general.

The larvae of Sericostoma sasbaddes Malicky 2010, S. maclachlanianum Costa 1884, S. clypeatum Hagen 1864, and S. siculum McLachlan 1876 (Trichoptera: Sericostomatidae), including a preliminary key to the Sericostomatidae larvae of European Ecoregion 3 (Italy, Corsica, and Malta)

Caddisfly samples collected from Corsica, Sardinia, and the Italian mainland included the adults of Sericostoma sasbaddes Malicky 2010, S. maclachlanianum Costa 1884, S. clypeatum Hagen 1864, and S. siculum McLachlan 1876. This material, combined with the unique distribution patterns of the four species, enabled the association of the final instar Sericostoma larvae collected at the same locations. Larvae of the two species from Sardinia (S. sasbaddes, S. maclachlanianum) can be separated by the number of bifurcate setae on each pleuron VIII. The two other species are geographically well separated: S. clypeatum is the only Sericostoma species present in Corsica, whereas S. siculum is confined to the Southern Italian Provinces including Sicily. Morphological characters from the larval descriptions have been used for constructing a preliminary larval key to the hitherto known Sericostomatidae species from European Ecoregion 3 (Italy, Corsica, and Malta).